http://2014.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=50&target=Leighla&year=&month=2014.igem.org - User contributions [en]2024-03-28T23:54:58ZFrom 2014.igem.orgMediaWiki 1.16.5http://2014.igem.org/Team:CU-Boulder/Project/DesignTeam:CU-Boulder/Project/Design2014-10-18T00:00:31Z<p>Leighla: </p>
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==Design==<br />
<br />
To test the concept of CRISPR-Cas9 mediated phage therapy, a three step experiment was needed.<br />
<br><br />
===CRISPR-Cas Vector Synthesis=== <br />
The first step was to design a spacer RNA sequence to specifically bind within a target gene but no where else in the ''E. coli'' genome. An RNA sequence was designed to be complementary to a unique sequence found within the neomycin phosphotransferase gene. Using restriction digestion and ligation the spacer sequence in BBa_K1218011(CRISPR-Cas9 plasmid) was replaced with the new targeting spacer sequence.<br />
<br />
<br><br />
===Verification of CRISPR specificity===<br />
The second phase of the project was to transform the targeting CRISPR-Cas9 plasmid into BW23115(::kan) ''E. coli'' to verify that Cas9 can be directed to a specific sequence determined by the spacer and that double stranded breaks in the genome are sufficient to kill a bacterium. The CRISPR-Cas plasmid has been shown to effectively kill bacterial strains when expressed within targeted bacterial cells if the spacer is complementary to the target cell’s genome¹ ² ³. The specificity of CRISPR-Cas9 killing has been demonstrated to be specifically cytotoxic to a single nucleotide mutation, meaning strains of bacteria differing in a single SNP can be differentiated by the CRISPR guide RNA². However, creating an effective delivery mechanism for the CRISPR-Cas9 plasmid remains a large obstacle. <br />
<br />
<br><br />
===Phage delivery of CRISPR-Cas9===<br />
The third part of the project attempts to solve the delivery mechanism problem by using non-replicating recombinant bacteriophage. The non-replicating phage are created by infecting cells containing helper phagemids (plasmids coding for phage structural proteins), M13K07, with the CRISPR-Cas9 phagemid (a plasmid containing a phage packaging signal), BBa_K1445001. Similar projects have been published in Nature Biotechnology this year by Professor Marrafini of the Laboratory of Bacteriology at the Rockefeller University in New York¹ and Professor Timothy Lu of the MIT Microbiology Program at the Massachusetts Institute of Technology². The results of these studies are promising; however, further refinement of the phage delivery is required to increase virulency rates². <br />
<br />
<br />
<br><br />
===References:===<br />
:1. David Bikard, Chad W Euler, Wenyan Jiang, Philip M Nussenzweig, Gregory W Goldberg, Xavier Duportet, Vincent A Fischetti, Luciano A Marraffini. 2014. Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials. doi:10.1038/nbt.3043<br />
:2. Robert J Citorik, Mark Mimee, Timothy K Lu. 2014. Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases. doi:10.1038/nbt.3011<br />
:3. Gomaa AA, Klumpe HE, Luo ML, Selle K, Barrangou R, Beisel CL. 2014. Programmable removal of bacterial strains by use of genome-targeting CRISPR-Cas systems. mBio 5(1):e00928-13. doi:10.1128/mBio.00928-13.<br />
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{{Template:UCB-Footer}}</div>Leighlahttp://2014.igem.org/Team:CU-Boulder/Project/MotivationTeam:CU-Boulder/Project/Motivation2014-10-17T23:59:18Z<p>Leighla: </p>
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==Motivation==<br />
<br><br />
Since the discovery of Penicillin in 1928, antibiotics have been successfully used to treat bacterial infections. In the past several decades, bacteria have begun evolving resistances to the antibiotics. The emergence of carbapenem-resistant ''enterobacteriaceae'' has been declared a serious threat to public health by the CDC, while other pan-resistant species, such MRSA and resistant variants of tuberculosis still pose imminent risk to public health. Due to the ability of bacteria to share advantageous mutations through conjugation, antibiotic resistance is spreading rapidly between bacterial species and pressuring companies to produce new antibacterials. Unfortunately, antibiotics have a poor return on investment, encouraging many companies to abandon antibiotic research in favor of more profitable drugs. One such alternative to antibiotics is the less expensive and more adaptive treatment option of phage therapy. Whereas, antibiotics target conserved metabolic pathways that are shared by many bacterial species and results in damage to commensal bacteria in a patient, phage therapy can specifically target a species. But even this method does not provide the specificity to distinguish between individuals in a species.<br />
<br><br><br />
A new therapeutic treatment for bacterial infections is necessary. A paper by Gomaa ''et al'' showed that a type I-E CRISPR-Cas could be used to specifically target and kill bacteria. The CRISPR-Cas system consists of two important parts: the Cas endonuclease and guide RNA (gRNA). The Cas endonuclease and CRISPR gRNA form a complex that searches genomic DNA for PAM motifs with upstream sequences that share a complementary region with the gRNA. If found, the Cas cleaves the DNA, resulting in a double stranded break. The gRNA is coded for by a spacer sequence that can be modified to direct the Cas protein to a different target sequence. Goma ''et al'' found that if this spacer sequence complimented the genome of the host cell adjacent to a PAM sequence, then the DNA would be targeted by the Cas, resulting in cell death. They then designed a spacer that would target one strain of bacteria but not another. Transforming the CRISPR-Cas machinery into a mixed population of both bacteria, they showed that the strain containing the target sequence was killed while the other strain was unharmed. Their experiment demonstrated the antibacterial potential of CRISPR-Cas9 but their experimental design lacked a delivery mechanism that could be used therapeutically. <br />
<br><br><br />
To address the delivery problem posed by Gomaa ''et al'', our experiment highlights the potential for non-replicating phage to be utilized as a delivery mechanism. During the laboratory experiment phase of this project, two papers were published in Nature Biotechnology that further demonstrate the potential of this system. Professor Marrafini of the Laboratory of Bacteriology at the Rockefeller University in New York² and Professor Timothy Lu of the MIT Microbiology Program at the Massachusetts Institute of Technology³ have demonstrated the specificity and effective delivery of CRISPR-Cas phage.<br />
<br />
<br><br />
===References:===<br />
:1. Gomaa AA, Klumpe HE, Luo ML, Selle K, Barrangou R, Beisel CL. 2014. Programmable removal of bacterial strains by use of genome-targeting CRISPR-Cas systems. mBio 5(1):e00928-13. doi:10.1128/mBio.00928-13.<br />
:2. David Bikard, Chad W Euler, Wenyan Jiang, Philip M Nussenzweig, Gregory W Goldberg, Xavier Duportet, Vincent A Fischetti, Luciano A Marraffini. 2014. Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials. doi:10.1038/nbt.3043<br />
:3. Robert J Citorik, Mark Mimee, Timothy K Lu. 2014. Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases. doi:10.1038/nbt.3011<br />
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{{Template:UCB-Footer}}</div>Leighlahttp://2014.igem.org/Team:CU-Boulder/MedalTeam:CU-Boulder/Medal2014-10-17T23:15:43Z<p>Leighla: </p>
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#silver {background-image: url('https://static.igem.org/mediawiki/2014/6/63/UCB-SILVER.png')}<br />
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</style><br />
<body><br />
<div id="contentMedal"><br />
<div class="medal" id="bronze"><br />
<h2>Bronze</h2><br />
<ul><br />
<li><a href="https://igem.org/Team.cgi">Team Registration</a></li><br />
<li>Completed <a href="https://igem.org/2014_Judging_Form?id=1445">Judging Form</a></li><br />
<li>Team Wiki</li><br />
<li>Poster and Presentation are Ready for iGem Jamboree</li><br />
<li>Documented new BioBrick Parts</li><br />
<ul><br />
<li><a href="http://parts.igem.org/Part:BBa_K1445000">Part: BBa_K1445000</a></li><br />
<li><a href="http://parts.igem.org/Part:BBa_K1445001">Part: BBa_K1445001</a></li><br />
<li><a href="http://parts.igem.org/Part:BBa_K1445002">Part: BBa_K1445002</a></li><br />
</ul><br />
</ul><br />
</div><br />
<div class="medal" id="silver"><br />
<h2>Silver</h2><br />
<ul><br />
<li>Experimentally Validated BioBrick Parts</li><br />
<li>Documented the Characterization of those Parts in the "Main Page" Section of the Part's/Device's Registry Entry</li><br />
<li>New Parts have been Submitted to the iGem Parts Registry</li><br />
<ul><br />
<li><a href="http://parts.igem.org/Part:BBa_K1445000">Part: BBa_K1445000</a></li><br />
<li><a href="http://parts.igem.org/Part:BBa_K1445001">Part: BBa_K1445001</a></li><br />
<li><a href="http://parts.igem.org/Part:BBa_K1445002">Part: BBa_K1445002</a></li><br />
</ul><br />
<li>Our project has implications for safety and ethics. We described more ways in which these have been taken into consideration in the design and execution of our project on our <a href="https://2014.igem.org/Team:CU-Boulder/Notebook/Safety">Safety</a> page.<br />
</ul><br />
</div><br />
<div class="medal" id="gold"><br />
<h2>Gold</h2><br />
<ul><br />
<li>Improved the Function and Characterized Existing BioBrick Part</li><br />
<ul><br />
<li>We changed the spacer of BBa_K1218011 and characterized the specificity of the endogenous CRISPR-Cas9 system </li><br />
<li>We added the M13ori and submitted the new part <a href="http://parts.igem.org/Part:BBa_K1445001"> BBa_K1445001</a></li><br />
</ul><br />
<li><a href="https://2014.igem.org/Team:CU-Boulder/Collaboration">Collaborated</a> with CSU Fort Collins</li><br />
</ul><br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
{{Template:UCB-Footer}}</div>Leighlahttp://2014.igem.org/Team:CU-Boulder/MedalTeam:CU-Boulder/Medal2014-10-17T23:14:43Z<p>Leighla: </p>
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.medal {display: block; float: left; padding-top: 0; height: 736px; width: 300px; margin: auto; background-repeat: no-repeat;} <br />
#bronze {background-image: url('https://static.igem.org/mediawiki/2014/3/36/UCB-BRONZE.png')}<br />
#silver {background-image: url('https://static.igem.org/mediawiki/2014/6/63/UCB-SILVER.png')}<br />
#gold {background-image: url('https://static.igem.org/mediawiki/2014/3/38/UCB-GOLD.png');}<br />
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</style><br />
<body><br />
<div id="contentMedal"><br />
<div class="medal" id="bronze"><br />
<h2>Bronze</h2><br />
<ul><br />
<li><a href="https://igem.org/Team.cgi">Team Registration</a></li><br />
<li>Completed <a href="https://igem.org/2014_Judging_Form?id=1445">Judging Form</a></li><br />
<li>Team Wiki</li><br />
<li>Poster and Presentation are Ready for iGem Jamboree</li><br />
<li>Documented new BioBrick Parts</li><br />
<ul><br />
<li><a href="http://parts.igem.org/Part:BBa_K1445000">Part: BBa_K1445000</a></li><br />
<li><a href="http://parts.igem.org/Part:BBa_K1445001">Part: BBa_K1445001</a></li><br />
<li><a href="http://parts.igem.org/Part:BBa_K1445002">Part: BBa_K1445002</a></li><br />
</ul><br />
</ul><br />
</div><br />
<div class="medal" id="silver"><br />
<h2>Silver</h2><br />
<ul><br />
<li>Experimentally Validated BioBrick Parts</li><br />
<li>Documented the Characterization of those Parts in the "Main Page" Section of the Part's/Device's Registry Entry</li><br />
<li>New Parts have been Submitted to the iGem Parts Registry</li><br />
<ul><br />
<li><a href="http://parts.igem.org/Part:BBa_K1445000">Part: BBa_K1445000</a></li><br />
<li><a href="http://parts.igem.org/Part:BBa_K1445001">Part: BBa_K1445001</a></li><br />
<li><a href="http://parts.igem.org/Part:BBa_K1445002">Part: BBa_K1445002</a></li><br />
</ul><br />
<li>Our project may have implications for safety and ethics. We described more ways in which these have been taken into consideration in the design and execution of our project on our <a href="https://2014.igem.org/Team:CU-Boulder/Notebook/Safety">Safety</a> page.<br />
</ul><br />
</div><br />
<div class="medal" id="gold"><br />
<h2>Gold</h2><br />
<ul><br />
<li>Improved the Function and Characterized Existing BioBrick Part</li><br />
<ul><br />
<li>We changed the spacer of BBa_K1218011 and characterized the specificity of the endogenous CRISPR-Cas9 system </li><br />
<li>We added the M13ori and submitted the new part <a href="http://parts.igem.org/Part:BBa_K1445001"> BBa_K1445001</a></li><br />
</ul><br />
<li><a href="https://2014.igem.org/Team:CU-Boulder/Collaboration">Collaborated</a> with CSU Fort Collins</li><br />
</ul><br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
{{Template:UCB-Footer}}</div>Leighlahttp://2014.igem.org/Team:CU-Boulder/MedalTeam:CU-Boulder/Medal2014-10-17T23:11:31Z<p>Leighla: </p>
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<style><br />
body {font-family: Source Sans Pro;}<br />
h2 {display: block; font-weight: bold; font-size: 2em; text-align: center; border: none;}<br />
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.medal {display: block; float: left; padding-top: 0; height: 736px; width: 300px; margin: auto; background-repeat: no-repeat;} <br />
#bronze {background-image: url('https://static.igem.org/mediawiki/2014/3/36/UCB-BRONZE.png')}<br />
#silver {background-image: url('https://static.igem.org/mediawiki/2014/6/63/UCB-SILVER.png')}<br />
#gold {background-image: url('https://static.igem.org/mediawiki/2014/3/38/UCB-GOLD.png');}<br />
div.medal {margin-right: 20px;}<br />
</style><br />
<body><br />
<div id="contentMedal"><br />
<div class="medal" id="bronze"><br />
<h2>Bronze</h2><br />
<ul><br />
<li><a href="https://igem.org/Team.cgi">Team Registration</a></li><br />
<li>Completed <a href="https://igem.org/2014_Judging_Form?id=1445">Judging Form</a></li><br />
<li>Team Wiki</li><br />
<li>Poster and Presentation are Ready for iGem Jamboree</li><br />
<li>Documented new BioBrick Parts</li><br />
<ul><br />
<li><a href="http://parts.igem.org/Part:BBa_K1445000">Part: BBa_K1445000</a></li><br />
<li><a href="http://parts.igem.org/Part:BBa_K1445001">Part: BBa_K1445001</a></li><br />
<li><a href="http://parts.igem.org/Part:BBa_K1445002">Part: BBa_K1445002</a></li><br />
</ul><br />
</ul><br />
</div><br />
<div class="medal" id="silver"><br />
<h2>Silver</h2><br />
<ul><br />
<li>Experimentally Validated BioBrick Parts</li><br />
<li>Documented the Characterization of those Parts in the "Main Page" Section of the Part's/Device's Registry Entry</li><br />
<li>New Parts have been Submitted to the iGem Parts Registry</li><br />
<ul><br />
<li><a href="http://parts.igem.org/Part:BBa_K1445000">Part: BBa_K1445000</a></li><br />
<li><a href="http://parts.igem.org/Part:BBa_K1445001">Part: BBa_K1445001</a></li><br />
<li><a href="http://parts.igem.org/Part:BBa_K1445002">Part: BBa_K1445002</a></li><br />
</ul><br />
<li>Our project may have implications for the environment, security, safety and ethics and/or ownership and sharing. We described more ways in which these have been taken into consideration in the design and execution of our project on our <a href="https://2014.igem.org/Team:CU-Boulder/Notebook/Safety">Safety</a> page.<br />
</ul><br />
</div><br />
<div class="medal" id="gold"><br />
<h2>Gold</h2><br />
<ul><br />
<li>Improved the Function and Characterized Existing BioBrick Part</li><br />
<ul><br />
<li>We changed the spacer of BBa_K1218011 (the SB part) and characterized the specificity of the endogenous CRISPR-Cas9 system </li><br />
<li>We added the M13ori and submitted the new part <a href="http://parts.igem.org/Part:BBa_K1445001"> BBa_K1445001</a></li><br />
</ul><br />
<li><a href="https://2014.igem.org/Team:CU-Boulder/Collaboration">Collaborated</a> with CSU Fort Collins</li><br />
</ul><br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
{{Template:UCB-Footer}}</div>Leighlahttp://2014.igem.org/Team:CU-Boulder/MedalTeam:CU-Boulder/Medal2014-10-17T23:07:19Z<p>Leighla: </p>
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<link href='http://fonts.googleapis.com/css?family=Source+Sans+Pro' rel='stylesheet' type='text/css'><br />
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<style><br />
body {font-family: Source Sans Pro;}<br />
h2 {display: block; font-weight: bold; font-size: 2em; text-align: center; border: none;}<br />
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a:hover {text-decoration: underline;}<br />
#contentMedal {position: relative; width: 1100px; height: 740px; margin-top: 100px; margin-left: -100px;}<br />
.medal {display: block; float: left; padding-top: 0; height: 736px; width: 300px; margin: auto; background-repeat: no-repeat;} <br />
#bronze {background-image: url('https://static.igem.org/mediawiki/2014/3/36/UCB-BRONZE.png')}<br />
#silver {background-image: url('https://static.igem.org/mediawiki/2014/6/63/UCB-SILVER.png')}<br />
#gold {background-image: url('https://static.igem.org/mediawiki/2014/3/38/UCB-GOLD.png');}<br />
div.medal {margin-right: 20px;}<br />
</style><br />
<body><br />
<div id="contentMedal"><br />
<div class="medal" id="bronze"><br />
<h2>Bronze</h2><br />
<ul><br />
<li><a href="https://igem.org/Team.cgi">Team Registration</a></li><br />
<li>Completed <a href="https://igem.org/2014_Judging_Form?id=1445">Judging Form</a></li><br />
<li>Team Wiki</li><br />
<li>Poster and Presentation are Ready for iGem Jamboree</li><br />
<li>Documented new BioBrick Parts</li><br />
<ul><br />
<li><a href="http://parts.igem.org/Part:BBa_K1445000">Part: BBa_K1445000</a></li><br />
<li><a href="http://parts.igem.org/Part:BBa_K1445001">Part: BBa_K1445001</a></li><br />
<li><a href="http://parts.igem.org/Part:BBa_K1445002">Part: BBa_K1445002</a></li><br />
</ul><br />
</ul><br />
</div><br />
<div class="medal" id="silver"><br />
<h2>Silver</h2><br />
<ul><br />
<li>Experimentally Validated BioBrick Parts</li><br />
<li>Documented the Characterization of those Parts in the "Main Page" Section of the Part's/Device's Registry Entry</li><br />
<li>New Parts have been Submitted to the iGem Parts Registry</li><br />
<ul><br />
<li><a href="http://parts.igem.org/Part:BBa_K1445000">Part: BBa_K1445000</a></li><br />
<li><a href="http://parts.igem.org/Part:BBa_K1445001">Part: BBa_K1445001</a></li><br />
<li><a href="http://parts.igem.org/Part:BBa_K1445002">Part: BBa_K1445002</a></li><br />
</ul><br />
<li>Our project may have implications for the environment, security, safety and ethics and/or ownership and sharing. We described more ways in which these have been taken into consideration in the design and execution of our project on our <a href="https://2014.igem.org/Team:CU-Boulder/Notebook/Safety">Safety</a> page.<br />
</ul><br />
</div><br />
<div class="medal" id="gold"><br />
<h2>Gold</h2><br />
<ul><br />
<li>Improved the Function and Characterized Existing BioBrick Part(s)</li><br />
<ul><br />
<li>We changed the spacer and tested its targetability of BBa_K1218011 (the SB part)</li><br />
<li>We added the M13ori and submitted the new part <a href="http://parts.igem.org/Part:BBa_K1445001"> BBa_K1445001</a></li><br />
</ul><br />
<li><a href="https://2014.igem.org/Team:CU-Boulder/Collaboration">Collaborated</a> with Another Registered iGem Team</li><br />
</ul><br />
</div><br />
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<br />
{{Template:UCB-Footer}}</div>Leighlahttp://2014.igem.org/Team:CU-Boulder/BiobricksTeam:CU-Boulder/Biobricks2014-10-17T22:56:04Z<p>Leighla: </p>
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== '''Part Summaries''' ==<br />
<br />
<br />
For detailed characterization, see the parts page.<br />
<br><br />
<br><br />
===Part: BBa_K1445000 (M13ori)===<br />
<br />
The M13 origin of replication (M13ori) has been documented as the packaging signal for the M13 phage. When single stranded, the M13ori forms unique hairpins that signal packaging into a phage capsid. It was experimentally demonstrated that this part is both necessary and sufficient for plasmid packaging into M13 phage. This part does not code for phage coat proteins; therefore, cannot produce phage.<br />
<br />
<br><br />
<br />
===Part: BBa_K1445001 (Endogenous Type II CRISPR-Cas9 phagemid)===<br />
<br />
This composite part consists of parts BBa_K1445000 (M13ori) and BBa_K1218011 (cas9).<br />
The cas9 construct includes the native trcrRNA and promoter region upstream of the type II Cas9 endonuclease. Downstream of the Cas9 protein is a minimal CRISPR array that contains the spacer region that determines the DNA sequence that is targeted by the Cas9 endonuclease. The spacer sequence can be easily replaced through a digestion-ligation reaction using BsaI. The addition of the M13 origin of replication (M13ori) allows for the uptake of the CRISPR-Cas9 machinery into M13 phage.<br />
<br />
<br />
'''[[File:UCB-Biobrick-01-141016.JPG]]'''<br />
<br />
<br />
===Part: BBa_K1445002 (High copy BioBricked M13 phagemid vector)===<br />
<br />
This biobrick backbone has the high copy number pUC19 origin of replication and an ampicillin resistance marker. It also contains the M13 origin of replication which allows for the phagemid to be packaged into the M13 phage. It was experimentally validated that vector retains its ability to be packaged into M13 phage.<br />
<br />
'''[[File:UCB-Biobrick-02-141016.JPG]]'''<br />
<br><br />
<br><br />
<br><br />
<br><br />
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{{Template:UCB-Footer}}</div>Leighlahttp://2014.igem.org/Team:CU-Boulder/BiobricksTeam:CU-Boulder/Biobricks2014-10-17T22:53:36Z<p>Leighla: </p>
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== '''Part Summaries''' ==<br />
<br />
<br />
For detailed characterization, see the parts page.<br />
<br><br />
<br><br />
===1.1 Part: BBa_K1445000 (M13ori)===<br />
<br />
The M13 origin of replication (M13ori) has been documented as the packaging signal for the M13 phage. When single stranded, the M13ori forms unique hairpins that signal packaging into a phage capsid. It was experimentally demonstrated that this part is both necessary and sufficient for plasmid packaging into M13 phage. This part does not code for phage coat proteins; therefore, cannot produce phage.<br />
<br />
<br><br />
<br />
===1.2 Part: BBa_K1445001 (Endogenous Type II CRISPR-Cas9 phagemid)===<br />
<br />
This composite part consists of parts BBa_K1445000 (M13ori) and BBa_K1218011 (cas9).<br />
The cas9 construct includes the native trcrRNA and promoter region upstream of the type II Cas9 endonuclease. Downstream of the Cas9 protein is a minimal CRISPR array that contains the spacer region that determines the DNA sequence that is targeted by the Cas9 endonuclease. The spacer sequence can be easily replaced through a digestion-ligation reaction using BsaI. The addition of the M13 origin of replication (M13ori) allows for the uptake of the CRISPR-Cas9 machinery into M13 phage.<br />
<br />
<br />
'''[[File:UCB-Biobrick-01-141016.JPG]]'''<br />
<br />
<br />
===1.3 Part: BBa_K1445002 (High copy BioBricked M13 phagemid vector)===<br />
<br />
This biobrick backbone has the high copy number pUC19 origin of replication and an ampicillin resistance marker. It also contains the M13 origin of replication which allows for the phagemid to be packaged into the M13 phage. It was experimentally validated that vector retains its ability to be packaged into M13 phage.<br />
<br />
'''[[File:UCB-Biobrick-02-141016.JPG]]'''<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
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{{Template:UCB-Footer}}</div>Leighlahttp://2014.igem.org/Team:CU-Boulder/MedalTeam:CU-Boulder/Medal2014-10-17T22:02:25Z<p>Leighla: </p>
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<html><br />
<link href='http://fonts.googleapis.com/css?family=Source+Sans+Pro' rel='stylesheet' type='text/css'><br />
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#bronze {background-image: url('https://static.igem.org/mediawiki/2014/3/36/UCB-BRONZE.png')}<br />
#silver {background-image: url('https://static.igem.org/mediawiki/2014/6/63/UCB-SILVER.png')}<br />
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<body><br />
<div id="contentMedal"><br />
<div class="medal" id="bronze"><br />
<h2>Bronze</h2><br />
<ul><br />
<li><a href="https://igem.org/Team.cgi">Team Registration</a></li><br />
<li>Completed <a href="https://igem.org/2014_Judging_Form?id=1445">Judging Form</a></li><br />
<li>Team Wiki</li><br />
<li>Poster and Presentation are Ready for iGem Jamboree</li><br />
<li>Documented new BioBrick Parts</li><br />
<ul><br />
<li><a href="http://parts.igem.org/Part:BBa_K1445000">Part: BBa_K1445000</a></li><br />
<li><a href="http://parts.igem.org/Part:BBa_K1445001">Part: BBa_K1445001</a></li><br />
<li><a href="http://parts.igem.org/Part:BBa_K1445002">Part: BBa_K1445002</a></li><br />
</ul><br />
</ul><br />
</div><br />
<div class="medal" id="silver"><br />
<h2>Silver</h2><br />
<ul><br />
<li>Experimentally Validated BioBrick Parts</li><br />
<li>Documented the Characterization of those Parts in the "Main Page" Section of the Part's/Device's Registry Entry</li><br />
<li>New Parts have been Submitted to the iGem Parts Registry</li><br />
<ul><br />
<li><a href="http://parts.igem.org/Part:BBa_K1445000">Part: BBa_K1445000</a></li><br />
<li><a href="http://parts.igem.org/Part:BBa_K1445001">Part: BBa_K1445001</a></li><br />
<li><a href="http://parts.igem.org/Part:BBa_K1445002">Part: BBa_K1445002</a></li><br />
</ul><br />
<li>Our project may have implications for the environment, security, safety and ethics and/or ownership and sharing. We described more ways in which these have been taken into consideration in the design and execution of our project on our <a href="https://2014.igem.org/Team:CU-Boulder/Notebook/Safety">Safety</a> page.<br />
</ul><br />
</div><br />
<div class="medal" id="gold"><br />
<h2>Gold</h2><br />
<ul><br />
<li>Improved the Function or Characterization of an Existing BioBrick Part(s)</li><br />
<ul><br />
<li>We changed the spacer and tested its targetability of BBa_K1218011 (the SB part)</li><br />
<li>We added the M13ori and submitted the new part <a href="http://parts.igem.org/Part:BBa_K1445001"> BBa_K1445001</a></li><br />
</ul><br />
<li><a href="https://2014.igem.org/Team:CU-Boulder/Collaboration">Collaborated</a> with Another Registered iGem Team</li><br />
</ul><br />
</div><br />
</div><br />
</body><br />
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<br />
{{Template:UCB-Footer}}</div>Leighlahttp://2014.igem.org/Team:CU-Boulder/Notebook/SafetyTeam:CU-Boulder/Notebook/Safety2014-10-17T21:37:48Z<p>Leighla: </p>
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<h2> Safety </h2><br />
<br><br />
<br />
<h3>Organism and Biological Part Safety </h3><br />
<ul><ul><br />
<br><br />
<h4>Escherichia coli K12 variants</h4><br />
K12 strains of E. coli are commonly used, biosafety level 1 organisms. They pose little risk to lab members or the environment. They can be potentially harmful if ingested so basic lab safety such as gloves and hand washing were implemented to ensure lab member safety.<br />
<br><br><br />
<h4>M13 phage</h4><br />
M13 phage are biosafety level 1 viruses. Phage only infect bacteria so the risk to humans is negligible. The helper phagemid have a weakened packaging signal which reduces their replication efficiency, however, these phage could infect E. coli and other bacterial species containing the F’ episome if the phage escaped laboratory containment. M13 phage are not synthetic organisms; their environmental impact if they escaped laboratory containment is small due to their disrupted packaging signal so they would be quickly outcompeted by wild-type phage. Precautions such as hand washing and use of gloves were implemented to reduce risk to lab members. Proper disposal of samples was implemented to avoid environmental contamination.<br />
<br><br><br />
<h4>Endogenous CRISPR-Cas(BBa_K1218011)</h4><br />
Part BBa_K1218011 was apart of the 2014 iGEM distribution. The encoded CRISPR-Cas9 system is derived from Streptococcus pyogenes, a biosafety level 1 organism, and is widely used in gene editing. Malicious use of the CRISPR-Cas9 system is theoretically possible as it is a system that targets specific genetic sequences and could be used to target certain alleles. However, this technology has been used for several years for the purpose of gene editing and no health risks have been reported.<br />
</ul></ul><br />
<br />
<br><br />
<br><br />
<h3>Risks Associated with Synthetic and Recombinant Biological Parts</h3><br />
No organism used in these experiments pose any significant risk to health or the environment. The Cas9 can only target DNA sequences that are adjacent to a PAM site and are complementary to its guide RNA. The CRISPR-Cas9 plasmid contains the M13 packaging signal but no other phage genes; therefore, this part can be packaged into, but cannot make, phage. A separate helper phagemid is needed to assemble the phage coat but the packaging signal on this part is disrupted; thus, it cannot be efficiently packaged into a phage. This reduces the chances of uncontrolled phage replication. Some phage may still contain the helper phagemid but they can only infect bacteria with an F’ episome.<br />
<br />
<br><br />
<br><br />
<h3>Institutional Safety</h3><br />
The University of Colorado has an Institutional Biosafety Committee(IBC) that oversees the safety and ethics of projects at the university. The IBC has not raised any concerns regarding the safety protocols or synthesized organisms used in this experiment. All reagents and organisms used in this experiment were disposed of by the University Environmental Health and Safety services in accordance with institutional, national and international safety standards.<br />
<br />
<br><br />
<br><br />
<br />
<h3>Safety Concerns when using Antibacterials</h3><br />
The overuse of antibiotics is a major cause of emerging antibiotic resistance. This project is aimed at developing a system that enables physicians to counter bacterial resistance to therapeutics at a rate that is more competitive to that of evolution. The health and safety of humanity is threatened by gene flow through bacterial species by conjugation and other methods. Through bioengineering, strain-specific CRISPR-Cas therapeutics can be designed within several days to target pathogenic strains with new mutations, while simultaneously leaving the symbiotic species in the microbiome unaffected. The end result is safer, more adaptive, and more versatile treatment methods.<br />
<br><br />
<br><br />
<br />
Online safety form found <a href="https://igem.org/Safety/Safety_Form?team_id=1445">here</a>.<br />
<br />
<br><br />
<br><br />
<br><br />
<h2>Ethics of CRISPR-Cas Mediated Phage Therapy</h2><br />
An ethical question encountered during the experiment design concerns the manipulation of genetic material in pathogenic organisms. Concerns were raised regarding the possibility that genome cleavage by CRISPR-Cas9 could result in accelerated mutation rates in pathogenic organisms. Through limited experimentation, it appears that cells that survive CRISPR-Cas9 cleavage received a non-targetable copy of the plasmid, rather than acquiring new mutations that disrupts the target sequence in the bacterial genome. Therefore, it does not appear that CRISPR-Cas9 selects for mutations.</div>Leighlahttp://2014.igem.org/Team:CU-Boulder/Notebook/ProtocolsTeam:CU-Boulder/Notebook/Protocols2014-10-17T09:15:17Z<p>Leighla: /* Mini-prep */</p>
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__FORCETOC__<br />
<br />
==Phage Amplification==<br />
<br />
'''Need'''<br />
:*Plate of infectable cells that contain F’ episome<br />
:*2.5M NaCl/20% PEG-8000<br />
:*1x TBS<br />
<br />
'''Day 1'''<br />
:1.Add a fresh colony of infectable cells to 50mL LB in 125mL flask. <br />
::a. Include phagemid antibiotic only. <br />
::b. Grow at 37°C, 250rpm until OD is between 0.03 and 0.05<br />
:2.Add the helper phage to a final concentration of 1 x10^8 phage/mL<br />
:3.Incubate for 60-90 minutes, shaking<br />
:4.Add Helper Phagemid antibiotic to a high concentration<br />
:5.Grow for 14-18 hours at 37°C, shaking<br />
<br />
'''Day 2'''<br />
#Spin culture at 4,000 x g for 10 minutes<br />
#Transfer supernatant to a fresh conical. Repeat spin on supernatant<br />
#Transfer the upper 90% of supernatant to a new conical<br />
#Add 0.2 volume of 2.5M NaCl/20% PEG-8000 to the new conical. Gently mix<br />
#Incubate at 4°C for at least 60 minutes<br />
#Centrifuge at 12,000 x g for 10 minutes. Carefully decant<br />
#Spin again briefly<br />
#Gently resuspend pellet in 1.6mL 1x TBS<br />
#Aliquot 800ul into 2 microfuge tubes. Proceed with both tubes<br />
#Spin sample for 1 minute to pellet remaining cells. Transfer supernatant to fresh tubes<br />
#Add 160ul of 2.5M NaCl/20% PEG-8000 solution to each<br />
#Let sit at room temperature for 5 minutes<br />
#Spin at 1300 x g for 10 minutes<br />
#Decant the supernatant<br />
#Spin briefly. Remove supernatant with pipet<br />
#Resuspend pellet in 200ul 1x TBS. <br />
::a. If desired, combine contents of both tubes into one<br />
<br />
'''2.5M NaCl/20% PEG-8000 (5x)'''<br />
:100 g PEG-8000<br />
:75 g NaCl<br />
:400 mL H<sub>2</sub>O<br />
:Bring final volume to 500 mL<br />
<br />
'''TBS (1x)'''<br />
:6.05 g Tris<br />
:8.76 g NaCl<br />
:800 mL H<sub>2</sub>O<br />
:Adjust pH to 7.6 with 1M HCL<br />
:Adjust volume to 1 L<br />
<br><br />
<br><br />
<br><br />
<br />
==Heat Shock Transformation==<br />
<br />
'''Before you start'''<br />
*Heat hot plate or water bath to 42°C<br />
*Warm selection plates to 37°C<br />
<br />
'''Transformation'''<br />
:1.Thaw chemically competent cells on ice for 10-15 minutes<br />
:2.Add 40ul cells to fresh 1.7mL tube<br />
:3.Add DNA<br />
::a. If using a ligation product add up to 10ul of sample<br />
::b. If using supercoiled plasmid add 100ng<br />
:4.Incubate on ice for 30 minutes<br />
:5.Heat shock cells on hot plate (or water bath) for 30-45s* @ 42°C<br />
:6.Incubate on ice for 2-5 minutes<br />
:7.Add 200 μL SOC and shake gently for 1-2 hours @ 37° C<br />
::a.('''Note''': Can also recover in 960ul. After recovery, gently spin cells and remove supernatant. Resuspend in 200ul LB)<br />
:8.Plate 100-200ul cells onto selection plates<br />
::a. If high efficiency is expected, we suggest also plating a 1:10 dilution<br />
:9.Once dry, turn upside down (agar on top) and incubate overnight @ 37° C<br />
<br />
::*Optimal timing depends on cells<br />
<br />
<br />
'''SOC (1L)'''<br />
:20 g Tryptone<br />
:5 g YeastExtract<br />
:4.8 g MgSO4 <br />
:3.6 g Dextrose <br />
:0.5 g NaCl <br />
:0.186 g KCL<br />
<br><br />
<br><br />
<br><br />
<br />
==Bacterial Conjugation==<br />
<br />
'''Need'''<br />
*Donor cells: Cells already containing F’ episome<br />
*Recipient cells: Cells with a resistance marker that is absent from donor cells<br />
*Double selection plate containing antibiotic to select for F’ episome and a second antibiotic to select for the recipient cells<br />
<br />
'''Day 1'''<br />
#Set up liquid overnight of '''donor''' cells. Include antibiotic<br />
#Set up liquid overnight of '''recipient''' cells. Include antibiotic<br />
<br />
'''Day 2'''<br />
:1.Mix 500ul of each overnight sample in a new tube. Mix by pipetting or flicking<br />
:2.Incubate for 30 minutes at 37°C, shaking<br />
:3.Plate 100ul onto double selection plate<br />
::a.We advise also plating a 1:10 dilution<br />
:4.Incubate at 37°C<br />
<br />
<br><br />
<br><br />
<br><br />
<br />
==CaCl2 Competent Cells==<br />
<br />
'''To prepare for Day 2'''<br />
* Set centrifuge to 4°C<br />
* MgCl2 and CaCl2 solutions<br />
* Thaw DMSO<br />
* Chill tubes<br />
* Acquire liquid nitrogen or dry ice<br />
<br />
'''Day 1'''<br />
#Grow cells O/N<br />
<br />
'''Day 2'''<br />
:1.Add 0.5mL of the overnight culture to 50mL LB<br />
:2.Grow until OD is between 0.2 and 0.4<br />
:3.Incubate on ice for 30 minutes<br />
:4.Centrifuge for 10 minutes at 2700 x g and 4°C<br />
:5.Decant. Dry upside down on a paper towel for 1 minute<br />
:6.Completely resuspend in 30mL 0.8M MgCl2, 0.2M CaCl2<br />
::a. Gently vortex<br />
:7.Centrifuge for 10 minutes at 2700 x g and 4°C<br />
:8.Decant. Dry upside down on a paper towel for 1 minute<br />
:9.Fully resuspend in 2mL of 0.1M CaCl2<br />
:10.Chill sample on ice. Add 70ul DMSO, keeping the sample tube on ice<br />
:11.Swirl to mix<br />
:12.Incubate on ice for 15 minutes<br />
:13.Add 70ul DMSO, swirl to mix, keeping the sample tube on ice<br />
:14.Dispense 200ul into pre-chilled 1.7mL tubes<br />
:15.Snap freeze with liquid nitrogen or dry ice<br />
:16.Store at -80°C until ready to use<br />
<br><br />
<br><br />
<br><br />
<br />
==Phagemid DNA Isolation==<br />
<br />
<br />
'''Need'''<br />
<br />
*Fresh plate of infectable cells (contain F’ episome)<br />
*2.5M NaCl/20% PEG-8000<br />
*TBS, TE, phenol, phenol/chloroform, chloroform<br />
<br />
'''Day 1'''<br />
<br />
:1.Add a fresh colony to 50mL LB in 125mL flask. Grow at 37°C, 250rpm until OD is between 0.03 and 0.05<br />
:2.Add M13KO7 helper phage to a final concentration of 1 x10^8 phage/mL*<br />
:3.Continue shaking for 60-90 minutes<br />
:4.Add Kanamycin to final concentration of 70ug/mL<br />
:5.Grow for 14-18 hours at 37°C, 250rpm<br />
<br />
:*Can use a different helper phage if needed. In step 4, add the antibiotic specific to the Helper Phagemid<br />
<br />
'''Day 2'''<br />
<br />
:1.Spin culture at 4,000 x g for 10 minutes<br />
:2.Transfer supernatant to a fresh conical. Repeat spin on supernatant<br />
:3.Transfer the upper 90% of supernatant to a new conical<br />
:4.Add 0.2 volume of 2.5M NaCl/20% PEG-8000 to the new conical. Gently mix<br />
:5.Incubate at 4°C for at least 60 minutes<br />
:6.Centrifuge at 12,000 x g for 10 minutes. Carefully decant<br />
:7.Spin again briefly<br />
:8.Gently resuspend pellet in 1.6mL 1x TBS<br />
:9.Aliquot 800ul into 2 microfuge tubes. Proceed with both tubes<br />
:10.Spin sample for 1 minute to pellet remaining cells. Transfer supernatant to fresh tubes<br />
:11.Add 160ul of 2.5M NaCl/20% PEG-8000 solution to each<br />
:12.Let sit at room temperature for 5 minutes<br />
:13.Spin at 1300 x g for 10 minutes<br />
:14.Decant the supernatant<br />
:15.Spin briefly. Remove supernatant with pipet<br />
:16.Resuspend pellets in 300ul TE<br />
:17.Phenol extraction: add 300ul phenol. Vortex for 15 seconds<br />
::a.Let sit for 15 minutes. Spin for 10 minutes<br />
:18.Add H2O so volume samples is about 300ul<br />
:19.Phenol/chloroform extraction*: add 300ul PCIA<br />
::a.Vortex for 15 seconds. Spin for 10 minutes<br />
:20.Repeat Phenol/Chloroform extraction<br />
:21.Chloroform extraction*: add 300ul chloroform<br />
::a.Vortex 15 seconds. Spin for 10 minutes<br />
:22.Add 30ul 2.5M NaAc (pH 4.8)<br />
:23.Add 2-2.5 volumes ethanol<br />
:24.Let precipitate for ~2 hours at -20°C<br />
:25.Spin for 1 minute<br />
:26.Decant supernatant<br />
:27.Resuspend in 25-50ul TE<br />
<br />
:*Performing steps at 4°C helps with separation<br />
<br />
<br />
'''2.5M NaCl/20% PEG-8000 (5x)'''<br />
<br />
:100 g PEG-800<br />
:75 g NaCl<br />
:400 mL H<sub>2</sub>O<br />
:: *Bring final volume to 500 mL <br />
<br />
'''TBS (1x)'''<br />
:6.05 g Tris<br />
:8.76 g NaCl<br />
:800 mL H<sub>2</sub>O<br />
<br />
:: *Adjust pH to 7.6 with 1M HCL<br />
<br />
:: *Adjust volume to 1L<br />
<br><br />
<br><br />
<br><br />
<br />
==M13 Amplification==<br />
<br><br />
This protocol is to make more M13 phages.<br />
<br />
'''Need'''<br />
<br />
*Fresh plate of infectable cells (contain F’ episome)<br />
<br />
'''Day 1'''<br />
:1.Grow liquid overnight culture of infectable cells<br />
<br />
'''Day 2'''<br />
<br />
:1.Add 200ul overnight culture to 20mL LB in a 250mL flask<br />
:2.Add 1ul phage suspension<br />
:3.Incubate for 4-5 hours at 37°C, 250rpm<br />
:4.Centrifuge for 10 minutes at 4500 x g<br />
:5.Transfer supernatant to a new tube.<br />
:6.Repeat centrifugation on supernatant<br />
:7.Transfer top 16mL of supernatant to a new tube<br />
:8.Add 4mL of 2.5M NaCl/20% PEG-8000. Briefly mix<br />
:9.Precipitate phage for 1 hour or overnight at 4°C<br />
:10.Centrifuge for 15 minutes at 12000 x g. Decant supernatant<br />
::a. Spin briefly. Remove residual supernatant with pipet<br />
:11.Resuspend pellet in 1mL 1x TBS. Transfer to 1.7mL tube<br />
:12.Spin briefly to remove cell debris<br />
:13.Transfer supernatant to a new tube<br />
:14.Add 200ul of 2.5M NaCl/20% PEG-8000<br />
:15.Incubate on ice for 15-60 minutes<br />
:16.Spin for 10 minutes at 12000-14000 rpm. Discard supernatant<br />
:17.Briefly spin. Remove supernatant with pipette<br />
:18.Resuspend pellet in 200uL TBS<br />
:19.For long term storage at -20C, add 200uL glycerol<br />
<br />
<br />
'''2.5M NaCl/20% PEG-8000 (5x)'''<br />
<br />
:100 g PEG-8000 <br />
:75 g NaCl <br />
:400 mL H<sub>2</sub>O <br />
::Bring final volume to 500 mL<br />
<br />
'''TBS (1x)'''<br />
:6.05 g Tris<br />
:8.76 g NaCl <br />
:800 mL H<sub>2</sub>O <br />
<br />
::Adjust pH to 7.6 with 1M HCl<br />
::Adjust volume to 1 L<br />
::Store at 3C for up to 3 months<br />
<br><br />
<br><br />
<br><br />
<br />
==Mini-prep==<br />
<br />
: This protocol is taken from the Qiagen Mini-prep Kit and is used to isolate plasmid DNA from a bacterial overnight.<br />
<br />
'''Notes before starting'''<br />
<br />
: '''Optional:''' Add LyseBlue reagent to Buffer P1 at a ratio of 1 to 1000<br />
<br />
*Add the provided RNase A solution to Buffer P1, mix, '''store bottle at 2-8°C'''<br />
<br />
*Add ethanol (96-100%) to Buffer PE before use<br />
<br />
*All centrifugation steps are carried out at 13,000 rpm (~17,900xg) in a conventional table-top microcentrifuge<br />
<br />
<br />
<br />
:1.Centrifuge 1-6mL bacterial overnight culture at >8000 rpm (6800xg) for 3 minutes at room temperature (15-25C)<br />
:2.Resuspend pellet in 250ul Buffer P1 and transfer to microcentrifuge tube<br />
:3.Add 250ul Buffer P2 and mix thoroughly by inverting the tube 4-6 times until the solution becomes clear.<br />
::a.DO NOT allow lysis reaction to proceed for more than 5 minutes<br />
::b.If using LyseBlue reagent, the solution will turn blue<br />
:4.Add 350ul Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.<br />
::a.If using LyseBlue reagent, the solution will turn colorless<br />
:5.Centrifuge for 10 minutes<br />
:6.Apply supernatant from step 5 to the QIAprep spin column by decanting or pipetting. Centrifuge for 30-60 s and discard the flow-through<br />
:7.Recommended: Wash the QIAprep spin column by adding 500 ul Buffer PB. Centrifuge for 30-60 s and discard the flow-through<br />
::a.Only required when using endA+ strains or other bacterial strains with high nuclease activity or carbohydrate content<br />
:8.Wash the QIAprep spin column by adding 750ul of Buffer PE. Centrifuge for 30-60 s and discard the flow-through<br />
:9.Centrifuge for 1 minutes to remove residual wash buffer<br />
:10.Place the QIAprep column in a clean 1.5mL microcentrifuge tube. To elute DNA, add 30ul Buffer EB. Let stand for 1 min, and centrifuge for 1 minute<br />
::a.Can elute DNA in 50ul but this will decrease DNA concentration<br />
::b.To increase yield, let sit for up to 4 minutes<br />
<br />
'''Buffer Recipes'''<br />
<br />
'''P1'''<br />
50 mM Tris-Cl, pH 8.0<br />
10 mM EDTA<br />
100 ug/mL RNase A<br />
<br />
: *After RNase A addition, the buffer should be stored at 2-8C<br />
<br />
'''P2 (Lysis buffer)'''<br />
:200 mM NaOH<br />
:1% SDS (w/v)<br />
<br />
'''N3*'''<br />
:4.2 M Gu-HCl<br />
:0.9 M KAc, pH 4.8<br />
<br />
'''PB*'''<br />
:5 M Gu-HCl<br />
:30% isopropanol<br />
<br />
'''PE*'''<br />
:10 mM Tris-HCl pH 7.5<br />
:80% ethanol<br />
<br />
'''Elution Buffer (EB)'''<br />
:10 mM Tris-CL, pH 8.5<br />
<br />
:''Recipes from OpenWetWare''<br />
<br />
<br><br />
<br><br />
<br><br />
<br />
==Bacterial Infection==<br />
<br />
'''Need'''<br />
:Fresh plate of cells that contain the F’ episome<br />
<br />
'''Day 1'''<br />
:1.Pick colony from plate and grow overnight<br />
<br />
'''Day 2'''<br />
:1.Dilute overnight to OD600 0.1<br />
:2.Incubate until OD600 ~ 0.5* <br />
:3.Add phage to a final concentration of 1 x108 phage/mL<br />
:4.Incubate for 30 minutes to infect<br />
:5.Plate samples under selection<br />
::a.We recommend trying a range of dilutions<br />
<br />
''Protocol from “Eliminating Helper phage from Phage Display''<br />
<br />
:'''Note:''' All incubations are done at 37°C and shaking at 225rpm<br />
:'''Note:''' If multiple samples are to be done in parallel<br />
:*If using the same cells, we suggest growing one large batch of cells. Once the OD has reached ~0.5, divide into 50mL samples then add phage.<br />
:*If using different cells, grow each sample to ~0.5 then store on ice. Once all samples have reached ~0.5, incubate on ice for another 30 minutes. Warm for 20-30 minutes at 37C, 250 rpm. Measure OD again to check that samples are comparable (yes, they will have grown some). Add phage<br />
<br />
==Cloning a new spacer into pCas9==<br />
<br />
'''[[File:UCB-lab protocol cloning-141010-01.jpg]]'''<br />
<br />
'''Design the new spacer'''<br />
<br />
'''[[File:UCB-lab protocol cloning-141010-02.jpg]]'''<br />
<br />
:1. Find a PAM site (5’- NGG -3’). This will be on the 3’ end of the targeting sequence<br />
:2. The 30bps upstream of NGG motif (on the same strand) will be the spacer<br />
:3. Oligo I (fwd. primer): To the spacer sequence, add AAAC to the 5’ end and G to the 3’ end<br />
::a. NOTE: These nucleotides are for ligation purposes and are NOT used in targeting. Therefore, they do not have to be present in the target sequence and do not count towards the 30bps<br />
:4. Oligo II (rvs. primer): Find the reverse complement of the spacer sequence. Add CAAAA to the 5’ end.<br />
<br />
'''Phosphorylation'''<br />
:1.Mix the following reactants<br />
{| class="wikitable"<br />
|-<br />
!Reactant<br />
!Amount<br />
|-<br />
|Oligo I (100uM)<br />
|1 ul<br />
|-<br />
|Oligo II (100uM)<br />
|1 ul<br />
|-<br />
|10x T4 Ligase Buffer<br />
|5 ul<br />
|-<br />
|T4 PNK<br />
|1 ul<br />
|-<br />
|H2O<br />
|42 ul<br />
|}<br />
<br />
:2.Incubate for 60 minutes at 37C then 20 minutes at 65C<br />
<br />
'''Anneal Oligos'''<br />
:1. Add 2.5 ul of 1M NaCl to the phosphorylation product<br />
:2. Incubate for 5 minutes at 95C then let slowly cool to room temperature.<br />
::a. It is recommended to use a thermocycler that either cools at a gradient or steps down in temperature over the course of ~2 hours.<br />
::b. Alternatively, incubate the sample at 95C for 5 minutes then remove the heat block from the heater and let it cool naturally for 2 hours<br />
:3. Dilute the annealed oligos 1:10<br />
<br />
'''Vector digest with BsaI'''<br />
:1. Mix the following reactants<br />
<br />
{| class="wikitable"<br />
|-<br />
!Reactant<br />
!Amount<br />
|-<br />
|Cutsmart Buffer<br />
|5 ul<br />
|-<br />
|BsaI enzyme<br />
|1 ul<br />
|-<br />
|DNA<br />
|1-2ug<br />
|-<br />
|H2O<br />
|Bring volume to 50ul<br />
|}<br />
:2. Incubate for 90 minutes at 37C then 20 minutes at 65C<br />
:3. Gel extract digested pCas9<br />
<br />
'''Ligation'''<br />
:1. Mix the following reactants<br />
{| class="wikitable"<br />
|-<br />
!Reactant<br />
!Amount<br />
|-<br />
|Gel extracted vector<br />
|1 ul<br />
|-<br />
|Diluted annealed oligos<br />
|2 ul<br />
|-<br />
|10x T4 Ligase Buffer<br />
|2 ul<br />
|-<br />
|T4 Ligase<br />
|1 ul<br />
|-<br />
|H2O<br />
|14 ul<br />
|}<br />
:2. Incubate overnight (8-12 hours) at 16C<br />
<br />
'''Transform'''<br />
:1. Transform into DH5alpha<br />
<br />
<br />
''Protocol from Marraffini Lab''<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{Template:UCB-Footer}}</div>Leighlahttp://2014.igem.org/Team:CU-Boulder/Notebook/ProtocolsTeam:CU-Boulder/Notebook/Protocols2014-10-17T09:14:36Z<p>Leighla: /* M13 Amplification */</p>
<hr />
<div>{{Template:UCB-Main}}<br />
{{UCB-NavBar}}<br />
__FORCETOC__<br />
<br />
==Phage Amplification==<br />
<br />
'''Need'''<br />
:*Plate of infectable cells that contain F’ episome<br />
:*2.5M NaCl/20% PEG-8000<br />
:*1x TBS<br />
<br />
'''Day 1'''<br />
:1.Add a fresh colony of infectable cells to 50mL LB in 125mL flask. <br />
::a. Include phagemid antibiotic only. <br />
::b. Grow at 37°C, 250rpm until OD is between 0.03 and 0.05<br />
:2.Add the helper phage to a final concentration of 1 x10^8 phage/mL<br />
:3.Incubate for 60-90 minutes, shaking<br />
:4.Add Helper Phagemid antibiotic to a high concentration<br />
:5.Grow for 14-18 hours at 37°C, shaking<br />
<br />
'''Day 2'''<br />
#Spin culture at 4,000 x g for 10 minutes<br />
#Transfer supernatant to a fresh conical. Repeat spin on supernatant<br />
#Transfer the upper 90% of supernatant to a new conical<br />
#Add 0.2 volume of 2.5M NaCl/20% PEG-8000 to the new conical. Gently mix<br />
#Incubate at 4°C for at least 60 minutes<br />
#Centrifuge at 12,000 x g for 10 minutes. Carefully decant<br />
#Spin again briefly<br />
#Gently resuspend pellet in 1.6mL 1x TBS<br />
#Aliquot 800ul into 2 microfuge tubes. Proceed with both tubes<br />
#Spin sample for 1 minute to pellet remaining cells. Transfer supernatant to fresh tubes<br />
#Add 160ul of 2.5M NaCl/20% PEG-8000 solution to each<br />
#Let sit at room temperature for 5 minutes<br />
#Spin at 1300 x g for 10 minutes<br />
#Decant the supernatant<br />
#Spin briefly. Remove supernatant with pipet<br />
#Resuspend pellet in 200ul 1x TBS. <br />
::a. If desired, combine contents of both tubes into one<br />
<br />
'''2.5M NaCl/20% PEG-8000 (5x)'''<br />
:100 g PEG-8000<br />
:75 g NaCl<br />
:400 mL H<sub>2</sub>O<br />
:Bring final volume to 500 mL<br />
<br />
'''TBS (1x)'''<br />
:6.05 g Tris<br />
:8.76 g NaCl<br />
:800 mL H<sub>2</sub>O<br />
:Adjust pH to 7.6 with 1M HCL<br />
:Adjust volume to 1 L<br />
<br><br />
<br><br />
<br><br />
<br />
==Heat Shock Transformation==<br />
<br />
'''Before you start'''<br />
*Heat hot plate or water bath to 42°C<br />
*Warm selection plates to 37°C<br />
<br />
'''Transformation'''<br />
:1.Thaw chemically competent cells on ice for 10-15 minutes<br />
:2.Add 40ul cells to fresh 1.7mL tube<br />
:3.Add DNA<br />
::a. If using a ligation product add up to 10ul of sample<br />
::b. If using supercoiled plasmid add 100ng<br />
:4.Incubate on ice for 30 minutes<br />
:5.Heat shock cells on hot plate (or water bath) for 30-45s* @ 42°C<br />
:6.Incubate on ice for 2-5 minutes<br />
:7.Add 200 μL SOC and shake gently for 1-2 hours @ 37° C<br />
::a.('''Note''': Can also recover in 960ul. After recovery, gently spin cells and remove supernatant. Resuspend in 200ul LB)<br />
:8.Plate 100-200ul cells onto selection plates<br />
::a. If high efficiency is expected, we suggest also plating a 1:10 dilution<br />
:9.Once dry, turn upside down (agar on top) and incubate overnight @ 37° C<br />
<br />
::*Optimal timing depends on cells<br />
<br />
<br />
'''SOC (1L)'''<br />
:20 g Tryptone<br />
:5 g YeastExtract<br />
:4.8 g MgSO4 <br />
:3.6 g Dextrose <br />
:0.5 g NaCl <br />
:0.186 g KCL<br />
<br><br />
<br><br />
<br><br />
<br />
==Bacterial Conjugation==<br />
<br />
'''Need'''<br />
*Donor cells: Cells already containing F’ episome<br />
*Recipient cells: Cells with a resistance marker that is absent from donor cells<br />
*Double selection plate containing antibiotic to select for F’ episome and a second antibiotic to select for the recipient cells<br />
<br />
'''Day 1'''<br />
#Set up liquid overnight of '''donor''' cells. Include antibiotic<br />
#Set up liquid overnight of '''recipient''' cells. Include antibiotic<br />
<br />
'''Day 2'''<br />
:1.Mix 500ul of each overnight sample in a new tube. Mix by pipetting or flicking<br />
:2.Incubate for 30 minutes at 37°C, shaking<br />
:3.Plate 100ul onto double selection plate<br />
::a.We advise also plating a 1:10 dilution<br />
:4.Incubate at 37°C<br />
<br />
<br><br />
<br><br />
<br><br />
<br />
==CaCl2 Competent Cells==<br />
<br />
'''To prepare for Day 2'''<br />
* Set centrifuge to 4°C<br />
* MgCl2 and CaCl2 solutions<br />
* Thaw DMSO<br />
* Chill tubes<br />
* Acquire liquid nitrogen or dry ice<br />
<br />
'''Day 1'''<br />
#Grow cells O/N<br />
<br />
'''Day 2'''<br />
:1.Add 0.5mL of the overnight culture to 50mL LB<br />
:2.Grow until OD is between 0.2 and 0.4<br />
:3.Incubate on ice for 30 minutes<br />
:4.Centrifuge for 10 minutes at 2700 x g and 4°C<br />
:5.Decant. Dry upside down on a paper towel for 1 minute<br />
:6.Completely resuspend in 30mL 0.8M MgCl2, 0.2M CaCl2<br />
::a. Gently vortex<br />
:7.Centrifuge for 10 minutes at 2700 x g and 4°C<br />
:8.Decant. Dry upside down on a paper towel for 1 minute<br />
:9.Fully resuspend in 2mL of 0.1M CaCl2<br />
:10.Chill sample on ice. Add 70ul DMSO, keeping the sample tube on ice<br />
:11.Swirl to mix<br />
:12.Incubate on ice for 15 minutes<br />
:13.Add 70ul DMSO, swirl to mix, keeping the sample tube on ice<br />
:14.Dispense 200ul into pre-chilled 1.7mL tubes<br />
:15.Snap freeze with liquid nitrogen or dry ice<br />
:16.Store at -80°C until ready to use<br />
<br><br />
<br><br />
<br><br />
<br />
==Phagemid DNA Isolation==<br />
<br />
<br />
'''Need'''<br />
<br />
*Fresh plate of infectable cells (contain F’ episome)<br />
*2.5M NaCl/20% PEG-8000<br />
*TBS, TE, phenol, phenol/chloroform, chloroform<br />
<br />
'''Day 1'''<br />
<br />
:1.Add a fresh colony to 50mL LB in 125mL flask. Grow at 37°C, 250rpm until OD is between 0.03 and 0.05<br />
:2.Add M13KO7 helper phage to a final concentration of 1 x10^8 phage/mL*<br />
:3.Continue shaking for 60-90 minutes<br />
:4.Add Kanamycin to final concentration of 70ug/mL<br />
:5.Grow for 14-18 hours at 37°C, 250rpm<br />
<br />
:*Can use a different helper phage if needed. In step 4, add the antibiotic specific to the Helper Phagemid<br />
<br />
'''Day 2'''<br />
<br />
:1.Spin culture at 4,000 x g for 10 minutes<br />
:2.Transfer supernatant to a fresh conical. Repeat spin on supernatant<br />
:3.Transfer the upper 90% of supernatant to a new conical<br />
:4.Add 0.2 volume of 2.5M NaCl/20% PEG-8000 to the new conical. Gently mix<br />
:5.Incubate at 4°C for at least 60 minutes<br />
:6.Centrifuge at 12,000 x g for 10 minutes. Carefully decant<br />
:7.Spin again briefly<br />
:8.Gently resuspend pellet in 1.6mL 1x TBS<br />
:9.Aliquot 800ul into 2 microfuge tubes. Proceed with both tubes<br />
:10.Spin sample for 1 minute to pellet remaining cells. Transfer supernatant to fresh tubes<br />
:11.Add 160ul of 2.5M NaCl/20% PEG-8000 solution to each<br />
:12.Let sit at room temperature for 5 minutes<br />
:13.Spin at 1300 x g for 10 minutes<br />
:14.Decant the supernatant<br />
:15.Spin briefly. Remove supernatant with pipet<br />
:16.Resuspend pellets in 300ul TE<br />
:17.Phenol extraction: add 300ul phenol. Vortex for 15 seconds<br />
::a.Let sit for 15 minutes. Spin for 10 minutes<br />
:18.Add H2O so volume samples is about 300ul<br />
:19.Phenol/chloroform extraction*: add 300ul PCIA<br />
::a.Vortex for 15 seconds. Spin for 10 minutes<br />
:20.Repeat Phenol/Chloroform extraction<br />
:21.Chloroform extraction*: add 300ul chloroform<br />
::a.Vortex 15 seconds. Spin for 10 minutes<br />
:22.Add 30ul 2.5M NaAc (pH 4.8)<br />
:23.Add 2-2.5 volumes ethanol<br />
:24.Let precipitate for ~2 hours at -20°C<br />
:25.Spin for 1 minute<br />
:26.Decant supernatant<br />
:27.Resuspend in 25-50ul TE<br />
<br />
:*Performing steps at 4°C helps with separation<br />
<br />
<br />
'''2.5M NaCl/20% PEG-8000 (5x)'''<br />
<br />
:100 g PEG-800<br />
:75 g NaCl<br />
:400 mL H<sub>2</sub>O<br />
:: *Bring final volume to 500 mL <br />
<br />
'''TBS (1x)'''<br />
:6.05 g Tris<br />
:8.76 g NaCl<br />
:800 mL H<sub>2</sub>O<br />
<br />
:: *Adjust pH to 7.6 with 1M HCL<br />
<br />
:: *Adjust volume to 1L<br />
<br><br />
<br><br />
<br><br />
<br />
==M13 Amplification==<br />
<br><br />
This protocol is to make more M13 phages.<br />
<br />
'''Need'''<br />
<br />
*Fresh plate of infectable cells (contain F’ episome)<br />
<br />
'''Day 1'''<br />
:1.Grow liquid overnight culture of infectable cells<br />
<br />
'''Day 2'''<br />
<br />
:1.Add 200ul overnight culture to 20mL LB in a 250mL flask<br />
:2.Add 1ul phage suspension<br />
:3.Incubate for 4-5 hours at 37°C, 250rpm<br />
:4.Centrifuge for 10 minutes at 4500 x g<br />
:5.Transfer supernatant to a new tube.<br />
:6.Repeat centrifugation on supernatant<br />
:7.Transfer top 16mL of supernatant to a new tube<br />
:8.Add 4mL of 2.5M NaCl/20% PEG-8000. Briefly mix<br />
:9.Precipitate phage for 1 hour or overnight at 4°C<br />
:10.Centrifuge for 15 minutes at 12000 x g. Decant supernatant<br />
::a. Spin briefly. Remove residual supernatant with pipet<br />
:11.Resuspend pellet in 1mL 1x TBS. Transfer to 1.7mL tube<br />
:12.Spin briefly to remove cell debris<br />
:13.Transfer supernatant to a new tube<br />
:14.Add 200ul of 2.5M NaCl/20% PEG-8000<br />
:15.Incubate on ice for 15-60 minutes<br />
:16.Spin for 10 minutes at 12000-14000 rpm. Discard supernatant<br />
:17.Briefly spin. Remove supernatant with pipette<br />
:18.Resuspend pellet in 200uL TBS<br />
:19.For long term storage at -20C, add 200uL glycerol<br />
<br />
<br />
'''2.5M NaCl/20% PEG-8000 (5x)'''<br />
<br />
:100 g PEG-8000 <br />
:75 g NaCl <br />
:400 mL H<sub>2</sub>O <br />
::Bring final volume to 500 mL<br />
<br />
'''TBS (1x)'''<br />
:6.05 g Tris<br />
:8.76 g NaCl <br />
:800 mL H<sub>2</sub>O <br />
<br />
::Adjust pH to 7.6 with 1M HCl<br />
::Adjust volume to 1 L<br />
::Store at 3C for up to 3 months<br />
<br><br />
<br><br />
<br><br />
<br />
==Mini-prep==<br />
<br />
: This protocol is taken from the Qiagen Mini-prep Kit and is used to isolate plasmid DNA from a bacterial overnight.<br />
<br />
'''Notes before starting'''<br />
<br />
: '''Optional:''' Add LyseBlue reagent to Buffer P1 at a ratio of 1 to 1000<br />
<br />
*Add the provided RNase A solution to Buffer P1, mix, '''store bottle at 2-8°C'''<br />
<br />
*Add ethanol (96-100%) to Buffer PE before use<br />
<br />
*All centrifugation steps are carried out at 13,000 rpm (~17,900xg) in a conventional table-top microcentrifuge<br />
<br />
<br />
<br />
:1.Centrifuge 1-6mL bacterial overnight culture at >8000 rpm (6800xg) for 3 minutes at room temperature (15-25C)<br />
:2.Resuspend pellet in 250ul Buffer P1 and transfer to microcentrifuge tube<br />
:3.Add 250ul Buffer P2 and mix thoroughly by inverting the tube 4-6 times until the solution becomes clear.<br />
:a.DO NOT allow lysis reaction to proceed for more than 5 minutes<br />
:b.If using LyseBlue reagent, the solution will turn blue<br />
:4.Add 350ul Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.<br />
:a.If using LyseBlue reagent, the solution will turn colorless<br />
:5.Centrifuge for 10 minutes<br />
:6.Apply supernatant from step 5 to the QIAprep spin column by decanting or pipetting. Centrifuge for 30-60 s and discard the flow-through<br />
:7.Recommended: Wash the QIAprep spin column by adding 500 ul Buffer PB. Centrifuge for 30-60 s and discard the flow-through<br />
:a.Only required when using endA+ strains or other bacterial strains with high nuclease activity or carbohydrate content<br />
:8.Wash the QIAprep spin column by adding 750ul of Buffer PE. Centrifuge for 30-60 s and discard the flow-through<br />
:9.Centrifuge for 1 minutes to remove residual wash buffer<br />
:10.Place the QIAprep column in a clean 1.5mL microcentrifuge tube. To elute DNA, add 30ul Buffer EB. Let stand for 1 min, and centrifuge for 1 minute<br />
::a.Can elute DNA in 50ul but this will decrease DNA concentration<br />
::b.To increase yield, let sit for up to 4 minutes<br />
<br />
'''Buffer Recipes'''<br />
<br />
'''P1'''<br />
50 mM Tris-Cl, pH 8.0<br />
10 mM EDTA<br />
100 ug/mL RNase A<br />
<br />
: *After RNase A addition, the buffer should be stored at 2-8C<br />
<br />
'''P2 (Lysis buffer)'''<br />
:200 mM NaOH<br />
:1% SDS (w/v)<br />
<br />
'''N3*'''<br />
:4.2 M Gu-HCl<br />
:0.9 M KAc, pH 4.8<br />
<br />
'''PB*'''<br />
:5 M Gu-HCl<br />
:30% isopropanol<br />
<br />
'''PE*'''<br />
:10 mM Tris-HCl pH 7.5<br />
:80% ethanol<br />
<br />
'''Elution Buffer (EB)'''<br />
:10 mM Tris-CL, pH 8.5<br />
<br />
:''Recipes from OpenWetWare''<br />
<br />
<br><br />
<br><br />
<br><br />
<br />
==Bacterial Infection==<br />
<br />
'''Need'''<br />
:Fresh plate of cells that contain the F’ episome<br />
<br />
'''Day 1'''<br />
:1.Pick colony from plate and grow overnight<br />
<br />
'''Day 2'''<br />
:1.Dilute overnight to OD600 0.1<br />
:2.Incubate until OD600 ~ 0.5* <br />
:3.Add phage to a final concentration of 1 x108 phage/mL<br />
:4.Incubate for 30 minutes to infect<br />
:5.Plate samples under selection<br />
::a.We recommend trying a range of dilutions<br />
<br />
''Protocol from “Eliminating Helper phage from Phage Display''<br />
<br />
:'''Note:''' All incubations are done at 37°C and shaking at 225rpm<br />
:'''Note:''' If multiple samples are to be done in parallel<br />
:*If using the same cells, we suggest growing one large batch of cells. Once the OD has reached ~0.5, divide into 50mL samples then add phage.<br />
:*If using different cells, grow each sample to ~0.5 then store on ice. Once all samples have reached ~0.5, incubate on ice for another 30 minutes. Warm for 20-30 minutes at 37C, 250 rpm. Measure OD again to check that samples are comparable (yes, they will have grown some). Add phage<br />
<br />
==Cloning a new spacer into pCas9==<br />
<br />
'''[[File:UCB-lab protocol cloning-141010-01.jpg]]'''<br />
<br />
'''Design the new spacer'''<br />
<br />
'''[[File:UCB-lab protocol cloning-141010-02.jpg]]'''<br />
<br />
:1. Find a PAM site (5’- NGG -3’). This will be on the 3’ end of the targeting sequence<br />
:2. The 30bps upstream of NGG motif (on the same strand) will be the spacer<br />
:3. Oligo I (fwd. primer): To the spacer sequence, add AAAC to the 5’ end and G to the 3’ end<br />
::a. NOTE: These nucleotides are for ligation purposes and are NOT used in targeting. Therefore, they do not have to be present in the target sequence and do not count towards the 30bps<br />
:4. Oligo II (rvs. primer): Find the reverse complement of the spacer sequence. Add CAAAA to the 5’ end.<br />
<br />
'''Phosphorylation'''<br />
:1.Mix the following reactants<br />
{| class="wikitable"<br />
|-<br />
!Reactant<br />
!Amount<br />
|-<br />
|Oligo I (100uM)<br />
|1 ul<br />
|-<br />
|Oligo II (100uM)<br />
|1 ul<br />
|-<br />
|10x T4 Ligase Buffer<br />
|5 ul<br />
|-<br />
|T4 PNK<br />
|1 ul<br />
|-<br />
|H2O<br />
|42 ul<br />
|}<br />
<br />
:2.Incubate for 60 minutes at 37C then 20 minutes at 65C<br />
<br />
'''Anneal Oligos'''<br />
:1. Add 2.5 ul of 1M NaCl to the phosphorylation product<br />
:2. Incubate for 5 minutes at 95C then let slowly cool to room temperature.<br />
::a. It is recommended to use a thermocycler that either cools at a gradient or steps down in temperature over the course of ~2 hours.<br />
::b. Alternatively, incubate the sample at 95C for 5 minutes then remove the heat block from the heater and let it cool naturally for 2 hours<br />
:3. Dilute the annealed oligos 1:10<br />
<br />
'''Vector digest with BsaI'''<br />
:1. Mix the following reactants<br />
<br />
{| class="wikitable"<br />
|-<br />
!Reactant<br />
!Amount<br />
|-<br />
|Cutsmart Buffer<br />
|5 ul<br />
|-<br />
|BsaI enzyme<br />
|1 ul<br />
|-<br />
|DNA<br />
|1-2ug<br />
|-<br />
|H2O<br />
|Bring volume to 50ul<br />
|}<br />
:2. Incubate for 90 minutes at 37C then 20 minutes at 65C<br />
:3. Gel extract digested pCas9<br />
<br />
'''Ligation'''<br />
:1. Mix the following reactants<br />
{| class="wikitable"<br />
|-<br />
!Reactant<br />
!Amount<br />
|-<br />
|Gel extracted vector<br />
|1 ul<br />
|-<br />
|Diluted annealed oligos<br />
|2 ul<br />
|-<br />
|10x T4 Ligase Buffer<br />
|2 ul<br />
|-<br />
|T4 Ligase<br />
|1 ul<br />
|-<br />
|H2O<br />
|14 ul<br />
|}<br />
:2. Incubate overnight (8-12 hours) at 16C<br />
<br />
'''Transform'''<br />
:1. Transform into DH5alpha<br />
<br />
<br />
''Protocol from Marraffini Lab''<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{Template:UCB-Footer}}</div>Leighlahttp://2014.igem.org/Team:CU-Boulder/Notebook/CC9_Team/ConstitutiveTeam:CU-Boulder/Notebook/CC9 Team/Constitutive2014-10-17T06:57:45Z<p>Leighla: /* Week 13/14 */</p>
<hr />
<div>{{Template:UCB-Main}}<br />
{{UCB-NavBar}}<br />
__FORCETOC__<br />
<br />
==Week 1==<br />
<br />
'''6/4'''<br />
*Resuspended BBa_K1218011 (biobricked pCas9) from Distribution Kit (4M)<br />
::-Part biobricked by Stanford-Brown 2013<br />
::-Part contains native tracrRNA, promoter, and Cas9 (the Cas9 was modified to remove an EcoRI digestion site) along with a minimal CRISPR array<br />
*Made chemically competent DH3alpha cells<br />
*Tested new competent cells through transformation<br />
:::-DNA<br />
:::*1ul p11+RBS(2) (4/23)<br />
:::*2ul 2B<br />
:::*2ul 2P<br />
:::*3ul 2P<br />
:::*No DNA<br />
::-Plated onto Chlor<br />
::-Also, thawed 5a cells for 45 minutes then returned to freezer<br />
::*Tomorrow, we will transform and test competency<br />
::*Also include non-competent control<br />
<br />
'''6/5'''<br />
*Transformation results from 6/4<br />
::- No growth on negative (no DNA) control: no contamination<br />
::- > 400 colonies for positive (p11+RBS (2), diluted 1:10) control. Lawn when not diluted<br />
::- ~100 colonies for 2B (2ul) not diluted, 18 colonies on 1:10 dilution<br />
::- 3 colonies for 2P (2ul) not diluted, 0 on 1:10 dilution<br />
::- 20 colonies for 2P (3ul) not diluted, 1 on 1:10 dilution<br />
*Made overnights of 2B and 2P to make freeze downs tomorrow<br />
<br />
'''6/7'''<br />
*Made 6mL O/N cultures<br />
::-4 from (3) cas9 plate<br />
<br />
==Week 2==<br />
<br />
'''6/8'''<br />
*Check colonies for presence of Cas9 plasmid<br />
::-Mini-prepped O/Ns from 6/7<br />
::-Digest with EcoRI and PstI (10ul reactions)<br />
::-Run results on a gel<br />
:::*All 4 samples had the expected bands of 2000 and 5000bp showing that we had Cas9 safely inside cells<br />
'''[[File:UCB-Consitiutive Crispr-140609.jpg]]'''<br />
:Gel of mini-preps containing Cas9<br />
::-2-log ladder<br />
<br />
'''6/9'''<br />
*Freeze downs<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Top Label<br />
! Side label<br />
! What?<br />
<br />
|-<br />
| Cas9 6/9<br />
| From dis kit 1<br />
| Cas9 construct from Sanford-Brown team<br />
<br />
|-<br />
| -> K1218011 (tracrRNA, Cas9, minimal CRISPR)<br />
<br />
|-<br />
| Cas9 6/9<br />
| From dis kit 2<br />
| “ “<br />
<br />
|-<br />
|}<br />
*Overnight cultures of Cas9 (Qi wrCas9 #P44250) and gRNA (Qi gRNA #P44251 cells from Andrew<br />
<br />
==Week 7==<br />
<br />
'''7/11'''<br />
*Trying to get pSB1C3-pCas9 (BBa_K1218011) to function<br />
::-Ordered 2 primers pairs so we could make 2 targeting guide RNAs<br />
:::*These guide RNAs target the Neocin (also Kanamycin) resistance gene in our BW strain.<br />
*Vector Digest<br />
***Digest 1-2ul of pCas9 with BsaI-HF<br />
'''[[File:UCB-Constitutive Crisper-140711.jpg]]'''<br />
:-Sample was divided between two lanes<br />
:-Gel extracted digestion but poor yield<br />
'''[[File:UCB-Constitutive Crisper-140711-02.jpg]]'''<br />
:-Redigest with less DNA<br />
::1. pCas9 from above gel<br />
::2. Different sample of pCas9<br />
:-Repeated, doing everything we could to increase gel extraction yield-- no luck<br />
<br />
*Phosphorylation<br />
::-phosphorylated primer pairs together<br />
::-1 hr at 37C then 20min at 65C<br />
*Annealing<br />
::-(forgot to add 2.5ul of 1M NaCl)<br />
::-Incubated in Thermocycler for 5 minutes at 95C<br />
::-Tried to decrement temp 0.1C/s until temp reached 21, then jumped to 4C<br />
:::*Took less than 30 minutes so we think the samples cooled too fast<br />
<br />
'''7/12'''<br />
*Trying to switch the gRNA in the CRISPR<br />
::-Phosphorylation<br />
:::-Phosphorylated primer pairs same as 7/11<br />
::-Annealing<br />
:::-Remembered to add 2.5ul of 1M NaCl<br />
:::-1. Heat block<br />
::::*5min at 95C-> removed from head to let cool 2 hours<br />
::::*Problem-> water condenses on lid<br />
:::-2. Thermocycler<br />
::::*Was supposed to cool slowly… not the case<br />
::-Vector Digest<br />
:::-Digested pCas9<br />
::::*1. 60 min at 37, 20 min at 80 (see #4 on gel below)<br />
::::*2. 90 min at 37, 20 min at 80 (see #5 on gel below)<br />
:::-Compared to un cut on a gel…. all bands looked pretty. Also, included gel extractions from 7/11 to test whether DNA was present<br />
'''[[File:UCB-Constitutive Crisper-140712.jpg]]'''<br />
:1. Gel extract 1 from 7/11<br />
:2. Gel extrac 2 from 7/11<br />
:3. Uncut pCas9<br />
:4. Digested pCas9 for 60 minutes<br />
:5. Digested pCas9 for 90 minutes<br />
:::-Did not gel extract sample<br />
::-Phosphorylation #3<br />
:::-Because I did not like my previous annealing attempts, so I decided to start over<br />
::-Annealing<br />
:::-Added 2.5ul 1M NaCl<br />
<br />
{| class = "wikitable"<br />
|-<br />
! 95C<br />
! 94.8<br />
! 94.6<br />
! 94.4<br />
! 94.2<br />
! 94.0<br />
! 21<br />
<br />
|-<br />
| 5 min<br />
| :20<br />
| :20<br />
| :20<br />
| :20<br />
| :20<br />
| hold<br />
<br />
|-<br />
| decrement<br />
| -1<br />
| -1<br />
| -1<br />
| -1<br />
| -1<br />
| <br />
<br />
|-<br />
|}<br />
::-font cycled 74 times. After each step, the temp would decrement by 1 to produce a consistent cooling rate of 0.2C/20s (about equivalent to letting a heat block cool<br />
::-Diluted 1:10<br />
::-Ligation<br />
:::-4 reactions total<br />
::::gRNA1- gel extracted pCas9<br />
::::gRNA1- non-gel extracted pCas9<br />
::::gRNA2- gel extracted pCas9<br />
::::gRNA2- non-gel extracted pCas9<br />
<br />
==Week8==<br />
<br />
'''7/13'''<br />
*Transformation of 7/12 ligations (attempts to swap gRNAs in the CRISPR)<br />
::-Plated on low Chlor (34ug/mL) and high Chlor (170ug/mL)<br />
<br />
'''7/14'''<br />
*Did get colonies for pCas9 with new gRNAs (some for each primer pair)<br />
::-Colony PCR using VR and Fwd gRNA primer<br />
:::-Dream taq<br />
::-Gel had big black spot<br />
:::-(Realized this the next day that the imager was set to high exposure so could not see bands)<br />
::-Set up O/N of colonies in despair<br />
<br />
'''7/15'''<br />
*Check colonies for pSB1C3-pCas9 using mini-prepped DNA<br />
::-Mini-prepped O/Ns<br />
::-Repeated PCR using VR and gRNA primer<br />
'''[[File:UCB-Constitutive Crisper-140715.jpg]]'''<br />
::-Found some samples with new gRNA<br />
::-Tried to make O/Ns of correct samples….. turns out there was a miscommunication and DNA, not living cells, were added to the liquid cultures…<br />
*First proof of concept: Transformation of pSB1C3-pCas9 containing targeting gRNAs into BW23115 chemically competent cells and 5alphas<br />
::-DNA<br />
:::pSB1C3-pCas9(unmodified)--> non-targeting<br />
:::pSB1C3-pCas9(gRNA1)--> targeting<br />
:::pSB1C3-pCas9(gRNA2) --> targeting<br />
::-Controls<br />
:::-5alpha cells. These cells do not contain kanamycin resistance in their genome so should not be targeted<br />
:::-pSB1C3-pCas9(unmodified): gRNA does not target anywhere in either E. coli genome so is we expect no targeted killing; hence no death<br />
::-Hypothesis: gRNA1 and gRNA2 are complimentary to the kanamycin resistance gene in the BW23115 strain. Therefore, we expect the samples that contain pCas9 with these targeting gRNAs to target and cleave the resistance gene and cause either direct cell death or a loss of function of the kanamycin resistance gene, resulting in cell death. <br />
<br />
'''7/17'''<br />
*Results of 7/15 pCas9 transformation experiment<br />
::-5alphas<br />
:::*No growth for No DNA control on Chlor<br />
:::*Much growth for non-targeting gRNA and targeting gRNA1<br />
:::*Half as much growth for targeting gRNA2<br />
::-BW23115<br />
:::*No growth for No DNA control on Chlor+Kan<br />
:::*Fewer, but larger colonies for non-targeting sample<br />
:::*More growth for targeting gRNA1 and gRNA2? large range of sizes<br />
::-Conclusions<br />
:::*Targeting the Kanamycin resistance gene did not kill the bacteria. <br />
::-Repeat the transformation with remaining DNA samples<br />
<br />
'''7/18'''<br />
*Made liquid O/Ns of gRNAs to remake pSB1C3-pCas9(gRNA1) and pSB1C3-pCas9(gRNA2)<br />
<br />
'''7/19'''<br />
*Tested overnights from 7/18 pSB1C3-pCas9 with gRNAs 1 and 2<br />
::-Mini-prepped<br />
::-Repeated PCR with DreamTaq<br />
:::*used VR (reverse primer for iGEM biobricks (amplifies insert from end)) and fwd primer used to make gRNA<br />
::::*There should only be a product if the fwd primer can anneal to DNA. Product should be about 400bp<br />
::-Found some samples with correct band at 400bp- these were the correct sizes so are likely correct<br />
<br />
==Week 9==<br />
<br />
'''7/20'''<br />
*Freeze downs of samples from 7/19 that looked right<br />
::-gRNA1 targets nt 23-53 of KanR gene <br />
::-gRNA2 targets ny 551-582 of KanR gene<br />
<br />
'''7/21'''<br />
*Sent samples for sequencing<br />
::-pSB1C3-pCas9(gRNA1)<br />
::-pSB1C3-pCas9(gRNA3)<br />
<br />
'''7/22'''<br />
*Pricked colony from pSB1C3-pCas9 plate<br />
::-Mini-prepped DNA to isolate DNA<br />
<br />
'''7/23'''<br />
*Sequencing samples from 7/21 were lost in the mail. Resent samples<br />
*Primers came in for new guide RNAs<br />
::-Note: Upon review of the protocol, we realized that the first round of primers we bought were incorrectly made, resulting in a 1nt shift away from the PAM site. The NGG motif requirement was not met so binding was absent. We bought new primers to correct this problem<br />
::-gRNA3: Targets same place as gRNA1 but is corrected to bind the correct distance from the PAM site<br />
::-gRNA4: Targets same place as gRNA2 but is corrected to bind the correct distance from the PAM site<br />
*To swap gRNAs on pCas9 (gRNA3 and gRNA4)<br />
::-Phosphorylate primers 1hr @ 37C, 20 min @ 65C<br />
::-Annealed primers<br />
:::*Used the thermocycler like on 7/12<br />
'''[[File:UCB-Constitutive Crisper-140723.jpg]]'''<br />
::-Digest vector DNA (pCas9 DNA with BsaI)<br />
:::-Did not get complete digestion<br />
<br />
'''7/24'''<br />
*To swap gRNAs on pSB1C3-pCas9 (gRNA3 and gRNA4)<br />
::-Used digested pSB1C3-pCas9 from 7/12. Same one used the last time we swapped gRNAs<br />
::-Ligated the above pSB1C3-pCas9 to annealed oligos<br />
<br />
'''7/25'''<br />
*Transformed ligation from 7/24 (gRNA1/gRNA2 into pSB1C3-pCas9) into 5alpha cells<br />
<br />
'''7/26'''<br />
*No growth for 7/25 transformation<br />
::-Retried transformation<br />
::-pCas9 was digested a week earlier- possible source of problem<br />
*Retried making pCas9 with gRNA3 and gRNA4<br />
::-Digestion (so we have freshly cut samples)<br />
:::pSB1C3-pCas9 with BsaI<br />
'''[[File:UCB-Constitutive Crisper-140726.jpg]]'''<br />
:::-gel extracted linearlized pCas9 (smaller band)<br />
::-Ligation (10hrs at 16C, 10min at 80C)<br />
:::*pSB1C3-pCas9 + gRNA3<br />
:::*pSB1C3-pCas9 + gRNA4<br />
<br />
==Week 10==<br />
<br />
'''7/27'''<br />
*Transformation results<br />
::-No growth for no DNA controls--> on Chlor<br />
::-No growth for gRNA4 --> on Chlor<br />
::-3 colonies for gRNA3 --> on Chlor<br />
*Picked the 3 colonies for gRNA3 for O/Ns<br />
<br />
'''7/28'''<br />
*All O/Ns grew<br />
*To test the overnights for gRNA3<br />
::-Mini-prep samples -> good yields<br />
::-PCR with Dream-Taq<br />
:::-Diluted all samples 1:100<br />
'''[[File:UCB-Constitutive Crisper-140728.jpg]]'''<br />
<br />
:1. pSB1C3-pCas9(unmod.)<br />
:2. Sample 1<br />
:3. Sample 2<br />
:4. Sample 3<br />
:5. No DNA control<br />
::-All samples contained a band of the expected <400bp, including the Stanford-Brown Cas9 unmodified and no DNA control<br />
*Transformations<br />
::-pSB1C3-pCas9(gRNA3)<br />
::-SB1C3-pCas9(gRNA4)<br />
::-Positive control with pSB1C3-pCas9(unmod.)<br />
::-No DNA control<br />
<br />
'''7/30'''<br />
*Sent samples for sequencing<br />
::-pSB1C3(gRNA3) (5) VR<br />
::-pSB1C3(gRNA3) (6) VR<br />
::-pSB1C3(gRNA3) (7) VR<br />
<br />
'''7/31'''<br />
*Sequencing results came back positive<br />
::-Had correct gRNA3 insert<br />
*Transformation experiments with gNRAs: Repeat of 7/15 experiment<br />
::-Samples<br />
:::-Scramble- unmodified pCas9<br />
:::-non-targeting- gRNA1<br />
:::-targeting- gRNA3<br />
:::-no DNA control<br />
::-Added 578ng each<br />
::-Transformed into 5alpha and BW (F+, +KanR)<br />
<br />
'''8/1'''<br />
*Transformation results from 7/31<br />
::-Contamination for 5alpha samples<br />
::-BW cells on Chlor<br />
:::-Hundreds of colonies for all (unmodified, gRNA1, gRNA3)<br />
:::-No colonies on no DNA control<br />
::-BW cells on Chlor + Kan<br />
:::-Hundreds of colonies for unmodified and gRNA1<br />
:::-3-4 colonies for gRNA3<br />
:::-No colonies for no DNA control<br />
::-Notes:<br />
:::*Unexpected that for gRNA3, cells died on Chlor+Kan but survived at comparable amounts on Chlor<br />
::::-Suggests that targeting of an essential gene<br />
::::-Later, cells on gRNA3 plate looked sickly, maybe delayed killing?<br />
<br />
==Week 11==<br />
<br />
'''8/4'''<br />
*Spotted contamination in SOC-> remade SOC<br />
*Got new 5alpha (from NEB) from Mike to make comp cells (C2987 by NEB)<br />
*Transformation experiment (~578ng)<br />
::-(barrier tips, new SOC, BW cells (5/30))<br />
::-unmodified pCas9<br />
::-gRNA1<br />
::-gRNA3<br />
::-No DNA control<br />
*Test pCas9 for functional Cas9 rather than dCas9<br />
::-Digestion to confirm<br />
::-pCas9 EcoRV no extraction- just image on gel<br />
'''[[File:UCB-Constitutive Crisper-140804.jpg]]'''<br />
*To add gRNA4 to pSB1C3-pCas9<br />
::-Digestion<br />
:::*pCas9 BsaI extracted entire plasmid<br />
:::*insert gel<br />
::-Ligation (10 hrs at 16C, 10 min at 80C)<br />
:::*pSB1C3-pCas9 + gRNA4<br />
:::*No DNA control to test for contamination in reagents<br />
<br />
'''8/5'''<br />
*Transformation into ER2738 (7/18), 5ul of ligation products<br />
:1. Litmus28ibb-pCas9gRNA3<br />
:2. pSB1C3-Cas9 (gRNA4)<br />
:3. No DNA in ligation mix<br />
:No DNA control<br />
:Variations on protocol<br />
::-Added 200ul SOC to 1.7ul epi-tubes, incubated in small tubes<br />
::-Incubated at ~200rpm<br />
::-Added only 5ul Ligation products<br />
*Make chem comp 5alpha cells<br />
::-Got new 5alpha cells from Mike (chem comp from NEB) on 8/4<br />
::-grew O/N in 5mL LB<br />
::-Made fresh .8M MgCl2 and .2M CaCl2<br />
*Transformation results from 8/4 [not targeted killing experiment]<br />
::-No growth when only SOC was plate<br />
:::*SOC is not contaminated with cells ChlorR or ChlorR+kanR<br />
::-No growth for No DNA controls on A+T or C+T<br />
:::*Negative Ligation control<br />
:::*Negative transformation control<br />
::-No growth for M13g-3C5 or LCR ligations (8/3)<br />
::-Lawn for amilCP on pSB1C3 transformation<br />
*Targeted killing experiment Transformation results<br />
::-Images of plates can be found in the results section<br />
::-Colony count on chlor (34ug/mL) and kan (25ug/mL) - kanR is essential<br />
<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! 1:1<br />
! 1:10<br />
<br />
|-<br />
| unmodified<br />
| lawn<br />
| 2760<br />
<br />
|-<br />
| gRNA1<br />
| lawn<br />
| 2316<br />
<br />
|-<br />
| gRNA3<br />
| 23<br />
| 1<br />
<br />
|-<br />
| no DNA control<br />
| 0<br />
| NA<br />
<br />
|-<br />
|}<br />
**Colony count on chlor (170ug/mL) - kanR is non-essential<br />
<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! 1:1<br />
! 1:10<br />
<br />
|-<br />
| unmodified<br />
| 1920<br />
| 226<br />
<br />
|-<br />
| gRNA1<br />
| 960<br />
| 71<br />
<br />
|-<br />
| gRNA3<br />
| 8<br />
| 1<br />
<br />
|-<br />
| no DNA control<br />
| 0<br />
| NA<br />
<br />
|-<br />
|}<br />
::-Shows successful killing when KanR is essential and when KanR is nonessential<br />
<br />
'''8/6'''<br />
<br />
*Transformation results from 8/5<br />
::-No colonies for pCas9(gRNA4) on Tet+reduced Chlor<br />
:::-BLASTed sequence against MG1655. The 10nts at the 3’ end of spacer match a sequence in MG1655 that is adjacet to a PAM site. We think gRNA is targeting the production strain.<br />
::::*To compare, the spacer of gRNA3 matches 8nts on its 3’ end.<br />
::-No growth for no DNA control of ligation or transformation<br />
:::-Ligation reactants are not cause of recent contamination on Amp or Chlor<br />
<br />
==Week 13/14==<br />
<br />
'''8/12'''<br />
*Targeted Killing Transformation<br />
::-Transform into 3alpha (7/28) and BWF’ (5/30)<br />
::-DNA<br />
:::*pSB1C3-pCas9(unmod.)<br />
:::*pSB1C3-pCas9(gRNA1)<br />
:::*pSB1C3-pCas9(gRNA3)<br />
<br />
'''8/16'''<br />
Results of 8/12 transformation of BWF’ with gRNA(unmod, 1, or 3) targeted killing<br />
<br />
No growth on Chlor (34ug/mL) or Chlor (34ug/mL)+Kan(25ug/mL)<br />
<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! Chlor+Kan<br />
! Chor<br />
<br />
<br />
|-<br />
| Scramble<br />
| Healthy lawn<br />
| Healthy lawn<br />
<br />
|-<br />
| gRNA1<br />
| Healthy lawn (thinner that scramble)<br />
| Healthy lawn (thinner that scramble)<br />
<br />
|-<br />
| gRNA3<br />
| Shadowy, flat lawn, 42 healthy colonies<br />
| Shadowy, flat lawn, 42 healthy colonies<br />
|}<br />
<br />
'''8/19'''<br />
<br />
Sequence survivor colonies from transformation experiments. These are the colonies from gRNA3 samples.<br />
<br><br />
<br><br />
<br />
{{Template:UCB-Footer}}</div>Leighlahttp://2014.igem.org/Team:CU-Boulder/Notebook/Phage_TeamTeam:CU-Boulder/Notebook/Phage Team2014-10-17T06:56:14Z<p>Leighla: /* Week 19 */</p>
<hr />
<div>{{Template:UCB-Main}}<br />
{{UCB-NavBar}}<br />
__FORCETOC__<br />
<br />
'''Phage Delivery'''<br />
==Week 1==<br />
'''Notes:''' Unless stated otherwise, all gels contain 2-log ladder<br />
<br />
'''5/9'''<br />
*Obtained BW23115 KanR cells- BW23115 cells that had their native CRISPR-Cas system knocked out by the insertion of a Kanamycin resistance gene<br />
::-Will also be called BW23115 or BW<br />
::-Conjugated BW23115 KanR cells with contain F’ notation (ex. BWF’)<br />
*Obtained ER2738 cells that contain the F’ episome (no changes from NEB sample)<br />
::-Will also be called ER. Assume that all ER samples contain the F’ episome<br />
::-Streaked sample onto LB+Tet (20ug/mL) to select for colonies containing F’ episome<br />
<br />
'''5/10'''<br />
*Did receive colonies from 5/9 selection<br />
<br />
==Week 2==<br />
'''5/12'''<br />
*Need to conjugate BW23115 KanR cells with the F’ episome<br />
::-Set up overnight cultures of ER2738 and BW23115 KanR<br />
::-When mixed, ER2738 will donate it’s F’ episome and BW23115 KanR will receive the F’ episome. F’ episome confers Tetracycline resistance<br />
<br />
'''5/13'''<br />
*Started M13 Amplification: Amplify M13 phage using the M13K07 Helper Phage<br />
::-Let precipitated in NaCl/PEG solution overnight<br />
::-Possible sources of error<br />
:::*Did not sterilize 2.5M NaCl/20% PEG-8000 solution<br />
:::*Added 4-fold PEG solution<br />
::::Compensated by adding more LB<br />
:::*During precipitation, put sample in -20C for 30 minutes before realizing mistake and moving to it to 4C. Sample partially froze<br />
*Conjugated BW23115 with F’ episome<br />
::-Added 1mL BW23115 to 1mL ER2738 overnight culture<br />
::-Incubated at 37C for 30 minutes, shaking<br />
::-Plated on LB+Kan(50ug/mL)+Tet(20ug/mL)<br />
:::*To select for BW cells that took the F’ episome (containing Tet resistance)<br />
<br />
'''5/14'''<br />
*Finished the M13 Amplification<br />
::-Visualized product on UV-vis. There was a tall spike at 269nm indicating that DNA was present. Did not test at 320nm.<br />
*Results of BW23115 Conjugation<br />
::-Many colonies indicating successful conjugation of F’ episome into BW23115<br />
::-Set up overnight to make freeze down tomorrow<br />
*Set up overnight of ER2738 to make chemically competent tomorrow<br />
<br />
'''5/15'''<br />
*Made freeze down of BW23115 KanR F’<br />
::-BW23115 E. coli strain with Kanamycin resistance gene inserted into genome and with F’ episome<br />
*Made chemically competent ER2738 cells<br />
*Transformation of Litmus28i (from NEB) into chemically competent ER2738 cells<br />
::-Added 1ul Litmus28i plasmid to 40ul competent cells<br />
::-Plated on LB + Amp(100ug/mL)<br />
::-Purpose: To make M13 phage that package Litmus28i DNA. Need phagemid (Litmus28i) DNA in infectable cells (cells containing F’ episome) to introduce M13K07 Helper Phage and make phage. <br />
<br />
'''5/16'''<br />
*Results of 5/15 transformation<br />
::-No growth for No DNA control<br />
::-Many colonies for sample<br />
<br />
==Week 3==<br />
'''5/19'''<br />
*M13 Amplification to isolate M13-Litmus28i phage<br />
::-Cells: ER2738 cells containing Litmus28i phagemid<br />
::-Helper Phage: M13K07<br />
::-Not much phage was precipitated<br />
*Set up overnight culture of ER2738 to infect tomorrow<br />
<br />
'''5/20'''<br />
*Infected ER2738 cells with M13-Litmus28i phage<br />
::-Plated only on Ampicillin(100ug/mL) (should have also plated on kanamycin)<br />
::-Infected for 4-5 hours-> should have only infected for 30 minutes maximum. This extra time gives the cells that were infected with M13-M13K07 the time to produced M13-M13K07 phage and reinfect<br />
<br />
'''5/21'''<br />
*Results from M13-Litmus28i infection of ER2738<br />
::-Solid lawn of growth for diluted and non-diluted<br />
::-Also sickly looking growth<br />
*Set up overnights<br />
::-ER2738 cells containing Litmus28i for freeze down<br />
::-BW23115 with F’ episome to make chemically competent cells <br />
::-ER2738 to redo infection<br />
<br />
'''5/22'''<br />
*Tested absorbance of phage produced through M13 amplification on 5/19<br />
::-Low absorbance of 0.018 at 269nm but no detection at wavelength 320nm<br />
::-Decided to redo M13 amplification<br />
*Made chemically competent BW23115 with f-episome<br />
*Made freeze down of ER2738 containing Litmus28i<br />
*Set up overnight of ER2738 containing Litmus28i to redo M13 amplification tomorrow<br />
<br />
'''5/23'''<br />
*Protocol switch to make phage using phagemid<br />
::-“M13 Amplification” protocol should only be used to make more M13-M13K07, not to make M13 phage containing a different phagemid<br />
::-Switched to new protocol (“Use of M13K07 Helper Phage for isolation of single stranded phagemid DNA” by NEB. Made modifications (see our protocols) to isolate phage rather than single-stranded DNA)<br />
::-Making phage….<br />
{| class = "wikitable"<br />
|-<br />
! Helper phage<br />
! Phagemid<br />
! Cells<br />
! Notes<br />
<br />
|-<br />
| M13K07<br />
| None<br />
| ER2738<br />
| Make more M13-M13K07<br />
<br />
|-<br />
| M13K07<br />
| Litmus28i<br />
| ER2738<br />
| Test packaging of Litmus28i<br />
<br />
|-<br />
|}<br />
*Made fresh antibiotics<br />
::-Chloramphenicol (34 ng/mL)<br />
:::*1.44g chloramphenicol into 42mL EtOH<br />
::-Ampicillin (50 ng/mL)<br />
:::*4g ampicillin into 80mL mili-Q H2O<br />
<br />
'''5/24'''<br />
*Isolated phage using new protocol<br />
::-Resuspended pellet in 200ul TBS and 200ul 30% glycerol<br />
::-Measured absorbance with UV-vis<br />
:::*concentration (phage/mL) = 6x10^16 x (A269-A320)/ (#of base pairs in the phage genome)<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! Abs (269nm)<br />
! Abs (320nm)<br />
! Genome size<br />
! Concentration (phage/mL)<br />
<br />
|-<br />
| M13-M13K07<br />
| 0.721<br />
| 0.060<br />
| <br />
| 4.57 x10^12<br />
<br />
|-<br />
| M13-Litmus28i<br />
| 0.250<br />
| 0.028<br />
| 2823<br />
| 4.72 x10^12<br />
<br />
|-<br />
|}<br />
*Infect ER2738 cells with M13-Litmus28i<br />
::-Wanted 1:10 phage:cell ratio. Math….<br />
:::*At 1 OD (e.coli), cell/mL = 5x10^8<br />
:::*5x10^7 phage * (1mL/4.72x10^12 phage) = 0.011ul phage<br />
*Set up overnights<br />
::-ER2738 for infection with M13-Litmus28i<br />
::-BW23115 F’ for infection with M13-Litmus28i to test infectivity of conjugated strain<br />
<br />
==Week 4==<br />
'''5/25'''<br />
*Infect ER and BWF’ cells with M13-Litmus28i<br />
::*Made 5mL culture of ER and BW that was at 1 OD<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! OD<br />
! mL sample for 1OD in 5mL<br />
! mL LB to 5mL<br />
<br />
|-<br />
| ER2738<br />
| 2.5<br />
| 2 mL<br />
| 3 mL<br />
<br />
|-<br />
| BW23115<br />
| 2.0<br />
| 2.5 mL<br />
| 2.5 mL<br />
<br />
|-<br />
|}<br />
::-Based on calculations from 5/24, we needed to add 0.011 ul phage per 1 mL of cells at 1 OD. This equates to 0.055 ul of phage into 5 mL cells; therefore we made a 1:10 dilution so we could add 0.5ul. Unfortunately, the pipet would not take up 0.5ul so we added 0.8ul of M13-Litmus28i phage<br />
::-Grew the cells for 20 minutes at 37C<br />
::-Plated 300ul onto Kanamycin (50ug/mL) and 300ul onto Ampicillin (100ug/mL) for each sample<br />
:::*Incubated overnight at 37C<br />
*'''Note:''' During the production of phage, the phagemid SHOULD be packaged preferentially over the Helper Phagemid but some Helper Phagemid will still be packaged. We plated on Amp to select for cells that were infected with phage containing Phagemid. We plated on Kan to select for cells that were infected with phage containing Helper Phagemid. This allows us to compare the packaging efficiency of Helper Phagemid: Phagemid.<br />
'''5/26'''<br />
*Results from 5/25 infection with M13-Litmus28i<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Result<br />
! Significance<br />
<br />
|-<br />
| ER2738 on Amp<br />
| Lawn<br />
| Litmus28i phagemid was successfully packaged into the M13 phage and is infectable<br />
<br />
|-<br />
| ER2738 on Kan<br />
| 100-200 colonies<br />
| Some M13 helper phage is packaged into the M13 phage but at a much lower rate than Litmus28i<br />
<br />
|-<br />
| BW23115 on Amp<br />
| Lawn<br />
| BW23115 is ‘equally’ infectable by M13 as ER2738<br />
<br />
|-<br />
| BW23115 on Kan<br />
| Lawn<br />
| BW23115 contains Kan resistance in its genome so this tells us nothing<br />
<br />
|-<br />
|}<br />
:*Conclusions:<br />
::-Cells grew on Ampicillin; therefore, Litmus28i phagemid was successfully packaged into M13 phage. <br />
::-For ER2738 samples, there was significant growth on Ampicillin compared to Kanamycin; therefore, Litmus28i phagemid is packaged preferentially over M13K07 Helper Phagemid<br />
::-M13-Litmus28i retains its infectivity of cells containing the F’ episome<br />
:*Because we received lawns, we have to redo the infection and plate less cells so we can calculate the uptake ratio between the phagemid and helper phage based on the number of colonies<br />
*Started 50 mL overnight of K12 ER2738 and BW23115<br />
<br />
'''5/27'''<br />
*Redo the infection done on 5/25<br />
::-Infectable cells: ER2738 and BW23115<br />
:::*Plated non-infected samples of each (non-diluted) to check for contaminants<br />
::-Diluted M13-Litmus28i (1) phage by a factor of 10. Added 5.5ul to each sample<br />
::-Grew samples for 20 minutes at 37C, 250rpm<br />
::-Plated 100ul of onto an Ampicillin (100ug/mL) plate and onto a Kanamycin (50ug/mL) plate. Incubated overnight at 37C.<br />
:::*Dilutions = 1:10; 1:100; and 1:1000<br />
'''5/28'''<br />
*Results from 5/27<br />
::-Controls were as expected<br />
:::*No growth for ER2738 non-infected grown on Amp, ER2738 non-infected grown on Kan, or BW23115 non-infected grown on Amp<br />
:::*Growth for BW23115 non-infected grown on Kan (BW23115 has Kan R in genome)<br />
::-Many colonies were received for all dilutions (1:10, 1:100, and 1:1000) of the following<br />
:::*ER2738 infected and plated on Amp<br />
:::*BW23115 infected and plated on Amp<br />
:::*BW23115 infected and plated on Kan<br />
::-Many (100s to 1000?) colonies grew on 1:10 and 1:100 dilutions of ER2738. 50-100 colonies grew on the 1:1000 dilution of ER2738<br />
:::*Compare this to the 100-200 colonies that grew from 2/25 infection (which was 300ul non-diluted, infected cells)<br />
:::*Reasons for increased yield<br />
::::*Added too much phage?<br />
::::*volume changed between experiment (5mL to 50mL)<br />
::::*Overnight culture may not have been saturated. If still in log phase, the cells would continue to grow<br />
*Made 50mL O/N cultures of ER2738 and BW23115 so we can repeat the infection tomorrow and plate further dilutions starting at 1:1000<br />
::-Carry out infection in 5mL and 50mL to test volume effect?<br />
<br />
'''5/29'''<br />
*Measured OD of overnights<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! OD<br />
! mL to have .1OD in 50mL<br />
! mL to have 1OD in 5mL<br />
<br />
|-<br />
| K12 ER2738<br />
| 3.0<br />
| 1.7<br />
| 1.7<br />
<br />
|-<br />
| BW23115<br />
| 2.9<br />
| 1.7<br />
| 1.7<br />
<br />
|-<br />
|}<br />
<br />
<br />
*Experiment 1: Infect cells using same method as 5/25 (in a 5mL culture)<br />
::-Started with 1OD cells in 5mL<br />
::-Added about 0.7ul (inaccuracies in pipet) of 1:10 diluted M13-Litmus28i phage<br />
::-Incubated (rotating) for 20 minutes<br />
::-Made 1:1,000 and 1:10,000 dilutions<br />
::-Plated 100ul on Ampicillin (100ug/mL) plates and on Kanamycin (50ugmL) plates<br />
:::*Included non-infected samples diluted by 1:1000<br />
::::*This negative control can be used for Experiment 2 since the non-infected parent solution is the same<br />
::-Incubate overnight at 37C<br />
*Experiment 2: Infect cells using protocol from “Eliminating helper phage from phage display”<br />
::-Diluted O/Ns to OD of 0.1 in 50 mL culture<br />
::-Grew samples until of OD of ER2728 = 0.59 and the OD of BW23115 = 0.60<br />
:::*Missed OD of 0.5 mark, but the two samples are close to each other<br />
::-Chilled samples on ice for 30 minutes<br />
::-Warmed in incubator for 35 minutes (should have been 30)<br />
::-Amount of phage. Rather than use 1:1 as mentioned in protocol, we used multiplicity of 1:10 (phage:cell)<br />
:::*Added 3.3ul of 1:10 diluted M13-Litmus28i (1) phage<br />
::::*(On 5/27 we added 5.5ul of diluted phage to 50mL of cells at OD of 1. Our cells were at OD of .6; therefore, 5.5*.6 = 3.3ul)<br />
::-Incubated for 30 minutes at 37C, not shaking<br />
:::*We later change this to shaking<br />
::-Dilutions<br />
:::*1:1,000; 1:5,000; 1:10,000; 1:50,000; 1:100,000; 1:1,000,000<br />
:::*Plated ER2738 and BW23115 on Ampicillin (100ug/mL)<br />
:::*Plated ER2738 on Kanamycin (50ug/mL)<br />
*Experiment 3: Growth Test (for growth curve)<br />
::-We were concerned by the low OD of the Overnights from the last few days. Wanted to be sure that 2.0-3.0 was not still in log phase. Cultures looked saturated but the OD seemed low.<br />
{| class = "wikitable"<br />
|-<br />
! Time<br />
! Elapsed time (min)<br />
! ER2738 (no antibiotic)<br />
! ER2738 (Tetracycline (20ug/mL))<br />
! BW23115 (with F’ episome) no antibiotic<br />
<br />
|-<br />
| 10:08<br />
| 0<br />
| 0.1<br />
| 0.1<br />
| 0.1<br />
<br />
|-<br />
| 11:10<br />
| 62<br />
| 0.24<br />
| 0.21<br />
| 0.21<br />
<br />
|-<br />
| 12:00<br />
| 112<br />
| 0.49<br />
| 0.45<br />
| 0.44<br />
<br />
|-<br />
| 13:00<br />
| 172<br />
| 1.00<br />
| 0.93<br />
| 0.98<br />
<br />
|-<br />
| 14:15<br />
| 217<br />
| 1.29<br />
| 1.21<br />
| 1.31<br />
<br />
|-<br />
| 15:40<br />
| 302<br />
| 2.1<br />
| 1.8<br />
| 2.3<br />
<br />
|-<br />
| 16:47<br />
| 369<br />
| 2.5<br />
| 1.9<br />
| 3.4<br />
<br />
|-<br />
| 18:05<br />
| 447<br />
| 2.6<br />
| 2.2<br />
| 2.5<br />
<br />
|-<br />
| 19:00<br />
| 502<br />
| 2.9<br />
| 2.3<br />
| 2.5<br />
<br />
|-<br />
| 20:00<br />
| 562<br />
| 3.0<br />
| 2.6<br />
| 2.8<br />
<br />
|-<br />
|}<br />
<br />
:::*The time point at 16:47 (369 minutes elapsed) for BW23115 conjugated (without antibiotics) is most likely an error. It has been removed from the growth plot<br />
'''[[File:UCB-Phage Delivery-140529.JPG]]'''<br />
*Other<br />
::-Made Amp and Kan plates (1 sleeve of each)<br />
::-Made 50mL O/N of ER2738 and BW23115F’ in case we need further dilutions<br />
::-Made 5mL O/N of ER2738, BW23115F’, and BW23115 (without F’ episome) to make chemically competent tomorrow<br />
:::*Did not have plate of BW23115 (without F’ episome) so used freeze down. Hoping to get O/N of a picked colony from CRISPR Team tomorrow morning<br />
<br />
'''5/30'''<br />
*Made chemically competent cells of…<br />
::-ER2738<br />
::-BW23115F’ (conjugated with F’ episome)<br />
::-BW23115 (not conjugated- without F’ episome)<br />
:::*Culture started from plate<br />
::-BW23115* not conjugated (without F’ episome)<br />
:::*Culture started from freeze down<br />
*Results from infections<br />
::-Negative Controls (cells were not infected; cells were diluted 1:1000)<br />
'''[[File:UCB-Phage Delivery-140530.JPG]]'''<br />
::-Results from Experiment 1 (5/29)<br />
'''[[File:UCB-Phage Delivery-140530-02.JPG]]'''<br />
::-Results from Experiment 2 (5/29)<br />
'''[[File:UCB-Phage Delivery-140530-03.JPG]]'''<br />
*Math<br />
::-If there are 5.00E+8 cells in 1mL of culture at OD of 1, then in 1mL of culture at OD of 0.59, there are 2.95E+8 cells. In a 50mL culture at OD of 0.59, there are 1.48E+10 cells.<br />
::-We added 3.3ul (0.0033mL) phage at concentration 4.62E+11 phage/mL which amounts to 1.52E+9 total phage<br />
::-Assuming that 1 phage infects 1 bacterium, we can assume that 1.52E+9 bacterial have the potential to be infected in the 50mL culture<br />
::-We plated 100ul of culture at various dilutions. If not diluted, the number of cells that can be potentially infected in 0.1mL equals 1.52E+9/500, or 3.05E+06 cells. We then accounted for the dilutions (for 1:1000 dilution, we divided 3.05E+06 by 1000 to receive 3.05E+03)<br />
::-The following table contains the number of cells with the potential to be infected assuming a 100% infectivity rate by M13 phage and that 1 cell is infected only once.<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Dilution<br />
! Potential infected cells<br />
! Colonies on Amp<br />
! % Potential (Amp)<br />
! Colonies on Kan<br />
! % Potential(Kan)<br />
! Kan:Amp<br />
<br />
|-<br />
| 1:1000<br />
| 1.52E+06<br />
| 476<br />
| 15.62%<br />
| 15<br />
| 0.820%<br />
| 1:19.04<br />
<br />
|-<br />
| 1:5000<br />
| 3.05E+05<br />
| 131<br />
| 21.49%<br />
| 13<br />
| 2.133%<br />
| 1:10.08<br />
<br />
|-<br />
| 1:10000<br />
| 1.52E+05<br />
| 93<br />
| 30.51%<br />
| 5<br />
| 1.640%<br />
| 1:18.60<br />
<br />
|-<br />
| 1:50000<br />
| 3.05E+04<br />
| 17<br />
| 44.29%<br />
| 0<br />
| 0.000%<br />
| <br />
<br />
|-<br />
|}<br />
<br />
*Conclusions from infections<br />
::-Results between and within the three trials are inconsistent. For example, the number of colonies received in experiments 1 and 2 from 5/29 differ greatly. Due to the differences in protocol, variation was expected but not to this extent. <br />
::-Our dilutions did not yield the expected 10 fold (or 5 fold) decrease in growth that was expected.<br />
::-Plates from 5/29 could be plated better to reduce dense areas of growth and growth around the rim.<br />
::-Though the experiment contained many errors we can say that the phagemid (Litmus 28i) is preferentially packaged compared to the helper phage (M13K07) but not to the degree we expected.<br />
::-Could receive increased occurrences of cells containing M13k07 due to infection, phage production, further infection<br />
<br />
==Week 5==<br />
<br />
'''6/2'''<br />
*Tested chemically competent cells through transformation<br />
::-Are cells contaminated?<br />
::-Are cells competent?<br />
*The samples for transformation<br />
{| class = "wikitable"<br />
|-<br />
! #<br />
! Cells (Tube label)<br />
! DNA (Tube label)<br />
! Resistance before transformation<br />
! Resistance after transformation<br />
<br />
|-<br />
| 1<br />
| K12 ER2738 5/20<br />
| p110+RBS (2) 4/23<br />
| Tet<br />
| Tet, Chlor<br />
<br />
|-<br />
| 2<br />
| BW (-f) 5/30<br />
| p110+RBS (2) 4/23<br />
| Kan<br />
| Kan, Chlor<br />
<br />
|-<br />
| 3<br />
| BW f-ep comp 5/22<br />
| p110+RBS (2) 4/23<br />
| Kan, Tet<br />
| Kan, Tet, Chlor<br />
<br />
|-<br />
| 4<br />
| BW (+f) 5/30<br />
| p110+RBS (2) 4/23<br />
| Kan, Tet<br />
| Kan, Tet, Chlor<br />
<br />
|-<br />
| 5<br />
| *BW23115 5/30<br />
| p110+RBS (2) 4/23<br />
| Kan<br />
| Kan, Chlor<br />
<br />
|-<br />
| 2B<br />
| K12 ER2738<br />
| 2B [from dis. kit]<br />
| Tet<br />
| Tet, Chlor<br />
<br />
|-<br />
| 2P<br />
| BW f-ep comp 5/22<br />
| 2P [from dis. kit]<br />
| Kan, Tet<br />
| Kan, Tet, Chlor<br />
<br />
|-<br />
|}<br />
<br />
'''6/3'''<br />
*Results from 6/2 Transformation<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Growth on Chlor<br />
! Growth on Kan<br />
! Growth on Kan+Tet<br />
! Growth on Amp<br />
<br />
|-<br />
| 1 N<br />
| X<br />
| X<br />
| X<br />
| X<br />
<br />
|-<br />
| 2 N<br />
| X<br />
| +<br />
| X<br />
| X<br />
<br />
|-<br />
| 3 N<br />
| X<br />
| +<br />
| +<br />
| X<br />
<br />
|-<br />
| 4 N<br />
| X<br />
| +<br />
| +<br />
| X<br />
<br />
|-<br />
| 5 N<br />
| X<br />
| +<br />
| X<br />
| X<br />
<br />
|-<br />
| 1<br />
| +<br />
| X<br />
| X<br />
| <br />
<br />
|-<br />
| 2<br />
| +<br />
| +<br />
| X<br />
| <br />
<br />
|-<br />
| 3<br />
| +<br />
| +<br />
| +<br />
| <br />
<br />
|-<br />
| 4<br />
| +<br />
| +<br />
| +<br />
| <br />
<br />
|-<br />
| 5<br />
| +<br />
| +<br />
| Many colonies but close to samp. 4<br />
| <br />
<br />
|-<br />
| 2P<br />
| + (~100)<br />
| +<br />
| +<br />
| <br />
<br />
|-<br />
| 1- JW<br />
| +<br />
| X<br />
| X<br />
| <br />
<br />
|-<br />
| 2- JW<br />
| +<br />
| +<br />
| X<br />
| <br />
<br />
|-<br />
| 3- JW<br />
| +<br />
| +<br />
| +<br />
| <br />
<br />
|-<br />
| 4- JW<br />
| +<br />
| +<br />
| +<br />
| <br />
<br />
|-<br />
| 5-JW<br />
| +<br />
| +<br />
| X<br />
| <br />
<br />
|-<br />
| 2B- JW<br />
| + (24)<br />
| X<br />
| X<br />
| <br />
<br />
|-<br />
|}<br />
*The transformations with DNA from the well (B2 and P2) had lower efficiencies than those with DNA from a mini-prep. Most likely this is due to the differences in DNA concentration (p110+RBS (2) 4/23 was at 254.4ng/ul)<br />
*Conclusions<br />
::-None of the competent cells were contaminated<br />
::-All of the competent cells are in fact, competent<br />
*Set up O/N of DH5-alpha cells to make competent tomorrow<br />
<br />
'''6/4'''<br />
*Isolation of single-stranded phagemid DNA using M13K07<br />
::-Added ER2738 colony to 50mL LB<br />
:::*Plate was cold. Next time warm plate before pricking<br />
::::*Best to use freshly grown plate<br />
::-After 4 hours, OD was at 0.02. Waited 45 minutes and OD was at 0.08. Therefore, we infected at OD 0.08<br />
::-Had started another culture when we did not think the first was growing. In incubator for about 1 hour. OD was 0.00. We infected anyway because last time it worked.<br />
::-Let infection proceed for 60 minutes then added 70ul of Kanamycin to be a final concentration of 70ug/mL<br />
*Primers came in to biobrick M13ori (packaging signal on Litmus28i)<br />
::-Resuspended primers and diluted 1:10<br />
<br />
'''6/5'''<br />
*Isolated single-stranded M13K07 DNA<br />
::-Final concentration = 5724 ng/ul (calculated from a 1:10 dilution)<br />
::-For second sample in pair, we resuspended it in TE but did not proceed to DNA extraction teps<br />
::-For the second culture we started 6/4, we resuspended pellet in TBS and glycerol to preserve the M13 phage. Measured absorbances (before glycerol was added)<br />
: #1<br />
::269 => 1.690A<br />
::320 => 0.103A<br />
: #2<br />
::269 => 1.453<br />
::320 => 0.059<br />
*For our first biobrick, we wanted to isolate the M13 origin, a segment ~500bp that allows for packaging into the M13 phage. We tried to achieve this by biobrick assembly and by Gibson Assembly.<br />
::-To biobrick M13 ori through biobrick assembly (the old-school way)<br />
:::PCR on Litmus 28i to amplify/biobrick M13ori<br />
::::*Used primers Gem003 F & R<br />
::::*Diluted Litmus 28i DNA 1:10<br />
:::Digestion of p11+RBS (1) to digest pSB1C3 bb with EcoRI-HF and PstI-HF<br />
:::Ran samples on gel and gel extracted pieces. We recieved very low yields (out of range for nano drop)<br />
::::*M13 ori: 4.0 ng/ul<br />
::::*pSB1C3: 1.8 ng/ul<br />
:::Digested M13 ori fragment despite poor extraction yield with EcoRI-HF and PstI-HF<br />
::::*Used 1.5x as much DNA as instructed based on inaccurate concentration<br />
'''[[File:UCB-Phage Delivery-140605.JPG]]'''<br />
::-Gel extracted red rectangles<br />
::-Ligation<br />
:::*10hr @ 16C, 10min @ 65C, 4ever @ 4C<br />
*To biobrick M13 ori through Gibson Assembly (the cool-kids way)<br />
::-PCR on Litmus 28i<br />
:::*Used primers Gem002 F & R<br />
:::*Diluted Litmus 28i DNA 1:10<br />
::-PCR on pSB1C3 (p11+RBS (1))<br />
:::*Used primers Gem001 F & R<br />
:::*Diluted pSB1C3 DNA 1:3<br />
<br />
'''6/6'''<br />
*Ran gel of PCR products from (6/5). Products will be used for Gibson Assembly<br />
::-Recieved bands for pSB1C3 around 2000bp and M13ori around 500bp<br />
::-No contamination in pSB1C3 PCR negative control<br />
::-Band in M13ori negative control that is the same size as sample. Contaminated by sample?<br />
::-Gel of PCR products #1 and #2 from 6/5<br />
'''[[File:UCB-Phage Delivery-140606.JPG]]'''<br />
:1. pSB1C3 with promoter+RBS as insert. Amplified with Gem001<br />
:2. No DNA control for Gem001<br />
:3. M13ori amplified with Gem002 from Litmus28i<br />
:4. No DNA control for Gem002<br />
*Gibson Assembly<br />
{| class = "wikitable"<br />
|-<br />
! Total Amount of Frag.<br />
! .02-.5pmol<br />
! 10 ul total<br />
<br />
|-<br />
| Gibson Assembly MM (2x)<br />
| 10 ul<br />
| 10<br />
<br />
|-<br />
| Dionized H2O<br />
| 10-x<br />
| <br />
<br />
|-<br />
|}<br />
::-Diluted pSB1C3 and M13ori PCR products 1:10<br />
::-Incubated 60min @ 50C<br />
::-Also used provided pUC16 as positive control<br />
*Transformation<br />
:1. p110+RBS Positive control<br />
:2. No DNA Negative control<br />
:3. Cas9 from distribution kit so we can have more<br />
:4. Thaw and refreeze cells Test competency of comp cells after thawed<br />
:5. Not chem comp cells Negative control for the above<br />
:6. Ligation Product<br />
:7. Gibson product<br />
::*7.2. Gibson product diluted 1:4<br />
:8. Gibson positive control<br />
::*7.2. Gibson positive control diluted 1:4<br />
::-For the Gibson product and the positive control, we transformed 2ul of product and 2ul of 1:4 diluted product. NEB recommends the first if using their competent cells and the second if using cells from other companies. Our cells are from NEB but we made them competent ourselves so we tried both ways<br />
:::*Plated on Chlor at concentrations of 170, 85, and 33 ug/mL<br />
*Primers came in<br />
::-Resuspended and made 1:10 dilutions<br />
<br />
'''6/7'''<br />
*Results from 6/6 transformation<br />
:1. Positive control<br />
::*Lots of growth, ~300-400 on 1:10 dilution<br />
:2. No DNA negative control<br />
::*No growth<br />
:3. Cas9 from distribution kit<br />
::*7 potential colonies (some are close to edges through) on non-diluted<br />
:4. Thawed then refroze cells<br />
::*Looks like (1)<br />
:5. Not chemically competent cells<br />
::*No growth<br />
:6. Ligation product<br />
::*13 potential colonies (some are close to edge)<br />
:7. Gibson Assembly Product<br />
::*170 -> No colonies<br />
::*85 -> No colonies<br />
::*33 -> 3 specks<br />
:7.2. Gibson Assembly product diluted 1:10<br />
::*170 -> 1 speck<br />
::*85 -> 3 colonies<br />
::*33 -> 13 colonies<br />
:8. Gibson positive control<br />
::*No colonies<br />
:8.2. Gibson positive control diluted 1:4<br />
::*No colonies<br />
*Made 6mL O/N cultures<br />
::-4 from (3) cas9 plate<br />
:::See Constitutive CRISPR notebook for more info on these samples<br />
::-7 from (6) Ligation product<br />
::-5 from (7.2 [85]) Diluted Gibson product on 85 ug/mL Chlor<br />
::-8 from (7.2 [33]) Diluted Gibson product on 33 ug/mL Chlor<br />
<br />
==Week 6==<br />
'''6/8'''<br />
*Check colonies for correct constructs.<br />
::-Mini-prepped all 24 O/Ns<br />
:::*Yielded low concentrations for samples 12, 15, 19, and 22<br />
::-Digested all with EcoRI and PstI (10ul reactions)<br />
::-Ran results on gel<br />
:::*All 4 cas9 samples had the expected bands of 2000 and 5000bp<br />
:::*All 7 ligation products have expected bands of 2000 and ~570bp<br />
:::*3 of 5 Gibson assemblies from 85ug/mL Chlor plate had expected bands of 2000 and ~500bp<br />
:::*3 of 8 Gibson assemblies from 33ug/mL Chlor plate had expected bands of 2000 and ~500bp<br />
'''[[File:UCB-Phage Delivery-140608-01.JPG]]'''<br />
<br />
'''[[File:UCB-Phage Delivery-140608-02.JPG]]'''<br />
::1-4: Cas9 from Stanford-Brown team<br />
::5-11: pSB1C3-M13ori cloned through ligation<br />
::12-24: pSB1C3-M13ori cloned through Gibson Assembly<br />
:::*12-16: Grown with 85 ug/mL Chlor<br />
:::*17-24: Grown with 33 ug/mL Chlor<br />
*Conclusions from gel<br />
::-We have cas9 safely in cells<br />
::-Our ligation reactions successfully yielded M13ori on pSB1C3<br />
::-Combined, we had a 46% success rate for the Gibson Assembly in yielding M13ori on pSB1C3<br />
:::*The 4 samples that had the lowest concentration after being mini-prepped (12,15, 19, and 22) correlate with samples that had the correct band pattern<br />
*We selected 4 samples from each (4 total between the two Gibson reactions) type<br />
::-For non-Gibson Assembled samples<br />
:::Plated 25ul on 170ug/mL Chlor<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Our usual method<br />
! Gibson Method<br />
<br />
|-<br />
| 1. Thaw on ice<br />
| 1. Thaw on ice <br />
<br />
|-<br />
| 2. Transfer 40ul cells to tube<br />
| 2. Transfer 50ul cell to tube<br />
<br />
|-<br />
| 3. Add DNA. 1ul for mini-prep OR up to 10ul for ligation<br />
| 3. Add 2ul to NEB cells OR 2ul of 1:4 diluted to other cells<br />
<br />
|-<br />
| 4. Mix by pipet Let sit 30min on ice<br />
| 4. Mix by pipet or flicking Let sit 30 min. on ice<br />
<br />
|-<br />
| 5. Heat shock: 42C for 45s<br />
| 5. Heat shock: 42C for 30s<br />
<br />
|-<br />
| 6. Ice for 5 minutes<br />
| 6. Ice for 2 min.<br />
<br />
|-<br />
| 7. Transfer to culture tube; Add 200ul SOC<br />
| 7. Add 950ul SOC to tube<br />
<br />
|-<br />
| 8. Shake or rotate for 60-120min at 37C<br />
| 8. Shake (250rpm) or rotate for 60 min. at 37C<br />
<br />
|-<br />
| 9.<br />
| 9. Warm plates to 37C<br />
<br />
|-<br />
| 10. Plate 100ul onto selection plate<br />
| 10. Plate 100ul onto plate<br />
<br />
|-<br />
| 11. Incubate O/N @ 37C<br />
| 11. Incubate O/N @ 37C<br />
<br />
|-<br />
|}<br />
::-Added 6mL LB and Chlor at concentration of 170ug/mL to grow O/N<br />
:*For Gibson Assembled samples<br />
::-Plated 25ul onto 170, 85, and 33ug/mL Chlor<br />
::-Samples from 85ng/mL plate<br />
:::Transferred 100ul to new tube, added media, added Chlor at 170ng/mL<br />
::-Samples from 33ng/mL plate<br />
:::Tranfered 100ul to new tubes, added media, added Chlor at 85ng/mL to one and 33ng/mL to the other<br />
:*Tomorrow, may send for sequencing and make freeze downs<br />
<br />
*Transformation<br />
:1. Positive control (p110+RBS diluted 1:10)<br />
:2. Non-diluted Gibson product<br />
:3. Gibson product diluted 1:4<br />
:4. Gibson product diluted 1:10<br />
::-Transformed each sample using our usual method and using the protocol given by Gibson<br />
<br />
::-Due to not have plates ready before transformation, in step 4, the samples sat for about 50 minutes. Then in step 8, they both recovered for about 150 minutes. Though not specified in our protocol, we did warm the plates to 37C. In step 10 for our protocol, since we only added 200ul SOC and wanted to plate on 3 selection plates (see below), we only plated 50ul (except for the positive control). <br />
::-Plated on three concentrations of Chloramphenicol (33 ug/mL, 85 ug/mL, and 170 ug/mL) to determine the differences in yield due to differences in concentration.Obvious hypothesis: more colonies will grow on plates that have a lower concentration of chlor.<br />
<br />
<br />
'''6/9'''<br />
*Made chemically competent 5alpha cells with Dan and Alex from main campus<br />
::-Waiting to hear results on competency<br />
*Will eventually make phage containing CRISPR-Cas9 that targets Kanamycin resistance. M13K07 has Kanamycin resistance so we need to switch the resistance on the M13 genes.<br />
::-PCR on pwp 2.po (plasmid that Sam gave us that contains the zeoR gene adjacent to ori) to amplify zeoR and ori. Zeo is on EM7 promoter<br />
:::-Primers: Gem008 R & R<br />
:::-Anneal temp from NEBuilder: 63.7C<br />
:::-Extension time: 90s<br />
:::-Expected band size on gel: 1300bp<br />
:::-Used phusion polymerase<br />
*PCR on M13K07 DNA to amplify M13 phage genes (also removes majority of M13 ori, all of KanR, and all of p15 ori)<br />
:::-Primers: Gem007 F & R<br />
:::-Anneal temp from NEBuilder: 60.2C<br />
:::-Extension time: 4:30<br />
:::-Expected band size on gel: about 6000bp<br />
:::-Used phusion polymerase<br />
*Freeze downs<br />
::-Note: Phagemid 1C3 was the original name for ‘pSB1C3-M13ori’<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Top Label<br />
! Side label<br />
! What?<br />
<br />
|-<br />
| Phagemid 1C3 6/9<br />
| From Lig<br />
| M13 ori inserted into 1C3 (biobricked); Done through ligation; Contains extra bases as spacer between biobrick prefrix/suffix and part for primer design<br />
<br />
|-<br />
| Phagemid 1C3 6/9<br />
| From Lig<br />
| “ “<br />
<br />
|-<br />
| Phagemid 1C3 6/9<br />
| Gibson<br />
| M13 ori inserted into 1C3 (biobricked); Done through Gibson cloning<br />
<br />
|-<br />
| Phagemid 1C3 6/9<br />
| Gibson<br />
| “ “<br />
<br />
|-<br />
|}<br />
'''6/10'''<br />
*Ran gel of PCRs from 6/9<br />
<br />
'''[[File:UCB-Phage Delivery-140610.JPG]]'''<br />
:1. Amplification of M13 genes from M13K07 (~6000bp)<br />
:2. No DNA control for (1) amplification<br />
:3. Amplification of Zeo resistance gene + plasmid ori (~1300bp)<br />
:4. No DNA control for (3) amplification<br />
*Gibson Assembly of above parts (did not gel extract)<br />
::-Diluted the PCR products 1:10 then added 3ul of M13K07 genes product and 7ul of ZeoR+ori product<br />
::-Incubated at 50C for 60 min.<br />
::-Transformed Gibson Assembly product into 5alpha cells<br />
:::*Used our usual protocol<br />
:::*Add 2ul of DNA<br />
::::-In one sample, diluted DNA 1:4 and in the other, we diluted DNA 1:10<br />
*Started Phage Amplification Protocol<br />
::-ER2738 transformed with Litmus 28i<br />
:::*Grew for ~2.5hr before reaching an OD of 0.04<br />
::-ER2738 transformed with pSB1C3-M13ori (M13ori on pSB1C3)<br />
:::*Grew for ~ 3.5hr before reaching an OD of 0.01, then in the next 1.5 hours, spiked to 0.19<br />
:::*We gave up and went home, and will restart tomorrow<br />
*Analyzed transformation results from 6/8<br />
<br />
'''6/11'''<br />
*Made chemically competent 5alpha cells<br />
*Restarted Phage Amplification Protocol<br />
::-Forgot to add phagemid antibiotic at start of growth. Added phagemid antibiotic when we added phage. Incubated for 90 minutes before adding Kanamycin (to select for cells that were infected by M13K07)<br />
::-Phage is at concentration 4.57x10^12 phage/mL<br />
:::Protocol calls for final concentration of 1 x10^8 phage/mL<br />
::::*(4.57 x10^12)*V = (1 x10^8)(50mL)<br />
::::*V = 0.00109mL<br />
:::Added 1.1ul of phage<br />
*Transformations<br />
{| class="wikitable"<br />
|-<br />
!DNA<br />
!Plate Selection<br />
|-<br />
|No DNA control<br />
|(all)<br />
|-<br />
|Positive control (p110+RBS)<br />
|(C)<br />
|-<br />
|M13 genes + ZeoR ori<br />
|(Z)<br />
|-<br />
|M13 genes +ZeoR ori diluted 1:4<br />
|(z)<br />
|} <br />
<br />
'''6/12'''<br />
*Transformation Results from 6/10 [Took ~36 hours to be clearly visible]<br />
::No DNA control<br />
:::-Amp: 0 <br />
:::-Zeo (50ug/mL): 200 colonies<br />
::M13 genes + ZeoR ori (1:4 dilution) <br />
:::-Zeo (25ug/mL): 300<br />
:::-Zeo (50ug/mL): 200<br />
:::-Zeo (100ug/mL): 100<br />
::M13 genes + ZeoR ori (1:10 dilution) <br />
:::-Zeo (25ug/mL): 300<br />
:::-Zeo (50ug/mL): 150<br />
:::-Zeo (100ug/mL): 150<br />
*Transformation Results for 6/11<br />
::-Positive (p110+RBS) on Chloramphenicol: 500 colonies<br />
::-No DNA on Amp: 0 colonies Zeo (100 ug/mL): specks<br />
::-No apparent growth on any other plate<br />
:::*Realized later that we grew our samples on the wrong plates. Will repeat transformation today<br />
*Transformation #1<br />
::This morning there were no colonies on positive (p110+RBS) coltrol from 6/11 even though we observed fast growth in the past. Without waiting for colonies to appear, we started a control transformation<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Diluted<br />
! Time at 42C<br />
<br />
|-<br />
| p110+RBS<br />
| No<br />
| 45s<br />
<br />
|-<br />
| p110+RBS<br />
| 1:10<br />
| 45s<br />
<br />
|-<br />
| p110+RBS<br />
| 1:10<br />
| 30s<br />
<br />
|-<br />
| p110+RBS<br />
| 1:10<br />
| No timer. ~43s<br />
<br />
|-<br />
| No DNA control<br />
| No<br />
| 45<br />
<br />
|-<br />
|}<br />
*Because ‘No DNA control 6/10’ yielded colonies, we researched Zeocin plates<br />
::-According to Life Technologies (Invitrogen), Zeocin requires low salt medium and a pH of 7.5<br />
::-Low Salt LB Medium (1L)<br />
:::*Ingredients<br />
::::10g Tryptone<br />
::::5g NaCl<br />
::::5g Yeast Extract<br />
:::-Mix ingredients<br />
:::-Adjust pH to 7.5 using NaOH (If go over, use HCl)<br />
:::-Add agar for plates at 15g/L. Autoclave<br />
:::-Thaw Zeocin on ice. Vortex<br />
:::-Add Zeocin to final concentration of 25ug/mL<br />
*Transformation #2<br />
::-Repeat of transformation on 6/11 but this time we will plate on the correct plates<br />
::-Also remade Zeocin plates<br />
*Finished isolation of M13 Litmus phage and M13 pSB1C3-M13ori phage<br />
::*Note: Phagemid 1C3 was the original name for ‘pSB1C3-M13ori’<br />
{| class = "wikitable"<br />
|-<br />
! Phage Sample<br />
! A269<br />
! A320<br />
! Concentration (phage/mL)<br />
<br />
|-<br />
| Litmus phage (1)<br />
| 0.181<br />
| 0.034<br />
| 3.12 x10^12<br />
<br />
|-<br />
| Litmus phage (2)<br />
| 0.227<br />
| 0.047<br />
| 3.83 x10^12<br />
<br />
|-<br />
| Phagemid 1C3 phage (1)<br />
| 0.101<br />
| 0.020<br />
| 1.87 x10^12<br />
<br />
|-<br />
| Phagemid 1C3 phage (2)<br />
| 0.126<br />
| 0.021<br />
| 2.42 x10^12<br />
<br />
|-<br />
|}<br />
::::phage/mL = 6x10^16 x (A269-A320)/ (#of base pairs in the phage genome)<br />
*Set up 50mL O/N of K12 ER2738 (containing f-episome) for infection tomorrow with Litmus phage and pSB1C3-M13ori phage<br />
<br />
<br />
'''6/13'''<br />
*Transformation Results for 6/11<br />
::-Several hundred colonies on Positive Control (p110+RBS) on Chlor<br />
::-No colonies for GA positive control on Amp<br />
::-No colonies for M13ori + ZeoR mistakenly plated on Amp<br />
::-No colonies for No DNA control on Amp<br />
::-100-ish colonies for No DNA control on Zeo<br />
::-100-ish colonies for cas9+AmpR+gRNA mistakenly plated on Zeo<br />
::-These last 3 points suggest/confirm that Zeo plates are no good<br />
*Transformation Results from 6/12 control test<br />
::-Colonies grew is about equal amounts on all plates, including No DNA control<br />
:::Either plates don’t contain Chlor or competent cells are contaminated<br />
::-Streaked competent cells onto new and old Chlor plates<br />
*Infection test of ER2738: Is Litmus preferentially packaged over M13K07 Helper phage? Is pSB1C3-M13ori preferentially packaged?<br />
::-Infect ER2738 with phage produced 6/12<br />
:::*Phage should have packaged Litmus 28i phagemid or pSB1C3-M13ori<br />
:::*Cells infected with phage packaging Litmus 28i will grow on Amp<br />
:::*Cells infected with phage packaging pSB1C3-M13ori will grow on Chlor<br />
:::*Cells infected with phage packaging M13K07 will grow on Kan<br />
::-After we plate, we can count the colonies and calculate a ratio of Litmus28i: M13K07 or pSB1C3-M13ori:M13K07 packaging<br />
<br />
'''6/14'''<br />
*Results of contamination test (streaked competent cells onto new and old Chlor plates)<br />
::-Colonies grew in low amounts on both plates. most likely the cells are contaminated<br />
*Transformation results for 6/12<br />
::-Many colonies for No DNA control on Zeo<br />
:::-Some colonies are turning pinkish-red<br />
::-Many colonies for diluted and non-diluted M13 genes+ZeoR on Zeo<br />
:::-Some colonies are turning pinkish-red<br />
*Could the white colonies be the designed colonies and can we kill the red colonies with Zeocin before killing the white colonies (aka. Use high Zeocin concentrations to select for the correct construct)<br />
::-Selected 1 red colony from No DNA Zeo control plate and 1 white colony from M13genes+ZeoR sample plate<br />
::-Added 100ul H2O then divided amongst 5 culture tubes each (with 5mL of low-salt LB, pH 7.5)<br />
::-Then added Zeocin to a final concentration of: 0, 25, 50, 75, 100ug/mL.<br />
*Results of 6/13 infection<br />
::-Litmus 28i infected cells<br />
:::*on Kan: Individual colonies for 1:10, 1:100, and 1:1000. No colonies on 1:5000<br />
:::*on Amp: lawn for 1:10, 1:100, and near lawn for 1:1000. Single colonies for 1:5000<br />
::-pSB1C3-M13ori infected cells<br />
:::*on Kan: Same as Litmus 28i samples on Kan<br />
:::*on Chlor: Lawn for all dilutions. Must discredit due to recent Chlor contamination<br />
<br />
==Week 7==<br />
<br />
'''6/15'''<br />
*Results of Zeocin experiment on 6/14<br />
::-Healthy growth for both at 0ug/mL Zeo<br />
::-No growth for white colony with any Zeo<br />
::-Good growth for red colony even at 100ug/mL Zeo<br />
::-This suggests that the red colonies are naturally resistant to Zeocin. Also, our plates must not contain active Neocin. We are sure that we are adding enough. Possibly, we add it while the media is too hot or leave the plates at room temperature (decondensing) for too long and this deactivates the antibiotic? Perhaps we are not attaining the correct pH<br />
*In light of recent contamination problems on both Chlor and Zeo, we made new competent cells<br />
::-5alpha<br />
::-BW23115<br />
::-BW23115 conjugated (contains f-episome)<br />
<br />
'''6/16'''<br />
*Made new Chlor antibiotic. Made to Chlor plates<br />
*Contamination Test: Streaked Zeo and new Chlor plates each with<br />
::-Colony from Zeo contaminated plate<br />
::-Colony from Chlor contaminated plate<br />
::-Non-transformed OLD chemically competent cells<br />
::-Non-transformed NEW chemically competent cells<br />
*Redid bacterial infection<br />
::-For both samples, we used ER2738 (not from the same ‘O/N’ (also, not a real O/N))<br />
::-Infected one sample with isolated ‘Litmus28i phage’ and one with ‘pSB1C3-M13ori’<br />
::-Alterations to protocol<br />
:::*Did not started from saturated O/N. Started each with a colony, waited several hours until at OD ~1. We then added these cells to fresh 50mL LB to have an OD of 0.1.<br />
:::*Missed the 30 minute infection mark. Infected for ~45 minutes.<br />
::-Plated on Kan and Amp (Litmus28i sample) or Chlor (pSB1C3-M13ori sample) at dilutions of<br />
:::*1:100, 1:1k, 1:10k, and 1:100k<br />
<br />
'''6/17'''<br />
*Q5 PCR to replace KanR with ZeoR in M13K07<br />
::-Used recommendations<br />
::-Unfortunately, I (Jo) don’t know the difference between tightening and loosening the thermocycler lid; therefore, our M13K07 sample to amplify the M13K07 genes evaporated. But that’s ok, because we were sick of Zeocin anyway and decided mid-PCR to not waste our time with a Gibson Assembly and transformation. Instead we will be using the Old-School method of digestion and ligation because let’s face it, it’s a classic (and Gibson sucks) =)<br />
*Results from infection Test<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Litmus 28i<br />
! 1:100<br />
! 1:1k<br />
! 1:10k<br />
! 1:100k<br />
<br />
|-<br />
| Amp<br />
| ~6000?<br />
| ~2056<br />
| 377<br />
| 36<br />
<br />
|-<br />
| Kan<br />
| 130<br />
| 8<br />
| 3<br />
| 0<br />
<br />
|-<br />
|}<br />
{| class = "wikitable"<br />
|-<br />
! pSB1C3-M13ori<br />
! 1:100<br />
! 1:1k<br />
! 1:10k<br />
! 1:100k<br />
<br />
|-<br />
| Chlor<br />
| 8<br />
| 1<br />
| 0<br />
| 0<br />
<br />
|-<br />
| Kan<br />
| 97<br />
| 9<br />
| 0<br />
| 0<br />
<br />
|-<br />
|}<br />
::-We consider the Litmus 28i to have been a success. The pSB1C3-M13ori …. not so much. Looking back at the Litmus28i and the M13K07, we noticed that the M13ori is facing the opposite direction as the plasmid ori. Ours faces the same direction as the plasmid ori. Because we are working with phagemids that are single stranded, we think that by flipping the M13ori, we may be able to recover functionality. We will also look into other reasons.<br />
*Results from contamination test (6/16)<br />
::-Old Chlor plate (6/12)<br />
:::-Non-transformed chemically competent cells<br />
::::*Old: some colonies<br />
::::*New: No colonies<br />
::-New Chlor plate (6/16)<br />
:::-Colony from<br />
::::*Zeo contaminated plate: No growth<br />
::::*Chlor contaminated plate: Much growth<br />
:::-Non-transformed chemically competent cells<br />
::::*Old: some colonies<br />
::::*New: No growth<br />
::-Zeo (25ug/mL, Low NaCl, pH 7.5) (6/12)<br />
:::-Colony from<br />
::::*Zeo contaminated plate: Much growth<br />
::::*Chlor contaminated plate: No growth<br />
:::-Non-transformed chemically competent cells<br />
::::*Old: some specks<br />
::::*New: some specks<br />
<br />
'''6/18'''<br />
*Ordered primers to…<br />
::-biobrick M13ori (in other direction)<br />
::-biobrick M13K07 genes<br />
'''6/19'''<br />
*Waited for primers<br />
*Set up O/N cultures to test last infection (6/16) for colonies containing pSB1C3-M13ori<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample #<br />
! Selection<br />
! Dilution<br />
! Presumably<br />
! Notes<br />
<br />
|-<br />
| 1-8<br />
| Chlor<br />
| 1:100<br />
| pSB1C3-M13ori<br />
| <br />
<br />
|-<br />
| 9<br />
| Chlor<br />
| 1:1k<br />
| pSB1C3-M13ori<br />
| <br />
<br />
|-<br />
| 10-14<br />
| Chlor<br />
| 1:1k<br />
| pSB1C3-M13ori<br />
| These colonies were not present on plate during initial counting on 6/17<br />
<br />
|-<br />
| 15-16<br />
| Kan<br />
| 1:1k<br />
| M13K07<br />
| <br />
<br />
|-<br />
|}<br />
:::-Selection and dilution refer to the plate. Cells were then grown under selection.<br />
:::-More colonies were seen on all Chlor plates. No new colonies appeared on Kan plates<br />
*Tomorrow, we will mini-prep, digest, and run the samples on a gel to verify gene transfer.<br />
<br />
'''6/20'''<br />
*Primers are in!<br />
*Cloning of pSB1C3-M13ori(New)<br />
::-PCR to amplify M13ori (packaging signal) from Litmus 28i to be in the other direction<br />
:::-Primers (Gem011 F&R) add cut sites to make part biobrick compatible<br />
:::-Was able to gel extract<br />
::::*Band at the same size as sample….. contaminated primers?<br />
:::-Then digested with E and P<br />
::-Digested pSB1C3 plasmid with E and P to linearize backbone<br />
::::*Was able to gel extract<br />
'''[[File:UCB-Phage Delivery-140620.JPG]]'''<br />
<br />
:1. Spill over from 2<br />
:2 and 3. pSB1C3 digested with EcoRI and PstI<br />
::-Insert <100bp so cannot be seen<br />
:4. M13ori<br />
*Ligation of M13ori (packaging signal) into pSB1C3<br />
::10h at 16C<br />
::20m at 80C<br />
::Hold at 4C<br />
<br />
*PCR to amplify M13K07 genes from M13K07 DNA isolated from phage<br />
::-Primers (Gem012 F&R) add cut sites to make part biobrick compatible<br />
::-Very light bands and not band might correlate with 6000pb but not enough resolution on gel to be certain. Bands were too light so did not extract<br />
::-Set up PCR again using Q5 to run O/N<br />
:::*Received only a feint, smudgy band that was too large. We did not bother with extracting the DNA<br />
*Test of 6/19 O/Ns (14 that are presumably pSB1C3-M13ori and 2 that are presumably M13K07)<br />
::-Mini-prepped all O/Ns<br />
::-Digested all with Pst-I (common RE between pSB1C3-M13ori and M13K07)<br />
:::-Even though these are phagemids, we assumed that because they were still in the cell, the plasmids were still double stranded so could be recognized by RE. Our assumption was valid<br />
:::-Results of digestion<br />
::::*1,2,4-14: all had bands that corresponded to pSB1C3-M13ori cut once<br />
::::*3: Slight band at expected size but very feint<br />
::::*15, 16: as expected, they contain bands of ~9000bp, correlating with the M13K07 phagemid. Interestingly, sample 15 also contained a (brighter) band that corresponds with pSB1C3-M13ori. We assume that this was an incident of double infection (chance or did this occur at high frequency?)<br />
'''[[File:UCB-Phage Delivery-140620-02.JPG]]'''<br />
::Check table from 6/19 for more details<br />
:::-1-14: pSB1C3-M13ori<br />
:::-15-16: M13K07 from 1:1k diluted plate<br />
<br />
'''6/21'''<br />
*Needed to transform our pSB1C3-M13ori(New) (6/20) into cells. First, we transformed into 5alpha cells; however, we need to infect these cells later in order to make phage. Our 5alphas are not competent so we repeated the transformation, this time using ER2738 cells which contain the F’ episome, allowing us to infect with the M13 phage.<br />
::-Also transformed sample #1 from 6/20 mini-preps (pSB1C3-M13ori(Old) into ER2738 cells. Do not see any notes about this plasmid being in infectable cells during the initial experiment. Reason for experiment failure???<br />
*Ran 6/20 O/N PCR (to amplify M13K07 genes) on gel (also re-ran the previous sample from the day with more DNA)<br />
::-Still, band looks too big. Brightest band still occurs between 8000 and 9000bp. Lighter band around 7000bp-- might be ~6000pb but too light to tell<br />
*Alternative plan for amplifying M13K07 genes<br />
::-Digest sample with PstI (cuts outside of the target region)<br />
'''[[File:UCB-Phage Delivery-140621.JPG]]'''<br />
::Used sample #16 from 6/20 b/c it is double stranded so will cut<br />
:-PCR amplified the linearized digestion product<br />
:::1. PCR of M13genes<br />
:::2. PCR of M13genes + DMSO<br />
::::Gel extracted pieces boxed in red<br />
::-Used primers Gem012 F&R<br />
::-Gel extracted piece but received very low yield<br />
*Alternative to alternative plan for amplifying M13K07 genes<br />
::-Digested with PstI and AgeI<br />
'''[[File:UCB-Phage Delivery-140621-02.JPG]]'''<br />
:1. Cut with PstI and AgeI<br />
:2. Uncut<br />
:3. Cut with PstI<br />
:4. Just PCR<br />
::-Gel extracted ~6000pb band after double digestion<br />
::-PCR (Phusion)<br />
:::-Digestion (A+P) product<br />
:::-Gel extraction product<br />
:::-No DNA control<br />
<br />
==Week 8==<br />
<br />
'''6/22'''<br />
*Did receive colonies from 6/21 transformations. Selected colonies for overnight<br />
{|class="wikitable"<br />
|-<br />
!number<br />
!colonies<br />
|-<br />
|1-7<br />
|pSB1C3-M13ori(NEW) in 5alpha<br />
|-<br />
|8-14<br />
|pSB1C3-M13ori(NEW) in ER2738<br />
|-<br />
|15-18<br />
|pSB1C3-M13ori(OLD) in ER2738<br />
|-<br />
|19<br />
|p110+RBS in 5alpha as control<br />
|-<br />
|20<br />
|p110+RBS in ER2738 as control<br />
|}<br />
<br />
*Gel of 6/21 O/N PCR<br />
::Digestion (AgeI+PstI) product ? Yielded the three bands that appear with every PCR of M13K07<br />
'''[[File:UCB-Phage Delivery-140622.JPG]]'''<br />
<br />
:1. M13K07-> digested (AgeI + PstI) -> PCR<br />
:2. M13K07-> digested (AgeI+ PstI) -> extraction-> PCR<br />
:3. No DNA control for PCR<br />
::-Gel extraction product ? No bands<br />
::-No DNA control ? No bands<br />
*PCR purified the above PCR (Digestion (AgeI+PstI) product) and PCR from 6/21<br />
*Digested PCR purified sample with EcoRI+PstI and ran gel<br />
'''[[File:UCB-Phage Delivery-140622-02.JPG]]'''<br />
:1. PCR-> PCR purified<br />
:2. Sample after DpnI digest<br />
:3. Sample after digestion with EcoRI and PstI<br />
:4. Samples after digestion with DpnI then EcoRI and PstI<br />
*Ligation of above digestion (M13K07 genes) with pSB1C3<br />
<br />
<br />
'''6/23'''<br />
*Transformed pSB1C3-M13ori(New) (6/22 ligation)<br />
*Check overnights from 6/22 for the correct insert<br />
::-Mini-prep O/Ns<br />
::-Digested with EcoRI and PstI<br />
'''[[File:UCB-Phage Delivery-140622-03.JPG]]'''<br />
:::1-7: pSB1C3-M13ori(New) in 5alpha<br />
:::8-14: pSB1C3-M13ori(New) in ER2738<br />
:::15-18: pSB1C3-M13ori(Old)<br />
::-All were the expected size (though gel wiggled)<br />
::-Sent 4 samples for sequencing (iGEM primers: VF2 and VR)<br />
:::# 4: Divergent (new) phagemid 1C3 transformed into 5alpha<br />
:::# 11, 13: Divergent (new) phagemid 1C3 transformed into ER2738<br />
:::# 16: Convergent (old) phagemid 1C3 transformed into ER2738<br />
<br />
'''6/24'''<br />
*Results of 6/23 transformation (pSB1C3-M13genes) from 6/22 ligation)<br />
::-Received ~100 colonies<br />
::-Set up O/N cultures for 8 of the colonies<br />
::-Time passed…..<br />
::-Mini-prepped the 8 O/N samples mentioned above (yes, it was a long day)<br />
:::*Digested samples (E+P)<br />
*Started phage amplification protocol<br />
::-Amount of phage added = 10.9ul of 1:10 diluted phage (M13 phage (1) from 5/24 (4.575 x10^12 phage/mL)) to a final concentration of 1x10^8 phage/mL in 50mL<br />
::-Managed to get both samples to the 14-28hr incubation<br />
<br />
'''6/25'''<br />
*Checked mini-prep samples from 6/24 (pSB1C3-M13genes)<br />
::Ran the digestion overnight (6/24 to 6/25)<br />
'''[[File:UCB-Phage Delivery-140625.JPG]]'''<br />
::-All contained a band at 2000bp. Mostly empty vector or contained small, light band. Sample 1 had prominent band at ~1250bp.<br />
::-None of samples contained M13genes<br />
*M13K07 is on P15A ori (10-12 copy number) whereas the pSB1C3 ori is on pUC19 (500-700 copies). It’s possible that this overexpression is detrimental to cell<br />
::Alternative low copy plasmids found in distribution kit<br />
:::*2013 (plate 5)<br />
::::pSB6A1 (1K)<br />
::::pSB3C5 (3C)<br />
::::pSB3K3 (5E)<br />
:::*2014 (plate 4)<br />
::::pSB3C5 (4D)<br />
::::pSB6A1 (2L)<br />
::-Suspended and transformed the above plasmids into 5alphas<br />
::-Next, we will select colonies, mini-prep, digest, gel extract, ligate with M13genes<br />
*Started phage amplification protocol <br />
::-Phagemids in ER2738<br />
:::pSB1C3-M13ori(New)<br />
:::pSB1C3-M13ori(Old) <br />
::-Set up O/N of ER2738 for infection tomorrow<br />
<br />
'''6/26'''<br />
*Results of transformation of plasmids from the distribution kit<br />
::-Only received colonies pSB6A1 (2L from 2014) and pSB3C5 (4D from 2014)<br />
::-Pricked colonies for O/N<br />
*PCR of M13 genes so we can ligate it into the above backbones tomorrow<br />
::-DNA: M13K07 digested with EcoRI+PstI (6/21)<br />
::-Primers: Gem012 F & R<br />
*Finished phage amplification protocol<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Samples<br />
! A269<br />
! A320<br />
! Concentration<br />
<br />
|-<br />
| Old phagemid 1C3 (1)<br />
| 0.832<br />
| 0.492<br />
| 7.828 x10^12<br />
<br />
|-<br />
| Old phagemid 1C3 (2)<br />
| 0.324<br />
| 0.083<br />
| 5.549 x10^12<br />
<br />
|-<br />
| New phagemid 1C3 (1)<br />
| 0.391<br />
| 0.077<br />
| 7.229 x10^12<br />
<br />
|-<br />
| New phagemid 1C3 (2)<br />
| 0.402<br />
| 0.639<br />
| -5.457 x10^12<br />
<br />
|-<br />
|}<br />
::::*While resuspending “New phagemid 1C3 (2)”, the tip fell off and we lost half of sample. Evidently, we lost most of phage so we tossed sample<br />
::-Prepped and Infected ER2738 with “Old phagemid 1C3 (1)” and “New phagemid 1C3 (2)”<br />
:::Added 1.6ul of 1:10 diluted “Old phagemid 1C3 (1) to ER2738 cells<br />
:::Added 1.8ul of 1:10 diluted “New phagemid 1C3 (1) to ER2738 cells<br />
::-Plated the infected cells at dilutions<br />
:::1:1 onto Chlor+Kan plates<br />
:::1:10 onto Chlor+Kan plates<br />
:::1:100 onto Chlor and onto Kan plates<br />
:::1:1000 onto Chlor and onto Kan plates<br />
:::1:10000 onto Chlor and onto Kan plates<br />
:::1:100000 onto Chlor and onto Kan plates<br />
<br />
'''6/27'''<br />
*To ligate M13genes onto different backbones<br />
::-Mini-prepped the O/Ns from 6/26 to get backbones with low copy number<br />
:::pSB6A1<br />
:::pSB3C5 <br />
::::*did not grow as well<br />
::-PCR purified PCR (to amplify M13 genes with Gem012 F & R) from 6/26<br />
::-Digestions (50ul)<br />
:::Digest pSB6A1 with E + P<br />
:::Digest pSB3C5 with E + P<br />
:::Digest PCR purification product with E + P<br />
'''[[File:UCB-Phage Delivery-140627.JPG]]'''<br />
:1. pSB6A1<br />
:2. pSB3C5<br />
:3. M13genes<br />
::-Gel extraction of the above digestions<br />
:::For each: Added 10ul of 6x loading dye to 50ul digestions and divided the total volume between 2 wells<br />
::-Ligation<br />
:::1) pSB6A1-M13genes<br />
:::2) pSB3C5-M13genes<br />
*Results from 6/26 infection (after 22hrs in incubator)<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Anti-Sense Phagemid 1C3<br />
! 1:100<br />
! 1:1000<br />
! 1:10000<br />
! 1:100000<br />
<br />
|-<br />
| Chlor<br />
| 85<br />
| 6<br />
| 2<br />
| 0<br />
<br />
|-<br />
| Kan<br />
| 405<br />
| 13<br />
| 2<br />
| 1<br />
<br />
|-<br />
|}<br />
{| class = "wikitable"<br />
|-<br />
! Sense Phagemid1C3<br />
! 1:100<br />
! 1:1000<br />
! 1:10000<br />
! 1:100000<br />
<br />
|-<br />
| Chlor<br />
| 121<br />
| 24<br />
| 3<br />
| 0<br />
<br />
|-<br />
| Kan<br />
| 25<br />
| 1<br />
| 0<br />
| 0<br />
<br />
|-<br />
|}<br />
-We also plated the infected cells on plates containing Chlor and Kan to test for the possibility of double infection<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Phagemid 1C3<br />
! 1:1<br />
! 1:10<br />
<br />
|-<br />
| Anti-Sense<br />
| 59<br />
| 0<br />
<br />
|-<br />
| Sense<br />
| 4<br />
| 0<br />
<br />
|-<br />
|}<br />
<br />
<br />
'''6/28'''<br />
*We noticed that there were more colonies on our infection plates from 6/26 than on 6/27; therefore, we recounted colonies<br />
::-No increase of colonies on Kan plates<br />
::-Significant increase of colonies on Chlor plates<br />
::-Sense refers to the first phagemid 1C3 where the M13ori is in the sense direction compared to the plasmid ori<br />
::-Anti-Sense refers to the new phagemid 1C3 where the M13ori is in the anti-sense direction compared to the plasmid ori<br />
'''[[File:UCB-Phage Delivery-140628.JPG]]'''<br />
::Numbers on the left are after 22 hours. Numbers on the right are after 38.75 hours<br />
*Results of transformation from 6/27 (pSB6A1-M13genes and pSB3C5-M13genes)<br />
::-We have many colonies. Unfortunately, some are red, suggesting that original insert (J04450) was not successfully separated from backbone through gel extraction<br />
::-Selected colonies to grow overnight in 5mL LB<br />
<br />
==Week 9==<br />
<br />
'''6/29'''<br />
*Check O/N cultures for correct constructs (pSB6A1-M13genes and pSB3C5-M13genes)<br />
::-Mini-prep samples<br />
::-Digested with EcoRI and PstI to check insert sizes<br />
::-Gel<br />
'''[[File:UCB-Phage Delivery-140629.JPG]]'''<br />
::lanes….<br />
:::1-11: pSB6A1-M13genes<br />
:::12-18: pSB3C5-M13genes<br />
::-Epic failure<br />
::-All pSB6A1 backbones are empty<br />
::-Half of pSB3C5 backbones were empty. The others contained random inserts (1700 OR 3500). We don’t know what these inserts are. 1700 band is likely the digestion product that appears when we digest M13genes<br />
*Question: Can we use empty vectors from these mini-preps as ligation vectors?<br />
::-Digest pSB6A1 mini-prep with…. (see if both cut sites were retained during re-ligation)<br />
:::no enzyme<br />
:::EcoRI<br />
:::PstI<br />
:::EcoRI + PstI<br />
'''[[File:UCB-Phage Delivery-140629-02.JPG]]'''<br />
::lanes....<br />
:::1-2: last two samples of pSB3C5-M13genes from above gel<br />
:::3. Uncut<br />
:::4. Cut with EcoRI<br />
:::5. Cut with PstI<br />
:::6. Cut with EcoRI and PstI<br />
:-Appears that two backbones are ligated together<br />
<br />
'''6/30'''<br />
*Made chemically competent ER2738 cells that contain Litmus28i DNA<br />
*To amplify M13genes in order to retry ligation<br />
::-PCR of M13K07 DNA (diluted 1:100) to amplify the M13 genes<br />
::-PCR purify PCR product<br />
::-Run on gel: PCR purification, PCR, noDNAcontrol<br />
:::*PCR and purification showed bands at ~9k; therefore, did not get product<br />
:::*No DNA control was clean<br />
*Made freeze downs of<br />
::pSB3C5-J04450<br />
::pSB6A1-J04450<br />
::‘empty’ pSB3C5<br />
::‘empty’ pSB6A1<br />
<br />
'''7/1'''<br />
*Digestion of PCR product from 6/30 to figure out where mistake is<br />
:-Not really useful<br />
'''[[File:UCB-Phage Delivery-140701.JPG]]'''<br />
:Lanes...<br />
:1. PCR pur -> digested with AgeI<br />
:2. PCR pur -> digested with NgoMIV<br />
:3. PCR pur -> digested with PstI<br />
:4. PCR pur -> uncut<br />
:5. PCR pur -> digested with dpnI<br />
:6. Uncut plasmid DNA<br />
*Talked to Mary: She says we were adding to much DNA<br />
::-PCR again. Used 1:100 dilution of 1:100 diluted M13K07 DNA. (aka 1:10000 dilution)<br />
'''[[File:UCB-Phage Delivery-140701-02.JPG]]'''<br />
*Received a beautiful band at 6k bp<br />
:lanes<br />
:1. 1:100 dilution<br />
:2. 1:1000 dilution<br />
:3. 1:10 000 dilution<br />
:4. No DNA control<br />
<br />
'''7/2'''<br />
*To biobrick M13 genes (pSB6A1-M13genes and pSB3C5-M13genes)<br />
::-PCR purified 7/1 PCR product (Primers = Gem012)<br />
::-Digested with EcoRI-HF and PstI-HF<br />
::-Ligation to<br />
::::pSBA61 (digested and gel extracted)<br />
::::pSB3C5 (digested and gel extracted)<br />
<br />
'''7/3'''<br />
*Transformation<br />
::-pSB6A1-M13genes into 5alpha cells and ER2738 with pSB1C3-M13ori<br />
::-pSB3C5-M13genes into 5alpha cells and ER2738 with Litmus28i<br />
::-Transformed into cells containing a phagemid in order to skip some steps<br />
<br />
'''7/3-7/7 Vacation!'''<br />
<br />
==Week 10==<br />
<br />
'''7/7'''<br />
*Made O/N cultures of 7/3 transformation colonies<br />
::-Transformation results were not recorded until 7/8 (see below)<br />
<br />
'''7/8'''<br />
*Transformation results ( CC = Chemically comp cells, ER = ER2738)<br />
::-No growth on No DNA controls<br />
:::ER-Litmus28i CC on AMP-Chlor-Tet<br />
:::ER-phagemid1C3 CC on AMP-Chlor-Tet <br />
:::5alpha CC on Amp <br />
:::5alpha CC on Chlor <br />
::-Lots of red colonies on positive controls, no white colonies<br />
:::ER-Litmus28i CC + pSB3C5 on AMP-Chlor-Tet<br />
:::ER-phagemid1C3 CC + pSB6A1 on AMP-Chlor-Tet<br />
::-Lots of white colonies on sample plates, some red colonies<br />
:::ER-Lit CC + M13genes-pSB3C5 on AMP-Chlor-Tet<br />
:::ER-phagemid1C3 CC + M13genes-pSB6A1 on AMP-Chlor-Tet<br />
:::5alpha CC + M13genes-pSB3C5 on Chlor<br />
:::5alpha CC + M13genes-pSB6A1 on Amp<br />
*Overnights from 7/7 look healthy<br />
::-Renamed O/N to have numbers instead of long names<br />
{| class = "wikitable"<br />
|-<br />
! Sample #<br />
! Cells<br />
! DNA<br />
<br />
|-<br />
| 1-5<br />
| ER-Lit<br />
| M13genes-pSB3C5<br />
<br />
|-<br />
| 6-10<br />
| ER-phagemid1C3<br />
| M13genes-pSB6A1<br />
<br />
|-<br />
| 11-15<br />
| 5alpha<br />
| M13genes-pSB3C5<br />
<br />
|-<br />
| 16-20<br />
| 5alpha<br />
| M13genes-pSB6A1<br />
<br />
|-<br />
|}<br />
::-Mini-prepped DNA<br />
::-Digest the mini-preps with EcoRI-HF and PstI-HF<br />
::-Run digestions on gel to check sizes<br />
'''[[File:UCB-Phage Delivery-140708.JPG]]'''<br />
<br />
:lanes<br />
::top-left: pSB3C5-M13genes in ER2738 with Litmus28i<br />
::top-right: pSB6A1-M13genes in ER2738 with pSB1C3-1C3<br />
::bottom-left: pSB3C5-M13genes in 5alpha<br />
::bottom-right: pSB6A1-M13genes in 5alpha<br />
:None are correct<br />
*Primers Gem013 came in. Resuspend and diluted primers<br />
::-O/N of pSB3C5 to use for PCR tomorrow<br />
<br />
'''7/9'''<br />
*Wanted to make Litmus28i biobrick compatible for use as a phagemid backbone for us and other iGEM teams<br />
::-Mini-prepped O/N of pSB3C5<br />
::-PCR of pSB3C5 to amplify J04450 with Litmus28i compatible cut sites<br />
:::Primers: Gem013<br />
::::*At the 3’ end, these primers are the same as VF2 and VR so will bind the region flanking J04450. This conserves the terminators that exist between the biobrick prefix and VF2 on one side and those between the biobrick suffix and VR on the other side. Therefore, we are amplifying, VF2 priming site, terminators, J04450, terminators, and VR priming site<br />
::::*At the 5’ end, these primers contain unique restriction sites found in the Litmus28i MCS<br />
*PCR of M13 genes…. again (did 4 samples)<br />
::-Used the 1:10000 dilutions<br />
<br />
'''7/10'''<br />
*Note: The following two projects were done in parallel when possible<br />
*To make Litmus28i biobrick compatible<br />
::-Ran gel of PCR from 7/9 (J04450 amplification with Gem013)<br />
'''[[File:UCB-Phage Delivery-140710.JPG]]'''<br />
<br />
:1-4: PCRs of M13genes<br />
:5. No DNA control for M13genes PCR<br />
:6. PCR of pSB3C5 backbone<br />
:7. No DNA control for pSB3C5 backbone<br />
::-PRC purified the PCR product<br />
::-Digestion #1<br />
:::Restriction enzymes had the same buffer conditions but different activation temperatures so we had to do a 2 part digestion<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Restriction enzymes<br />
! Notes<br />
<br />
|-<br />
| J04450<br />
| Sac1<br />
| PCR purified<br />
<br />
|-<br />
| Litmus 28i<br />
| Sac1<br />
| From NEB tube<br />
<br />
|-<br />
|}<br />
:::*Incubate 1hr at 37 C<br />
:::*Heat inactivated 20 minutes at 80C<br />
::-Digestion #2<br />
:::-Added 1ul BsmI to both samples<br />
:::-Incubated 1hr at 65C<br />
:::-Heat inactivated 20 minutes at 80C<br />
::Ran gel<br />
:::-See gel below<br />
:::-Tried to extract J04450 segment but received very low yield. Since band otherwise looked clean, we decided to redo digestion then skip straight to ligation<br />
::Repeat digestion #1 for J04450<br />
::Repeat digestion #2 for J04450<br />
::Ligation (10hr at 16C, 10 min at 80C)<br />
:::3. Litmus28i + J04450<br />
*To retry ligation to biobrick backbone<br />
::-Ran gel of PCR from 7/9 (M13genes amplified with Gem012)<br />
:::See gel below<br />
::-PCR purified the PCR product<br />
::-Digestion<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Restriction enzymes<br />
! Notes<br />
<br />
|-<br />
| M13 genes<br />
| EcoRI-HF + PstI-HF<br />
| PCR purified<br />
<br />
|-<br />
| 6A1<br />
| EcoRI-HF + PstI-HF<br />
| ‘empty’ pSB6A1<br />
<br />
|-<br />
| 3C5<br />
| EcoRI-HF + PstI-HF<br />
| contains J04450<br />
<br />
|-<br />
|}<br />
:Incubate 1hr at 37C<br />
:Heat inactivated 20 minutes at 80C<br />
::-Ran gel<br />
:::*See gel below<br />
:::*Tried to extract M13genes and pSB3C5 segments but received very low yields. Since bands otherwise looked clean, we decided to redo digestion then skip straight to ligation<br />
::-Repeat digestion for M13genes and pSB3C5<br />
::-Ligation (10h at 16C, 10m at 80C)<br />
:::1. pSB3C5 + M13genes<br />
:::2. pSB6A1 + M13genes<br />
*Gel<br />
'''[[File:UCB-Phage Delivery-140710-02.JPG]]'''<br />
:1. pSB1A3<br />
:2. Limtus28i<br />
:3. M13genes<br />
:4. pSB3C5<br />
:5. J04450<br />
<br />
'''7/11'''<br />
*Transformed ligations from 7/10 into 5alpha cells<br />
::1. pSB3C5 + M13genes<br />
::2. pSB6A1 + M13genes<br />
::3. Litmus28i + J04450<br />
<br />
<br />
'''7/12'''<br />
*Results of 7/11 transformation<br />
::-No growth on no DNA control (Amp or Chlor)<br />
::-Lots of growth on Lit-J04450 -> some red-> colonies are too close together to prick individual colonies<br />
:::-It’s possible that these red colonies are satellites =(<br />
:::-Swiped some and plated on new Amp plate (restreak)<br />
::-Lawn of positive control (Litmus 28i) on Amp<br />
::-Many colonies for L1 (pSB3C5-M13genes) and L2 (pSB6A1-M13genes)<br />
:::-Due to high number of red colonies on Lit-J04450 plate, we assumed that most of these colonies contain empty vector<br />
:::-Did not make O/N<br />
*Ligated M13 genes to pSB6A1<br />
::-Used the remained of our digested M13genes.<br />
::-Both digestions from 7/10<br />
*Infection Experiment<br />
::-had 3 cell stocks (each taken from a different colony the night before)<br />
::-Tested Litmus 28i, Tandem phagemid 1C3, and double infection<br />
:::*Therefore, there were 9 flasks total.<br />
::-negative control: Streaked parent cells (non-infected) onto Chlor, Amp, and kan<br />
::-Included double infection plates for Litmus 28i and phagemid 1C3<br />
<br />
==Week 11==<br />
<br />
'''7/13'''<br />
*Transformed 7/12 ligation (pSB6A1-M13genes)<br />
*Litmus28i-J04450<br />
::-All growth from 7/12 restreak was white<br />
::-Pricked some red colonies for liquid O/N-will hopefully see red tomorrow<br />
::-Pricked a few red colonies and put into 200ul H2O (a few colonies per tube- 2 tubes total).<br />
:::*Plated 150ul onto Amp plates<br />
::-Put original plate into incubator to hopefully get bigger colonies<br />
<br />
'''7/14'''<br />
*Finished TWIV ppt<br />
*Set up 2 liquid culture of red Litmus 28i colonies (Litmus28i-J04450)-- slow growth <br />
::-Mini-prepped red Litmus 28i ‘O/N’ from earlier in day<br />
::-Digested samples—to check for insert and correct cut sites<br />
*Made O/Ns of pSB6A1-M13genes colonies from 7/13 transformation<br />
<br />
<br />
'''7/15'''<br />
*Ran gel of Litmus28i-J04450 samples<br />
'''[[File:UCB-Phage Delivery-140715.JPG]]'''<br />
:1. J04450-Litmus28ibb #1 (EcoRI+PstI)<br />
:2. J04450-Litmus28ibb #1 (uncut)<br />
:3. J04450-Litmus28ibb #2 (EcoRI+PstI)<br />
:4. J04450-Litmus28ibb #2 (EcoRI+PstI)<br />
::*Accidentally added restriction enzymes<br />
:5. Litmus28i (EcoRI+PstI)<br />
::*Only has PstI site<br />
<br />
::-Verifies that Litmus28i-J00450 had correct cut sites<br />
::-From this point on, biobricked Litmus28i is called Litmus28ibb<br />
*Check 7/14 O/Ns for pSB6A1-M13genes<br />
::-Mini-prepped liquid cultures<br />
::-Digested with EcoRI and PstI<br />
::::Received bold bands of just under 4000bp. Some lanes had a very feint band ~330bp. None were the correct size<br />
*Transformed Litmus28ibb-J04450 into ER2738 cells<br />
*Remake phage packaging<br />
::-pSB1C3-M13ori(New)<br />
::-pSB1C3-M13ori(Old)<br />
<br />
'''7/16'''<br />
*Results of 7/15 transformation of Litmus28i into 6/30 ER2738 cells<br />
::-lawn on no DNA control-- most likely contaminated cells<br />
::::Streaked chem comp 5alpha and ER onto Amp and Amp+Tet plates to determine if problem is with cells or plates<br />
::-O/N culture of ER to make new chem comp cells<br />
<br />
'''7/17'''<br />
*Finished phage isolation<br />
::Note:<br />
:::*pSB1C3-M13ori (New): M13ori and plasmid ori are convergent<br />
:::*pSB1C3-M13ori (Old): M13ori and plasmid ori are tandem<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! 269nm<br />
! 320nm<br />
! [] phage/mL<br />
<br />
|-<br />
| pSB1C3-M13ori (New)<br />
| 0.386<br />
| 0.034<br />
| 8.126 E12<br />
<br />
|-<br />
| pSB1C3-M13ori (New)<br />
| 0.419<br />
| 0.036<br />
| 8.842 E12<br />
<br />
|-<br />
| pSB1C3-M13ori (Old)<br />
| 0.283<br />
| 0.025<br />
| 5.956 E12<br />
<br />
|-<br />
| pSB1C3-M13ori (Old)<br />
| 0.326<br />
| 0.024<br />
| 6.972 E12<br />
<br />
|-<br />
|}<br />
*Infection to compare the new and the old pSB1C3-M13ori<br />
::-Prepped cells for infection<br />
::-Infected cells with pSB1C3-M13ori (New) or pSB1C3-M13ori (New)<br />
::-and plated on dilutions of 1:100, 1:1000, and 1:10000 on Chlor and kan<br />
::-Also plated 1:1000 dilution on lowered Chlor concentration (34ug/mL)<br />
*Sent Litmus28ibb-J04450 for sequencing<br />
<br />
'''7/18'''<br />
*Results from contamination test<br />
::-5alpha on Amp = no growth<br />
::-5alpha on Amp+Tet = colonies<br />
::-ER on Amp = lawn<br />
::-ER on Amp+Tet = lawn<br />
*Made new chemically competent ER2738<br />
*Transformed Litmus28ibb-J04450 into ER2738 made on 3/30 and ER2738 made on 7/18<br />
::-Had two samples of Litmus28ibb-J04450 and two cell stocks, so 4 samples total<br />
<br />
'''7/19'''<br />
*Results of 7/17 infection<br />
::-pSB1C3-M13ori(Old) (tandem-when plasmid ori and M13ori point in the same direction) packages better than pSB1C3-M13ori(New) (when ori and M13ori are convergent), implying that directionality matters.<br />
'''[[File:UCB-phage lab7-19-141012.jpg]]'''<br />
<br />
==Week 12==<br />
<br />
'''7/20'''<br />
*Freeze downs<br />
:-Litmus28i-J04450 Litmus 28i is now biobrick compatible<br />
<br />
'''7/21'''<br />
*Send samples for sequencing<br />
::-pSB1C3-M13ori(Old)<br />
::-pSB1C3-M13ori(New)<br />
*To ligate M13ori to a kanamycin backbone (pSB1K3)<br />
::-Resuspended pSB1K3-J04450 from distribution kit (6B on plate 4)<br />
:::*Contains J04450 as insert (full RFP construct)<br />
::-Transform pSB1K3 into 5alpha cells<br />
<br />
'''7/22'''<br />
*Pricked colony from pSB1K3-J04450 transformation<br />
::-Mini-prepped DNA to get a supply of DNA<br />
*To put M13ori (M13 phackaging signal) onto Kanamycin resistance o we can test packaging ratios with M13K07 on the same antibiotic<br />
::-Digested pSB1K3 with EcoRI-HF and XbaI<br />
::-Digested pSB1C3-M13ori (Old) with EcoRI-HF and SpeI-HF<br />
:::*DNA was not sufficiently cut. Too much DNA? Problem with enzyme (SpeI-HF)?<br />
'''[[File:UCB-Phage Delivery-140722.JPG]]'''<br />
:1. pSB1K3<br />
:2. M13ori<br />
<br />
'''7/23'''<br />
*Sequencing samples from 7/21 were lost in the mail. Resent samples<br />
*Yet another attempt to biobrick M13genes<br />
::1. using primers that would amplify genes and M13 ori parts from M13K07<br />
::2. using primers that would amplify genes, M13 ori parts, and plasmid ori from M13K07<br />
'''[[File:UCB-Phage Delivery-140723.JPG]]'''<br />
:1. Amplified only the M13genes and M13ori<br />
:2. No DNA control for 1<br />
:3. Amplified M13genes, M13ori, and plasmid ori<br />
:4. No DNA control for 3<br />
::-When run on a gel, samples were clean with only one band at around 7kb. No contamination in no DNA controls<br />
*Digestions<br />
::-pSB1K3 with EcoRI-HF and PstI-HF to check for correct insert<br />
::-pSB1C3-M13ori (Old and new) with EcoRI-HF and SpeI-HF to test for efficient cutting with different stock of restriction enzyme<br />
'''[[File:UCB-Phage Delivery-140723-02.JPG]]'''<br />
:1-3: pSB1K3<br />
:4. pSB1C3-M13ori (Old)<br />
:5. pSB1C3-M13ori (New)<br />
*Still had inefficient cutting. Tested M13ori next to Litmus28i to disern the problem<br />
:*Uncut<br />
:*Cut once with (E, X, S, or P)<br />
:*Cut twice with (E+S or E+P)<br />
'''[[File:UCB-Phage Delivery-140723-03.JPG]]'''<br />
:1. M13ori uncut<br />
:2. M13ori Ecori-HF<br />
:3. M13ori SpeI-HF<br />
:4. M13ori XbaI<br />
:5. M13ori PstI-HF<br />
:6. M13ori EcoRI-HF + SpeI-HF<br />
:7. M13ori EcoRI-HF + PstI-HF<br />
:8. Litmus28i uncut<br />
:9. Litmus28i EcoRI-HF<br />
:10. Litmus28i SpeI-HF<br />
:11. Litmus28i XbaI<br />
:12. Litmus28i PstI-HF<br />
:13. Litmus28i EcoRI-HF + SpeI-HF<br />
:14. Litmus28i EcoRI-HF + PstI-HF<br />
*Only partial digest with only SpeI-HF for both. Complete digestion with all others, including E+S<br />
<br />
'''7/24'''<br />
*To Biobrick M13genes using pSB6A1<br />
::-PCR purified M13 genes (did both samples at the same time)<br />
::-Digested PCR purification and pSB6A1 with EcoRI-HF and PstI-HF<br />
'''[[File:UCB-Phage Delivery-140724.JPG]]'''<br />
*Gel extracted M13genes<br />
<br />
:1. M13genes<br />
:2. pSB6A1<br />
:-Bands of pSB6A1 were too light to gel extract<br />
*To swap the kanamycin resistance marker on M13K07 with ampicillin resistance<br />
::-PCR amplified AmpR from pSB6A1<br />
*made O/N culture of pSB6A1 from freeze down to mini-prep<br />
::-Also streaked cells onto plate<br />
<br />
'''7/25'''<br />
*To change resistance marker on M13K07<br />
:1. Biobrick method<br />
::*Tried to mini-prep O/N but pellet (after liquid culture was spun) was not red even after 16+ hours. Set up O/N from plate colonies<br />
:2. Swap only resistance method<br />
::*PCR purified 7/24 PCR<br />
::*Ran product on gel-> band of correct size<br />
'''[[File:UCB-Phage Delivery-140725.JPG]]'''<br />
<br />
:1. PCR product<br />
:2. PCR product-> PCR purified<br />
:3. No DNA control<br />
::-Digested the PCR purification products of M13genes+ori and AmpR with AgeI-HF and NotI-HF<br />
::*Bands on previous gel looked clean enough for both so did not gel extract<br />
::-Overnight ligation<br />
*Set up O/Ns of pSB1K3 colonies<br />
<br />
'''7/26'''<br />
*Transform<br />
::-pSB6A1+M13genes<br />
*To biobrick M13genes<br />
::-Digestion<br />
:::*pSB6A1 with EcoRI and PstI<br />
'''[[File:UCB-Phage Delivery-140726.JPG]]'''<br />
:Gel extracted backbone (band ~4000bp)<br />
*Ligations (10hrs 16C, 10min at 80C)<br />
::-pSB6A1+M13genes<br />
<br />
==Week 13==<br />
<br />
'''7/27'''<br />
*Transformation results from 7/26<br />
::-No growth for no DNA controls ''on Amp''<br />
::-No growth for M13 genes onto pSB6A1 ''on Amp''<br />
*Set up O/Ns of 5alpha and ER2738 to make competent cell tomorrow<br />
*Made freeze down of pSB1K3<br />
*Plated ER competent cells from 5/15 and 6/16 Onto Chlor plates to check for contamination<br />
<br />
'''7/28'''<br />
*No growth of ER2738 on Chlor (either sample)<br />
::-ER is not contaminated with ChlorR<br />
*Make new 5alpha chem comp cells <br />
*Transformations<br />
::-pSB6A1-M13genes<br />
::-positive control for Amp (pSB6A1)<br />
::-No DNA control<br />
<br />
'''7/29'''<br />
*Set up O/Ns of pSB6A1-M13genes<br />
::-Plated sample on reduced Amp and regular Tet<br />
::-Later in day…<br />
::-Mini-prepped O/Ns<br />
::-Digested and ran on a gel to check for insert sizes<br />
'''[[File:UCB-Phage Delivery-140729.JPG]]'''<br />
:*All but the 4th lane with 3 bands look correct<br />
<br />
'''7/30'''<br />
*Lawn on last night’s plating-> replate<br />
*Digest pSB3C5 and pSB6A1-M13genes with E and P to move M13genes to a Chlor backbone<br />
'''[[File:UCB-Phage Delivery-140730.JPG]]'''<br />
::-Extracted bands<br />
::-Ligations<br />
:::*pSB3C5 + M13genes<br />
*Sent samples for sequencing<br />
**M13 genes on pSB6A1 VF2<br />
**M13 genes on pSB6A1 VR<br />
<br />
'''Test experiment for high school kids coming to lab (7/29-7/31)'''<br />
*General idea: Have 2 strands of DNA, one has an EcoRI site while the other contains a SNP, abolishing the cut sight. We pretend that one is pathogenic (the one that is not cut) and tells kids to figure out which one is which <br />
*PCR with Dream-Taq<br />
::-Did not have special fastdigest enzyme<br />
::-Hypothesized that green dye will interfere with enzyme effectivity<br />
*PCR with Dream-Taq (no green dye)<br />
::-Digest with EcoRI fastdigest-> did not cut<br />
*Redid experiment several times<br />
::-Should digest with XbaI<br />
::-PCR purified PCR products<br />
<br />
'''7/31'''<br />
*Spent the morning with Heritage High School<br />
*Made phage<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Phage<br />
! A269<br />
! A320<br />
! genome size<br />
! [ ]<br />
<br />
|-<br />
| Litmus28ibb-J04450 (1)<br />
| 0.823<br />
| 0.197<br />
| <br />
| 9.35 x10^12<br />
<br />
|-<br />
| Litmus28ibb-J04450 (2)<br />
| 0.940<br />
| 0.168<br />
| <br />
| 1.14 x10^13<br />
<br />
|-<br />
|}<br />
<br />
'''8/2'''<br />
*Digestion<br />
::-pSB6A1-M13genes (E+P)<br />
::-pSB3C5 (E+P)<br />
<br />
==Week 14==<br />
<br />
'''8/3'''<br />
*Another attempt to clone M13genes onto pSB3C5<br />
::-Run 8/2 digestions on gel<br />
'''[[File:UCB-Phage Delivery-140803.JPG]]'''<br />
:1. M13genes (E+P)<br />
:2. pSB3C5 (E+P)<br />
::-Gel extracted <br />
:::*M13genes away from pSB6A1 backbone<br />
:::*pSB3C5 backbone away from J04450<br />
::-Ligations<br />
:::*pSB3C5 + M13genes<br />
:::*pSB3C5 + no insert<br />
::-Transformed ligations into ER2738<br />
<br />
'''8/5'''<br />
*Transformation of pSB3C5+M13genes failed<br />
*Made phage<br />
::-Fd-CAT DNA packaged with Fd-CAT<br />
::-phagemid 1C3 packaged with M13K07<br />
::-amilCP on pSB1C3 packaged with M13K07<br />
::-Litmus28ibb-J04450 packaged with M13K07<br />
<br />
'''8/6'''<br />
*Isolated phage<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Phage<br />
! [ ]<br />
<br />
|-<br />
| Fd CAT<br />
| 2.72 x10^12<br />
<br />
|-<br />
| Litmus28ibb-J04450<br />
| 8.37 x10^12<br />
<br />
|-<br />
| pSB1C3-amilCP<br />
| 1.42 x10^13<br />
<br />
|-<br />
| pSB1C3-M13ori<br />
| 4.32 x10^12<br />
<br />
|-<br />
|}<br />
<br />
'''8/6'''<br />
*Started making phage with Fd CAT the should contain Litmus28ibb-RFP<br />
*Infection<br />
::-Infected ER2738 cells with Litmus28ibb-RFP, pSB1C3-M13ori, or pSB1C3-amilCP<br />
::-Plated Litmus samples on 100ug/mL Amp and 50ug/mL Kan<br />
::-Plated other two samples on 34ug/mL Chlor and 25ug/mL Kan<br />
<br />
'''8/7'''<br />
*Isolated phage containing Litmus28ibb-RFP using helper phage<br />
::-M13K07 (as control)<br />
::-Fd-CAT (no phage pellet was observed-worried that there was no phage)<br />
*Went to CSU to have them test M13ori part compared to amilCP. Grew the samples in 5mL O/Ns then diluted to 0.5OD and grew 30 minutes rather than starting from a fresh colony<br />
<br />
'''8/8'''<br />
*At CSU<br />
::-Finished phage protocol (Test packaging of M13ori part)<br />
::-Only used 20mL infection samples<br />
::-grew to 0.55 OD<br />
::-When making phage (after 14 hour incubation), there was very little growth<br />
::-Plated non-diluted and diluted 1:1000 of M13ori and amilCP sample<br />
*Infected cells using phage isolated 8/7. Plated at dilutions of 1:100 and 1:100k<br />
*Want to test progeny Fd-CAT phage (made 8/7) for infectability<br />
::-Start phage isolation prodocol using Fd-CAT phage from 8/7 do deliver the helper phagemid<br />
:::*Phagemid:<br />
::::*None: will make Fd-CAT phage packaing Fd-CAT phagemid<br />
::::*Litmus28ibb-J04450: make Fd-CAT phage packaging Litmus28ibb-J04450 phagemid<br />
*Remake phage (Fd-CAT) using fresh stock from Mike<br />
<br />
<br />
'''8/9'''<br />
*Results of 8/8 infection<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Helper Phagemid<br />
! phagemid<br />
! Selection<br />
! 1:1000 dilution<br />
! 1:00k dilution<br />
<br />
|-<br />
| M13K07<br />
| Litmus28ibb-RFP<br />
| Kan+Tet<br />
| 400-500 (some red)<br />
| 61 white; 20 red<br />
<br />
|-<br />
| same<br />
| same<br />
| Amp+Tet<br />
| lawn<br />
| 2<br />
<br />
|-<br />
| Fd-CAT<br />
| Litmus28ibb-RFP<br />
| Chlor+Tet<br />
| 0<br />
| 0<br />
<br />
|-<br />
| same<br />
| same<br />
| Amp+Tet<br />
| 10<br />
| 707<br />
<br />
|-<br />
|}<br />
*NOTE: The same sample was plated on two different plates. For example, the sample using M13K07 as the helper phage was plated on Kan+Tet and Amp+Tet<br />
*Mini-prep<br />
::-pSC3C5-J04450 -- very little growth, low DNA yield<br />
::-Fd-CAT infected cells<br />
:::*Digest and run on a gel to verify presence of a band- there was a very feint band<br />
*Finished isolating phage…. messed up and used 0.8MgCl2, 0.2M NaCl instead of PEG during precipitation step. Also used the wrong phage<br />
::-Infected ER2738 cells anyway…. no growth by 8/12<br />
<br />
==Week 15==<br />
<br />
'''8/10'''<br />
*Are the progeny phage from 8/6 (original progeny from first Fd-Tet application) viable/ able to reproduce?<br />
::-Used these progeny to make phage that should amplify Fd-CAT phage containing either the Fd-CAT or Litmus28ibb-J04450 phagemid<br />
<br />
'''8/11'''<br />
*Finished isolating phage<br />
{| class = "wikitable"<br />
|-<br />
! Helper Phagemid<br />
! Phagemid<br />
! A269<br />
! A320<br />
! genome size<br />
! [ ]<br />
<br />
|-<br />
| Fd-CAT<br />
| Litmus28ibb (1)<br />
| 0.029<br />
| 0.013<br />
| 4080<br />
| 2.35 x10^11<br />
<br />
|-<br />
| Fd-CAT<br />
| Ltimus28ibb (2)<br />
| 0.033<br />
| 0.015<br />
| 4080<br />
| 2.65 x10^11<br />
<br />
|-<br />
| Fd-CAT<br />
| Fd-CAT (1)<br />
| 0.326<br />
| 0.082<br />
| 7775<br />
| 1.88 x10^12<br />
<br />
|-<br />
| Fd-CAT<br />
| Fd-CAT (2)<br />
| 0.320<br />
| 0.079<br />
| 7775<br />
| 1.86 x10^12<br />
<br />
|-<br />
| M13g6A1<br />
| M13g6A1 (1)<br />
| 0.322<br />
| 0.148<br />
| 10,029<br />
| 1.04 x10^12<br />
<br />
|-<br />
| M13g6A1<br />
| M13g6A1 (2)<br />
| 0.503<br />
| 0.130<br />
| 10,029<br />
| 2.23 x10^12<br />
<br />
|-<br />
|}<br />
<br />
'''8/13'''<br />
*Infected ER2738 with Fd-CAT packaging Litmus28ibb-J04450<br />
<br />
'''8/16'''<br />
*Results from Infection on 8/11<br />
{| class = "wikitable"<br />
|-<br />
! Helper Phagemid<br />
! Phagemid<br />
! 1:1<br />
! 1:10<br />
! 1:100<br />
! 1:1000<br />
<br />
|-<br />
| M13g6A1<br />
| M13g6A1<br />
| almost lawn<br />
| 728<br />
| 287<br />
| 55<br />
<br />
|-<br />
| Fd-CAT<br />
| Fd-CAT<br />
| lawn<br />
| almost lawn<br />
| 1260<br />
| 640<br />
<br />
|-<br />
| Fd-CAT<br />
| Litmus28ibb-J04450<br />
| lawn<br />
| almost lawn<br />
| 2004<br />
| 304<br />
<br />
|-<br />
|}<br />
<br />
Results: packaging of Litmus28ibb-J04450 using Fd-CAT helper phagemid from (8/15)<br />
<br />
{| class="wikitable"<br />
|-<br />
!Antibiotic<br />
!1:10 <br />
!1:100 <br />
!1:1k <br />
!1:10k <br />
!1:100k<br />
<br />
|-<br />
|Amp <br />
|Lawn<br />
|Almost lawn <br />
|1025 <br />
|217 <br />
|25<br />
<br />
|-<br />
|Kan <br />
|9 <br />
|2 <br />
|0 <br />
|0 <br />
|0<br />
|}<br />
<br />
==Week 16==<br />
<br />
'''8/19'''<br />
<br />
Sent samples for sequencing<br />
<br />
Litmus28ibb-J04450<br />
<br />
==Week 17==<br />
<br />
'''8/27'''<br />
<br />
Transform pSB1C3-M13ori into ER2738 cells<br />
<br />
'''8/28'''<br />
<br />
Make phage with pSB1C3-M13ori as phagemid and M13g6A1 as helper phagemid<br />
<br />
'''8/29'''<br />
<br />
Finish phage isolation of M13 phage containing pSB1C3-M13ori<br />
<br />
'''9/3'''<br />
<br />
digest pSB6A1-M13g and pSB3C5 with EcoRI and PstI. Did not receive a band for M13genes so did not proceed with ligation to pSB3C5<br />
<br />
==Week 18==<br />
<br />
'''9/4'''<br />
<br />
Mini-prepped new pSB6A1-M13genes (bad quality) and pSB3C5-J04550. Digested both plasmids with EcoRI and PstI and ran products on gel. Did receive a band for the M13genes but it was too feint to gel extract<br />
<br />
'''9/5'''<br />
<br />
Mini-prep pSB6A1-M13genes again. Digest this and pSB3C5 backbone from previous day with EcoRI and PstI. M14g band was barely bright enough to extract so we extracted M13genes and pSB3C5 backbone. Pieces were ligated together overnight at 16C for 10 hours.<br />
<br />
'''9/7'''<br />
<br />
Transformed 9/5 ligation (pSB3C5-M13genes into 5alpha cells). Plated sample onto low Chlor plate (34ug/mL)<br />
<br />
'''9/9'''<br />
<br />
Still no growth for 9/7 transformation of pSB3C5-M13genes ligation PCR M13genes to attempt another ligation into pSB3C5. Diluted DNA to ~1.17ng/ul<br />
<br />
'''9/10'''<br />
<br />
Gel of 9/9 PCR of M13genes looked clean (only ran 3ul of PCR sample on gel). PCR purified the remaining PCR sample. Digested with EcoRI and PstI. Ligated to previously digested pSB3C5.<br />
<br />
==Week 19==<br />
<br />
'''9/11'''<br />
<br />
Transform 9/10 ligation (pSB3C5-M13genes) into 5alpha cells<br />
<br />
'''9/13'''<br />
<br />
Colony PCR of colonies that grew for 9/11 transformation. Looking for pSB3C5-M13genes)<br />
<br />
'''9/15'''<br />
<br />
Gel of colony PCR showed failure. Set up overnight cultures anyway.<br />
<br />
'''9/17'''<br />
<br />
Submitted Part to iGEM Registry<br />
<br />
{| class="wikitable"<br />
|-<br />
|iGEM # <br />
|Description<br />
<br />
|-<br />
|BBa_K1445000 <br />
|M13ori- the packaging signal for the M13 and fd phage<br />
|}<br />
<br />
Mini-prepped overnights from 9/15 and sent samples for sequencing<br />
<br />
Sent Litmus28ibb-J04450 sample for sequencing to sequence the remaining section that was not reached before<br />
<br />
==Week 20==<br />
<br />
'''9/21'''<br />
<br />
Up until this point, very little luck was had with pSB3C5. Retried putting M13genes onto a chloramphenicol backbone, but this time used pSB1C3. Digested M13genes and pSB1C3 with EcoRI and PstI. Gel showed incomplete digestion so did not gel extract.<br />
<br />
'''9/22'''<br />
<br />
Digested pSB1C3-J04450 with EcoRI and PstI then gel extracted the backbone. PCR purified M13genes(PCR product was clean so did not bother to gel extract). Ligated the two pieces together.<br />
<br />
'''9/23'''<br />
<br />
Transform ligations from 9/22 (pSB1C3-M13genes) into 5 alpha cells<br />
<br />
'''9/27'''<br />
<br />
Transform pSB1C3-M13genes into 5alpha cells. Plate onto low Chlor (34ug/mL)<br />
<br />
Later in day.... Set up overnight cultures of colonies<br />
<br />
'''9/28'''<br />
<br />
Mini-prepped O/N cultures from 9/27. Digested samples with EcoRI and PstI. There were no bands of the correct size on the gel.<br />
<br><br />
<br><br />
<br />
{{Template:UCB-Footer}}<br />
<br />
the M13 and fd phage</div>Leighlahttp://2014.igem.org/Team:CU-Boulder/Notebook/Phage_TeamTeam:CU-Boulder/Notebook/Phage Team2014-10-17T06:55:35Z<p>Leighla: /* Week 18 */</p>
<hr />
<div>{{Template:UCB-Main}}<br />
{{UCB-NavBar}}<br />
__FORCETOC__<br />
<br />
'''Phage Delivery'''<br />
==Week 1==<br />
'''Notes:''' Unless stated otherwise, all gels contain 2-log ladder<br />
<br />
'''5/9'''<br />
*Obtained BW23115 KanR cells- BW23115 cells that had their native CRISPR-Cas system knocked out by the insertion of a Kanamycin resistance gene<br />
::-Will also be called BW23115 or BW<br />
::-Conjugated BW23115 KanR cells with contain F’ notation (ex. BWF’)<br />
*Obtained ER2738 cells that contain the F’ episome (no changes from NEB sample)<br />
::-Will also be called ER. Assume that all ER samples contain the F’ episome<br />
::-Streaked sample onto LB+Tet (20ug/mL) to select for colonies containing F’ episome<br />
<br />
'''5/10'''<br />
*Did receive colonies from 5/9 selection<br />
<br />
==Week 2==<br />
'''5/12'''<br />
*Need to conjugate BW23115 KanR cells with the F’ episome<br />
::-Set up overnight cultures of ER2738 and BW23115 KanR<br />
::-When mixed, ER2738 will donate it’s F’ episome and BW23115 KanR will receive the F’ episome. F’ episome confers Tetracycline resistance<br />
<br />
'''5/13'''<br />
*Started M13 Amplification: Amplify M13 phage using the M13K07 Helper Phage<br />
::-Let precipitated in NaCl/PEG solution overnight<br />
::-Possible sources of error<br />
:::*Did not sterilize 2.5M NaCl/20% PEG-8000 solution<br />
:::*Added 4-fold PEG solution<br />
::::Compensated by adding more LB<br />
:::*During precipitation, put sample in -20C for 30 minutes before realizing mistake and moving to it to 4C. Sample partially froze<br />
*Conjugated BW23115 with F’ episome<br />
::-Added 1mL BW23115 to 1mL ER2738 overnight culture<br />
::-Incubated at 37C for 30 minutes, shaking<br />
::-Plated on LB+Kan(50ug/mL)+Tet(20ug/mL)<br />
:::*To select for BW cells that took the F’ episome (containing Tet resistance)<br />
<br />
'''5/14'''<br />
*Finished the M13 Amplification<br />
::-Visualized product on UV-vis. There was a tall spike at 269nm indicating that DNA was present. Did not test at 320nm.<br />
*Results of BW23115 Conjugation<br />
::-Many colonies indicating successful conjugation of F’ episome into BW23115<br />
::-Set up overnight to make freeze down tomorrow<br />
*Set up overnight of ER2738 to make chemically competent tomorrow<br />
<br />
'''5/15'''<br />
*Made freeze down of BW23115 KanR F’<br />
::-BW23115 E. coli strain with Kanamycin resistance gene inserted into genome and with F’ episome<br />
*Made chemically competent ER2738 cells<br />
*Transformation of Litmus28i (from NEB) into chemically competent ER2738 cells<br />
::-Added 1ul Litmus28i plasmid to 40ul competent cells<br />
::-Plated on LB + Amp(100ug/mL)<br />
::-Purpose: To make M13 phage that package Litmus28i DNA. Need phagemid (Litmus28i) DNA in infectable cells (cells containing F’ episome) to introduce M13K07 Helper Phage and make phage. <br />
<br />
'''5/16'''<br />
*Results of 5/15 transformation<br />
::-No growth for No DNA control<br />
::-Many colonies for sample<br />
<br />
==Week 3==<br />
'''5/19'''<br />
*M13 Amplification to isolate M13-Litmus28i phage<br />
::-Cells: ER2738 cells containing Litmus28i phagemid<br />
::-Helper Phage: M13K07<br />
::-Not much phage was precipitated<br />
*Set up overnight culture of ER2738 to infect tomorrow<br />
<br />
'''5/20'''<br />
*Infected ER2738 cells with M13-Litmus28i phage<br />
::-Plated only on Ampicillin(100ug/mL) (should have also plated on kanamycin)<br />
::-Infected for 4-5 hours-> should have only infected for 30 minutes maximum. This extra time gives the cells that were infected with M13-M13K07 the time to produced M13-M13K07 phage and reinfect<br />
<br />
'''5/21'''<br />
*Results from M13-Litmus28i infection of ER2738<br />
::-Solid lawn of growth for diluted and non-diluted<br />
::-Also sickly looking growth<br />
*Set up overnights<br />
::-ER2738 cells containing Litmus28i for freeze down<br />
::-BW23115 with F’ episome to make chemically competent cells <br />
::-ER2738 to redo infection<br />
<br />
'''5/22'''<br />
*Tested absorbance of phage produced through M13 amplification on 5/19<br />
::-Low absorbance of 0.018 at 269nm but no detection at wavelength 320nm<br />
::-Decided to redo M13 amplification<br />
*Made chemically competent BW23115 with f-episome<br />
*Made freeze down of ER2738 containing Litmus28i<br />
*Set up overnight of ER2738 containing Litmus28i to redo M13 amplification tomorrow<br />
<br />
'''5/23'''<br />
*Protocol switch to make phage using phagemid<br />
::-“M13 Amplification” protocol should only be used to make more M13-M13K07, not to make M13 phage containing a different phagemid<br />
::-Switched to new protocol (“Use of M13K07 Helper Phage for isolation of single stranded phagemid DNA” by NEB. Made modifications (see our protocols) to isolate phage rather than single-stranded DNA)<br />
::-Making phage….<br />
{| class = "wikitable"<br />
|-<br />
! Helper phage<br />
! Phagemid<br />
! Cells<br />
! Notes<br />
<br />
|-<br />
| M13K07<br />
| None<br />
| ER2738<br />
| Make more M13-M13K07<br />
<br />
|-<br />
| M13K07<br />
| Litmus28i<br />
| ER2738<br />
| Test packaging of Litmus28i<br />
<br />
|-<br />
|}<br />
*Made fresh antibiotics<br />
::-Chloramphenicol (34 ng/mL)<br />
:::*1.44g chloramphenicol into 42mL EtOH<br />
::-Ampicillin (50 ng/mL)<br />
:::*4g ampicillin into 80mL mili-Q H2O<br />
<br />
'''5/24'''<br />
*Isolated phage using new protocol<br />
::-Resuspended pellet in 200ul TBS and 200ul 30% glycerol<br />
::-Measured absorbance with UV-vis<br />
:::*concentration (phage/mL) = 6x10^16 x (A269-A320)/ (#of base pairs in the phage genome)<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! Abs (269nm)<br />
! Abs (320nm)<br />
! Genome size<br />
! Concentration (phage/mL)<br />
<br />
|-<br />
| M13-M13K07<br />
| 0.721<br />
| 0.060<br />
| <br />
| 4.57 x10^12<br />
<br />
|-<br />
| M13-Litmus28i<br />
| 0.250<br />
| 0.028<br />
| 2823<br />
| 4.72 x10^12<br />
<br />
|-<br />
|}<br />
*Infect ER2738 cells with M13-Litmus28i<br />
::-Wanted 1:10 phage:cell ratio. Math….<br />
:::*At 1 OD (e.coli), cell/mL = 5x10^8<br />
:::*5x10^7 phage * (1mL/4.72x10^12 phage) = 0.011ul phage<br />
*Set up overnights<br />
::-ER2738 for infection with M13-Litmus28i<br />
::-BW23115 F’ for infection with M13-Litmus28i to test infectivity of conjugated strain<br />
<br />
==Week 4==<br />
'''5/25'''<br />
*Infect ER and BWF’ cells with M13-Litmus28i<br />
::*Made 5mL culture of ER and BW that was at 1 OD<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! OD<br />
! mL sample for 1OD in 5mL<br />
! mL LB to 5mL<br />
<br />
|-<br />
| ER2738<br />
| 2.5<br />
| 2 mL<br />
| 3 mL<br />
<br />
|-<br />
| BW23115<br />
| 2.0<br />
| 2.5 mL<br />
| 2.5 mL<br />
<br />
|-<br />
|}<br />
::-Based on calculations from 5/24, we needed to add 0.011 ul phage per 1 mL of cells at 1 OD. This equates to 0.055 ul of phage into 5 mL cells; therefore we made a 1:10 dilution so we could add 0.5ul. Unfortunately, the pipet would not take up 0.5ul so we added 0.8ul of M13-Litmus28i phage<br />
::-Grew the cells for 20 minutes at 37C<br />
::-Plated 300ul onto Kanamycin (50ug/mL) and 300ul onto Ampicillin (100ug/mL) for each sample<br />
:::*Incubated overnight at 37C<br />
*'''Note:''' During the production of phage, the phagemid SHOULD be packaged preferentially over the Helper Phagemid but some Helper Phagemid will still be packaged. We plated on Amp to select for cells that were infected with phage containing Phagemid. We plated on Kan to select for cells that were infected with phage containing Helper Phagemid. This allows us to compare the packaging efficiency of Helper Phagemid: Phagemid.<br />
'''5/26'''<br />
*Results from 5/25 infection with M13-Litmus28i<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Result<br />
! Significance<br />
<br />
|-<br />
| ER2738 on Amp<br />
| Lawn<br />
| Litmus28i phagemid was successfully packaged into the M13 phage and is infectable<br />
<br />
|-<br />
| ER2738 on Kan<br />
| 100-200 colonies<br />
| Some M13 helper phage is packaged into the M13 phage but at a much lower rate than Litmus28i<br />
<br />
|-<br />
| BW23115 on Amp<br />
| Lawn<br />
| BW23115 is ‘equally’ infectable by M13 as ER2738<br />
<br />
|-<br />
| BW23115 on Kan<br />
| Lawn<br />
| BW23115 contains Kan resistance in its genome so this tells us nothing<br />
<br />
|-<br />
|}<br />
:*Conclusions:<br />
::-Cells grew on Ampicillin; therefore, Litmus28i phagemid was successfully packaged into M13 phage. <br />
::-For ER2738 samples, there was significant growth on Ampicillin compared to Kanamycin; therefore, Litmus28i phagemid is packaged preferentially over M13K07 Helper Phagemid<br />
::-M13-Litmus28i retains its infectivity of cells containing the F’ episome<br />
:*Because we received lawns, we have to redo the infection and plate less cells so we can calculate the uptake ratio between the phagemid and helper phage based on the number of colonies<br />
*Started 50 mL overnight of K12 ER2738 and BW23115<br />
<br />
'''5/27'''<br />
*Redo the infection done on 5/25<br />
::-Infectable cells: ER2738 and BW23115<br />
:::*Plated non-infected samples of each (non-diluted) to check for contaminants<br />
::-Diluted M13-Litmus28i (1) phage by a factor of 10. Added 5.5ul to each sample<br />
::-Grew samples for 20 minutes at 37C, 250rpm<br />
::-Plated 100ul of onto an Ampicillin (100ug/mL) plate and onto a Kanamycin (50ug/mL) plate. Incubated overnight at 37C.<br />
:::*Dilutions = 1:10; 1:100; and 1:1000<br />
'''5/28'''<br />
*Results from 5/27<br />
::-Controls were as expected<br />
:::*No growth for ER2738 non-infected grown on Amp, ER2738 non-infected grown on Kan, or BW23115 non-infected grown on Amp<br />
:::*Growth for BW23115 non-infected grown on Kan (BW23115 has Kan R in genome)<br />
::-Many colonies were received for all dilutions (1:10, 1:100, and 1:1000) of the following<br />
:::*ER2738 infected and plated on Amp<br />
:::*BW23115 infected and plated on Amp<br />
:::*BW23115 infected and plated on Kan<br />
::-Many (100s to 1000?) colonies grew on 1:10 and 1:100 dilutions of ER2738. 50-100 colonies grew on the 1:1000 dilution of ER2738<br />
:::*Compare this to the 100-200 colonies that grew from 2/25 infection (which was 300ul non-diluted, infected cells)<br />
:::*Reasons for increased yield<br />
::::*Added too much phage?<br />
::::*volume changed between experiment (5mL to 50mL)<br />
::::*Overnight culture may not have been saturated. If still in log phase, the cells would continue to grow<br />
*Made 50mL O/N cultures of ER2738 and BW23115 so we can repeat the infection tomorrow and plate further dilutions starting at 1:1000<br />
::-Carry out infection in 5mL and 50mL to test volume effect?<br />
<br />
'''5/29'''<br />
*Measured OD of overnights<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! OD<br />
! mL to have .1OD in 50mL<br />
! mL to have 1OD in 5mL<br />
<br />
|-<br />
| K12 ER2738<br />
| 3.0<br />
| 1.7<br />
| 1.7<br />
<br />
|-<br />
| BW23115<br />
| 2.9<br />
| 1.7<br />
| 1.7<br />
<br />
|-<br />
|}<br />
<br />
<br />
*Experiment 1: Infect cells using same method as 5/25 (in a 5mL culture)<br />
::-Started with 1OD cells in 5mL<br />
::-Added about 0.7ul (inaccuracies in pipet) of 1:10 diluted M13-Litmus28i phage<br />
::-Incubated (rotating) for 20 minutes<br />
::-Made 1:1,000 and 1:10,000 dilutions<br />
::-Plated 100ul on Ampicillin (100ug/mL) plates and on Kanamycin (50ugmL) plates<br />
:::*Included non-infected samples diluted by 1:1000<br />
::::*This negative control can be used for Experiment 2 since the non-infected parent solution is the same<br />
::-Incubate overnight at 37C<br />
*Experiment 2: Infect cells using protocol from “Eliminating helper phage from phage display”<br />
::-Diluted O/Ns to OD of 0.1 in 50 mL culture<br />
::-Grew samples until of OD of ER2728 = 0.59 and the OD of BW23115 = 0.60<br />
:::*Missed OD of 0.5 mark, but the two samples are close to each other<br />
::-Chilled samples on ice for 30 minutes<br />
::-Warmed in incubator for 35 minutes (should have been 30)<br />
::-Amount of phage. Rather than use 1:1 as mentioned in protocol, we used multiplicity of 1:10 (phage:cell)<br />
:::*Added 3.3ul of 1:10 diluted M13-Litmus28i (1) phage<br />
::::*(On 5/27 we added 5.5ul of diluted phage to 50mL of cells at OD of 1. Our cells were at OD of .6; therefore, 5.5*.6 = 3.3ul)<br />
::-Incubated for 30 minutes at 37C, not shaking<br />
:::*We later change this to shaking<br />
::-Dilutions<br />
:::*1:1,000; 1:5,000; 1:10,000; 1:50,000; 1:100,000; 1:1,000,000<br />
:::*Plated ER2738 and BW23115 on Ampicillin (100ug/mL)<br />
:::*Plated ER2738 on Kanamycin (50ug/mL)<br />
*Experiment 3: Growth Test (for growth curve)<br />
::-We were concerned by the low OD of the Overnights from the last few days. Wanted to be sure that 2.0-3.0 was not still in log phase. Cultures looked saturated but the OD seemed low.<br />
{| class = "wikitable"<br />
|-<br />
! Time<br />
! Elapsed time (min)<br />
! ER2738 (no antibiotic)<br />
! ER2738 (Tetracycline (20ug/mL))<br />
! BW23115 (with F’ episome) no antibiotic<br />
<br />
|-<br />
| 10:08<br />
| 0<br />
| 0.1<br />
| 0.1<br />
| 0.1<br />
<br />
|-<br />
| 11:10<br />
| 62<br />
| 0.24<br />
| 0.21<br />
| 0.21<br />
<br />
|-<br />
| 12:00<br />
| 112<br />
| 0.49<br />
| 0.45<br />
| 0.44<br />
<br />
|-<br />
| 13:00<br />
| 172<br />
| 1.00<br />
| 0.93<br />
| 0.98<br />
<br />
|-<br />
| 14:15<br />
| 217<br />
| 1.29<br />
| 1.21<br />
| 1.31<br />
<br />
|-<br />
| 15:40<br />
| 302<br />
| 2.1<br />
| 1.8<br />
| 2.3<br />
<br />
|-<br />
| 16:47<br />
| 369<br />
| 2.5<br />
| 1.9<br />
| 3.4<br />
<br />
|-<br />
| 18:05<br />
| 447<br />
| 2.6<br />
| 2.2<br />
| 2.5<br />
<br />
|-<br />
| 19:00<br />
| 502<br />
| 2.9<br />
| 2.3<br />
| 2.5<br />
<br />
|-<br />
| 20:00<br />
| 562<br />
| 3.0<br />
| 2.6<br />
| 2.8<br />
<br />
|-<br />
|}<br />
<br />
:::*The time point at 16:47 (369 minutes elapsed) for BW23115 conjugated (without antibiotics) is most likely an error. It has been removed from the growth plot<br />
'''[[File:UCB-Phage Delivery-140529.JPG]]'''<br />
*Other<br />
::-Made Amp and Kan plates (1 sleeve of each)<br />
::-Made 50mL O/N of ER2738 and BW23115F’ in case we need further dilutions<br />
::-Made 5mL O/N of ER2738, BW23115F’, and BW23115 (without F’ episome) to make chemically competent tomorrow<br />
:::*Did not have plate of BW23115 (without F’ episome) so used freeze down. Hoping to get O/N of a picked colony from CRISPR Team tomorrow morning<br />
<br />
'''5/30'''<br />
*Made chemically competent cells of…<br />
::-ER2738<br />
::-BW23115F’ (conjugated with F’ episome)<br />
::-BW23115 (not conjugated- without F’ episome)<br />
:::*Culture started from plate<br />
::-BW23115* not conjugated (without F’ episome)<br />
:::*Culture started from freeze down<br />
*Results from infections<br />
::-Negative Controls (cells were not infected; cells were diluted 1:1000)<br />
'''[[File:UCB-Phage Delivery-140530.JPG]]'''<br />
::-Results from Experiment 1 (5/29)<br />
'''[[File:UCB-Phage Delivery-140530-02.JPG]]'''<br />
::-Results from Experiment 2 (5/29)<br />
'''[[File:UCB-Phage Delivery-140530-03.JPG]]'''<br />
*Math<br />
::-If there are 5.00E+8 cells in 1mL of culture at OD of 1, then in 1mL of culture at OD of 0.59, there are 2.95E+8 cells. In a 50mL culture at OD of 0.59, there are 1.48E+10 cells.<br />
::-We added 3.3ul (0.0033mL) phage at concentration 4.62E+11 phage/mL which amounts to 1.52E+9 total phage<br />
::-Assuming that 1 phage infects 1 bacterium, we can assume that 1.52E+9 bacterial have the potential to be infected in the 50mL culture<br />
::-We plated 100ul of culture at various dilutions. If not diluted, the number of cells that can be potentially infected in 0.1mL equals 1.52E+9/500, or 3.05E+06 cells. We then accounted for the dilutions (for 1:1000 dilution, we divided 3.05E+06 by 1000 to receive 3.05E+03)<br />
::-The following table contains the number of cells with the potential to be infected assuming a 100% infectivity rate by M13 phage and that 1 cell is infected only once.<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Dilution<br />
! Potential infected cells<br />
! Colonies on Amp<br />
! % Potential (Amp)<br />
! Colonies on Kan<br />
! % Potential(Kan)<br />
! Kan:Amp<br />
<br />
|-<br />
| 1:1000<br />
| 1.52E+06<br />
| 476<br />
| 15.62%<br />
| 15<br />
| 0.820%<br />
| 1:19.04<br />
<br />
|-<br />
| 1:5000<br />
| 3.05E+05<br />
| 131<br />
| 21.49%<br />
| 13<br />
| 2.133%<br />
| 1:10.08<br />
<br />
|-<br />
| 1:10000<br />
| 1.52E+05<br />
| 93<br />
| 30.51%<br />
| 5<br />
| 1.640%<br />
| 1:18.60<br />
<br />
|-<br />
| 1:50000<br />
| 3.05E+04<br />
| 17<br />
| 44.29%<br />
| 0<br />
| 0.000%<br />
| <br />
<br />
|-<br />
|}<br />
<br />
*Conclusions from infections<br />
::-Results between and within the three trials are inconsistent. For example, the number of colonies received in experiments 1 and 2 from 5/29 differ greatly. Due to the differences in protocol, variation was expected but not to this extent. <br />
::-Our dilutions did not yield the expected 10 fold (or 5 fold) decrease in growth that was expected.<br />
::-Plates from 5/29 could be plated better to reduce dense areas of growth and growth around the rim.<br />
::-Though the experiment contained many errors we can say that the phagemid (Litmus 28i) is preferentially packaged compared to the helper phage (M13K07) but not to the degree we expected.<br />
::-Could receive increased occurrences of cells containing M13k07 due to infection, phage production, further infection<br />
<br />
==Week 5==<br />
<br />
'''6/2'''<br />
*Tested chemically competent cells through transformation<br />
::-Are cells contaminated?<br />
::-Are cells competent?<br />
*The samples for transformation<br />
{| class = "wikitable"<br />
|-<br />
! #<br />
! Cells (Tube label)<br />
! DNA (Tube label)<br />
! Resistance before transformation<br />
! Resistance after transformation<br />
<br />
|-<br />
| 1<br />
| K12 ER2738 5/20<br />
| p110+RBS (2) 4/23<br />
| Tet<br />
| Tet, Chlor<br />
<br />
|-<br />
| 2<br />
| BW (-f) 5/30<br />
| p110+RBS (2) 4/23<br />
| Kan<br />
| Kan, Chlor<br />
<br />
|-<br />
| 3<br />
| BW f-ep comp 5/22<br />
| p110+RBS (2) 4/23<br />
| Kan, Tet<br />
| Kan, Tet, Chlor<br />
<br />
|-<br />
| 4<br />
| BW (+f) 5/30<br />
| p110+RBS (2) 4/23<br />
| Kan, Tet<br />
| Kan, Tet, Chlor<br />
<br />
|-<br />
| 5<br />
| *BW23115 5/30<br />
| p110+RBS (2) 4/23<br />
| Kan<br />
| Kan, Chlor<br />
<br />
|-<br />
| 2B<br />
| K12 ER2738<br />
| 2B [from dis. kit]<br />
| Tet<br />
| Tet, Chlor<br />
<br />
|-<br />
| 2P<br />
| BW f-ep comp 5/22<br />
| 2P [from dis. kit]<br />
| Kan, Tet<br />
| Kan, Tet, Chlor<br />
<br />
|-<br />
|}<br />
<br />
'''6/3'''<br />
*Results from 6/2 Transformation<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Growth on Chlor<br />
! Growth on Kan<br />
! Growth on Kan+Tet<br />
! Growth on Amp<br />
<br />
|-<br />
| 1 N<br />
| X<br />
| X<br />
| X<br />
| X<br />
<br />
|-<br />
| 2 N<br />
| X<br />
| +<br />
| X<br />
| X<br />
<br />
|-<br />
| 3 N<br />
| X<br />
| +<br />
| +<br />
| X<br />
<br />
|-<br />
| 4 N<br />
| X<br />
| +<br />
| +<br />
| X<br />
<br />
|-<br />
| 5 N<br />
| X<br />
| +<br />
| X<br />
| X<br />
<br />
|-<br />
| 1<br />
| +<br />
| X<br />
| X<br />
| <br />
<br />
|-<br />
| 2<br />
| +<br />
| +<br />
| X<br />
| <br />
<br />
|-<br />
| 3<br />
| +<br />
| +<br />
| +<br />
| <br />
<br />
|-<br />
| 4<br />
| +<br />
| +<br />
| +<br />
| <br />
<br />
|-<br />
| 5<br />
| +<br />
| +<br />
| Many colonies but close to samp. 4<br />
| <br />
<br />
|-<br />
| 2P<br />
| + (~100)<br />
| +<br />
| +<br />
| <br />
<br />
|-<br />
| 1- JW<br />
| +<br />
| X<br />
| X<br />
| <br />
<br />
|-<br />
| 2- JW<br />
| +<br />
| +<br />
| X<br />
| <br />
<br />
|-<br />
| 3- JW<br />
| +<br />
| +<br />
| +<br />
| <br />
<br />
|-<br />
| 4- JW<br />
| +<br />
| +<br />
| +<br />
| <br />
<br />
|-<br />
| 5-JW<br />
| +<br />
| +<br />
| X<br />
| <br />
<br />
|-<br />
| 2B- JW<br />
| + (24)<br />
| X<br />
| X<br />
| <br />
<br />
|-<br />
|}<br />
*The transformations with DNA from the well (B2 and P2) had lower efficiencies than those with DNA from a mini-prep. Most likely this is due to the differences in DNA concentration (p110+RBS (2) 4/23 was at 254.4ng/ul)<br />
*Conclusions<br />
::-None of the competent cells were contaminated<br />
::-All of the competent cells are in fact, competent<br />
*Set up O/N of DH5-alpha cells to make competent tomorrow<br />
<br />
'''6/4'''<br />
*Isolation of single-stranded phagemid DNA using M13K07<br />
::-Added ER2738 colony to 50mL LB<br />
:::*Plate was cold. Next time warm plate before pricking<br />
::::*Best to use freshly grown plate<br />
::-After 4 hours, OD was at 0.02. Waited 45 minutes and OD was at 0.08. Therefore, we infected at OD 0.08<br />
::-Had started another culture when we did not think the first was growing. In incubator for about 1 hour. OD was 0.00. We infected anyway because last time it worked.<br />
::-Let infection proceed for 60 minutes then added 70ul of Kanamycin to be a final concentration of 70ug/mL<br />
*Primers came in to biobrick M13ori (packaging signal on Litmus28i)<br />
::-Resuspended primers and diluted 1:10<br />
<br />
'''6/5'''<br />
*Isolated single-stranded M13K07 DNA<br />
::-Final concentration = 5724 ng/ul (calculated from a 1:10 dilution)<br />
::-For second sample in pair, we resuspended it in TE but did not proceed to DNA extraction teps<br />
::-For the second culture we started 6/4, we resuspended pellet in TBS and glycerol to preserve the M13 phage. Measured absorbances (before glycerol was added)<br />
: #1<br />
::269 => 1.690A<br />
::320 => 0.103A<br />
: #2<br />
::269 => 1.453<br />
::320 => 0.059<br />
*For our first biobrick, we wanted to isolate the M13 origin, a segment ~500bp that allows for packaging into the M13 phage. We tried to achieve this by biobrick assembly and by Gibson Assembly.<br />
::-To biobrick M13 ori through biobrick assembly (the old-school way)<br />
:::PCR on Litmus 28i to amplify/biobrick M13ori<br />
::::*Used primers Gem003 F & R<br />
::::*Diluted Litmus 28i DNA 1:10<br />
:::Digestion of p11+RBS (1) to digest pSB1C3 bb with EcoRI-HF and PstI-HF<br />
:::Ran samples on gel and gel extracted pieces. We recieved very low yields (out of range for nano drop)<br />
::::*M13 ori: 4.0 ng/ul<br />
::::*pSB1C3: 1.8 ng/ul<br />
:::Digested M13 ori fragment despite poor extraction yield with EcoRI-HF and PstI-HF<br />
::::*Used 1.5x as much DNA as instructed based on inaccurate concentration<br />
'''[[File:UCB-Phage Delivery-140605.JPG]]'''<br />
::-Gel extracted red rectangles<br />
::-Ligation<br />
:::*10hr @ 16C, 10min @ 65C, 4ever @ 4C<br />
*To biobrick M13 ori through Gibson Assembly (the cool-kids way)<br />
::-PCR on Litmus 28i<br />
:::*Used primers Gem002 F & R<br />
:::*Diluted Litmus 28i DNA 1:10<br />
::-PCR on pSB1C3 (p11+RBS (1))<br />
:::*Used primers Gem001 F & R<br />
:::*Diluted pSB1C3 DNA 1:3<br />
<br />
'''6/6'''<br />
*Ran gel of PCR products from (6/5). Products will be used for Gibson Assembly<br />
::-Recieved bands for pSB1C3 around 2000bp and M13ori around 500bp<br />
::-No contamination in pSB1C3 PCR negative control<br />
::-Band in M13ori negative control that is the same size as sample. Contaminated by sample?<br />
::-Gel of PCR products #1 and #2 from 6/5<br />
'''[[File:UCB-Phage Delivery-140606.JPG]]'''<br />
:1. pSB1C3 with promoter+RBS as insert. Amplified with Gem001<br />
:2. No DNA control for Gem001<br />
:3. M13ori amplified with Gem002 from Litmus28i<br />
:4. No DNA control for Gem002<br />
*Gibson Assembly<br />
{| class = "wikitable"<br />
|-<br />
! Total Amount of Frag.<br />
! .02-.5pmol<br />
! 10 ul total<br />
<br />
|-<br />
| Gibson Assembly MM (2x)<br />
| 10 ul<br />
| 10<br />
<br />
|-<br />
| Dionized H2O<br />
| 10-x<br />
| <br />
<br />
|-<br />
|}<br />
::-Diluted pSB1C3 and M13ori PCR products 1:10<br />
::-Incubated 60min @ 50C<br />
::-Also used provided pUC16 as positive control<br />
*Transformation<br />
:1. p110+RBS Positive control<br />
:2. No DNA Negative control<br />
:3. Cas9 from distribution kit so we can have more<br />
:4. Thaw and refreeze cells Test competency of comp cells after thawed<br />
:5. Not chem comp cells Negative control for the above<br />
:6. Ligation Product<br />
:7. Gibson product<br />
::*7.2. Gibson product diluted 1:4<br />
:8. Gibson positive control<br />
::*7.2. Gibson positive control diluted 1:4<br />
::-For the Gibson product and the positive control, we transformed 2ul of product and 2ul of 1:4 diluted product. NEB recommends the first if using their competent cells and the second if using cells from other companies. Our cells are from NEB but we made them competent ourselves so we tried both ways<br />
:::*Plated on Chlor at concentrations of 170, 85, and 33 ug/mL<br />
*Primers came in<br />
::-Resuspended and made 1:10 dilutions<br />
<br />
'''6/7'''<br />
*Results from 6/6 transformation<br />
:1. Positive control<br />
::*Lots of growth, ~300-400 on 1:10 dilution<br />
:2. No DNA negative control<br />
::*No growth<br />
:3. Cas9 from distribution kit<br />
::*7 potential colonies (some are close to edges through) on non-diluted<br />
:4. Thawed then refroze cells<br />
::*Looks like (1)<br />
:5. Not chemically competent cells<br />
::*No growth<br />
:6. Ligation product<br />
::*13 potential colonies (some are close to edge)<br />
:7. Gibson Assembly Product<br />
::*170 -> No colonies<br />
::*85 -> No colonies<br />
::*33 -> 3 specks<br />
:7.2. Gibson Assembly product diluted 1:10<br />
::*170 -> 1 speck<br />
::*85 -> 3 colonies<br />
::*33 -> 13 colonies<br />
:8. Gibson positive control<br />
::*No colonies<br />
:8.2. Gibson positive control diluted 1:4<br />
::*No colonies<br />
*Made 6mL O/N cultures<br />
::-4 from (3) cas9 plate<br />
:::See Constitutive CRISPR notebook for more info on these samples<br />
::-7 from (6) Ligation product<br />
::-5 from (7.2 [85]) Diluted Gibson product on 85 ug/mL Chlor<br />
::-8 from (7.2 [33]) Diluted Gibson product on 33 ug/mL Chlor<br />
<br />
==Week 6==<br />
'''6/8'''<br />
*Check colonies for correct constructs.<br />
::-Mini-prepped all 24 O/Ns<br />
:::*Yielded low concentrations for samples 12, 15, 19, and 22<br />
::-Digested all with EcoRI and PstI (10ul reactions)<br />
::-Ran results on gel<br />
:::*All 4 cas9 samples had the expected bands of 2000 and 5000bp<br />
:::*All 7 ligation products have expected bands of 2000 and ~570bp<br />
:::*3 of 5 Gibson assemblies from 85ug/mL Chlor plate had expected bands of 2000 and ~500bp<br />
:::*3 of 8 Gibson assemblies from 33ug/mL Chlor plate had expected bands of 2000 and ~500bp<br />
'''[[File:UCB-Phage Delivery-140608-01.JPG]]'''<br />
<br />
'''[[File:UCB-Phage Delivery-140608-02.JPG]]'''<br />
::1-4: Cas9 from Stanford-Brown team<br />
::5-11: pSB1C3-M13ori cloned through ligation<br />
::12-24: pSB1C3-M13ori cloned through Gibson Assembly<br />
:::*12-16: Grown with 85 ug/mL Chlor<br />
:::*17-24: Grown with 33 ug/mL Chlor<br />
*Conclusions from gel<br />
::-We have cas9 safely in cells<br />
::-Our ligation reactions successfully yielded M13ori on pSB1C3<br />
::-Combined, we had a 46% success rate for the Gibson Assembly in yielding M13ori on pSB1C3<br />
:::*The 4 samples that had the lowest concentration after being mini-prepped (12,15, 19, and 22) correlate with samples that had the correct band pattern<br />
*We selected 4 samples from each (4 total between the two Gibson reactions) type<br />
::-For non-Gibson Assembled samples<br />
:::Plated 25ul on 170ug/mL Chlor<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Our usual method<br />
! Gibson Method<br />
<br />
|-<br />
| 1. Thaw on ice<br />
| 1. Thaw on ice <br />
<br />
|-<br />
| 2. Transfer 40ul cells to tube<br />
| 2. Transfer 50ul cell to tube<br />
<br />
|-<br />
| 3. Add DNA. 1ul for mini-prep OR up to 10ul for ligation<br />
| 3. Add 2ul to NEB cells OR 2ul of 1:4 diluted to other cells<br />
<br />
|-<br />
| 4. Mix by pipet Let sit 30min on ice<br />
| 4. Mix by pipet or flicking Let sit 30 min. on ice<br />
<br />
|-<br />
| 5. Heat shock: 42C for 45s<br />
| 5. Heat shock: 42C for 30s<br />
<br />
|-<br />
| 6. Ice for 5 minutes<br />
| 6. Ice for 2 min.<br />
<br />
|-<br />
| 7. Transfer to culture tube; Add 200ul SOC<br />
| 7. Add 950ul SOC to tube<br />
<br />
|-<br />
| 8. Shake or rotate for 60-120min at 37C<br />
| 8. Shake (250rpm) or rotate for 60 min. at 37C<br />
<br />
|-<br />
| 9.<br />
| 9. Warm plates to 37C<br />
<br />
|-<br />
| 10. Plate 100ul onto selection plate<br />
| 10. Plate 100ul onto plate<br />
<br />
|-<br />
| 11. Incubate O/N @ 37C<br />
| 11. Incubate O/N @ 37C<br />
<br />
|-<br />
|}<br />
::-Added 6mL LB and Chlor at concentration of 170ug/mL to grow O/N<br />
:*For Gibson Assembled samples<br />
::-Plated 25ul onto 170, 85, and 33ug/mL Chlor<br />
::-Samples from 85ng/mL plate<br />
:::Transferred 100ul to new tube, added media, added Chlor at 170ng/mL<br />
::-Samples from 33ng/mL plate<br />
:::Tranfered 100ul to new tubes, added media, added Chlor at 85ng/mL to one and 33ng/mL to the other<br />
:*Tomorrow, may send for sequencing and make freeze downs<br />
<br />
*Transformation<br />
:1. Positive control (p110+RBS diluted 1:10)<br />
:2. Non-diluted Gibson product<br />
:3. Gibson product diluted 1:4<br />
:4. Gibson product diluted 1:10<br />
::-Transformed each sample using our usual method and using the protocol given by Gibson<br />
<br />
::-Due to not have plates ready before transformation, in step 4, the samples sat for about 50 minutes. Then in step 8, they both recovered for about 150 minutes. Though not specified in our protocol, we did warm the plates to 37C. In step 10 for our protocol, since we only added 200ul SOC and wanted to plate on 3 selection plates (see below), we only plated 50ul (except for the positive control). <br />
::-Plated on three concentrations of Chloramphenicol (33 ug/mL, 85 ug/mL, and 170 ug/mL) to determine the differences in yield due to differences in concentration.Obvious hypothesis: more colonies will grow on plates that have a lower concentration of chlor.<br />
<br />
<br />
'''6/9'''<br />
*Made chemically competent 5alpha cells with Dan and Alex from main campus<br />
::-Waiting to hear results on competency<br />
*Will eventually make phage containing CRISPR-Cas9 that targets Kanamycin resistance. M13K07 has Kanamycin resistance so we need to switch the resistance on the M13 genes.<br />
::-PCR on pwp 2.po (plasmid that Sam gave us that contains the zeoR gene adjacent to ori) to amplify zeoR and ori. Zeo is on EM7 promoter<br />
:::-Primers: Gem008 R & R<br />
:::-Anneal temp from NEBuilder: 63.7C<br />
:::-Extension time: 90s<br />
:::-Expected band size on gel: 1300bp<br />
:::-Used phusion polymerase<br />
*PCR on M13K07 DNA to amplify M13 phage genes (also removes majority of M13 ori, all of KanR, and all of p15 ori)<br />
:::-Primers: Gem007 F & R<br />
:::-Anneal temp from NEBuilder: 60.2C<br />
:::-Extension time: 4:30<br />
:::-Expected band size on gel: about 6000bp<br />
:::-Used phusion polymerase<br />
*Freeze downs<br />
::-Note: Phagemid 1C3 was the original name for ‘pSB1C3-M13ori’<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Top Label<br />
! Side label<br />
! What?<br />
<br />
|-<br />
| Phagemid 1C3 6/9<br />
| From Lig<br />
| M13 ori inserted into 1C3 (biobricked); Done through ligation; Contains extra bases as spacer between biobrick prefrix/suffix and part for primer design<br />
<br />
|-<br />
| Phagemid 1C3 6/9<br />
| From Lig<br />
| “ “<br />
<br />
|-<br />
| Phagemid 1C3 6/9<br />
| Gibson<br />
| M13 ori inserted into 1C3 (biobricked); Done through Gibson cloning<br />
<br />
|-<br />
| Phagemid 1C3 6/9<br />
| Gibson<br />
| “ “<br />
<br />
|-<br />
|}<br />
'''6/10'''<br />
*Ran gel of PCRs from 6/9<br />
<br />
'''[[File:UCB-Phage Delivery-140610.JPG]]'''<br />
:1. Amplification of M13 genes from M13K07 (~6000bp)<br />
:2. No DNA control for (1) amplification<br />
:3. Amplification of Zeo resistance gene + plasmid ori (~1300bp)<br />
:4. No DNA control for (3) amplification<br />
*Gibson Assembly of above parts (did not gel extract)<br />
::-Diluted the PCR products 1:10 then added 3ul of M13K07 genes product and 7ul of ZeoR+ori product<br />
::-Incubated at 50C for 60 min.<br />
::-Transformed Gibson Assembly product into 5alpha cells<br />
:::*Used our usual protocol<br />
:::*Add 2ul of DNA<br />
::::-In one sample, diluted DNA 1:4 and in the other, we diluted DNA 1:10<br />
*Started Phage Amplification Protocol<br />
::-ER2738 transformed with Litmus 28i<br />
:::*Grew for ~2.5hr before reaching an OD of 0.04<br />
::-ER2738 transformed with pSB1C3-M13ori (M13ori on pSB1C3)<br />
:::*Grew for ~ 3.5hr before reaching an OD of 0.01, then in the next 1.5 hours, spiked to 0.19<br />
:::*We gave up and went home, and will restart tomorrow<br />
*Analyzed transformation results from 6/8<br />
<br />
'''6/11'''<br />
*Made chemically competent 5alpha cells<br />
*Restarted Phage Amplification Protocol<br />
::-Forgot to add phagemid antibiotic at start of growth. Added phagemid antibiotic when we added phage. Incubated for 90 minutes before adding Kanamycin (to select for cells that were infected by M13K07)<br />
::-Phage is at concentration 4.57x10^12 phage/mL<br />
:::Protocol calls for final concentration of 1 x10^8 phage/mL<br />
::::*(4.57 x10^12)*V = (1 x10^8)(50mL)<br />
::::*V = 0.00109mL<br />
:::Added 1.1ul of phage<br />
*Transformations<br />
{| class="wikitable"<br />
|-<br />
!DNA<br />
!Plate Selection<br />
|-<br />
|No DNA control<br />
|(all)<br />
|-<br />
|Positive control (p110+RBS)<br />
|(C)<br />
|-<br />
|M13 genes + ZeoR ori<br />
|(Z)<br />
|-<br />
|M13 genes +ZeoR ori diluted 1:4<br />
|(z)<br />
|} <br />
<br />
'''6/12'''<br />
*Transformation Results from 6/10 [Took ~36 hours to be clearly visible]<br />
::No DNA control<br />
:::-Amp: 0 <br />
:::-Zeo (50ug/mL): 200 colonies<br />
::M13 genes + ZeoR ori (1:4 dilution) <br />
:::-Zeo (25ug/mL): 300<br />
:::-Zeo (50ug/mL): 200<br />
:::-Zeo (100ug/mL): 100<br />
::M13 genes + ZeoR ori (1:10 dilution) <br />
:::-Zeo (25ug/mL): 300<br />
:::-Zeo (50ug/mL): 150<br />
:::-Zeo (100ug/mL): 150<br />
*Transformation Results for 6/11<br />
::-Positive (p110+RBS) on Chloramphenicol: 500 colonies<br />
::-No DNA on Amp: 0 colonies Zeo (100 ug/mL): specks<br />
::-No apparent growth on any other plate<br />
:::*Realized later that we grew our samples on the wrong plates. Will repeat transformation today<br />
*Transformation #1<br />
::This morning there were no colonies on positive (p110+RBS) coltrol from 6/11 even though we observed fast growth in the past. Without waiting for colonies to appear, we started a control transformation<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Diluted<br />
! Time at 42C<br />
<br />
|-<br />
| p110+RBS<br />
| No<br />
| 45s<br />
<br />
|-<br />
| p110+RBS<br />
| 1:10<br />
| 45s<br />
<br />
|-<br />
| p110+RBS<br />
| 1:10<br />
| 30s<br />
<br />
|-<br />
| p110+RBS<br />
| 1:10<br />
| No timer. ~43s<br />
<br />
|-<br />
| No DNA control<br />
| No<br />
| 45<br />
<br />
|-<br />
|}<br />
*Because ‘No DNA control 6/10’ yielded colonies, we researched Zeocin plates<br />
::-According to Life Technologies (Invitrogen), Zeocin requires low salt medium and a pH of 7.5<br />
::-Low Salt LB Medium (1L)<br />
:::*Ingredients<br />
::::10g Tryptone<br />
::::5g NaCl<br />
::::5g Yeast Extract<br />
:::-Mix ingredients<br />
:::-Adjust pH to 7.5 using NaOH (If go over, use HCl)<br />
:::-Add agar for plates at 15g/L. Autoclave<br />
:::-Thaw Zeocin on ice. Vortex<br />
:::-Add Zeocin to final concentration of 25ug/mL<br />
*Transformation #2<br />
::-Repeat of transformation on 6/11 but this time we will plate on the correct plates<br />
::-Also remade Zeocin plates<br />
*Finished isolation of M13 Litmus phage and M13 pSB1C3-M13ori phage<br />
::*Note: Phagemid 1C3 was the original name for ‘pSB1C3-M13ori’<br />
{| class = "wikitable"<br />
|-<br />
! Phage Sample<br />
! A269<br />
! A320<br />
! Concentration (phage/mL)<br />
<br />
|-<br />
| Litmus phage (1)<br />
| 0.181<br />
| 0.034<br />
| 3.12 x10^12<br />
<br />
|-<br />
| Litmus phage (2)<br />
| 0.227<br />
| 0.047<br />
| 3.83 x10^12<br />
<br />
|-<br />
| Phagemid 1C3 phage (1)<br />
| 0.101<br />
| 0.020<br />
| 1.87 x10^12<br />
<br />
|-<br />
| Phagemid 1C3 phage (2)<br />
| 0.126<br />
| 0.021<br />
| 2.42 x10^12<br />
<br />
|-<br />
|}<br />
::::phage/mL = 6x10^16 x (A269-A320)/ (#of base pairs in the phage genome)<br />
*Set up 50mL O/N of K12 ER2738 (containing f-episome) for infection tomorrow with Litmus phage and pSB1C3-M13ori phage<br />
<br />
<br />
'''6/13'''<br />
*Transformation Results for 6/11<br />
::-Several hundred colonies on Positive Control (p110+RBS) on Chlor<br />
::-No colonies for GA positive control on Amp<br />
::-No colonies for M13ori + ZeoR mistakenly plated on Amp<br />
::-No colonies for No DNA control on Amp<br />
::-100-ish colonies for No DNA control on Zeo<br />
::-100-ish colonies for cas9+AmpR+gRNA mistakenly plated on Zeo<br />
::-These last 3 points suggest/confirm that Zeo plates are no good<br />
*Transformation Results from 6/12 control test<br />
::-Colonies grew is about equal amounts on all plates, including No DNA control<br />
:::Either plates don’t contain Chlor or competent cells are contaminated<br />
::-Streaked competent cells onto new and old Chlor plates<br />
*Infection test of ER2738: Is Litmus preferentially packaged over M13K07 Helper phage? Is pSB1C3-M13ori preferentially packaged?<br />
::-Infect ER2738 with phage produced 6/12<br />
:::*Phage should have packaged Litmus 28i phagemid or pSB1C3-M13ori<br />
:::*Cells infected with phage packaging Litmus 28i will grow on Amp<br />
:::*Cells infected with phage packaging pSB1C3-M13ori will grow on Chlor<br />
:::*Cells infected with phage packaging M13K07 will grow on Kan<br />
::-After we plate, we can count the colonies and calculate a ratio of Litmus28i: M13K07 or pSB1C3-M13ori:M13K07 packaging<br />
<br />
'''6/14'''<br />
*Results of contamination test (streaked competent cells onto new and old Chlor plates)<br />
::-Colonies grew in low amounts on both plates. most likely the cells are contaminated<br />
*Transformation results for 6/12<br />
::-Many colonies for No DNA control on Zeo<br />
:::-Some colonies are turning pinkish-red<br />
::-Many colonies for diluted and non-diluted M13 genes+ZeoR on Zeo<br />
:::-Some colonies are turning pinkish-red<br />
*Could the white colonies be the designed colonies and can we kill the red colonies with Zeocin before killing the white colonies (aka. Use high Zeocin concentrations to select for the correct construct)<br />
::-Selected 1 red colony from No DNA Zeo control plate and 1 white colony from M13genes+ZeoR sample plate<br />
::-Added 100ul H2O then divided amongst 5 culture tubes each (with 5mL of low-salt LB, pH 7.5)<br />
::-Then added Zeocin to a final concentration of: 0, 25, 50, 75, 100ug/mL.<br />
*Results of 6/13 infection<br />
::-Litmus 28i infected cells<br />
:::*on Kan: Individual colonies for 1:10, 1:100, and 1:1000. No colonies on 1:5000<br />
:::*on Amp: lawn for 1:10, 1:100, and near lawn for 1:1000. Single colonies for 1:5000<br />
::-pSB1C3-M13ori infected cells<br />
:::*on Kan: Same as Litmus 28i samples on Kan<br />
:::*on Chlor: Lawn for all dilutions. Must discredit due to recent Chlor contamination<br />
<br />
==Week 7==<br />
<br />
'''6/15'''<br />
*Results of Zeocin experiment on 6/14<br />
::-Healthy growth for both at 0ug/mL Zeo<br />
::-No growth for white colony with any Zeo<br />
::-Good growth for red colony even at 100ug/mL Zeo<br />
::-This suggests that the red colonies are naturally resistant to Zeocin. Also, our plates must not contain active Neocin. We are sure that we are adding enough. Possibly, we add it while the media is too hot or leave the plates at room temperature (decondensing) for too long and this deactivates the antibiotic? Perhaps we are not attaining the correct pH<br />
*In light of recent contamination problems on both Chlor and Zeo, we made new competent cells<br />
::-5alpha<br />
::-BW23115<br />
::-BW23115 conjugated (contains f-episome)<br />
<br />
'''6/16'''<br />
*Made new Chlor antibiotic. Made to Chlor plates<br />
*Contamination Test: Streaked Zeo and new Chlor plates each with<br />
::-Colony from Zeo contaminated plate<br />
::-Colony from Chlor contaminated plate<br />
::-Non-transformed OLD chemically competent cells<br />
::-Non-transformed NEW chemically competent cells<br />
*Redid bacterial infection<br />
::-For both samples, we used ER2738 (not from the same ‘O/N’ (also, not a real O/N))<br />
::-Infected one sample with isolated ‘Litmus28i phage’ and one with ‘pSB1C3-M13ori’<br />
::-Alterations to protocol<br />
:::*Did not started from saturated O/N. Started each with a colony, waited several hours until at OD ~1. We then added these cells to fresh 50mL LB to have an OD of 0.1.<br />
:::*Missed the 30 minute infection mark. Infected for ~45 minutes.<br />
::-Plated on Kan and Amp (Litmus28i sample) or Chlor (pSB1C3-M13ori sample) at dilutions of<br />
:::*1:100, 1:1k, 1:10k, and 1:100k<br />
<br />
'''6/17'''<br />
*Q5 PCR to replace KanR with ZeoR in M13K07<br />
::-Used recommendations<br />
::-Unfortunately, I (Jo) don’t know the difference between tightening and loosening the thermocycler lid; therefore, our M13K07 sample to amplify the M13K07 genes evaporated. But that’s ok, because we were sick of Zeocin anyway and decided mid-PCR to not waste our time with a Gibson Assembly and transformation. Instead we will be using the Old-School method of digestion and ligation because let’s face it, it’s a classic (and Gibson sucks) =)<br />
*Results from infection Test<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Litmus 28i<br />
! 1:100<br />
! 1:1k<br />
! 1:10k<br />
! 1:100k<br />
<br />
|-<br />
| Amp<br />
| ~6000?<br />
| ~2056<br />
| 377<br />
| 36<br />
<br />
|-<br />
| Kan<br />
| 130<br />
| 8<br />
| 3<br />
| 0<br />
<br />
|-<br />
|}<br />
{| class = "wikitable"<br />
|-<br />
! pSB1C3-M13ori<br />
! 1:100<br />
! 1:1k<br />
! 1:10k<br />
! 1:100k<br />
<br />
|-<br />
| Chlor<br />
| 8<br />
| 1<br />
| 0<br />
| 0<br />
<br />
|-<br />
| Kan<br />
| 97<br />
| 9<br />
| 0<br />
| 0<br />
<br />
|-<br />
|}<br />
::-We consider the Litmus 28i to have been a success. The pSB1C3-M13ori …. not so much. Looking back at the Litmus28i and the M13K07, we noticed that the M13ori is facing the opposite direction as the plasmid ori. Ours faces the same direction as the plasmid ori. Because we are working with phagemids that are single stranded, we think that by flipping the M13ori, we may be able to recover functionality. We will also look into other reasons.<br />
*Results from contamination test (6/16)<br />
::-Old Chlor plate (6/12)<br />
:::-Non-transformed chemically competent cells<br />
::::*Old: some colonies<br />
::::*New: No colonies<br />
::-New Chlor plate (6/16)<br />
:::-Colony from<br />
::::*Zeo contaminated plate: No growth<br />
::::*Chlor contaminated plate: Much growth<br />
:::-Non-transformed chemically competent cells<br />
::::*Old: some colonies<br />
::::*New: No growth<br />
::-Zeo (25ug/mL, Low NaCl, pH 7.5) (6/12)<br />
:::-Colony from<br />
::::*Zeo contaminated plate: Much growth<br />
::::*Chlor contaminated plate: No growth<br />
:::-Non-transformed chemically competent cells<br />
::::*Old: some specks<br />
::::*New: some specks<br />
<br />
'''6/18'''<br />
*Ordered primers to…<br />
::-biobrick M13ori (in other direction)<br />
::-biobrick M13K07 genes<br />
'''6/19'''<br />
*Waited for primers<br />
*Set up O/N cultures to test last infection (6/16) for colonies containing pSB1C3-M13ori<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample #<br />
! Selection<br />
! Dilution<br />
! Presumably<br />
! Notes<br />
<br />
|-<br />
| 1-8<br />
| Chlor<br />
| 1:100<br />
| pSB1C3-M13ori<br />
| <br />
<br />
|-<br />
| 9<br />
| Chlor<br />
| 1:1k<br />
| pSB1C3-M13ori<br />
| <br />
<br />
|-<br />
| 10-14<br />
| Chlor<br />
| 1:1k<br />
| pSB1C3-M13ori<br />
| These colonies were not present on plate during initial counting on 6/17<br />
<br />
|-<br />
| 15-16<br />
| Kan<br />
| 1:1k<br />
| M13K07<br />
| <br />
<br />
|-<br />
|}<br />
:::-Selection and dilution refer to the plate. Cells were then grown under selection.<br />
:::-More colonies were seen on all Chlor plates. No new colonies appeared on Kan plates<br />
*Tomorrow, we will mini-prep, digest, and run the samples on a gel to verify gene transfer.<br />
<br />
'''6/20'''<br />
*Primers are in!<br />
*Cloning of pSB1C3-M13ori(New)<br />
::-PCR to amplify M13ori (packaging signal) from Litmus 28i to be in the other direction<br />
:::-Primers (Gem011 F&R) add cut sites to make part biobrick compatible<br />
:::-Was able to gel extract<br />
::::*Band at the same size as sample….. contaminated primers?<br />
:::-Then digested with E and P<br />
::-Digested pSB1C3 plasmid with E and P to linearize backbone<br />
::::*Was able to gel extract<br />
'''[[File:UCB-Phage Delivery-140620.JPG]]'''<br />
<br />
:1. Spill over from 2<br />
:2 and 3. pSB1C3 digested with EcoRI and PstI<br />
::-Insert <100bp so cannot be seen<br />
:4. M13ori<br />
*Ligation of M13ori (packaging signal) into pSB1C3<br />
::10h at 16C<br />
::20m at 80C<br />
::Hold at 4C<br />
<br />
*PCR to amplify M13K07 genes from M13K07 DNA isolated from phage<br />
::-Primers (Gem012 F&R) add cut sites to make part biobrick compatible<br />
::-Very light bands and not band might correlate with 6000pb but not enough resolution on gel to be certain. Bands were too light so did not extract<br />
::-Set up PCR again using Q5 to run O/N<br />
:::*Received only a feint, smudgy band that was too large. We did not bother with extracting the DNA<br />
*Test of 6/19 O/Ns (14 that are presumably pSB1C3-M13ori and 2 that are presumably M13K07)<br />
::-Mini-prepped all O/Ns<br />
::-Digested all with Pst-I (common RE between pSB1C3-M13ori and M13K07)<br />
:::-Even though these are phagemids, we assumed that because they were still in the cell, the plasmids were still double stranded so could be recognized by RE. Our assumption was valid<br />
:::-Results of digestion<br />
::::*1,2,4-14: all had bands that corresponded to pSB1C3-M13ori cut once<br />
::::*3: Slight band at expected size but very feint<br />
::::*15, 16: as expected, they contain bands of ~9000bp, correlating with the M13K07 phagemid. Interestingly, sample 15 also contained a (brighter) band that corresponds with pSB1C3-M13ori. We assume that this was an incident of double infection (chance or did this occur at high frequency?)<br />
'''[[File:UCB-Phage Delivery-140620-02.JPG]]'''<br />
::Check table from 6/19 for more details<br />
:::-1-14: pSB1C3-M13ori<br />
:::-15-16: M13K07 from 1:1k diluted plate<br />
<br />
'''6/21'''<br />
*Needed to transform our pSB1C3-M13ori(New) (6/20) into cells. First, we transformed into 5alpha cells; however, we need to infect these cells later in order to make phage. Our 5alphas are not competent so we repeated the transformation, this time using ER2738 cells which contain the F’ episome, allowing us to infect with the M13 phage.<br />
::-Also transformed sample #1 from 6/20 mini-preps (pSB1C3-M13ori(Old) into ER2738 cells. Do not see any notes about this plasmid being in infectable cells during the initial experiment. Reason for experiment failure???<br />
*Ran 6/20 O/N PCR (to amplify M13K07 genes) on gel (also re-ran the previous sample from the day with more DNA)<br />
::-Still, band looks too big. Brightest band still occurs between 8000 and 9000bp. Lighter band around 7000bp-- might be ~6000pb but too light to tell<br />
*Alternative plan for amplifying M13K07 genes<br />
::-Digest sample with PstI (cuts outside of the target region)<br />
'''[[File:UCB-Phage Delivery-140621.JPG]]'''<br />
::Used sample #16 from 6/20 b/c it is double stranded so will cut<br />
:-PCR amplified the linearized digestion product<br />
:::1. PCR of M13genes<br />
:::2. PCR of M13genes + DMSO<br />
::::Gel extracted pieces boxed in red<br />
::-Used primers Gem012 F&R<br />
::-Gel extracted piece but received very low yield<br />
*Alternative to alternative plan for amplifying M13K07 genes<br />
::-Digested with PstI and AgeI<br />
'''[[File:UCB-Phage Delivery-140621-02.JPG]]'''<br />
:1. Cut with PstI and AgeI<br />
:2. Uncut<br />
:3. Cut with PstI<br />
:4. Just PCR<br />
::-Gel extracted ~6000pb band after double digestion<br />
::-PCR (Phusion)<br />
:::-Digestion (A+P) product<br />
:::-Gel extraction product<br />
:::-No DNA control<br />
<br />
==Week 8==<br />
<br />
'''6/22'''<br />
*Did receive colonies from 6/21 transformations. Selected colonies for overnight<br />
{|class="wikitable"<br />
|-<br />
!number<br />
!colonies<br />
|-<br />
|1-7<br />
|pSB1C3-M13ori(NEW) in 5alpha<br />
|-<br />
|8-14<br />
|pSB1C3-M13ori(NEW) in ER2738<br />
|-<br />
|15-18<br />
|pSB1C3-M13ori(OLD) in ER2738<br />
|-<br />
|19<br />
|p110+RBS in 5alpha as control<br />
|-<br />
|20<br />
|p110+RBS in ER2738 as control<br />
|}<br />
<br />
*Gel of 6/21 O/N PCR<br />
::Digestion (AgeI+PstI) product ? Yielded the three bands that appear with every PCR of M13K07<br />
'''[[File:UCB-Phage Delivery-140622.JPG]]'''<br />
<br />
:1. M13K07-> digested (AgeI + PstI) -> PCR<br />
:2. M13K07-> digested (AgeI+ PstI) -> extraction-> PCR<br />
:3. No DNA control for PCR<br />
::-Gel extraction product ? No bands<br />
::-No DNA control ? No bands<br />
*PCR purified the above PCR (Digestion (AgeI+PstI) product) and PCR from 6/21<br />
*Digested PCR purified sample with EcoRI+PstI and ran gel<br />
'''[[File:UCB-Phage Delivery-140622-02.JPG]]'''<br />
:1. PCR-> PCR purified<br />
:2. Sample after DpnI digest<br />
:3. Sample after digestion with EcoRI and PstI<br />
:4. Samples after digestion with DpnI then EcoRI and PstI<br />
*Ligation of above digestion (M13K07 genes) with pSB1C3<br />
<br />
<br />
'''6/23'''<br />
*Transformed pSB1C3-M13ori(New) (6/22 ligation)<br />
*Check overnights from 6/22 for the correct insert<br />
::-Mini-prep O/Ns<br />
::-Digested with EcoRI and PstI<br />
'''[[File:UCB-Phage Delivery-140622-03.JPG]]'''<br />
:::1-7: pSB1C3-M13ori(New) in 5alpha<br />
:::8-14: pSB1C3-M13ori(New) in ER2738<br />
:::15-18: pSB1C3-M13ori(Old)<br />
::-All were the expected size (though gel wiggled)<br />
::-Sent 4 samples for sequencing (iGEM primers: VF2 and VR)<br />
:::# 4: Divergent (new) phagemid 1C3 transformed into 5alpha<br />
:::# 11, 13: Divergent (new) phagemid 1C3 transformed into ER2738<br />
:::# 16: Convergent (old) phagemid 1C3 transformed into ER2738<br />
<br />
'''6/24'''<br />
*Results of 6/23 transformation (pSB1C3-M13genes) from 6/22 ligation)<br />
::-Received ~100 colonies<br />
::-Set up O/N cultures for 8 of the colonies<br />
::-Time passed…..<br />
::-Mini-prepped the 8 O/N samples mentioned above (yes, it was a long day)<br />
:::*Digested samples (E+P)<br />
*Started phage amplification protocol<br />
::-Amount of phage added = 10.9ul of 1:10 diluted phage (M13 phage (1) from 5/24 (4.575 x10^12 phage/mL)) to a final concentration of 1x10^8 phage/mL in 50mL<br />
::-Managed to get both samples to the 14-28hr incubation<br />
<br />
'''6/25'''<br />
*Checked mini-prep samples from 6/24 (pSB1C3-M13genes)<br />
::Ran the digestion overnight (6/24 to 6/25)<br />
'''[[File:UCB-Phage Delivery-140625.JPG]]'''<br />
::-All contained a band at 2000bp. Mostly empty vector or contained small, light band. Sample 1 had prominent band at ~1250bp.<br />
::-None of samples contained M13genes<br />
*M13K07 is on P15A ori (10-12 copy number) whereas the pSB1C3 ori is on pUC19 (500-700 copies). It’s possible that this overexpression is detrimental to cell<br />
::Alternative low copy plasmids found in distribution kit<br />
:::*2013 (plate 5)<br />
::::pSB6A1 (1K)<br />
::::pSB3C5 (3C)<br />
::::pSB3K3 (5E)<br />
:::*2014 (plate 4)<br />
::::pSB3C5 (4D)<br />
::::pSB6A1 (2L)<br />
::-Suspended and transformed the above plasmids into 5alphas<br />
::-Next, we will select colonies, mini-prep, digest, gel extract, ligate with M13genes<br />
*Started phage amplification protocol <br />
::-Phagemids in ER2738<br />
:::pSB1C3-M13ori(New)<br />
:::pSB1C3-M13ori(Old) <br />
::-Set up O/N of ER2738 for infection tomorrow<br />
<br />
'''6/26'''<br />
*Results of transformation of plasmids from the distribution kit<br />
::-Only received colonies pSB6A1 (2L from 2014) and pSB3C5 (4D from 2014)<br />
::-Pricked colonies for O/N<br />
*PCR of M13 genes so we can ligate it into the above backbones tomorrow<br />
::-DNA: M13K07 digested with EcoRI+PstI (6/21)<br />
::-Primers: Gem012 F & R<br />
*Finished phage amplification protocol<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Samples<br />
! A269<br />
! A320<br />
! Concentration<br />
<br />
|-<br />
| Old phagemid 1C3 (1)<br />
| 0.832<br />
| 0.492<br />
| 7.828 x10^12<br />
<br />
|-<br />
| Old phagemid 1C3 (2)<br />
| 0.324<br />
| 0.083<br />
| 5.549 x10^12<br />
<br />
|-<br />
| New phagemid 1C3 (1)<br />
| 0.391<br />
| 0.077<br />
| 7.229 x10^12<br />
<br />
|-<br />
| New phagemid 1C3 (2)<br />
| 0.402<br />
| 0.639<br />
| -5.457 x10^12<br />
<br />
|-<br />
|}<br />
::::*While resuspending “New phagemid 1C3 (2)”, the tip fell off and we lost half of sample. Evidently, we lost most of phage so we tossed sample<br />
::-Prepped and Infected ER2738 with “Old phagemid 1C3 (1)” and “New phagemid 1C3 (2)”<br />
:::Added 1.6ul of 1:10 diluted “Old phagemid 1C3 (1) to ER2738 cells<br />
:::Added 1.8ul of 1:10 diluted “New phagemid 1C3 (1) to ER2738 cells<br />
::-Plated the infected cells at dilutions<br />
:::1:1 onto Chlor+Kan plates<br />
:::1:10 onto Chlor+Kan plates<br />
:::1:100 onto Chlor and onto Kan plates<br />
:::1:1000 onto Chlor and onto Kan plates<br />
:::1:10000 onto Chlor and onto Kan plates<br />
:::1:100000 onto Chlor and onto Kan plates<br />
<br />
'''6/27'''<br />
*To ligate M13genes onto different backbones<br />
::-Mini-prepped the O/Ns from 6/26 to get backbones with low copy number<br />
:::pSB6A1<br />
:::pSB3C5 <br />
::::*did not grow as well<br />
::-PCR purified PCR (to amplify M13 genes with Gem012 F & R) from 6/26<br />
::-Digestions (50ul)<br />
:::Digest pSB6A1 with E + P<br />
:::Digest pSB3C5 with E + P<br />
:::Digest PCR purification product with E + P<br />
'''[[File:UCB-Phage Delivery-140627.JPG]]'''<br />
:1. pSB6A1<br />
:2. pSB3C5<br />
:3. M13genes<br />
::-Gel extraction of the above digestions<br />
:::For each: Added 10ul of 6x loading dye to 50ul digestions and divided the total volume between 2 wells<br />
::-Ligation<br />
:::1) pSB6A1-M13genes<br />
:::2) pSB3C5-M13genes<br />
*Results from 6/26 infection (after 22hrs in incubator)<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Anti-Sense Phagemid 1C3<br />
! 1:100<br />
! 1:1000<br />
! 1:10000<br />
! 1:100000<br />
<br />
|-<br />
| Chlor<br />
| 85<br />
| 6<br />
| 2<br />
| 0<br />
<br />
|-<br />
| Kan<br />
| 405<br />
| 13<br />
| 2<br />
| 1<br />
<br />
|-<br />
|}<br />
{| class = "wikitable"<br />
|-<br />
! Sense Phagemid1C3<br />
! 1:100<br />
! 1:1000<br />
! 1:10000<br />
! 1:100000<br />
<br />
|-<br />
| Chlor<br />
| 121<br />
| 24<br />
| 3<br />
| 0<br />
<br />
|-<br />
| Kan<br />
| 25<br />
| 1<br />
| 0<br />
| 0<br />
<br />
|-<br />
|}<br />
-We also plated the infected cells on plates containing Chlor and Kan to test for the possibility of double infection<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Phagemid 1C3<br />
! 1:1<br />
! 1:10<br />
<br />
|-<br />
| Anti-Sense<br />
| 59<br />
| 0<br />
<br />
|-<br />
| Sense<br />
| 4<br />
| 0<br />
<br />
|-<br />
|}<br />
<br />
<br />
'''6/28'''<br />
*We noticed that there were more colonies on our infection plates from 6/26 than on 6/27; therefore, we recounted colonies<br />
::-No increase of colonies on Kan plates<br />
::-Significant increase of colonies on Chlor plates<br />
::-Sense refers to the first phagemid 1C3 where the M13ori is in the sense direction compared to the plasmid ori<br />
::-Anti-Sense refers to the new phagemid 1C3 where the M13ori is in the anti-sense direction compared to the plasmid ori<br />
'''[[File:UCB-Phage Delivery-140628.JPG]]'''<br />
::Numbers on the left are after 22 hours. Numbers on the right are after 38.75 hours<br />
*Results of transformation from 6/27 (pSB6A1-M13genes and pSB3C5-M13genes)<br />
::-We have many colonies. Unfortunately, some are red, suggesting that original insert (J04450) was not successfully separated from backbone through gel extraction<br />
::-Selected colonies to grow overnight in 5mL LB<br />
<br />
==Week 9==<br />
<br />
'''6/29'''<br />
*Check O/N cultures for correct constructs (pSB6A1-M13genes and pSB3C5-M13genes)<br />
::-Mini-prep samples<br />
::-Digested with EcoRI and PstI to check insert sizes<br />
::-Gel<br />
'''[[File:UCB-Phage Delivery-140629.JPG]]'''<br />
::lanes….<br />
:::1-11: pSB6A1-M13genes<br />
:::12-18: pSB3C5-M13genes<br />
::-Epic failure<br />
::-All pSB6A1 backbones are empty<br />
::-Half of pSB3C5 backbones were empty. The others contained random inserts (1700 OR 3500). We don’t know what these inserts are. 1700 band is likely the digestion product that appears when we digest M13genes<br />
*Question: Can we use empty vectors from these mini-preps as ligation vectors?<br />
::-Digest pSB6A1 mini-prep with…. (see if both cut sites were retained during re-ligation)<br />
:::no enzyme<br />
:::EcoRI<br />
:::PstI<br />
:::EcoRI + PstI<br />
'''[[File:UCB-Phage Delivery-140629-02.JPG]]'''<br />
::lanes....<br />
:::1-2: last two samples of pSB3C5-M13genes from above gel<br />
:::3. Uncut<br />
:::4. Cut with EcoRI<br />
:::5. Cut with PstI<br />
:::6. Cut with EcoRI and PstI<br />
:-Appears that two backbones are ligated together<br />
<br />
'''6/30'''<br />
*Made chemically competent ER2738 cells that contain Litmus28i DNA<br />
*To amplify M13genes in order to retry ligation<br />
::-PCR of M13K07 DNA (diluted 1:100) to amplify the M13 genes<br />
::-PCR purify PCR product<br />
::-Run on gel: PCR purification, PCR, noDNAcontrol<br />
:::*PCR and purification showed bands at ~9k; therefore, did not get product<br />
:::*No DNA control was clean<br />
*Made freeze downs of<br />
::pSB3C5-J04450<br />
::pSB6A1-J04450<br />
::‘empty’ pSB3C5<br />
::‘empty’ pSB6A1<br />
<br />
'''7/1'''<br />
*Digestion of PCR product from 6/30 to figure out where mistake is<br />
:-Not really useful<br />
'''[[File:UCB-Phage Delivery-140701.JPG]]'''<br />
:Lanes...<br />
:1. PCR pur -> digested with AgeI<br />
:2. PCR pur -> digested with NgoMIV<br />
:3. PCR pur -> digested with PstI<br />
:4. PCR pur -> uncut<br />
:5. PCR pur -> digested with dpnI<br />
:6. Uncut plasmid DNA<br />
*Talked to Mary: She says we were adding to much DNA<br />
::-PCR again. Used 1:100 dilution of 1:100 diluted M13K07 DNA. (aka 1:10000 dilution)<br />
'''[[File:UCB-Phage Delivery-140701-02.JPG]]'''<br />
*Received a beautiful band at 6k bp<br />
:lanes<br />
:1. 1:100 dilution<br />
:2. 1:1000 dilution<br />
:3. 1:10 000 dilution<br />
:4. No DNA control<br />
<br />
'''7/2'''<br />
*To biobrick M13 genes (pSB6A1-M13genes and pSB3C5-M13genes)<br />
::-PCR purified 7/1 PCR product (Primers = Gem012)<br />
::-Digested with EcoRI-HF and PstI-HF<br />
::-Ligation to<br />
::::pSBA61 (digested and gel extracted)<br />
::::pSB3C5 (digested and gel extracted)<br />
<br />
'''7/3'''<br />
*Transformation<br />
::-pSB6A1-M13genes into 5alpha cells and ER2738 with pSB1C3-M13ori<br />
::-pSB3C5-M13genes into 5alpha cells and ER2738 with Litmus28i<br />
::-Transformed into cells containing a phagemid in order to skip some steps<br />
<br />
'''7/3-7/7 Vacation!'''<br />
<br />
==Week 10==<br />
<br />
'''7/7'''<br />
*Made O/N cultures of 7/3 transformation colonies<br />
::-Transformation results were not recorded until 7/8 (see below)<br />
<br />
'''7/8'''<br />
*Transformation results ( CC = Chemically comp cells, ER = ER2738)<br />
::-No growth on No DNA controls<br />
:::ER-Litmus28i CC on AMP-Chlor-Tet<br />
:::ER-phagemid1C3 CC on AMP-Chlor-Tet <br />
:::5alpha CC on Amp <br />
:::5alpha CC on Chlor <br />
::-Lots of red colonies on positive controls, no white colonies<br />
:::ER-Litmus28i CC + pSB3C5 on AMP-Chlor-Tet<br />
:::ER-phagemid1C3 CC + pSB6A1 on AMP-Chlor-Tet<br />
::-Lots of white colonies on sample plates, some red colonies<br />
:::ER-Lit CC + M13genes-pSB3C5 on AMP-Chlor-Tet<br />
:::ER-phagemid1C3 CC + M13genes-pSB6A1 on AMP-Chlor-Tet<br />
:::5alpha CC + M13genes-pSB3C5 on Chlor<br />
:::5alpha CC + M13genes-pSB6A1 on Amp<br />
*Overnights from 7/7 look healthy<br />
::-Renamed O/N to have numbers instead of long names<br />
{| class = "wikitable"<br />
|-<br />
! Sample #<br />
! Cells<br />
! DNA<br />
<br />
|-<br />
| 1-5<br />
| ER-Lit<br />
| M13genes-pSB3C5<br />
<br />
|-<br />
| 6-10<br />
| ER-phagemid1C3<br />
| M13genes-pSB6A1<br />
<br />
|-<br />
| 11-15<br />
| 5alpha<br />
| M13genes-pSB3C5<br />
<br />
|-<br />
| 16-20<br />
| 5alpha<br />
| M13genes-pSB6A1<br />
<br />
|-<br />
|}<br />
::-Mini-prepped DNA<br />
::-Digest the mini-preps with EcoRI-HF and PstI-HF<br />
::-Run digestions on gel to check sizes<br />
'''[[File:UCB-Phage Delivery-140708.JPG]]'''<br />
<br />
:lanes<br />
::top-left: pSB3C5-M13genes in ER2738 with Litmus28i<br />
::top-right: pSB6A1-M13genes in ER2738 with pSB1C3-1C3<br />
::bottom-left: pSB3C5-M13genes in 5alpha<br />
::bottom-right: pSB6A1-M13genes in 5alpha<br />
:None are correct<br />
*Primers Gem013 came in. Resuspend and diluted primers<br />
::-O/N of pSB3C5 to use for PCR tomorrow<br />
<br />
'''7/9'''<br />
*Wanted to make Litmus28i biobrick compatible for use as a phagemid backbone for us and other iGEM teams<br />
::-Mini-prepped O/N of pSB3C5<br />
::-PCR of pSB3C5 to amplify J04450 with Litmus28i compatible cut sites<br />
:::Primers: Gem013<br />
::::*At the 3’ end, these primers are the same as VF2 and VR so will bind the region flanking J04450. This conserves the terminators that exist between the biobrick prefix and VF2 on one side and those between the biobrick suffix and VR on the other side. Therefore, we are amplifying, VF2 priming site, terminators, J04450, terminators, and VR priming site<br />
::::*At the 5’ end, these primers contain unique restriction sites found in the Litmus28i MCS<br />
*PCR of M13 genes…. again (did 4 samples)<br />
::-Used the 1:10000 dilutions<br />
<br />
'''7/10'''<br />
*Note: The following two projects were done in parallel when possible<br />
*To make Litmus28i biobrick compatible<br />
::-Ran gel of PCR from 7/9 (J04450 amplification with Gem013)<br />
'''[[File:UCB-Phage Delivery-140710.JPG]]'''<br />
<br />
:1-4: PCRs of M13genes<br />
:5. No DNA control for M13genes PCR<br />
:6. PCR of pSB3C5 backbone<br />
:7. No DNA control for pSB3C5 backbone<br />
::-PRC purified the PCR product<br />
::-Digestion #1<br />
:::Restriction enzymes had the same buffer conditions but different activation temperatures so we had to do a 2 part digestion<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Restriction enzymes<br />
! Notes<br />
<br />
|-<br />
| J04450<br />
| Sac1<br />
| PCR purified<br />
<br />
|-<br />
| Litmus 28i<br />
| Sac1<br />
| From NEB tube<br />
<br />
|-<br />
|}<br />
:::*Incubate 1hr at 37 C<br />
:::*Heat inactivated 20 minutes at 80C<br />
::-Digestion #2<br />
:::-Added 1ul BsmI to both samples<br />
:::-Incubated 1hr at 65C<br />
:::-Heat inactivated 20 minutes at 80C<br />
::Ran gel<br />
:::-See gel below<br />
:::-Tried to extract J04450 segment but received very low yield. Since band otherwise looked clean, we decided to redo digestion then skip straight to ligation<br />
::Repeat digestion #1 for J04450<br />
::Repeat digestion #2 for J04450<br />
::Ligation (10hr at 16C, 10 min at 80C)<br />
:::3. Litmus28i + J04450<br />
*To retry ligation to biobrick backbone<br />
::-Ran gel of PCR from 7/9 (M13genes amplified with Gem012)<br />
:::See gel below<br />
::-PCR purified the PCR product<br />
::-Digestion<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Restriction enzymes<br />
! Notes<br />
<br />
|-<br />
| M13 genes<br />
| EcoRI-HF + PstI-HF<br />
| PCR purified<br />
<br />
|-<br />
| 6A1<br />
| EcoRI-HF + PstI-HF<br />
| ‘empty’ pSB6A1<br />
<br />
|-<br />
| 3C5<br />
| EcoRI-HF + PstI-HF<br />
| contains J04450<br />
<br />
|-<br />
|}<br />
:Incubate 1hr at 37C<br />
:Heat inactivated 20 minutes at 80C<br />
::-Ran gel<br />
:::*See gel below<br />
:::*Tried to extract M13genes and pSB3C5 segments but received very low yields. Since bands otherwise looked clean, we decided to redo digestion then skip straight to ligation<br />
::-Repeat digestion for M13genes and pSB3C5<br />
::-Ligation (10h at 16C, 10m at 80C)<br />
:::1. pSB3C5 + M13genes<br />
:::2. pSB6A1 + M13genes<br />
*Gel<br />
'''[[File:UCB-Phage Delivery-140710-02.JPG]]'''<br />
:1. pSB1A3<br />
:2. Limtus28i<br />
:3. M13genes<br />
:4. pSB3C5<br />
:5. J04450<br />
<br />
'''7/11'''<br />
*Transformed ligations from 7/10 into 5alpha cells<br />
::1. pSB3C5 + M13genes<br />
::2. pSB6A1 + M13genes<br />
::3. Litmus28i + J04450<br />
<br />
<br />
'''7/12'''<br />
*Results of 7/11 transformation<br />
::-No growth on no DNA control (Amp or Chlor)<br />
::-Lots of growth on Lit-J04450 -> some red-> colonies are too close together to prick individual colonies<br />
:::-It’s possible that these red colonies are satellites =(<br />
:::-Swiped some and plated on new Amp plate (restreak)<br />
::-Lawn of positive control (Litmus 28i) on Amp<br />
::-Many colonies for L1 (pSB3C5-M13genes) and L2 (pSB6A1-M13genes)<br />
:::-Due to high number of red colonies on Lit-J04450 plate, we assumed that most of these colonies contain empty vector<br />
:::-Did not make O/N<br />
*Ligated M13 genes to pSB6A1<br />
::-Used the remained of our digested M13genes.<br />
::-Both digestions from 7/10<br />
*Infection Experiment<br />
::-had 3 cell stocks (each taken from a different colony the night before)<br />
::-Tested Litmus 28i, Tandem phagemid 1C3, and double infection<br />
:::*Therefore, there were 9 flasks total.<br />
::-negative control: Streaked parent cells (non-infected) onto Chlor, Amp, and kan<br />
::-Included double infection plates for Litmus 28i and phagemid 1C3<br />
<br />
==Week 11==<br />
<br />
'''7/13'''<br />
*Transformed 7/12 ligation (pSB6A1-M13genes)<br />
*Litmus28i-J04450<br />
::-All growth from 7/12 restreak was white<br />
::-Pricked some red colonies for liquid O/N-will hopefully see red tomorrow<br />
::-Pricked a few red colonies and put into 200ul H2O (a few colonies per tube- 2 tubes total).<br />
:::*Plated 150ul onto Amp plates<br />
::-Put original plate into incubator to hopefully get bigger colonies<br />
<br />
'''7/14'''<br />
*Finished TWIV ppt<br />
*Set up 2 liquid culture of red Litmus 28i colonies (Litmus28i-J04450)-- slow growth <br />
::-Mini-prepped red Litmus 28i ‘O/N’ from earlier in day<br />
::-Digested samples—to check for insert and correct cut sites<br />
*Made O/Ns of pSB6A1-M13genes colonies from 7/13 transformation<br />
<br />
<br />
'''7/15'''<br />
*Ran gel of Litmus28i-J04450 samples<br />
'''[[File:UCB-Phage Delivery-140715.JPG]]'''<br />
:1. J04450-Litmus28ibb #1 (EcoRI+PstI)<br />
:2. J04450-Litmus28ibb #1 (uncut)<br />
:3. J04450-Litmus28ibb #2 (EcoRI+PstI)<br />
:4. J04450-Litmus28ibb #2 (EcoRI+PstI)<br />
::*Accidentally added restriction enzymes<br />
:5. Litmus28i (EcoRI+PstI)<br />
::*Only has PstI site<br />
<br />
::-Verifies that Litmus28i-J00450 had correct cut sites<br />
::-From this point on, biobricked Litmus28i is called Litmus28ibb<br />
*Check 7/14 O/Ns for pSB6A1-M13genes<br />
::-Mini-prepped liquid cultures<br />
::-Digested with EcoRI and PstI<br />
::::Received bold bands of just under 4000bp. Some lanes had a very feint band ~330bp. None were the correct size<br />
*Transformed Litmus28ibb-J04450 into ER2738 cells<br />
*Remake phage packaging<br />
::-pSB1C3-M13ori(New)<br />
::-pSB1C3-M13ori(Old)<br />
<br />
'''7/16'''<br />
*Results of 7/15 transformation of Litmus28i into 6/30 ER2738 cells<br />
::-lawn on no DNA control-- most likely contaminated cells<br />
::::Streaked chem comp 5alpha and ER onto Amp and Amp+Tet plates to determine if problem is with cells or plates<br />
::-O/N culture of ER to make new chem comp cells<br />
<br />
'''7/17'''<br />
*Finished phage isolation<br />
::Note:<br />
:::*pSB1C3-M13ori (New): M13ori and plasmid ori are convergent<br />
:::*pSB1C3-M13ori (Old): M13ori and plasmid ori are tandem<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! 269nm<br />
! 320nm<br />
! [] phage/mL<br />
<br />
|-<br />
| pSB1C3-M13ori (New)<br />
| 0.386<br />
| 0.034<br />
| 8.126 E12<br />
<br />
|-<br />
| pSB1C3-M13ori (New)<br />
| 0.419<br />
| 0.036<br />
| 8.842 E12<br />
<br />
|-<br />
| pSB1C3-M13ori (Old)<br />
| 0.283<br />
| 0.025<br />
| 5.956 E12<br />
<br />
|-<br />
| pSB1C3-M13ori (Old)<br />
| 0.326<br />
| 0.024<br />
| 6.972 E12<br />
<br />
|-<br />
|}<br />
*Infection to compare the new and the old pSB1C3-M13ori<br />
::-Prepped cells for infection<br />
::-Infected cells with pSB1C3-M13ori (New) or pSB1C3-M13ori (New)<br />
::-and plated on dilutions of 1:100, 1:1000, and 1:10000 on Chlor and kan<br />
::-Also plated 1:1000 dilution on lowered Chlor concentration (34ug/mL)<br />
*Sent Litmus28ibb-J04450 for sequencing<br />
<br />
'''7/18'''<br />
*Results from contamination test<br />
::-5alpha on Amp = no growth<br />
::-5alpha on Amp+Tet = colonies<br />
::-ER on Amp = lawn<br />
::-ER on Amp+Tet = lawn<br />
*Made new chemically competent ER2738<br />
*Transformed Litmus28ibb-J04450 into ER2738 made on 3/30 and ER2738 made on 7/18<br />
::-Had two samples of Litmus28ibb-J04450 and two cell stocks, so 4 samples total<br />
<br />
'''7/19'''<br />
*Results of 7/17 infection<br />
::-pSB1C3-M13ori(Old) (tandem-when plasmid ori and M13ori point in the same direction) packages better than pSB1C3-M13ori(New) (when ori and M13ori are convergent), implying that directionality matters.<br />
'''[[File:UCB-phage lab7-19-141012.jpg]]'''<br />
<br />
==Week 12==<br />
<br />
'''7/20'''<br />
*Freeze downs<br />
:-Litmus28i-J04450 Litmus 28i is now biobrick compatible<br />
<br />
'''7/21'''<br />
*Send samples for sequencing<br />
::-pSB1C3-M13ori(Old)<br />
::-pSB1C3-M13ori(New)<br />
*To ligate M13ori to a kanamycin backbone (pSB1K3)<br />
::-Resuspended pSB1K3-J04450 from distribution kit (6B on plate 4)<br />
:::*Contains J04450 as insert (full RFP construct)<br />
::-Transform pSB1K3 into 5alpha cells<br />
<br />
'''7/22'''<br />
*Pricked colony from pSB1K3-J04450 transformation<br />
::-Mini-prepped DNA to get a supply of DNA<br />
*To put M13ori (M13 phackaging signal) onto Kanamycin resistance o we can test packaging ratios with M13K07 on the same antibiotic<br />
::-Digested pSB1K3 with EcoRI-HF and XbaI<br />
::-Digested pSB1C3-M13ori (Old) with EcoRI-HF and SpeI-HF<br />
:::*DNA was not sufficiently cut. Too much DNA? Problem with enzyme (SpeI-HF)?<br />
'''[[File:UCB-Phage Delivery-140722.JPG]]'''<br />
:1. pSB1K3<br />
:2. M13ori<br />
<br />
'''7/23'''<br />
*Sequencing samples from 7/21 were lost in the mail. Resent samples<br />
*Yet another attempt to biobrick M13genes<br />
::1. using primers that would amplify genes and M13 ori parts from M13K07<br />
::2. using primers that would amplify genes, M13 ori parts, and plasmid ori from M13K07<br />
'''[[File:UCB-Phage Delivery-140723.JPG]]'''<br />
:1. Amplified only the M13genes and M13ori<br />
:2. No DNA control for 1<br />
:3. Amplified M13genes, M13ori, and plasmid ori<br />
:4. No DNA control for 3<br />
::-When run on a gel, samples were clean with only one band at around 7kb. No contamination in no DNA controls<br />
*Digestions<br />
::-pSB1K3 with EcoRI-HF and PstI-HF to check for correct insert<br />
::-pSB1C3-M13ori (Old and new) with EcoRI-HF and SpeI-HF to test for efficient cutting with different stock of restriction enzyme<br />
'''[[File:UCB-Phage Delivery-140723-02.JPG]]'''<br />
:1-3: pSB1K3<br />
:4. pSB1C3-M13ori (Old)<br />
:5. pSB1C3-M13ori (New)<br />
*Still had inefficient cutting. Tested M13ori next to Litmus28i to disern the problem<br />
:*Uncut<br />
:*Cut once with (E, X, S, or P)<br />
:*Cut twice with (E+S or E+P)<br />
'''[[File:UCB-Phage Delivery-140723-03.JPG]]'''<br />
:1. M13ori uncut<br />
:2. M13ori Ecori-HF<br />
:3. M13ori SpeI-HF<br />
:4. M13ori XbaI<br />
:5. M13ori PstI-HF<br />
:6. M13ori EcoRI-HF + SpeI-HF<br />
:7. M13ori EcoRI-HF + PstI-HF<br />
:8. Litmus28i uncut<br />
:9. Litmus28i EcoRI-HF<br />
:10. Litmus28i SpeI-HF<br />
:11. Litmus28i XbaI<br />
:12. Litmus28i PstI-HF<br />
:13. Litmus28i EcoRI-HF + SpeI-HF<br />
:14. Litmus28i EcoRI-HF + PstI-HF<br />
*Only partial digest with only SpeI-HF for both. Complete digestion with all others, including E+S<br />
<br />
'''7/24'''<br />
*To Biobrick M13genes using pSB6A1<br />
::-PCR purified M13 genes (did both samples at the same time)<br />
::-Digested PCR purification and pSB6A1 with EcoRI-HF and PstI-HF<br />
'''[[File:UCB-Phage Delivery-140724.JPG]]'''<br />
*Gel extracted M13genes<br />
<br />
:1. M13genes<br />
:2. pSB6A1<br />
:-Bands of pSB6A1 were too light to gel extract<br />
*To swap the kanamycin resistance marker on M13K07 with ampicillin resistance<br />
::-PCR amplified AmpR from pSB6A1<br />
*made O/N culture of pSB6A1 from freeze down to mini-prep<br />
::-Also streaked cells onto plate<br />
<br />
'''7/25'''<br />
*To change resistance marker on M13K07<br />
:1. Biobrick method<br />
::*Tried to mini-prep O/N but pellet (after liquid culture was spun) was not red even after 16+ hours. Set up O/N from plate colonies<br />
:2. Swap only resistance method<br />
::*PCR purified 7/24 PCR<br />
::*Ran product on gel-> band of correct size<br />
'''[[File:UCB-Phage Delivery-140725.JPG]]'''<br />
<br />
:1. PCR product<br />
:2. PCR product-> PCR purified<br />
:3. No DNA control<br />
::-Digested the PCR purification products of M13genes+ori and AmpR with AgeI-HF and NotI-HF<br />
::*Bands on previous gel looked clean enough for both so did not gel extract<br />
::-Overnight ligation<br />
*Set up O/Ns of pSB1K3 colonies<br />
<br />
'''7/26'''<br />
*Transform<br />
::-pSB6A1+M13genes<br />
*To biobrick M13genes<br />
::-Digestion<br />
:::*pSB6A1 with EcoRI and PstI<br />
'''[[File:UCB-Phage Delivery-140726.JPG]]'''<br />
:Gel extracted backbone (band ~4000bp)<br />
*Ligations (10hrs 16C, 10min at 80C)<br />
::-pSB6A1+M13genes<br />
<br />
==Week 13==<br />
<br />
'''7/27'''<br />
*Transformation results from 7/26<br />
::-No growth for no DNA controls ''on Amp''<br />
::-No growth for M13 genes onto pSB6A1 ''on Amp''<br />
*Set up O/Ns of 5alpha and ER2738 to make competent cell tomorrow<br />
*Made freeze down of pSB1K3<br />
*Plated ER competent cells from 5/15 and 6/16 Onto Chlor plates to check for contamination<br />
<br />
'''7/28'''<br />
*No growth of ER2738 on Chlor (either sample)<br />
::-ER is not contaminated with ChlorR<br />
*Make new 5alpha chem comp cells <br />
*Transformations<br />
::-pSB6A1-M13genes<br />
::-positive control for Amp (pSB6A1)<br />
::-No DNA control<br />
<br />
'''7/29'''<br />
*Set up O/Ns of pSB6A1-M13genes<br />
::-Plated sample on reduced Amp and regular Tet<br />
::-Later in day…<br />
::-Mini-prepped O/Ns<br />
::-Digested and ran on a gel to check for insert sizes<br />
'''[[File:UCB-Phage Delivery-140729.JPG]]'''<br />
:*All but the 4th lane with 3 bands look correct<br />
<br />
'''7/30'''<br />
*Lawn on last night’s plating-> replate<br />
*Digest pSB3C5 and pSB6A1-M13genes with E and P to move M13genes to a Chlor backbone<br />
'''[[File:UCB-Phage Delivery-140730.JPG]]'''<br />
::-Extracted bands<br />
::-Ligations<br />
:::*pSB3C5 + M13genes<br />
*Sent samples for sequencing<br />
**M13 genes on pSB6A1 VF2<br />
**M13 genes on pSB6A1 VR<br />
<br />
'''Test experiment for high school kids coming to lab (7/29-7/31)'''<br />
*General idea: Have 2 strands of DNA, one has an EcoRI site while the other contains a SNP, abolishing the cut sight. We pretend that one is pathogenic (the one that is not cut) and tells kids to figure out which one is which <br />
*PCR with Dream-Taq<br />
::-Did not have special fastdigest enzyme<br />
::-Hypothesized that green dye will interfere with enzyme effectivity<br />
*PCR with Dream-Taq (no green dye)<br />
::-Digest with EcoRI fastdigest-> did not cut<br />
*Redid experiment several times<br />
::-Should digest with XbaI<br />
::-PCR purified PCR products<br />
<br />
'''7/31'''<br />
*Spent the morning with Heritage High School<br />
*Made phage<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Phage<br />
! A269<br />
! A320<br />
! genome size<br />
! [ ]<br />
<br />
|-<br />
| Litmus28ibb-J04450 (1)<br />
| 0.823<br />
| 0.197<br />
| <br />
| 9.35 x10^12<br />
<br />
|-<br />
| Litmus28ibb-J04450 (2)<br />
| 0.940<br />
| 0.168<br />
| <br />
| 1.14 x10^13<br />
<br />
|-<br />
|}<br />
<br />
'''8/2'''<br />
*Digestion<br />
::-pSB6A1-M13genes (E+P)<br />
::-pSB3C5 (E+P)<br />
<br />
==Week 14==<br />
<br />
'''8/3'''<br />
*Another attempt to clone M13genes onto pSB3C5<br />
::-Run 8/2 digestions on gel<br />
'''[[File:UCB-Phage Delivery-140803.JPG]]'''<br />
:1. M13genes (E+P)<br />
:2. pSB3C5 (E+P)<br />
::-Gel extracted <br />
:::*M13genes away from pSB6A1 backbone<br />
:::*pSB3C5 backbone away from J04450<br />
::-Ligations<br />
:::*pSB3C5 + M13genes<br />
:::*pSB3C5 + no insert<br />
::-Transformed ligations into ER2738<br />
<br />
'''8/5'''<br />
*Transformation of pSB3C5+M13genes failed<br />
*Made phage<br />
::-Fd-CAT DNA packaged with Fd-CAT<br />
::-phagemid 1C3 packaged with M13K07<br />
::-amilCP on pSB1C3 packaged with M13K07<br />
::-Litmus28ibb-J04450 packaged with M13K07<br />
<br />
'''8/6'''<br />
*Isolated phage<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Phage<br />
! [ ]<br />
<br />
|-<br />
| Fd CAT<br />
| 2.72 x10^12<br />
<br />
|-<br />
| Litmus28ibb-J04450<br />
| 8.37 x10^12<br />
<br />
|-<br />
| pSB1C3-amilCP<br />
| 1.42 x10^13<br />
<br />
|-<br />
| pSB1C3-M13ori<br />
| 4.32 x10^12<br />
<br />
|-<br />
|}<br />
<br />
'''8/6'''<br />
*Started making phage with Fd CAT the should contain Litmus28ibb-RFP<br />
*Infection<br />
::-Infected ER2738 cells with Litmus28ibb-RFP, pSB1C3-M13ori, or pSB1C3-amilCP<br />
::-Plated Litmus samples on 100ug/mL Amp and 50ug/mL Kan<br />
::-Plated other two samples on 34ug/mL Chlor and 25ug/mL Kan<br />
<br />
'''8/7'''<br />
*Isolated phage containing Litmus28ibb-RFP using helper phage<br />
::-M13K07 (as control)<br />
::-Fd-CAT (no phage pellet was observed-worried that there was no phage)<br />
*Went to CSU to have them test M13ori part compared to amilCP. Grew the samples in 5mL O/Ns then diluted to 0.5OD and grew 30 minutes rather than starting from a fresh colony<br />
<br />
'''8/8'''<br />
*At CSU<br />
::-Finished phage protocol (Test packaging of M13ori part)<br />
::-Only used 20mL infection samples<br />
::-grew to 0.55 OD<br />
::-When making phage (after 14 hour incubation), there was very little growth<br />
::-Plated non-diluted and diluted 1:1000 of M13ori and amilCP sample<br />
*Infected cells using phage isolated 8/7. Plated at dilutions of 1:100 and 1:100k<br />
*Want to test progeny Fd-CAT phage (made 8/7) for infectability<br />
::-Start phage isolation prodocol using Fd-CAT phage from 8/7 do deliver the helper phagemid<br />
:::*Phagemid:<br />
::::*None: will make Fd-CAT phage packaing Fd-CAT phagemid<br />
::::*Litmus28ibb-J04450: make Fd-CAT phage packaging Litmus28ibb-J04450 phagemid<br />
*Remake phage (Fd-CAT) using fresh stock from Mike<br />
<br />
<br />
'''8/9'''<br />
*Results of 8/8 infection<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Helper Phagemid<br />
! phagemid<br />
! Selection<br />
! 1:1000 dilution<br />
! 1:00k dilution<br />
<br />
|-<br />
| M13K07<br />
| Litmus28ibb-RFP<br />
| Kan+Tet<br />
| 400-500 (some red)<br />
| 61 white; 20 red<br />
<br />
|-<br />
| same<br />
| same<br />
| Amp+Tet<br />
| lawn<br />
| 2<br />
<br />
|-<br />
| Fd-CAT<br />
| Litmus28ibb-RFP<br />
| Chlor+Tet<br />
| 0<br />
| 0<br />
<br />
|-<br />
| same<br />
| same<br />
| Amp+Tet<br />
| 10<br />
| 707<br />
<br />
|-<br />
|}<br />
*NOTE: The same sample was plated on two different plates. For example, the sample using M13K07 as the helper phage was plated on Kan+Tet and Amp+Tet<br />
*Mini-prep<br />
::-pSC3C5-J04450 -- very little growth, low DNA yield<br />
::-Fd-CAT infected cells<br />
:::*Digest and run on a gel to verify presence of a band- there was a very feint band<br />
*Finished isolating phage…. messed up and used 0.8MgCl2, 0.2M NaCl instead of PEG during precipitation step. Also used the wrong phage<br />
::-Infected ER2738 cells anyway…. no growth by 8/12<br />
<br />
==Week 15==<br />
<br />
'''8/10'''<br />
*Are the progeny phage from 8/6 (original progeny from first Fd-Tet application) viable/ able to reproduce?<br />
::-Used these progeny to make phage that should amplify Fd-CAT phage containing either the Fd-CAT or Litmus28ibb-J04450 phagemid<br />
<br />
'''8/11'''<br />
*Finished isolating phage<br />
{| class = "wikitable"<br />
|-<br />
! Helper Phagemid<br />
! Phagemid<br />
! A269<br />
! A320<br />
! genome size<br />
! [ ]<br />
<br />
|-<br />
| Fd-CAT<br />
| Litmus28ibb (1)<br />
| 0.029<br />
| 0.013<br />
| 4080<br />
| 2.35 x10^11<br />
<br />
|-<br />
| Fd-CAT<br />
| Ltimus28ibb (2)<br />
| 0.033<br />
| 0.015<br />
| 4080<br />
| 2.65 x10^11<br />
<br />
|-<br />
| Fd-CAT<br />
| Fd-CAT (1)<br />
| 0.326<br />
| 0.082<br />
| 7775<br />
| 1.88 x10^12<br />
<br />
|-<br />
| Fd-CAT<br />
| Fd-CAT (2)<br />
| 0.320<br />
| 0.079<br />
| 7775<br />
| 1.86 x10^12<br />
<br />
|-<br />
| M13g6A1<br />
| M13g6A1 (1)<br />
| 0.322<br />
| 0.148<br />
| 10,029<br />
| 1.04 x10^12<br />
<br />
|-<br />
| M13g6A1<br />
| M13g6A1 (2)<br />
| 0.503<br />
| 0.130<br />
| 10,029<br />
| 2.23 x10^12<br />
<br />
|-<br />
|}<br />
<br />
'''8/13'''<br />
*Infected ER2738 with Fd-CAT packaging Litmus28ibb-J04450<br />
<br />
'''8/16'''<br />
*Results from Infection on 8/11<br />
{| class = "wikitable"<br />
|-<br />
! Helper Phagemid<br />
! Phagemid<br />
! 1:1<br />
! 1:10<br />
! 1:100<br />
! 1:1000<br />
<br />
|-<br />
| M13g6A1<br />
| M13g6A1<br />
| almost lawn<br />
| 728<br />
| 287<br />
| 55<br />
<br />
|-<br />
| Fd-CAT<br />
| Fd-CAT<br />
| lawn<br />
| almost lawn<br />
| 1260<br />
| 640<br />
<br />
|-<br />
| Fd-CAT<br />
| Litmus28ibb-J04450<br />
| lawn<br />
| almost lawn<br />
| 2004<br />
| 304<br />
<br />
|-<br />
|}<br />
<br />
Results: packaging of Litmus28ibb-J04450 using Fd-CAT helper phagemid from (8/15)<br />
<br />
{| class="wikitable"<br />
|-<br />
!Antibiotic<br />
!1:10 <br />
!1:100 <br />
!1:1k <br />
!1:10k <br />
!1:100k<br />
<br />
|-<br />
|Amp <br />
|Lawn<br />
|Almost lawn <br />
|1025 <br />
|217 <br />
|25<br />
<br />
|-<br />
|Kan <br />
|9 <br />
|2 <br />
|0 <br />
|0 <br />
|0<br />
|}<br />
<br />
==Week 16==<br />
<br />
'''8/19'''<br />
<br />
Sent samples for sequencing<br />
<br />
Litmus28ibb-J04450<br />
<br />
==Week 17==<br />
<br />
'''8/27'''<br />
<br />
Transform pSB1C3-M13ori into ER2738 cells<br />
<br />
'''8/28'''<br />
<br />
Make phage with pSB1C3-M13ori as phagemid and M13g6A1 as helper phagemid<br />
<br />
'''8/29'''<br />
<br />
Finish phage isolation of M13 phage containing pSB1C3-M13ori<br />
<br />
'''9/3'''<br />
<br />
digest pSB6A1-M13g and pSB3C5 with EcoRI and PstI. Did not receive a band for M13genes so did not proceed with ligation to pSB3C5<br />
<br />
==Week 18==<br />
<br />
'''9/4'''<br />
<br />
Mini-prepped new pSB6A1-M13genes (bad quality) and pSB3C5-J04550. Digested both plasmids with EcoRI and PstI and ran products on gel. Did receive a band for the M13genes but it was too feint to gel extract<br />
<br />
'''9/5'''<br />
<br />
Mini-prep pSB6A1-M13genes again. Digest this and pSB3C5 backbone from previous day with EcoRI and PstI. M14g band was barely bright enough to extract so we extracted M13genes and pSB3C5 backbone. Pieces were ligated together overnight at 16C for 10 hours.<br />
<br />
'''9/7'''<br />
<br />
Transformed 9/5 ligation (pSB3C5-M13genes into 5alpha cells). Plated sample onto low Chlor plate (34ug/mL)<br />
<br />
'''9/9'''<br />
<br />
Still no growth for 9/7 transformation of pSB3C5-M13genes ligation PCR M13genes to attempt another ligation into pSB3C5. Diluted DNA to ~1.17ng/ul<br />
<br />
'''9/10'''<br />
<br />
Gel of 9/9 PCR of M13genes looked clean (only ran 3ul of PCR sample on gel). PCR purified the remaining PCR sample. Digested with EcoRI and PstI. Ligated to previously digested pSB3C5.<br />
<br />
==Week 19==<br />
<br />
'''9/11'''<br />
<br />
Transform 9/10 ligation (pSB3C5-M13genes) into 5alpha cells<br />
<br />
'''9/13'''<br />
<br />
Colony PCR of colonies that grew for 9/11 transformation. Looking for pSB3C5-M13genes)<br />
<br />
'''9/15'''<br />
<br />
Gel of colony PCR showed failure. Set up overnight cultures anyway.<br />
<br />
'''9/17'''<br />
<br />
Submitted Part to iGEM Registry<br />
<br />
{| class="wikitable"<br />
|-<br />
|iGEM # <br />
|Description<br />
<br />
|-<br />
|BBa_K1445000 <br />
|M13ori- the packaging signal for the M13 and fd phage<br />
|}<br />
<br />
Mini-prepped overnights from 9/15 and sent samples for sequencing<br />
<br />
Sent Litmus28ibb-J04450 sample for sequencing to sequence the remaining section that was not reached before<br />
<br />
==Week 19==<br />
<br />
'''9/21'''<br />
<br />
Up until this point, very little luck was had with pSB3C5. Retried putting M13genes onto a chloramphenicol backbone, but this time used pSB1C3. Digested M13genes and pSB1C3 with EcoRI and PstI. Gel showed incomplete digestion so did not gel extract.<br />
<br />
'''9/22'''<br />
<br />
Digested pSB1C3-J04450 with EcoRI and PstI then gel extracted the backbone. PCR purified M13genes(PCR product was clean so did not bother to gel extract). Ligated the two pieces together.<br />
<br />
'''9/23'''<br />
<br />
Transform ligations from 9/22 (pSB1C3-M13genes) into 5 alpha cells<br />
<br />
'''9/27'''<br />
<br />
Transform pSB1C3-M13genes into 5alpha cells. Plate onto low Chlor (34ug/mL)<br />
<br />
Later in day.... Set up overnight cultures of colonies<br />
<br />
'''9/28'''<br />
<br />
Mini-prepped O/N cultures from 9/27. Digested samples with EcoRI and PstI. There were no bands of the correct size on the gel.<br />
<br />
the M13 and fd phage</div>Leighlahttp://2014.igem.org/Team:CU-Boulder/Notebook/Phage_TeamTeam:CU-Boulder/Notebook/Phage Team2014-10-17T06:55:10Z<p>Leighla: /* Week 18 */</p>
<hr />
<div>{{Template:UCB-Main}}<br />
{{UCB-NavBar}}<br />
__FORCETOC__<br />
<br />
'''Phage Delivery'''<br />
==Week 1==<br />
'''Notes:''' Unless stated otherwise, all gels contain 2-log ladder<br />
<br />
'''5/9'''<br />
*Obtained BW23115 KanR cells- BW23115 cells that had their native CRISPR-Cas system knocked out by the insertion of a Kanamycin resistance gene<br />
::-Will also be called BW23115 or BW<br />
::-Conjugated BW23115 KanR cells with contain F’ notation (ex. BWF’)<br />
*Obtained ER2738 cells that contain the F’ episome (no changes from NEB sample)<br />
::-Will also be called ER. Assume that all ER samples contain the F’ episome<br />
::-Streaked sample onto LB+Tet (20ug/mL) to select for colonies containing F’ episome<br />
<br />
'''5/10'''<br />
*Did receive colonies from 5/9 selection<br />
<br />
==Week 2==<br />
'''5/12'''<br />
*Need to conjugate BW23115 KanR cells with the F’ episome<br />
::-Set up overnight cultures of ER2738 and BW23115 KanR<br />
::-When mixed, ER2738 will donate it’s F’ episome and BW23115 KanR will receive the F’ episome. F’ episome confers Tetracycline resistance<br />
<br />
'''5/13'''<br />
*Started M13 Amplification: Amplify M13 phage using the M13K07 Helper Phage<br />
::-Let precipitated in NaCl/PEG solution overnight<br />
::-Possible sources of error<br />
:::*Did not sterilize 2.5M NaCl/20% PEG-8000 solution<br />
:::*Added 4-fold PEG solution<br />
::::Compensated by adding more LB<br />
:::*During precipitation, put sample in -20C for 30 minutes before realizing mistake and moving to it to 4C. Sample partially froze<br />
*Conjugated BW23115 with F’ episome<br />
::-Added 1mL BW23115 to 1mL ER2738 overnight culture<br />
::-Incubated at 37C for 30 minutes, shaking<br />
::-Plated on LB+Kan(50ug/mL)+Tet(20ug/mL)<br />
:::*To select for BW cells that took the F’ episome (containing Tet resistance)<br />
<br />
'''5/14'''<br />
*Finished the M13 Amplification<br />
::-Visualized product on UV-vis. There was a tall spike at 269nm indicating that DNA was present. Did not test at 320nm.<br />
*Results of BW23115 Conjugation<br />
::-Many colonies indicating successful conjugation of F’ episome into BW23115<br />
::-Set up overnight to make freeze down tomorrow<br />
*Set up overnight of ER2738 to make chemically competent tomorrow<br />
<br />
'''5/15'''<br />
*Made freeze down of BW23115 KanR F’<br />
::-BW23115 E. coli strain with Kanamycin resistance gene inserted into genome and with F’ episome<br />
*Made chemically competent ER2738 cells<br />
*Transformation of Litmus28i (from NEB) into chemically competent ER2738 cells<br />
::-Added 1ul Litmus28i plasmid to 40ul competent cells<br />
::-Plated on LB + Amp(100ug/mL)<br />
::-Purpose: To make M13 phage that package Litmus28i DNA. Need phagemid (Litmus28i) DNA in infectable cells (cells containing F’ episome) to introduce M13K07 Helper Phage and make phage. <br />
<br />
'''5/16'''<br />
*Results of 5/15 transformation<br />
::-No growth for No DNA control<br />
::-Many colonies for sample<br />
<br />
==Week 3==<br />
'''5/19'''<br />
*M13 Amplification to isolate M13-Litmus28i phage<br />
::-Cells: ER2738 cells containing Litmus28i phagemid<br />
::-Helper Phage: M13K07<br />
::-Not much phage was precipitated<br />
*Set up overnight culture of ER2738 to infect tomorrow<br />
<br />
'''5/20'''<br />
*Infected ER2738 cells with M13-Litmus28i phage<br />
::-Plated only on Ampicillin(100ug/mL) (should have also plated on kanamycin)<br />
::-Infected for 4-5 hours-> should have only infected for 30 minutes maximum. This extra time gives the cells that were infected with M13-M13K07 the time to produced M13-M13K07 phage and reinfect<br />
<br />
'''5/21'''<br />
*Results from M13-Litmus28i infection of ER2738<br />
::-Solid lawn of growth for diluted and non-diluted<br />
::-Also sickly looking growth<br />
*Set up overnights<br />
::-ER2738 cells containing Litmus28i for freeze down<br />
::-BW23115 with F’ episome to make chemically competent cells <br />
::-ER2738 to redo infection<br />
<br />
'''5/22'''<br />
*Tested absorbance of phage produced through M13 amplification on 5/19<br />
::-Low absorbance of 0.018 at 269nm but no detection at wavelength 320nm<br />
::-Decided to redo M13 amplification<br />
*Made chemically competent BW23115 with f-episome<br />
*Made freeze down of ER2738 containing Litmus28i<br />
*Set up overnight of ER2738 containing Litmus28i to redo M13 amplification tomorrow<br />
<br />
'''5/23'''<br />
*Protocol switch to make phage using phagemid<br />
::-“M13 Amplification” protocol should only be used to make more M13-M13K07, not to make M13 phage containing a different phagemid<br />
::-Switched to new protocol (“Use of M13K07 Helper Phage for isolation of single stranded phagemid DNA” by NEB. Made modifications (see our protocols) to isolate phage rather than single-stranded DNA)<br />
::-Making phage….<br />
{| class = "wikitable"<br />
|-<br />
! Helper phage<br />
! Phagemid<br />
! Cells<br />
! Notes<br />
<br />
|-<br />
| M13K07<br />
| None<br />
| ER2738<br />
| Make more M13-M13K07<br />
<br />
|-<br />
| M13K07<br />
| Litmus28i<br />
| ER2738<br />
| Test packaging of Litmus28i<br />
<br />
|-<br />
|}<br />
*Made fresh antibiotics<br />
::-Chloramphenicol (34 ng/mL)<br />
:::*1.44g chloramphenicol into 42mL EtOH<br />
::-Ampicillin (50 ng/mL)<br />
:::*4g ampicillin into 80mL mili-Q H2O<br />
<br />
'''5/24'''<br />
*Isolated phage using new protocol<br />
::-Resuspended pellet in 200ul TBS and 200ul 30% glycerol<br />
::-Measured absorbance with UV-vis<br />
:::*concentration (phage/mL) = 6x10^16 x (A269-A320)/ (#of base pairs in the phage genome)<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! Abs (269nm)<br />
! Abs (320nm)<br />
! Genome size<br />
! Concentration (phage/mL)<br />
<br />
|-<br />
| M13-M13K07<br />
| 0.721<br />
| 0.060<br />
| <br />
| 4.57 x10^12<br />
<br />
|-<br />
| M13-Litmus28i<br />
| 0.250<br />
| 0.028<br />
| 2823<br />
| 4.72 x10^12<br />
<br />
|-<br />
|}<br />
*Infect ER2738 cells with M13-Litmus28i<br />
::-Wanted 1:10 phage:cell ratio. Math….<br />
:::*At 1 OD (e.coli), cell/mL = 5x10^8<br />
:::*5x10^7 phage * (1mL/4.72x10^12 phage) = 0.011ul phage<br />
*Set up overnights<br />
::-ER2738 for infection with M13-Litmus28i<br />
::-BW23115 F’ for infection with M13-Litmus28i to test infectivity of conjugated strain<br />
<br />
==Week 4==<br />
'''5/25'''<br />
*Infect ER and BWF’ cells with M13-Litmus28i<br />
::*Made 5mL culture of ER and BW that was at 1 OD<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! OD<br />
! mL sample for 1OD in 5mL<br />
! mL LB to 5mL<br />
<br />
|-<br />
| ER2738<br />
| 2.5<br />
| 2 mL<br />
| 3 mL<br />
<br />
|-<br />
| BW23115<br />
| 2.0<br />
| 2.5 mL<br />
| 2.5 mL<br />
<br />
|-<br />
|}<br />
::-Based on calculations from 5/24, we needed to add 0.011 ul phage per 1 mL of cells at 1 OD. This equates to 0.055 ul of phage into 5 mL cells; therefore we made a 1:10 dilution so we could add 0.5ul. Unfortunately, the pipet would not take up 0.5ul so we added 0.8ul of M13-Litmus28i phage<br />
::-Grew the cells for 20 minutes at 37C<br />
::-Plated 300ul onto Kanamycin (50ug/mL) and 300ul onto Ampicillin (100ug/mL) for each sample<br />
:::*Incubated overnight at 37C<br />
*'''Note:''' During the production of phage, the phagemid SHOULD be packaged preferentially over the Helper Phagemid but some Helper Phagemid will still be packaged. We plated on Amp to select for cells that were infected with phage containing Phagemid. We plated on Kan to select for cells that were infected with phage containing Helper Phagemid. This allows us to compare the packaging efficiency of Helper Phagemid: Phagemid.<br />
'''5/26'''<br />
*Results from 5/25 infection with M13-Litmus28i<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Result<br />
! Significance<br />
<br />
|-<br />
| ER2738 on Amp<br />
| Lawn<br />
| Litmus28i phagemid was successfully packaged into the M13 phage and is infectable<br />
<br />
|-<br />
| ER2738 on Kan<br />
| 100-200 colonies<br />
| Some M13 helper phage is packaged into the M13 phage but at a much lower rate than Litmus28i<br />
<br />
|-<br />
| BW23115 on Amp<br />
| Lawn<br />
| BW23115 is ‘equally’ infectable by M13 as ER2738<br />
<br />
|-<br />
| BW23115 on Kan<br />
| Lawn<br />
| BW23115 contains Kan resistance in its genome so this tells us nothing<br />
<br />
|-<br />
|}<br />
:*Conclusions:<br />
::-Cells grew on Ampicillin; therefore, Litmus28i phagemid was successfully packaged into M13 phage. <br />
::-For ER2738 samples, there was significant growth on Ampicillin compared to Kanamycin; therefore, Litmus28i phagemid is packaged preferentially over M13K07 Helper Phagemid<br />
::-M13-Litmus28i retains its infectivity of cells containing the F’ episome<br />
:*Because we received lawns, we have to redo the infection and plate less cells so we can calculate the uptake ratio between the phagemid and helper phage based on the number of colonies<br />
*Started 50 mL overnight of K12 ER2738 and BW23115<br />
<br />
'''5/27'''<br />
*Redo the infection done on 5/25<br />
::-Infectable cells: ER2738 and BW23115<br />
:::*Plated non-infected samples of each (non-diluted) to check for contaminants<br />
::-Diluted M13-Litmus28i (1) phage by a factor of 10. Added 5.5ul to each sample<br />
::-Grew samples for 20 minutes at 37C, 250rpm<br />
::-Plated 100ul of onto an Ampicillin (100ug/mL) plate and onto a Kanamycin (50ug/mL) plate. Incubated overnight at 37C.<br />
:::*Dilutions = 1:10; 1:100; and 1:1000<br />
'''5/28'''<br />
*Results from 5/27<br />
::-Controls were as expected<br />
:::*No growth for ER2738 non-infected grown on Amp, ER2738 non-infected grown on Kan, or BW23115 non-infected grown on Amp<br />
:::*Growth for BW23115 non-infected grown on Kan (BW23115 has Kan R in genome)<br />
::-Many colonies were received for all dilutions (1:10, 1:100, and 1:1000) of the following<br />
:::*ER2738 infected and plated on Amp<br />
:::*BW23115 infected and plated on Amp<br />
:::*BW23115 infected and plated on Kan<br />
::-Many (100s to 1000?) colonies grew on 1:10 and 1:100 dilutions of ER2738. 50-100 colonies grew on the 1:1000 dilution of ER2738<br />
:::*Compare this to the 100-200 colonies that grew from 2/25 infection (which was 300ul non-diluted, infected cells)<br />
:::*Reasons for increased yield<br />
::::*Added too much phage?<br />
::::*volume changed between experiment (5mL to 50mL)<br />
::::*Overnight culture may not have been saturated. If still in log phase, the cells would continue to grow<br />
*Made 50mL O/N cultures of ER2738 and BW23115 so we can repeat the infection tomorrow and plate further dilutions starting at 1:1000<br />
::-Carry out infection in 5mL and 50mL to test volume effect?<br />
<br />
'''5/29'''<br />
*Measured OD of overnights<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! OD<br />
! mL to have .1OD in 50mL<br />
! mL to have 1OD in 5mL<br />
<br />
|-<br />
| K12 ER2738<br />
| 3.0<br />
| 1.7<br />
| 1.7<br />
<br />
|-<br />
| BW23115<br />
| 2.9<br />
| 1.7<br />
| 1.7<br />
<br />
|-<br />
|}<br />
<br />
<br />
*Experiment 1: Infect cells using same method as 5/25 (in a 5mL culture)<br />
::-Started with 1OD cells in 5mL<br />
::-Added about 0.7ul (inaccuracies in pipet) of 1:10 diluted M13-Litmus28i phage<br />
::-Incubated (rotating) for 20 minutes<br />
::-Made 1:1,000 and 1:10,000 dilutions<br />
::-Plated 100ul on Ampicillin (100ug/mL) plates and on Kanamycin (50ugmL) plates<br />
:::*Included non-infected samples diluted by 1:1000<br />
::::*This negative control can be used for Experiment 2 since the non-infected parent solution is the same<br />
::-Incubate overnight at 37C<br />
*Experiment 2: Infect cells using protocol from “Eliminating helper phage from phage display”<br />
::-Diluted O/Ns to OD of 0.1 in 50 mL culture<br />
::-Grew samples until of OD of ER2728 = 0.59 and the OD of BW23115 = 0.60<br />
:::*Missed OD of 0.5 mark, but the two samples are close to each other<br />
::-Chilled samples on ice for 30 minutes<br />
::-Warmed in incubator for 35 minutes (should have been 30)<br />
::-Amount of phage. Rather than use 1:1 as mentioned in protocol, we used multiplicity of 1:10 (phage:cell)<br />
:::*Added 3.3ul of 1:10 diluted M13-Litmus28i (1) phage<br />
::::*(On 5/27 we added 5.5ul of diluted phage to 50mL of cells at OD of 1. Our cells were at OD of .6; therefore, 5.5*.6 = 3.3ul)<br />
::-Incubated for 30 minutes at 37C, not shaking<br />
:::*We later change this to shaking<br />
::-Dilutions<br />
:::*1:1,000; 1:5,000; 1:10,000; 1:50,000; 1:100,000; 1:1,000,000<br />
:::*Plated ER2738 and BW23115 on Ampicillin (100ug/mL)<br />
:::*Plated ER2738 on Kanamycin (50ug/mL)<br />
*Experiment 3: Growth Test (for growth curve)<br />
::-We were concerned by the low OD of the Overnights from the last few days. Wanted to be sure that 2.0-3.0 was not still in log phase. Cultures looked saturated but the OD seemed low.<br />
{| class = "wikitable"<br />
|-<br />
! Time<br />
! Elapsed time (min)<br />
! ER2738 (no antibiotic)<br />
! ER2738 (Tetracycline (20ug/mL))<br />
! BW23115 (with F’ episome) no antibiotic<br />
<br />
|-<br />
| 10:08<br />
| 0<br />
| 0.1<br />
| 0.1<br />
| 0.1<br />
<br />
|-<br />
| 11:10<br />
| 62<br />
| 0.24<br />
| 0.21<br />
| 0.21<br />
<br />
|-<br />
| 12:00<br />
| 112<br />
| 0.49<br />
| 0.45<br />
| 0.44<br />
<br />
|-<br />
| 13:00<br />
| 172<br />
| 1.00<br />
| 0.93<br />
| 0.98<br />
<br />
|-<br />
| 14:15<br />
| 217<br />
| 1.29<br />
| 1.21<br />
| 1.31<br />
<br />
|-<br />
| 15:40<br />
| 302<br />
| 2.1<br />
| 1.8<br />
| 2.3<br />
<br />
|-<br />
| 16:47<br />
| 369<br />
| 2.5<br />
| 1.9<br />
| 3.4<br />
<br />
|-<br />
| 18:05<br />
| 447<br />
| 2.6<br />
| 2.2<br />
| 2.5<br />
<br />
|-<br />
| 19:00<br />
| 502<br />
| 2.9<br />
| 2.3<br />
| 2.5<br />
<br />
|-<br />
| 20:00<br />
| 562<br />
| 3.0<br />
| 2.6<br />
| 2.8<br />
<br />
|-<br />
|}<br />
<br />
:::*The time point at 16:47 (369 minutes elapsed) for BW23115 conjugated (without antibiotics) is most likely an error. It has been removed from the growth plot<br />
'''[[File:UCB-Phage Delivery-140529.JPG]]'''<br />
*Other<br />
::-Made Amp and Kan plates (1 sleeve of each)<br />
::-Made 50mL O/N of ER2738 and BW23115F’ in case we need further dilutions<br />
::-Made 5mL O/N of ER2738, BW23115F’, and BW23115 (without F’ episome) to make chemically competent tomorrow<br />
:::*Did not have plate of BW23115 (without F’ episome) so used freeze down. Hoping to get O/N of a picked colony from CRISPR Team tomorrow morning<br />
<br />
'''5/30'''<br />
*Made chemically competent cells of…<br />
::-ER2738<br />
::-BW23115F’ (conjugated with F’ episome)<br />
::-BW23115 (not conjugated- without F’ episome)<br />
:::*Culture started from plate<br />
::-BW23115* not conjugated (without F’ episome)<br />
:::*Culture started from freeze down<br />
*Results from infections<br />
::-Negative Controls (cells were not infected; cells were diluted 1:1000)<br />
'''[[File:UCB-Phage Delivery-140530.JPG]]'''<br />
::-Results from Experiment 1 (5/29)<br />
'''[[File:UCB-Phage Delivery-140530-02.JPG]]'''<br />
::-Results from Experiment 2 (5/29)<br />
'''[[File:UCB-Phage Delivery-140530-03.JPG]]'''<br />
*Math<br />
::-If there are 5.00E+8 cells in 1mL of culture at OD of 1, then in 1mL of culture at OD of 0.59, there are 2.95E+8 cells. In a 50mL culture at OD of 0.59, there are 1.48E+10 cells.<br />
::-We added 3.3ul (0.0033mL) phage at concentration 4.62E+11 phage/mL which amounts to 1.52E+9 total phage<br />
::-Assuming that 1 phage infects 1 bacterium, we can assume that 1.52E+9 bacterial have the potential to be infected in the 50mL culture<br />
::-We plated 100ul of culture at various dilutions. If not diluted, the number of cells that can be potentially infected in 0.1mL equals 1.52E+9/500, or 3.05E+06 cells. We then accounted for the dilutions (for 1:1000 dilution, we divided 3.05E+06 by 1000 to receive 3.05E+03)<br />
::-The following table contains the number of cells with the potential to be infected assuming a 100% infectivity rate by M13 phage and that 1 cell is infected only once.<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Dilution<br />
! Potential infected cells<br />
! Colonies on Amp<br />
! % Potential (Amp)<br />
! Colonies on Kan<br />
! % Potential(Kan)<br />
! Kan:Amp<br />
<br />
|-<br />
| 1:1000<br />
| 1.52E+06<br />
| 476<br />
| 15.62%<br />
| 15<br />
| 0.820%<br />
| 1:19.04<br />
<br />
|-<br />
| 1:5000<br />
| 3.05E+05<br />
| 131<br />
| 21.49%<br />
| 13<br />
| 2.133%<br />
| 1:10.08<br />
<br />
|-<br />
| 1:10000<br />
| 1.52E+05<br />
| 93<br />
| 30.51%<br />
| 5<br />
| 1.640%<br />
| 1:18.60<br />
<br />
|-<br />
| 1:50000<br />
| 3.05E+04<br />
| 17<br />
| 44.29%<br />
| 0<br />
| 0.000%<br />
| <br />
<br />
|-<br />
|}<br />
<br />
*Conclusions from infections<br />
::-Results between and within the three trials are inconsistent. For example, the number of colonies received in experiments 1 and 2 from 5/29 differ greatly. Due to the differences in protocol, variation was expected but not to this extent. <br />
::-Our dilutions did not yield the expected 10 fold (or 5 fold) decrease in growth that was expected.<br />
::-Plates from 5/29 could be plated better to reduce dense areas of growth and growth around the rim.<br />
::-Though the experiment contained many errors we can say that the phagemid (Litmus 28i) is preferentially packaged compared to the helper phage (M13K07) but not to the degree we expected.<br />
::-Could receive increased occurrences of cells containing M13k07 due to infection, phage production, further infection<br />
<br />
==Week 5==<br />
<br />
'''6/2'''<br />
*Tested chemically competent cells through transformation<br />
::-Are cells contaminated?<br />
::-Are cells competent?<br />
*The samples for transformation<br />
{| class = "wikitable"<br />
|-<br />
! #<br />
! Cells (Tube label)<br />
! DNA (Tube label)<br />
! Resistance before transformation<br />
! Resistance after transformation<br />
<br />
|-<br />
| 1<br />
| K12 ER2738 5/20<br />
| p110+RBS (2) 4/23<br />
| Tet<br />
| Tet, Chlor<br />
<br />
|-<br />
| 2<br />
| BW (-f) 5/30<br />
| p110+RBS (2) 4/23<br />
| Kan<br />
| Kan, Chlor<br />
<br />
|-<br />
| 3<br />
| BW f-ep comp 5/22<br />
| p110+RBS (2) 4/23<br />
| Kan, Tet<br />
| Kan, Tet, Chlor<br />
<br />
|-<br />
| 4<br />
| BW (+f) 5/30<br />
| p110+RBS (2) 4/23<br />
| Kan, Tet<br />
| Kan, Tet, Chlor<br />
<br />
|-<br />
| 5<br />
| *BW23115 5/30<br />
| p110+RBS (2) 4/23<br />
| Kan<br />
| Kan, Chlor<br />
<br />
|-<br />
| 2B<br />
| K12 ER2738<br />
| 2B [from dis. kit]<br />
| Tet<br />
| Tet, Chlor<br />
<br />
|-<br />
| 2P<br />
| BW f-ep comp 5/22<br />
| 2P [from dis. kit]<br />
| Kan, Tet<br />
| Kan, Tet, Chlor<br />
<br />
|-<br />
|}<br />
<br />
'''6/3'''<br />
*Results from 6/2 Transformation<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Growth on Chlor<br />
! Growth on Kan<br />
! Growth on Kan+Tet<br />
! Growth on Amp<br />
<br />
|-<br />
| 1 N<br />
| X<br />
| X<br />
| X<br />
| X<br />
<br />
|-<br />
| 2 N<br />
| X<br />
| +<br />
| X<br />
| X<br />
<br />
|-<br />
| 3 N<br />
| X<br />
| +<br />
| +<br />
| X<br />
<br />
|-<br />
| 4 N<br />
| X<br />
| +<br />
| +<br />
| X<br />
<br />
|-<br />
| 5 N<br />
| X<br />
| +<br />
| X<br />
| X<br />
<br />
|-<br />
| 1<br />
| +<br />
| X<br />
| X<br />
| <br />
<br />
|-<br />
| 2<br />
| +<br />
| +<br />
| X<br />
| <br />
<br />
|-<br />
| 3<br />
| +<br />
| +<br />
| +<br />
| <br />
<br />
|-<br />
| 4<br />
| +<br />
| +<br />
| +<br />
| <br />
<br />
|-<br />
| 5<br />
| +<br />
| +<br />
| Many colonies but close to samp. 4<br />
| <br />
<br />
|-<br />
| 2P<br />
| + (~100)<br />
| +<br />
| +<br />
| <br />
<br />
|-<br />
| 1- JW<br />
| +<br />
| X<br />
| X<br />
| <br />
<br />
|-<br />
| 2- JW<br />
| +<br />
| +<br />
| X<br />
| <br />
<br />
|-<br />
| 3- JW<br />
| +<br />
| +<br />
| +<br />
| <br />
<br />
|-<br />
| 4- JW<br />
| +<br />
| +<br />
| +<br />
| <br />
<br />
|-<br />
| 5-JW<br />
| +<br />
| +<br />
| X<br />
| <br />
<br />
|-<br />
| 2B- JW<br />
| + (24)<br />
| X<br />
| X<br />
| <br />
<br />
|-<br />
|}<br />
*The transformations with DNA from the well (B2 and P2) had lower efficiencies than those with DNA from a mini-prep. Most likely this is due to the differences in DNA concentration (p110+RBS (2) 4/23 was at 254.4ng/ul)<br />
*Conclusions<br />
::-None of the competent cells were contaminated<br />
::-All of the competent cells are in fact, competent<br />
*Set up O/N of DH5-alpha cells to make competent tomorrow<br />
<br />
'''6/4'''<br />
*Isolation of single-stranded phagemid DNA using M13K07<br />
::-Added ER2738 colony to 50mL LB<br />
:::*Plate was cold. Next time warm plate before pricking<br />
::::*Best to use freshly grown plate<br />
::-After 4 hours, OD was at 0.02. Waited 45 minutes and OD was at 0.08. Therefore, we infected at OD 0.08<br />
::-Had started another culture when we did not think the first was growing. In incubator for about 1 hour. OD was 0.00. We infected anyway because last time it worked.<br />
::-Let infection proceed for 60 minutes then added 70ul of Kanamycin to be a final concentration of 70ug/mL<br />
*Primers came in to biobrick M13ori (packaging signal on Litmus28i)<br />
::-Resuspended primers and diluted 1:10<br />
<br />
'''6/5'''<br />
*Isolated single-stranded M13K07 DNA<br />
::-Final concentration = 5724 ng/ul (calculated from a 1:10 dilution)<br />
::-For second sample in pair, we resuspended it in TE but did not proceed to DNA extraction teps<br />
::-For the second culture we started 6/4, we resuspended pellet in TBS and glycerol to preserve the M13 phage. Measured absorbances (before glycerol was added)<br />
: #1<br />
::269 => 1.690A<br />
::320 => 0.103A<br />
: #2<br />
::269 => 1.453<br />
::320 => 0.059<br />
*For our first biobrick, we wanted to isolate the M13 origin, a segment ~500bp that allows for packaging into the M13 phage. We tried to achieve this by biobrick assembly and by Gibson Assembly.<br />
::-To biobrick M13 ori through biobrick assembly (the old-school way)<br />
:::PCR on Litmus 28i to amplify/biobrick M13ori<br />
::::*Used primers Gem003 F & R<br />
::::*Diluted Litmus 28i DNA 1:10<br />
:::Digestion of p11+RBS (1) to digest pSB1C3 bb with EcoRI-HF and PstI-HF<br />
:::Ran samples on gel and gel extracted pieces. We recieved very low yields (out of range for nano drop)<br />
::::*M13 ori: 4.0 ng/ul<br />
::::*pSB1C3: 1.8 ng/ul<br />
:::Digested M13 ori fragment despite poor extraction yield with EcoRI-HF and PstI-HF<br />
::::*Used 1.5x as much DNA as instructed based on inaccurate concentration<br />
'''[[File:UCB-Phage Delivery-140605.JPG]]'''<br />
::-Gel extracted red rectangles<br />
::-Ligation<br />
:::*10hr @ 16C, 10min @ 65C, 4ever @ 4C<br />
*To biobrick M13 ori through Gibson Assembly (the cool-kids way)<br />
::-PCR on Litmus 28i<br />
:::*Used primers Gem002 F & R<br />
:::*Diluted Litmus 28i DNA 1:10<br />
::-PCR on pSB1C3 (p11+RBS (1))<br />
:::*Used primers Gem001 F & R<br />
:::*Diluted pSB1C3 DNA 1:3<br />
<br />
'''6/6'''<br />
*Ran gel of PCR products from (6/5). Products will be used for Gibson Assembly<br />
::-Recieved bands for pSB1C3 around 2000bp and M13ori around 500bp<br />
::-No contamination in pSB1C3 PCR negative control<br />
::-Band in M13ori negative control that is the same size as sample. Contaminated by sample?<br />
::-Gel of PCR products #1 and #2 from 6/5<br />
'''[[File:UCB-Phage Delivery-140606.JPG]]'''<br />
:1. pSB1C3 with promoter+RBS as insert. Amplified with Gem001<br />
:2. No DNA control for Gem001<br />
:3. M13ori amplified with Gem002 from Litmus28i<br />
:4. No DNA control for Gem002<br />
*Gibson Assembly<br />
{| class = "wikitable"<br />
|-<br />
! Total Amount of Frag.<br />
! .02-.5pmol<br />
! 10 ul total<br />
<br />
|-<br />
| Gibson Assembly MM (2x)<br />
| 10 ul<br />
| 10<br />
<br />
|-<br />
| Dionized H2O<br />
| 10-x<br />
| <br />
<br />
|-<br />
|}<br />
::-Diluted pSB1C3 and M13ori PCR products 1:10<br />
::-Incubated 60min @ 50C<br />
::-Also used provided pUC16 as positive control<br />
*Transformation<br />
:1. p110+RBS Positive control<br />
:2. No DNA Negative control<br />
:3. Cas9 from distribution kit so we can have more<br />
:4. Thaw and refreeze cells Test competency of comp cells after thawed<br />
:5. Not chem comp cells Negative control for the above<br />
:6. Ligation Product<br />
:7. Gibson product<br />
::*7.2. Gibson product diluted 1:4<br />
:8. Gibson positive control<br />
::*7.2. Gibson positive control diluted 1:4<br />
::-For the Gibson product and the positive control, we transformed 2ul of product and 2ul of 1:4 diluted product. NEB recommends the first if using their competent cells and the second if using cells from other companies. Our cells are from NEB but we made them competent ourselves so we tried both ways<br />
:::*Plated on Chlor at concentrations of 170, 85, and 33 ug/mL<br />
*Primers came in<br />
::-Resuspended and made 1:10 dilutions<br />
<br />
'''6/7'''<br />
*Results from 6/6 transformation<br />
:1. Positive control<br />
::*Lots of growth, ~300-400 on 1:10 dilution<br />
:2. No DNA negative control<br />
::*No growth<br />
:3. Cas9 from distribution kit<br />
::*7 potential colonies (some are close to edges through) on non-diluted<br />
:4. Thawed then refroze cells<br />
::*Looks like (1)<br />
:5. Not chemically competent cells<br />
::*No growth<br />
:6. Ligation product<br />
::*13 potential colonies (some are close to edge)<br />
:7. Gibson Assembly Product<br />
::*170 -> No colonies<br />
::*85 -> No colonies<br />
::*33 -> 3 specks<br />
:7.2. Gibson Assembly product diluted 1:10<br />
::*170 -> 1 speck<br />
::*85 -> 3 colonies<br />
::*33 -> 13 colonies<br />
:8. Gibson positive control<br />
::*No colonies<br />
:8.2. Gibson positive control diluted 1:4<br />
::*No colonies<br />
*Made 6mL O/N cultures<br />
::-4 from (3) cas9 plate<br />
:::See Constitutive CRISPR notebook for more info on these samples<br />
::-7 from (6) Ligation product<br />
::-5 from (7.2 [85]) Diluted Gibson product on 85 ug/mL Chlor<br />
::-8 from (7.2 [33]) Diluted Gibson product on 33 ug/mL Chlor<br />
<br />
==Week 6==<br />
'''6/8'''<br />
*Check colonies for correct constructs.<br />
::-Mini-prepped all 24 O/Ns<br />
:::*Yielded low concentrations for samples 12, 15, 19, and 22<br />
::-Digested all with EcoRI and PstI (10ul reactions)<br />
::-Ran results on gel<br />
:::*All 4 cas9 samples had the expected bands of 2000 and 5000bp<br />
:::*All 7 ligation products have expected bands of 2000 and ~570bp<br />
:::*3 of 5 Gibson assemblies from 85ug/mL Chlor plate had expected bands of 2000 and ~500bp<br />
:::*3 of 8 Gibson assemblies from 33ug/mL Chlor plate had expected bands of 2000 and ~500bp<br />
'''[[File:UCB-Phage Delivery-140608-01.JPG]]'''<br />
<br />
'''[[File:UCB-Phage Delivery-140608-02.JPG]]'''<br />
::1-4: Cas9 from Stanford-Brown team<br />
::5-11: pSB1C3-M13ori cloned through ligation<br />
::12-24: pSB1C3-M13ori cloned through Gibson Assembly<br />
:::*12-16: Grown with 85 ug/mL Chlor<br />
:::*17-24: Grown with 33 ug/mL Chlor<br />
*Conclusions from gel<br />
::-We have cas9 safely in cells<br />
::-Our ligation reactions successfully yielded M13ori on pSB1C3<br />
::-Combined, we had a 46% success rate for the Gibson Assembly in yielding M13ori on pSB1C3<br />
:::*The 4 samples that had the lowest concentration after being mini-prepped (12,15, 19, and 22) correlate with samples that had the correct band pattern<br />
*We selected 4 samples from each (4 total between the two Gibson reactions) type<br />
::-For non-Gibson Assembled samples<br />
:::Plated 25ul on 170ug/mL Chlor<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Our usual method<br />
! Gibson Method<br />
<br />
|-<br />
| 1. Thaw on ice<br />
| 1. Thaw on ice <br />
<br />
|-<br />
| 2. Transfer 40ul cells to tube<br />
| 2. Transfer 50ul cell to tube<br />
<br />
|-<br />
| 3. Add DNA. 1ul for mini-prep OR up to 10ul for ligation<br />
| 3. Add 2ul to NEB cells OR 2ul of 1:4 diluted to other cells<br />
<br />
|-<br />
| 4. Mix by pipet Let sit 30min on ice<br />
| 4. Mix by pipet or flicking Let sit 30 min. on ice<br />
<br />
|-<br />
| 5. Heat shock: 42C for 45s<br />
| 5. Heat shock: 42C for 30s<br />
<br />
|-<br />
| 6. Ice for 5 minutes<br />
| 6. Ice for 2 min.<br />
<br />
|-<br />
| 7. Transfer to culture tube; Add 200ul SOC<br />
| 7. Add 950ul SOC to tube<br />
<br />
|-<br />
| 8. Shake or rotate for 60-120min at 37C<br />
| 8. Shake (250rpm) or rotate for 60 min. at 37C<br />
<br />
|-<br />
| 9.<br />
| 9. Warm plates to 37C<br />
<br />
|-<br />
| 10. Plate 100ul onto selection plate<br />
| 10. Plate 100ul onto plate<br />
<br />
|-<br />
| 11. Incubate O/N @ 37C<br />
| 11. Incubate O/N @ 37C<br />
<br />
|-<br />
|}<br />
::-Added 6mL LB and Chlor at concentration of 170ug/mL to grow O/N<br />
:*For Gibson Assembled samples<br />
::-Plated 25ul onto 170, 85, and 33ug/mL Chlor<br />
::-Samples from 85ng/mL plate<br />
:::Transferred 100ul to new tube, added media, added Chlor at 170ng/mL<br />
::-Samples from 33ng/mL plate<br />
:::Tranfered 100ul to new tubes, added media, added Chlor at 85ng/mL to one and 33ng/mL to the other<br />
:*Tomorrow, may send for sequencing and make freeze downs<br />
<br />
*Transformation<br />
:1. Positive control (p110+RBS diluted 1:10)<br />
:2. Non-diluted Gibson product<br />
:3. Gibson product diluted 1:4<br />
:4. Gibson product diluted 1:10<br />
::-Transformed each sample using our usual method and using the protocol given by Gibson<br />
<br />
::-Due to not have plates ready before transformation, in step 4, the samples sat for about 50 minutes. Then in step 8, they both recovered for about 150 minutes. Though not specified in our protocol, we did warm the plates to 37C. In step 10 for our protocol, since we only added 200ul SOC and wanted to plate on 3 selection plates (see below), we only plated 50ul (except for the positive control). <br />
::-Plated on three concentrations of Chloramphenicol (33 ug/mL, 85 ug/mL, and 170 ug/mL) to determine the differences in yield due to differences in concentration.Obvious hypothesis: more colonies will grow on plates that have a lower concentration of chlor.<br />
<br />
<br />
'''6/9'''<br />
*Made chemically competent 5alpha cells with Dan and Alex from main campus<br />
::-Waiting to hear results on competency<br />
*Will eventually make phage containing CRISPR-Cas9 that targets Kanamycin resistance. M13K07 has Kanamycin resistance so we need to switch the resistance on the M13 genes.<br />
::-PCR on pwp 2.po (plasmid that Sam gave us that contains the zeoR gene adjacent to ori) to amplify zeoR and ori. Zeo is on EM7 promoter<br />
:::-Primers: Gem008 R & R<br />
:::-Anneal temp from NEBuilder: 63.7C<br />
:::-Extension time: 90s<br />
:::-Expected band size on gel: 1300bp<br />
:::-Used phusion polymerase<br />
*PCR on M13K07 DNA to amplify M13 phage genes (also removes majority of M13 ori, all of KanR, and all of p15 ori)<br />
:::-Primers: Gem007 F & R<br />
:::-Anneal temp from NEBuilder: 60.2C<br />
:::-Extension time: 4:30<br />
:::-Expected band size on gel: about 6000bp<br />
:::-Used phusion polymerase<br />
*Freeze downs<br />
::-Note: Phagemid 1C3 was the original name for ‘pSB1C3-M13ori’<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Top Label<br />
! Side label<br />
! What?<br />
<br />
|-<br />
| Phagemid 1C3 6/9<br />
| From Lig<br />
| M13 ori inserted into 1C3 (biobricked); Done through ligation; Contains extra bases as spacer between biobrick prefrix/suffix and part for primer design<br />
<br />
|-<br />
| Phagemid 1C3 6/9<br />
| From Lig<br />
| “ “<br />
<br />
|-<br />
| Phagemid 1C3 6/9<br />
| Gibson<br />
| M13 ori inserted into 1C3 (biobricked); Done through Gibson cloning<br />
<br />
|-<br />
| Phagemid 1C3 6/9<br />
| Gibson<br />
| “ “<br />
<br />
|-<br />
|}<br />
'''6/10'''<br />
*Ran gel of PCRs from 6/9<br />
<br />
'''[[File:UCB-Phage Delivery-140610.JPG]]'''<br />
:1. Amplification of M13 genes from M13K07 (~6000bp)<br />
:2. No DNA control for (1) amplification<br />
:3. Amplification of Zeo resistance gene + plasmid ori (~1300bp)<br />
:4. No DNA control for (3) amplification<br />
*Gibson Assembly of above parts (did not gel extract)<br />
::-Diluted the PCR products 1:10 then added 3ul of M13K07 genes product and 7ul of ZeoR+ori product<br />
::-Incubated at 50C for 60 min.<br />
::-Transformed Gibson Assembly product into 5alpha cells<br />
:::*Used our usual protocol<br />
:::*Add 2ul of DNA<br />
::::-In one sample, diluted DNA 1:4 and in the other, we diluted DNA 1:10<br />
*Started Phage Amplification Protocol<br />
::-ER2738 transformed with Litmus 28i<br />
:::*Grew for ~2.5hr before reaching an OD of 0.04<br />
::-ER2738 transformed with pSB1C3-M13ori (M13ori on pSB1C3)<br />
:::*Grew for ~ 3.5hr before reaching an OD of 0.01, then in the next 1.5 hours, spiked to 0.19<br />
:::*We gave up and went home, and will restart tomorrow<br />
*Analyzed transformation results from 6/8<br />
<br />
'''6/11'''<br />
*Made chemically competent 5alpha cells<br />
*Restarted Phage Amplification Protocol<br />
::-Forgot to add phagemid antibiotic at start of growth. Added phagemid antibiotic when we added phage. Incubated for 90 minutes before adding Kanamycin (to select for cells that were infected by M13K07)<br />
::-Phage is at concentration 4.57x10^12 phage/mL<br />
:::Protocol calls for final concentration of 1 x10^8 phage/mL<br />
::::*(4.57 x10^12)*V = (1 x10^8)(50mL)<br />
::::*V = 0.00109mL<br />
:::Added 1.1ul of phage<br />
*Transformations<br />
{| class="wikitable"<br />
|-<br />
!DNA<br />
!Plate Selection<br />
|-<br />
|No DNA control<br />
|(all)<br />
|-<br />
|Positive control (p110+RBS)<br />
|(C)<br />
|-<br />
|M13 genes + ZeoR ori<br />
|(Z)<br />
|-<br />
|M13 genes +ZeoR ori diluted 1:4<br />
|(z)<br />
|} <br />
<br />
'''6/12'''<br />
*Transformation Results from 6/10 [Took ~36 hours to be clearly visible]<br />
::No DNA control<br />
:::-Amp: 0 <br />
:::-Zeo (50ug/mL): 200 colonies<br />
::M13 genes + ZeoR ori (1:4 dilution) <br />
:::-Zeo (25ug/mL): 300<br />
:::-Zeo (50ug/mL): 200<br />
:::-Zeo (100ug/mL): 100<br />
::M13 genes + ZeoR ori (1:10 dilution) <br />
:::-Zeo (25ug/mL): 300<br />
:::-Zeo (50ug/mL): 150<br />
:::-Zeo (100ug/mL): 150<br />
*Transformation Results for 6/11<br />
::-Positive (p110+RBS) on Chloramphenicol: 500 colonies<br />
::-No DNA on Amp: 0 colonies Zeo (100 ug/mL): specks<br />
::-No apparent growth on any other plate<br />
:::*Realized later that we grew our samples on the wrong plates. Will repeat transformation today<br />
*Transformation #1<br />
::This morning there were no colonies on positive (p110+RBS) coltrol from 6/11 even though we observed fast growth in the past. Without waiting for colonies to appear, we started a control transformation<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Diluted<br />
! Time at 42C<br />
<br />
|-<br />
| p110+RBS<br />
| No<br />
| 45s<br />
<br />
|-<br />
| p110+RBS<br />
| 1:10<br />
| 45s<br />
<br />
|-<br />
| p110+RBS<br />
| 1:10<br />
| 30s<br />
<br />
|-<br />
| p110+RBS<br />
| 1:10<br />
| No timer. ~43s<br />
<br />
|-<br />
| No DNA control<br />
| No<br />
| 45<br />
<br />
|-<br />
|}<br />
*Because ‘No DNA control 6/10’ yielded colonies, we researched Zeocin plates<br />
::-According to Life Technologies (Invitrogen), Zeocin requires low salt medium and a pH of 7.5<br />
::-Low Salt LB Medium (1L)<br />
:::*Ingredients<br />
::::10g Tryptone<br />
::::5g NaCl<br />
::::5g Yeast Extract<br />
:::-Mix ingredients<br />
:::-Adjust pH to 7.5 using NaOH (If go over, use HCl)<br />
:::-Add agar for plates at 15g/L. Autoclave<br />
:::-Thaw Zeocin on ice. Vortex<br />
:::-Add Zeocin to final concentration of 25ug/mL<br />
*Transformation #2<br />
::-Repeat of transformation on 6/11 but this time we will plate on the correct plates<br />
::-Also remade Zeocin plates<br />
*Finished isolation of M13 Litmus phage and M13 pSB1C3-M13ori phage<br />
::*Note: Phagemid 1C3 was the original name for ‘pSB1C3-M13ori’<br />
{| class = "wikitable"<br />
|-<br />
! Phage Sample<br />
! A269<br />
! A320<br />
! Concentration (phage/mL)<br />
<br />
|-<br />
| Litmus phage (1)<br />
| 0.181<br />
| 0.034<br />
| 3.12 x10^12<br />
<br />
|-<br />
| Litmus phage (2)<br />
| 0.227<br />
| 0.047<br />
| 3.83 x10^12<br />
<br />
|-<br />
| Phagemid 1C3 phage (1)<br />
| 0.101<br />
| 0.020<br />
| 1.87 x10^12<br />
<br />
|-<br />
| Phagemid 1C3 phage (2)<br />
| 0.126<br />
| 0.021<br />
| 2.42 x10^12<br />
<br />
|-<br />
|}<br />
::::phage/mL = 6x10^16 x (A269-A320)/ (#of base pairs in the phage genome)<br />
*Set up 50mL O/N of K12 ER2738 (containing f-episome) for infection tomorrow with Litmus phage and pSB1C3-M13ori phage<br />
<br />
<br />
'''6/13'''<br />
*Transformation Results for 6/11<br />
::-Several hundred colonies on Positive Control (p110+RBS) on Chlor<br />
::-No colonies for GA positive control on Amp<br />
::-No colonies for M13ori + ZeoR mistakenly plated on Amp<br />
::-No colonies for No DNA control on Amp<br />
::-100-ish colonies for No DNA control on Zeo<br />
::-100-ish colonies for cas9+AmpR+gRNA mistakenly plated on Zeo<br />
::-These last 3 points suggest/confirm that Zeo plates are no good<br />
*Transformation Results from 6/12 control test<br />
::-Colonies grew is about equal amounts on all plates, including No DNA control<br />
:::Either plates don’t contain Chlor or competent cells are contaminated<br />
::-Streaked competent cells onto new and old Chlor plates<br />
*Infection test of ER2738: Is Litmus preferentially packaged over M13K07 Helper phage? Is pSB1C3-M13ori preferentially packaged?<br />
::-Infect ER2738 with phage produced 6/12<br />
:::*Phage should have packaged Litmus 28i phagemid or pSB1C3-M13ori<br />
:::*Cells infected with phage packaging Litmus 28i will grow on Amp<br />
:::*Cells infected with phage packaging pSB1C3-M13ori will grow on Chlor<br />
:::*Cells infected with phage packaging M13K07 will grow on Kan<br />
::-After we plate, we can count the colonies and calculate a ratio of Litmus28i: M13K07 or pSB1C3-M13ori:M13K07 packaging<br />
<br />
'''6/14'''<br />
*Results of contamination test (streaked competent cells onto new and old Chlor plates)<br />
::-Colonies grew in low amounts on both plates. most likely the cells are contaminated<br />
*Transformation results for 6/12<br />
::-Many colonies for No DNA control on Zeo<br />
:::-Some colonies are turning pinkish-red<br />
::-Many colonies for diluted and non-diluted M13 genes+ZeoR on Zeo<br />
:::-Some colonies are turning pinkish-red<br />
*Could the white colonies be the designed colonies and can we kill the red colonies with Zeocin before killing the white colonies (aka. Use high Zeocin concentrations to select for the correct construct)<br />
::-Selected 1 red colony from No DNA Zeo control plate and 1 white colony from M13genes+ZeoR sample plate<br />
::-Added 100ul H2O then divided amongst 5 culture tubes each (with 5mL of low-salt LB, pH 7.5)<br />
::-Then added Zeocin to a final concentration of: 0, 25, 50, 75, 100ug/mL.<br />
*Results of 6/13 infection<br />
::-Litmus 28i infected cells<br />
:::*on Kan: Individual colonies for 1:10, 1:100, and 1:1000. No colonies on 1:5000<br />
:::*on Amp: lawn for 1:10, 1:100, and near lawn for 1:1000. Single colonies for 1:5000<br />
::-pSB1C3-M13ori infected cells<br />
:::*on Kan: Same as Litmus 28i samples on Kan<br />
:::*on Chlor: Lawn for all dilutions. Must discredit due to recent Chlor contamination<br />
<br />
==Week 7==<br />
<br />
'''6/15'''<br />
*Results of Zeocin experiment on 6/14<br />
::-Healthy growth for both at 0ug/mL Zeo<br />
::-No growth for white colony with any Zeo<br />
::-Good growth for red colony even at 100ug/mL Zeo<br />
::-This suggests that the red colonies are naturally resistant to Zeocin. Also, our plates must not contain active Neocin. We are sure that we are adding enough. Possibly, we add it while the media is too hot or leave the plates at room temperature (decondensing) for too long and this deactivates the antibiotic? Perhaps we are not attaining the correct pH<br />
*In light of recent contamination problems on both Chlor and Zeo, we made new competent cells<br />
::-5alpha<br />
::-BW23115<br />
::-BW23115 conjugated (contains f-episome)<br />
<br />
'''6/16'''<br />
*Made new Chlor antibiotic. Made to Chlor plates<br />
*Contamination Test: Streaked Zeo and new Chlor plates each with<br />
::-Colony from Zeo contaminated plate<br />
::-Colony from Chlor contaminated plate<br />
::-Non-transformed OLD chemically competent cells<br />
::-Non-transformed NEW chemically competent cells<br />
*Redid bacterial infection<br />
::-For both samples, we used ER2738 (not from the same ‘O/N’ (also, not a real O/N))<br />
::-Infected one sample with isolated ‘Litmus28i phage’ and one with ‘pSB1C3-M13ori’<br />
::-Alterations to protocol<br />
:::*Did not started from saturated O/N. Started each with a colony, waited several hours until at OD ~1. We then added these cells to fresh 50mL LB to have an OD of 0.1.<br />
:::*Missed the 30 minute infection mark. Infected for ~45 minutes.<br />
::-Plated on Kan and Amp (Litmus28i sample) or Chlor (pSB1C3-M13ori sample) at dilutions of<br />
:::*1:100, 1:1k, 1:10k, and 1:100k<br />
<br />
'''6/17'''<br />
*Q5 PCR to replace KanR with ZeoR in M13K07<br />
::-Used recommendations<br />
::-Unfortunately, I (Jo) don’t know the difference between tightening and loosening the thermocycler lid; therefore, our M13K07 sample to amplify the M13K07 genes evaporated. But that’s ok, because we were sick of Zeocin anyway and decided mid-PCR to not waste our time with a Gibson Assembly and transformation. Instead we will be using the Old-School method of digestion and ligation because let’s face it, it’s a classic (and Gibson sucks) =)<br />
*Results from infection Test<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Litmus 28i<br />
! 1:100<br />
! 1:1k<br />
! 1:10k<br />
! 1:100k<br />
<br />
|-<br />
| Amp<br />
| ~6000?<br />
| ~2056<br />
| 377<br />
| 36<br />
<br />
|-<br />
| Kan<br />
| 130<br />
| 8<br />
| 3<br />
| 0<br />
<br />
|-<br />
|}<br />
{| class = "wikitable"<br />
|-<br />
! pSB1C3-M13ori<br />
! 1:100<br />
! 1:1k<br />
! 1:10k<br />
! 1:100k<br />
<br />
|-<br />
| Chlor<br />
| 8<br />
| 1<br />
| 0<br />
| 0<br />
<br />
|-<br />
| Kan<br />
| 97<br />
| 9<br />
| 0<br />
| 0<br />
<br />
|-<br />
|}<br />
::-We consider the Litmus 28i to have been a success. The pSB1C3-M13ori …. not so much. Looking back at the Litmus28i and the M13K07, we noticed that the M13ori is facing the opposite direction as the plasmid ori. Ours faces the same direction as the plasmid ori. Because we are working with phagemids that are single stranded, we think that by flipping the M13ori, we may be able to recover functionality. We will also look into other reasons.<br />
*Results from contamination test (6/16)<br />
::-Old Chlor plate (6/12)<br />
:::-Non-transformed chemically competent cells<br />
::::*Old: some colonies<br />
::::*New: No colonies<br />
::-New Chlor plate (6/16)<br />
:::-Colony from<br />
::::*Zeo contaminated plate: No growth<br />
::::*Chlor contaminated plate: Much growth<br />
:::-Non-transformed chemically competent cells<br />
::::*Old: some colonies<br />
::::*New: No growth<br />
::-Zeo (25ug/mL, Low NaCl, pH 7.5) (6/12)<br />
:::-Colony from<br />
::::*Zeo contaminated plate: Much growth<br />
::::*Chlor contaminated plate: No growth<br />
:::-Non-transformed chemically competent cells<br />
::::*Old: some specks<br />
::::*New: some specks<br />
<br />
'''6/18'''<br />
*Ordered primers to…<br />
::-biobrick M13ori (in other direction)<br />
::-biobrick M13K07 genes<br />
'''6/19'''<br />
*Waited for primers<br />
*Set up O/N cultures to test last infection (6/16) for colonies containing pSB1C3-M13ori<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample #<br />
! Selection<br />
! Dilution<br />
! Presumably<br />
! Notes<br />
<br />
|-<br />
| 1-8<br />
| Chlor<br />
| 1:100<br />
| pSB1C3-M13ori<br />
| <br />
<br />
|-<br />
| 9<br />
| Chlor<br />
| 1:1k<br />
| pSB1C3-M13ori<br />
| <br />
<br />
|-<br />
| 10-14<br />
| Chlor<br />
| 1:1k<br />
| pSB1C3-M13ori<br />
| These colonies were not present on plate during initial counting on 6/17<br />
<br />
|-<br />
| 15-16<br />
| Kan<br />
| 1:1k<br />
| M13K07<br />
| <br />
<br />
|-<br />
|}<br />
:::-Selection and dilution refer to the plate. Cells were then grown under selection.<br />
:::-More colonies were seen on all Chlor plates. No new colonies appeared on Kan plates<br />
*Tomorrow, we will mini-prep, digest, and run the samples on a gel to verify gene transfer.<br />
<br />
'''6/20'''<br />
*Primers are in!<br />
*Cloning of pSB1C3-M13ori(New)<br />
::-PCR to amplify M13ori (packaging signal) from Litmus 28i to be in the other direction<br />
:::-Primers (Gem011 F&R) add cut sites to make part biobrick compatible<br />
:::-Was able to gel extract<br />
::::*Band at the same size as sample….. contaminated primers?<br />
:::-Then digested with E and P<br />
::-Digested pSB1C3 plasmid with E and P to linearize backbone<br />
::::*Was able to gel extract<br />
'''[[File:UCB-Phage Delivery-140620.JPG]]'''<br />
<br />
:1. Spill over from 2<br />
:2 and 3. pSB1C3 digested with EcoRI and PstI<br />
::-Insert <100bp so cannot be seen<br />
:4. M13ori<br />
*Ligation of M13ori (packaging signal) into pSB1C3<br />
::10h at 16C<br />
::20m at 80C<br />
::Hold at 4C<br />
<br />
*PCR to amplify M13K07 genes from M13K07 DNA isolated from phage<br />
::-Primers (Gem012 F&R) add cut sites to make part biobrick compatible<br />
::-Very light bands and not band might correlate with 6000pb but not enough resolution on gel to be certain. Bands were too light so did not extract<br />
::-Set up PCR again using Q5 to run O/N<br />
:::*Received only a feint, smudgy band that was too large. We did not bother with extracting the DNA<br />
*Test of 6/19 O/Ns (14 that are presumably pSB1C3-M13ori and 2 that are presumably M13K07)<br />
::-Mini-prepped all O/Ns<br />
::-Digested all with Pst-I (common RE between pSB1C3-M13ori and M13K07)<br />
:::-Even though these are phagemids, we assumed that because they were still in the cell, the plasmids were still double stranded so could be recognized by RE. Our assumption was valid<br />
:::-Results of digestion<br />
::::*1,2,4-14: all had bands that corresponded to pSB1C3-M13ori cut once<br />
::::*3: Slight band at expected size but very feint<br />
::::*15, 16: as expected, they contain bands of ~9000bp, correlating with the M13K07 phagemid. Interestingly, sample 15 also contained a (brighter) band that corresponds with pSB1C3-M13ori. We assume that this was an incident of double infection (chance or did this occur at high frequency?)<br />
'''[[File:UCB-Phage Delivery-140620-02.JPG]]'''<br />
::Check table from 6/19 for more details<br />
:::-1-14: pSB1C3-M13ori<br />
:::-15-16: M13K07 from 1:1k diluted plate<br />
<br />
'''6/21'''<br />
*Needed to transform our pSB1C3-M13ori(New) (6/20) into cells. First, we transformed into 5alpha cells; however, we need to infect these cells later in order to make phage. Our 5alphas are not competent so we repeated the transformation, this time using ER2738 cells which contain the F’ episome, allowing us to infect with the M13 phage.<br />
::-Also transformed sample #1 from 6/20 mini-preps (pSB1C3-M13ori(Old) into ER2738 cells. Do not see any notes about this plasmid being in infectable cells during the initial experiment. Reason for experiment failure???<br />
*Ran 6/20 O/N PCR (to amplify M13K07 genes) on gel (also re-ran the previous sample from the day with more DNA)<br />
::-Still, band looks too big. Brightest band still occurs between 8000 and 9000bp. Lighter band around 7000bp-- might be ~6000pb but too light to tell<br />
*Alternative plan for amplifying M13K07 genes<br />
::-Digest sample with PstI (cuts outside of the target region)<br />
'''[[File:UCB-Phage Delivery-140621.JPG]]'''<br />
::Used sample #16 from 6/20 b/c it is double stranded so will cut<br />
:-PCR amplified the linearized digestion product<br />
:::1. PCR of M13genes<br />
:::2. PCR of M13genes + DMSO<br />
::::Gel extracted pieces boxed in red<br />
::-Used primers Gem012 F&R<br />
::-Gel extracted piece but received very low yield<br />
*Alternative to alternative plan for amplifying M13K07 genes<br />
::-Digested with PstI and AgeI<br />
'''[[File:UCB-Phage Delivery-140621-02.JPG]]'''<br />
:1. Cut with PstI and AgeI<br />
:2. Uncut<br />
:3. Cut with PstI<br />
:4. Just PCR<br />
::-Gel extracted ~6000pb band after double digestion<br />
::-PCR (Phusion)<br />
:::-Digestion (A+P) product<br />
:::-Gel extraction product<br />
:::-No DNA control<br />
<br />
==Week 8==<br />
<br />
'''6/22'''<br />
*Did receive colonies from 6/21 transformations. Selected colonies for overnight<br />
{|class="wikitable"<br />
|-<br />
!number<br />
!colonies<br />
|-<br />
|1-7<br />
|pSB1C3-M13ori(NEW) in 5alpha<br />
|-<br />
|8-14<br />
|pSB1C3-M13ori(NEW) in ER2738<br />
|-<br />
|15-18<br />
|pSB1C3-M13ori(OLD) in ER2738<br />
|-<br />
|19<br />
|p110+RBS in 5alpha as control<br />
|-<br />
|20<br />
|p110+RBS in ER2738 as control<br />
|}<br />
<br />
*Gel of 6/21 O/N PCR<br />
::Digestion (AgeI+PstI) product ? Yielded the three bands that appear with every PCR of M13K07<br />
'''[[File:UCB-Phage Delivery-140622.JPG]]'''<br />
<br />
:1. M13K07-> digested (AgeI + PstI) -> PCR<br />
:2. M13K07-> digested (AgeI+ PstI) -> extraction-> PCR<br />
:3. No DNA control for PCR<br />
::-Gel extraction product ? No bands<br />
::-No DNA control ? No bands<br />
*PCR purified the above PCR (Digestion (AgeI+PstI) product) and PCR from 6/21<br />
*Digested PCR purified sample with EcoRI+PstI and ran gel<br />
'''[[File:UCB-Phage Delivery-140622-02.JPG]]'''<br />
:1. PCR-> PCR purified<br />
:2. Sample after DpnI digest<br />
:3. Sample after digestion with EcoRI and PstI<br />
:4. Samples after digestion with DpnI then EcoRI and PstI<br />
*Ligation of above digestion (M13K07 genes) with pSB1C3<br />
<br />
<br />
'''6/23'''<br />
*Transformed pSB1C3-M13ori(New) (6/22 ligation)<br />
*Check overnights from 6/22 for the correct insert<br />
::-Mini-prep O/Ns<br />
::-Digested with EcoRI and PstI<br />
'''[[File:UCB-Phage Delivery-140622-03.JPG]]'''<br />
:::1-7: pSB1C3-M13ori(New) in 5alpha<br />
:::8-14: pSB1C3-M13ori(New) in ER2738<br />
:::15-18: pSB1C3-M13ori(Old)<br />
::-All were the expected size (though gel wiggled)<br />
::-Sent 4 samples for sequencing (iGEM primers: VF2 and VR)<br />
:::# 4: Divergent (new) phagemid 1C3 transformed into 5alpha<br />
:::# 11, 13: Divergent (new) phagemid 1C3 transformed into ER2738<br />
:::# 16: Convergent (old) phagemid 1C3 transformed into ER2738<br />
<br />
'''6/24'''<br />
*Results of 6/23 transformation (pSB1C3-M13genes) from 6/22 ligation)<br />
::-Received ~100 colonies<br />
::-Set up O/N cultures for 8 of the colonies<br />
::-Time passed…..<br />
::-Mini-prepped the 8 O/N samples mentioned above (yes, it was a long day)<br />
:::*Digested samples (E+P)<br />
*Started phage amplification protocol<br />
::-Amount of phage added = 10.9ul of 1:10 diluted phage (M13 phage (1) from 5/24 (4.575 x10^12 phage/mL)) to a final concentration of 1x10^8 phage/mL in 50mL<br />
::-Managed to get both samples to the 14-28hr incubation<br />
<br />
'''6/25'''<br />
*Checked mini-prep samples from 6/24 (pSB1C3-M13genes)<br />
::Ran the digestion overnight (6/24 to 6/25)<br />
'''[[File:UCB-Phage Delivery-140625.JPG]]'''<br />
::-All contained a band at 2000bp. Mostly empty vector or contained small, light band. Sample 1 had prominent band at ~1250bp.<br />
::-None of samples contained M13genes<br />
*M13K07 is on P15A ori (10-12 copy number) whereas the pSB1C3 ori is on pUC19 (500-700 copies). It’s possible that this overexpression is detrimental to cell<br />
::Alternative low copy plasmids found in distribution kit<br />
:::*2013 (plate 5)<br />
::::pSB6A1 (1K)<br />
::::pSB3C5 (3C)<br />
::::pSB3K3 (5E)<br />
:::*2014 (plate 4)<br />
::::pSB3C5 (4D)<br />
::::pSB6A1 (2L)<br />
::-Suspended and transformed the above plasmids into 5alphas<br />
::-Next, we will select colonies, mini-prep, digest, gel extract, ligate with M13genes<br />
*Started phage amplification protocol <br />
::-Phagemids in ER2738<br />
:::pSB1C3-M13ori(New)<br />
:::pSB1C3-M13ori(Old) <br />
::-Set up O/N of ER2738 for infection tomorrow<br />
<br />
'''6/26'''<br />
*Results of transformation of plasmids from the distribution kit<br />
::-Only received colonies pSB6A1 (2L from 2014) and pSB3C5 (4D from 2014)<br />
::-Pricked colonies for O/N<br />
*PCR of M13 genes so we can ligate it into the above backbones tomorrow<br />
::-DNA: M13K07 digested with EcoRI+PstI (6/21)<br />
::-Primers: Gem012 F & R<br />
*Finished phage amplification protocol<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Samples<br />
! A269<br />
! A320<br />
! Concentration<br />
<br />
|-<br />
| Old phagemid 1C3 (1)<br />
| 0.832<br />
| 0.492<br />
| 7.828 x10^12<br />
<br />
|-<br />
| Old phagemid 1C3 (2)<br />
| 0.324<br />
| 0.083<br />
| 5.549 x10^12<br />
<br />
|-<br />
| New phagemid 1C3 (1)<br />
| 0.391<br />
| 0.077<br />
| 7.229 x10^12<br />
<br />
|-<br />
| New phagemid 1C3 (2)<br />
| 0.402<br />
| 0.639<br />
| -5.457 x10^12<br />
<br />
|-<br />
|}<br />
::::*While resuspending “New phagemid 1C3 (2)”, the tip fell off and we lost half of sample. Evidently, we lost most of phage so we tossed sample<br />
::-Prepped and Infected ER2738 with “Old phagemid 1C3 (1)” and “New phagemid 1C3 (2)”<br />
:::Added 1.6ul of 1:10 diluted “Old phagemid 1C3 (1) to ER2738 cells<br />
:::Added 1.8ul of 1:10 diluted “New phagemid 1C3 (1) to ER2738 cells<br />
::-Plated the infected cells at dilutions<br />
:::1:1 onto Chlor+Kan plates<br />
:::1:10 onto Chlor+Kan plates<br />
:::1:100 onto Chlor and onto Kan plates<br />
:::1:1000 onto Chlor and onto Kan plates<br />
:::1:10000 onto Chlor and onto Kan plates<br />
:::1:100000 onto Chlor and onto Kan plates<br />
<br />
'''6/27'''<br />
*To ligate M13genes onto different backbones<br />
::-Mini-prepped the O/Ns from 6/26 to get backbones with low copy number<br />
:::pSB6A1<br />
:::pSB3C5 <br />
::::*did not grow as well<br />
::-PCR purified PCR (to amplify M13 genes with Gem012 F & R) from 6/26<br />
::-Digestions (50ul)<br />
:::Digest pSB6A1 with E + P<br />
:::Digest pSB3C5 with E + P<br />
:::Digest PCR purification product with E + P<br />
'''[[File:UCB-Phage Delivery-140627.JPG]]'''<br />
:1. pSB6A1<br />
:2. pSB3C5<br />
:3. M13genes<br />
::-Gel extraction of the above digestions<br />
:::For each: Added 10ul of 6x loading dye to 50ul digestions and divided the total volume between 2 wells<br />
::-Ligation<br />
:::1) pSB6A1-M13genes<br />
:::2) pSB3C5-M13genes<br />
*Results from 6/26 infection (after 22hrs in incubator)<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Anti-Sense Phagemid 1C3<br />
! 1:100<br />
! 1:1000<br />
! 1:10000<br />
! 1:100000<br />
<br />
|-<br />
| Chlor<br />
| 85<br />
| 6<br />
| 2<br />
| 0<br />
<br />
|-<br />
| Kan<br />
| 405<br />
| 13<br />
| 2<br />
| 1<br />
<br />
|-<br />
|}<br />
{| class = "wikitable"<br />
|-<br />
! Sense Phagemid1C3<br />
! 1:100<br />
! 1:1000<br />
! 1:10000<br />
! 1:100000<br />
<br />
|-<br />
| Chlor<br />
| 121<br />
| 24<br />
| 3<br />
| 0<br />
<br />
|-<br />
| Kan<br />
| 25<br />
| 1<br />
| 0<br />
| 0<br />
<br />
|-<br />
|}<br />
-We also plated the infected cells on plates containing Chlor and Kan to test for the possibility of double infection<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Phagemid 1C3<br />
! 1:1<br />
! 1:10<br />
<br />
|-<br />
| Anti-Sense<br />
| 59<br />
| 0<br />
<br />
|-<br />
| Sense<br />
| 4<br />
| 0<br />
<br />
|-<br />
|}<br />
<br />
<br />
'''6/28'''<br />
*We noticed that there were more colonies on our infection plates from 6/26 than on 6/27; therefore, we recounted colonies<br />
::-No increase of colonies on Kan plates<br />
::-Significant increase of colonies on Chlor plates<br />
::-Sense refers to the first phagemid 1C3 where the M13ori is in the sense direction compared to the plasmid ori<br />
::-Anti-Sense refers to the new phagemid 1C3 where the M13ori is in the anti-sense direction compared to the plasmid ori<br />
'''[[File:UCB-Phage Delivery-140628.JPG]]'''<br />
::Numbers on the left are after 22 hours. Numbers on the right are after 38.75 hours<br />
*Results of transformation from 6/27 (pSB6A1-M13genes and pSB3C5-M13genes)<br />
::-We have many colonies. Unfortunately, some are red, suggesting that original insert (J04450) was not successfully separated from backbone through gel extraction<br />
::-Selected colonies to grow overnight in 5mL LB<br />
<br />
==Week 9==<br />
<br />
'''6/29'''<br />
*Check O/N cultures for correct constructs (pSB6A1-M13genes and pSB3C5-M13genes)<br />
::-Mini-prep samples<br />
::-Digested with EcoRI and PstI to check insert sizes<br />
::-Gel<br />
'''[[File:UCB-Phage Delivery-140629.JPG]]'''<br />
::lanes….<br />
:::1-11: pSB6A1-M13genes<br />
:::12-18: pSB3C5-M13genes<br />
::-Epic failure<br />
::-All pSB6A1 backbones are empty<br />
::-Half of pSB3C5 backbones were empty. The others contained random inserts (1700 OR 3500). We don’t know what these inserts are. 1700 band is likely the digestion product that appears when we digest M13genes<br />
*Question: Can we use empty vectors from these mini-preps as ligation vectors?<br />
::-Digest pSB6A1 mini-prep with…. (see if both cut sites were retained during re-ligation)<br />
:::no enzyme<br />
:::EcoRI<br />
:::PstI<br />
:::EcoRI + PstI<br />
'''[[File:UCB-Phage Delivery-140629-02.JPG]]'''<br />
::lanes....<br />
:::1-2: last two samples of pSB3C5-M13genes from above gel<br />
:::3. Uncut<br />
:::4. Cut with EcoRI<br />
:::5. Cut with PstI<br />
:::6. Cut with EcoRI and PstI<br />
:-Appears that two backbones are ligated together<br />
<br />
'''6/30'''<br />
*Made chemically competent ER2738 cells that contain Litmus28i DNA<br />
*To amplify M13genes in order to retry ligation<br />
::-PCR of M13K07 DNA (diluted 1:100) to amplify the M13 genes<br />
::-PCR purify PCR product<br />
::-Run on gel: PCR purification, PCR, noDNAcontrol<br />
:::*PCR and purification showed bands at ~9k; therefore, did not get product<br />
:::*No DNA control was clean<br />
*Made freeze downs of<br />
::pSB3C5-J04450<br />
::pSB6A1-J04450<br />
::‘empty’ pSB3C5<br />
::‘empty’ pSB6A1<br />
<br />
'''7/1'''<br />
*Digestion of PCR product from 6/30 to figure out where mistake is<br />
:-Not really useful<br />
'''[[File:UCB-Phage Delivery-140701.JPG]]'''<br />
:Lanes...<br />
:1. PCR pur -> digested with AgeI<br />
:2. PCR pur -> digested with NgoMIV<br />
:3. PCR pur -> digested with PstI<br />
:4. PCR pur -> uncut<br />
:5. PCR pur -> digested with dpnI<br />
:6. Uncut plasmid DNA<br />
*Talked to Mary: She says we were adding to much DNA<br />
::-PCR again. Used 1:100 dilution of 1:100 diluted M13K07 DNA. (aka 1:10000 dilution)<br />
'''[[File:UCB-Phage Delivery-140701-02.JPG]]'''<br />
*Received a beautiful band at 6k bp<br />
:lanes<br />
:1. 1:100 dilution<br />
:2. 1:1000 dilution<br />
:3. 1:10 000 dilution<br />
:4. No DNA control<br />
<br />
'''7/2'''<br />
*To biobrick M13 genes (pSB6A1-M13genes and pSB3C5-M13genes)<br />
::-PCR purified 7/1 PCR product (Primers = Gem012)<br />
::-Digested with EcoRI-HF and PstI-HF<br />
::-Ligation to<br />
::::pSBA61 (digested and gel extracted)<br />
::::pSB3C5 (digested and gel extracted)<br />
<br />
'''7/3'''<br />
*Transformation<br />
::-pSB6A1-M13genes into 5alpha cells and ER2738 with pSB1C3-M13ori<br />
::-pSB3C5-M13genes into 5alpha cells and ER2738 with Litmus28i<br />
::-Transformed into cells containing a phagemid in order to skip some steps<br />
<br />
'''7/3-7/7 Vacation!'''<br />
<br />
==Week 10==<br />
<br />
'''7/7'''<br />
*Made O/N cultures of 7/3 transformation colonies<br />
::-Transformation results were not recorded until 7/8 (see below)<br />
<br />
'''7/8'''<br />
*Transformation results ( CC = Chemically comp cells, ER = ER2738)<br />
::-No growth on No DNA controls<br />
:::ER-Litmus28i CC on AMP-Chlor-Tet<br />
:::ER-phagemid1C3 CC on AMP-Chlor-Tet <br />
:::5alpha CC on Amp <br />
:::5alpha CC on Chlor <br />
::-Lots of red colonies on positive controls, no white colonies<br />
:::ER-Litmus28i CC + pSB3C5 on AMP-Chlor-Tet<br />
:::ER-phagemid1C3 CC + pSB6A1 on AMP-Chlor-Tet<br />
::-Lots of white colonies on sample plates, some red colonies<br />
:::ER-Lit CC + M13genes-pSB3C5 on AMP-Chlor-Tet<br />
:::ER-phagemid1C3 CC + M13genes-pSB6A1 on AMP-Chlor-Tet<br />
:::5alpha CC + M13genes-pSB3C5 on Chlor<br />
:::5alpha CC + M13genes-pSB6A1 on Amp<br />
*Overnights from 7/7 look healthy<br />
::-Renamed O/N to have numbers instead of long names<br />
{| class = "wikitable"<br />
|-<br />
! Sample #<br />
! Cells<br />
! DNA<br />
<br />
|-<br />
| 1-5<br />
| ER-Lit<br />
| M13genes-pSB3C5<br />
<br />
|-<br />
| 6-10<br />
| ER-phagemid1C3<br />
| M13genes-pSB6A1<br />
<br />
|-<br />
| 11-15<br />
| 5alpha<br />
| M13genes-pSB3C5<br />
<br />
|-<br />
| 16-20<br />
| 5alpha<br />
| M13genes-pSB6A1<br />
<br />
|-<br />
|}<br />
::-Mini-prepped DNA<br />
::-Digest the mini-preps with EcoRI-HF and PstI-HF<br />
::-Run digestions on gel to check sizes<br />
'''[[File:UCB-Phage Delivery-140708.JPG]]'''<br />
<br />
:lanes<br />
::top-left: pSB3C5-M13genes in ER2738 with Litmus28i<br />
::top-right: pSB6A1-M13genes in ER2738 with pSB1C3-1C3<br />
::bottom-left: pSB3C5-M13genes in 5alpha<br />
::bottom-right: pSB6A1-M13genes in 5alpha<br />
:None are correct<br />
*Primers Gem013 came in. Resuspend and diluted primers<br />
::-O/N of pSB3C5 to use for PCR tomorrow<br />
<br />
'''7/9'''<br />
*Wanted to make Litmus28i biobrick compatible for use as a phagemid backbone for us and other iGEM teams<br />
::-Mini-prepped O/N of pSB3C5<br />
::-PCR of pSB3C5 to amplify J04450 with Litmus28i compatible cut sites<br />
:::Primers: Gem013<br />
::::*At the 3’ end, these primers are the same as VF2 and VR so will bind the region flanking J04450. This conserves the terminators that exist between the biobrick prefix and VF2 on one side and those between the biobrick suffix and VR on the other side. Therefore, we are amplifying, VF2 priming site, terminators, J04450, terminators, and VR priming site<br />
::::*At the 5’ end, these primers contain unique restriction sites found in the Litmus28i MCS<br />
*PCR of M13 genes…. again (did 4 samples)<br />
::-Used the 1:10000 dilutions<br />
<br />
'''7/10'''<br />
*Note: The following two projects were done in parallel when possible<br />
*To make Litmus28i biobrick compatible<br />
::-Ran gel of PCR from 7/9 (J04450 amplification with Gem013)<br />
'''[[File:UCB-Phage Delivery-140710.JPG]]'''<br />
<br />
:1-4: PCRs of M13genes<br />
:5. No DNA control for M13genes PCR<br />
:6. PCR of pSB3C5 backbone<br />
:7. No DNA control for pSB3C5 backbone<br />
::-PRC purified the PCR product<br />
::-Digestion #1<br />
:::Restriction enzymes had the same buffer conditions but different activation temperatures so we had to do a 2 part digestion<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Restriction enzymes<br />
! Notes<br />
<br />
|-<br />
| J04450<br />
| Sac1<br />
| PCR purified<br />
<br />
|-<br />
| Litmus 28i<br />
| Sac1<br />
| From NEB tube<br />
<br />
|-<br />
|}<br />
:::*Incubate 1hr at 37 C<br />
:::*Heat inactivated 20 minutes at 80C<br />
::-Digestion #2<br />
:::-Added 1ul BsmI to both samples<br />
:::-Incubated 1hr at 65C<br />
:::-Heat inactivated 20 minutes at 80C<br />
::Ran gel<br />
:::-See gel below<br />
:::-Tried to extract J04450 segment but received very low yield. Since band otherwise looked clean, we decided to redo digestion then skip straight to ligation<br />
::Repeat digestion #1 for J04450<br />
::Repeat digestion #2 for J04450<br />
::Ligation (10hr at 16C, 10 min at 80C)<br />
:::3. Litmus28i + J04450<br />
*To retry ligation to biobrick backbone<br />
::-Ran gel of PCR from 7/9 (M13genes amplified with Gem012)<br />
:::See gel below<br />
::-PCR purified the PCR product<br />
::-Digestion<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Restriction enzymes<br />
! Notes<br />
<br />
|-<br />
| M13 genes<br />
| EcoRI-HF + PstI-HF<br />
| PCR purified<br />
<br />
|-<br />
| 6A1<br />
| EcoRI-HF + PstI-HF<br />
| ‘empty’ pSB6A1<br />
<br />
|-<br />
| 3C5<br />
| EcoRI-HF + PstI-HF<br />
| contains J04450<br />
<br />
|-<br />
|}<br />
:Incubate 1hr at 37C<br />
:Heat inactivated 20 minutes at 80C<br />
::-Ran gel<br />
:::*See gel below<br />
:::*Tried to extract M13genes and pSB3C5 segments but received very low yields. Since bands otherwise looked clean, we decided to redo digestion then skip straight to ligation<br />
::-Repeat digestion for M13genes and pSB3C5<br />
::-Ligation (10h at 16C, 10m at 80C)<br />
:::1. pSB3C5 + M13genes<br />
:::2. pSB6A1 + M13genes<br />
*Gel<br />
'''[[File:UCB-Phage Delivery-140710-02.JPG]]'''<br />
:1. pSB1A3<br />
:2. Limtus28i<br />
:3. M13genes<br />
:4. pSB3C5<br />
:5. J04450<br />
<br />
'''7/11'''<br />
*Transformed ligations from 7/10 into 5alpha cells<br />
::1. pSB3C5 + M13genes<br />
::2. pSB6A1 + M13genes<br />
::3. Litmus28i + J04450<br />
<br />
<br />
'''7/12'''<br />
*Results of 7/11 transformation<br />
::-No growth on no DNA control (Amp or Chlor)<br />
::-Lots of growth on Lit-J04450 -> some red-> colonies are too close together to prick individual colonies<br />
:::-It’s possible that these red colonies are satellites =(<br />
:::-Swiped some and plated on new Amp plate (restreak)<br />
::-Lawn of positive control (Litmus 28i) on Amp<br />
::-Many colonies for L1 (pSB3C5-M13genes) and L2 (pSB6A1-M13genes)<br />
:::-Due to high number of red colonies on Lit-J04450 plate, we assumed that most of these colonies contain empty vector<br />
:::-Did not make O/N<br />
*Ligated M13 genes to pSB6A1<br />
::-Used the remained of our digested M13genes.<br />
::-Both digestions from 7/10<br />
*Infection Experiment<br />
::-had 3 cell stocks (each taken from a different colony the night before)<br />
::-Tested Litmus 28i, Tandem phagemid 1C3, and double infection<br />
:::*Therefore, there were 9 flasks total.<br />
::-negative control: Streaked parent cells (non-infected) onto Chlor, Amp, and kan<br />
::-Included double infection plates for Litmus 28i and phagemid 1C3<br />
<br />
==Week 11==<br />
<br />
'''7/13'''<br />
*Transformed 7/12 ligation (pSB6A1-M13genes)<br />
*Litmus28i-J04450<br />
::-All growth from 7/12 restreak was white<br />
::-Pricked some red colonies for liquid O/N-will hopefully see red tomorrow<br />
::-Pricked a few red colonies and put into 200ul H2O (a few colonies per tube- 2 tubes total).<br />
:::*Plated 150ul onto Amp plates<br />
::-Put original plate into incubator to hopefully get bigger colonies<br />
<br />
'''7/14'''<br />
*Finished TWIV ppt<br />
*Set up 2 liquid culture of red Litmus 28i colonies (Litmus28i-J04450)-- slow growth <br />
::-Mini-prepped red Litmus 28i ‘O/N’ from earlier in day<br />
::-Digested samples—to check for insert and correct cut sites<br />
*Made O/Ns of pSB6A1-M13genes colonies from 7/13 transformation<br />
<br />
<br />
'''7/15'''<br />
*Ran gel of Litmus28i-J04450 samples<br />
'''[[File:UCB-Phage Delivery-140715.JPG]]'''<br />
:1. J04450-Litmus28ibb #1 (EcoRI+PstI)<br />
:2. J04450-Litmus28ibb #1 (uncut)<br />
:3. J04450-Litmus28ibb #2 (EcoRI+PstI)<br />
:4. J04450-Litmus28ibb #2 (EcoRI+PstI)<br />
::*Accidentally added restriction enzymes<br />
:5. Litmus28i (EcoRI+PstI)<br />
::*Only has PstI site<br />
<br />
::-Verifies that Litmus28i-J00450 had correct cut sites<br />
::-From this point on, biobricked Litmus28i is called Litmus28ibb<br />
*Check 7/14 O/Ns for pSB6A1-M13genes<br />
::-Mini-prepped liquid cultures<br />
::-Digested with EcoRI and PstI<br />
::::Received bold bands of just under 4000bp. Some lanes had a very feint band ~330bp. None were the correct size<br />
*Transformed Litmus28ibb-J04450 into ER2738 cells<br />
*Remake phage packaging<br />
::-pSB1C3-M13ori(New)<br />
::-pSB1C3-M13ori(Old)<br />
<br />
'''7/16'''<br />
*Results of 7/15 transformation of Litmus28i into 6/30 ER2738 cells<br />
::-lawn on no DNA control-- most likely contaminated cells<br />
::::Streaked chem comp 5alpha and ER onto Amp and Amp+Tet plates to determine if problem is with cells or plates<br />
::-O/N culture of ER to make new chem comp cells<br />
<br />
'''7/17'''<br />
*Finished phage isolation<br />
::Note:<br />
:::*pSB1C3-M13ori (New): M13ori and plasmid ori are convergent<br />
:::*pSB1C3-M13ori (Old): M13ori and plasmid ori are tandem<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! 269nm<br />
! 320nm<br />
! [] phage/mL<br />
<br />
|-<br />
| pSB1C3-M13ori (New)<br />
| 0.386<br />
| 0.034<br />
| 8.126 E12<br />
<br />
|-<br />
| pSB1C3-M13ori (New)<br />
| 0.419<br />
| 0.036<br />
| 8.842 E12<br />
<br />
|-<br />
| pSB1C3-M13ori (Old)<br />
| 0.283<br />
| 0.025<br />
| 5.956 E12<br />
<br />
|-<br />
| pSB1C3-M13ori (Old)<br />
| 0.326<br />
| 0.024<br />
| 6.972 E12<br />
<br />
|-<br />
|}<br />
*Infection to compare the new and the old pSB1C3-M13ori<br />
::-Prepped cells for infection<br />
::-Infected cells with pSB1C3-M13ori (New) or pSB1C3-M13ori (New)<br />
::-and plated on dilutions of 1:100, 1:1000, and 1:10000 on Chlor and kan<br />
::-Also plated 1:1000 dilution on lowered Chlor concentration (34ug/mL)<br />
*Sent Litmus28ibb-J04450 for sequencing<br />
<br />
'''7/18'''<br />
*Results from contamination test<br />
::-5alpha on Amp = no growth<br />
::-5alpha on Amp+Tet = colonies<br />
::-ER on Amp = lawn<br />
::-ER on Amp+Tet = lawn<br />
*Made new chemically competent ER2738<br />
*Transformed Litmus28ibb-J04450 into ER2738 made on 3/30 and ER2738 made on 7/18<br />
::-Had two samples of Litmus28ibb-J04450 and two cell stocks, so 4 samples total<br />
<br />
'''7/19'''<br />
*Results of 7/17 infection<br />
::-pSB1C3-M13ori(Old) (tandem-when plasmid ori and M13ori point in the same direction) packages better than pSB1C3-M13ori(New) (when ori and M13ori are convergent), implying that directionality matters.<br />
'''[[File:UCB-phage lab7-19-141012.jpg]]'''<br />
<br />
==Week 12==<br />
<br />
'''7/20'''<br />
*Freeze downs<br />
:-Litmus28i-J04450 Litmus 28i is now biobrick compatible<br />
<br />
'''7/21'''<br />
*Send samples for sequencing<br />
::-pSB1C3-M13ori(Old)<br />
::-pSB1C3-M13ori(New)<br />
*To ligate M13ori to a kanamycin backbone (pSB1K3)<br />
::-Resuspended pSB1K3-J04450 from distribution kit (6B on plate 4)<br />
:::*Contains J04450 as insert (full RFP construct)<br />
::-Transform pSB1K3 into 5alpha cells<br />
<br />
'''7/22'''<br />
*Pricked colony from pSB1K3-J04450 transformation<br />
::-Mini-prepped DNA to get a supply of DNA<br />
*To put M13ori (M13 phackaging signal) onto Kanamycin resistance o we can test packaging ratios with M13K07 on the same antibiotic<br />
::-Digested pSB1K3 with EcoRI-HF and XbaI<br />
::-Digested pSB1C3-M13ori (Old) with EcoRI-HF and SpeI-HF<br />
:::*DNA was not sufficiently cut. Too much DNA? Problem with enzyme (SpeI-HF)?<br />
'''[[File:UCB-Phage Delivery-140722.JPG]]'''<br />
:1. pSB1K3<br />
:2. M13ori<br />
<br />
'''7/23'''<br />
*Sequencing samples from 7/21 were lost in the mail. Resent samples<br />
*Yet another attempt to biobrick M13genes<br />
::1. using primers that would amplify genes and M13 ori parts from M13K07<br />
::2. using primers that would amplify genes, M13 ori parts, and plasmid ori from M13K07<br />
'''[[File:UCB-Phage Delivery-140723.JPG]]'''<br />
:1. Amplified only the M13genes and M13ori<br />
:2. No DNA control for 1<br />
:3. Amplified M13genes, M13ori, and plasmid ori<br />
:4. No DNA control for 3<br />
::-When run on a gel, samples were clean with only one band at around 7kb. No contamination in no DNA controls<br />
*Digestions<br />
::-pSB1K3 with EcoRI-HF and PstI-HF to check for correct insert<br />
::-pSB1C3-M13ori (Old and new) with EcoRI-HF and SpeI-HF to test for efficient cutting with different stock of restriction enzyme<br />
'''[[File:UCB-Phage Delivery-140723-02.JPG]]'''<br />
:1-3: pSB1K3<br />
:4. pSB1C3-M13ori (Old)<br />
:5. pSB1C3-M13ori (New)<br />
*Still had inefficient cutting. Tested M13ori next to Litmus28i to disern the problem<br />
:*Uncut<br />
:*Cut once with (E, X, S, or P)<br />
:*Cut twice with (E+S or E+P)<br />
'''[[File:UCB-Phage Delivery-140723-03.JPG]]'''<br />
:1. M13ori uncut<br />
:2. M13ori Ecori-HF<br />
:3. M13ori SpeI-HF<br />
:4. M13ori XbaI<br />
:5. M13ori PstI-HF<br />
:6. M13ori EcoRI-HF + SpeI-HF<br />
:7. M13ori EcoRI-HF + PstI-HF<br />
:8. Litmus28i uncut<br />
:9. Litmus28i EcoRI-HF<br />
:10. Litmus28i SpeI-HF<br />
:11. Litmus28i XbaI<br />
:12. Litmus28i PstI-HF<br />
:13. Litmus28i EcoRI-HF + SpeI-HF<br />
:14. Litmus28i EcoRI-HF + PstI-HF<br />
*Only partial digest with only SpeI-HF for both. Complete digestion with all others, including E+S<br />
<br />
'''7/24'''<br />
*To Biobrick M13genes using pSB6A1<br />
::-PCR purified M13 genes (did both samples at the same time)<br />
::-Digested PCR purification and pSB6A1 with EcoRI-HF and PstI-HF<br />
'''[[File:UCB-Phage Delivery-140724.JPG]]'''<br />
*Gel extracted M13genes<br />
<br />
:1. M13genes<br />
:2. pSB6A1<br />
:-Bands of pSB6A1 were too light to gel extract<br />
*To swap the kanamycin resistance marker on M13K07 with ampicillin resistance<br />
::-PCR amplified AmpR from pSB6A1<br />
*made O/N culture of pSB6A1 from freeze down to mini-prep<br />
::-Also streaked cells onto plate<br />
<br />
'''7/25'''<br />
*To change resistance marker on M13K07<br />
:1. Biobrick method<br />
::*Tried to mini-prep O/N but pellet (after liquid culture was spun) was not red even after 16+ hours. Set up O/N from plate colonies<br />
:2. Swap only resistance method<br />
::*PCR purified 7/24 PCR<br />
::*Ran product on gel-> band of correct size<br />
'''[[File:UCB-Phage Delivery-140725.JPG]]'''<br />
<br />
:1. PCR product<br />
:2. PCR product-> PCR purified<br />
:3. No DNA control<br />
::-Digested the PCR purification products of M13genes+ori and AmpR with AgeI-HF and NotI-HF<br />
::*Bands on previous gel looked clean enough for both so did not gel extract<br />
::-Overnight ligation<br />
*Set up O/Ns of pSB1K3 colonies<br />
<br />
'''7/26'''<br />
*Transform<br />
::-pSB6A1+M13genes<br />
*To biobrick M13genes<br />
::-Digestion<br />
:::*pSB6A1 with EcoRI and PstI<br />
'''[[File:UCB-Phage Delivery-140726.JPG]]'''<br />
:Gel extracted backbone (band ~4000bp)<br />
*Ligations (10hrs 16C, 10min at 80C)<br />
::-pSB6A1+M13genes<br />
<br />
==Week 13==<br />
<br />
'''7/27'''<br />
*Transformation results from 7/26<br />
::-No growth for no DNA controls ''on Amp''<br />
::-No growth for M13 genes onto pSB6A1 ''on Amp''<br />
*Set up O/Ns of 5alpha and ER2738 to make competent cell tomorrow<br />
*Made freeze down of pSB1K3<br />
*Plated ER competent cells from 5/15 and 6/16 Onto Chlor plates to check for contamination<br />
<br />
'''7/28'''<br />
*No growth of ER2738 on Chlor (either sample)<br />
::-ER is not contaminated with ChlorR<br />
*Make new 5alpha chem comp cells <br />
*Transformations<br />
::-pSB6A1-M13genes<br />
::-positive control for Amp (pSB6A1)<br />
::-No DNA control<br />
<br />
'''7/29'''<br />
*Set up O/Ns of pSB6A1-M13genes<br />
::-Plated sample on reduced Amp and regular Tet<br />
::-Later in day…<br />
::-Mini-prepped O/Ns<br />
::-Digested and ran on a gel to check for insert sizes<br />
'''[[File:UCB-Phage Delivery-140729.JPG]]'''<br />
:*All but the 4th lane with 3 bands look correct<br />
<br />
'''7/30'''<br />
*Lawn on last night’s plating-> replate<br />
*Digest pSB3C5 and pSB6A1-M13genes with E and P to move M13genes to a Chlor backbone<br />
'''[[File:UCB-Phage Delivery-140730.JPG]]'''<br />
::-Extracted bands<br />
::-Ligations<br />
:::*pSB3C5 + M13genes<br />
*Sent samples for sequencing<br />
**M13 genes on pSB6A1 VF2<br />
**M13 genes on pSB6A1 VR<br />
<br />
'''Test experiment for high school kids coming to lab (7/29-7/31)'''<br />
*General idea: Have 2 strands of DNA, one has an EcoRI site while the other contains a SNP, abolishing the cut sight. We pretend that one is pathogenic (the one that is not cut) and tells kids to figure out which one is which <br />
*PCR with Dream-Taq<br />
::-Did not have special fastdigest enzyme<br />
::-Hypothesized that green dye will interfere with enzyme effectivity<br />
*PCR with Dream-Taq (no green dye)<br />
::-Digest with EcoRI fastdigest-> did not cut<br />
*Redid experiment several times<br />
::-Should digest with XbaI<br />
::-PCR purified PCR products<br />
<br />
'''7/31'''<br />
*Spent the morning with Heritage High School<br />
*Made phage<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Phage<br />
! A269<br />
! A320<br />
! genome size<br />
! [ ]<br />
<br />
|-<br />
| Litmus28ibb-J04450 (1)<br />
| 0.823<br />
| 0.197<br />
| <br />
| 9.35 x10^12<br />
<br />
|-<br />
| Litmus28ibb-J04450 (2)<br />
| 0.940<br />
| 0.168<br />
| <br />
| 1.14 x10^13<br />
<br />
|-<br />
|}<br />
<br />
'''8/2'''<br />
*Digestion<br />
::-pSB6A1-M13genes (E+P)<br />
::-pSB3C5 (E+P)<br />
<br />
==Week 14==<br />
<br />
'''8/3'''<br />
*Another attempt to clone M13genes onto pSB3C5<br />
::-Run 8/2 digestions on gel<br />
'''[[File:UCB-Phage Delivery-140803.JPG]]'''<br />
:1. M13genes (E+P)<br />
:2. pSB3C5 (E+P)<br />
::-Gel extracted <br />
:::*M13genes away from pSB6A1 backbone<br />
:::*pSB3C5 backbone away from J04450<br />
::-Ligations<br />
:::*pSB3C5 + M13genes<br />
:::*pSB3C5 + no insert<br />
::-Transformed ligations into ER2738<br />
<br />
'''8/5'''<br />
*Transformation of pSB3C5+M13genes failed<br />
*Made phage<br />
::-Fd-CAT DNA packaged with Fd-CAT<br />
::-phagemid 1C3 packaged with M13K07<br />
::-amilCP on pSB1C3 packaged with M13K07<br />
::-Litmus28ibb-J04450 packaged with M13K07<br />
<br />
'''8/6'''<br />
*Isolated phage<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Phage<br />
! [ ]<br />
<br />
|-<br />
| Fd CAT<br />
| 2.72 x10^12<br />
<br />
|-<br />
| Litmus28ibb-J04450<br />
| 8.37 x10^12<br />
<br />
|-<br />
| pSB1C3-amilCP<br />
| 1.42 x10^13<br />
<br />
|-<br />
| pSB1C3-M13ori<br />
| 4.32 x10^12<br />
<br />
|-<br />
|}<br />
<br />
'''8/6'''<br />
*Started making phage with Fd CAT the should contain Litmus28ibb-RFP<br />
*Infection<br />
::-Infected ER2738 cells with Litmus28ibb-RFP, pSB1C3-M13ori, or pSB1C3-amilCP<br />
::-Plated Litmus samples on 100ug/mL Amp and 50ug/mL Kan<br />
::-Plated other two samples on 34ug/mL Chlor and 25ug/mL Kan<br />
<br />
'''8/7'''<br />
*Isolated phage containing Litmus28ibb-RFP using helper phage<br />
::-M13K07 (as control)<br />
::-Fd-CAT (no phage pellet was observed-worried that there was no phage)<br />
*Went to CSU to have them test M13ori part compared to amilCP. Grew the samples in 5mL O/Ns then diluted to 0.5OD and grew 30 minutes rather than starting from a fresh colony<br />
<br />
'''8/8'''<br />
*At CSU<br />
::-Finished phage protocol (Test packaging of M13ori part)<br />
::-Only used 20mL infection samples<br />
::-grew to 0.55 OD<br />
::-When making phage (after 14 hour incubation), there was very little growth<br />
::-Plated non-diluted and diluted 1:1000 of M13ori and amilCP sample<br />
*Infected cells using phage isolated 8/7. Plated at dilutions of 1:100 and 1:100k<br />
*Want to test progeny Fd-CAT phage (made 8/7) for infectability<br />
::-Start phage isolation prodocol using Fd-CAT phage from 8/7 do deliver the helper phagemid<br />
:::*Phagemid:<br />
::::*None: will make Fd-CAT phage packaing Fd-CAT phagemid<br />
::::*Litmus28ibb-J04450: make Fd-CAT phage packaging Litmus28ibb-J04450 phagemid<br />
*Remake phage (Fd-CAT) using fresh stock from Mike<br />
<br />
<br />
'''8/9'''<br />
*Results of 8/8 infection<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Helper Phagemid<br />
! phagemid<br />
! Selection<br />
! 1:1000 dilution<br />
! 1:00k dilution<br />
<br />
|-<br />
| M13K07<br />
| Litmus28ibb-RFP<br />
| Kan+Tet<br />
| 400-500 (some red)<br />
| 61 white; 20 red<br />
<br />
|-<br />
| same<br />
| same<br />
| Amp+Tet<br />
| lawn<br />
| 2<br />
<br />
|-<br />
| Fd-CAT<br />
| Litmus28ibb-RFP<br />
| Chlor+Tet<br />
| 0<br />
| 0<br />
<br />
|-<br />
| same<br />
| same<br />
| Amp+Tet<br />
| 10<br />
| 707<br />
<br />
|-<br />
|}<br />
*NOTE: The same sample was plated on two different plates. For example, the sample using M13K07 as the helper phage was plated on Kan+Tet and Amp+Tet<br />
*Mini-prep<br />
::-pSC3C5-J04450 -- very little growth, low DNA yield<br />
::-Fd-CAT infected cells<br />
:::*Digest and run on a gel to verify presence of a band- there was a very feint band<br />
*Finished isolating phage…. messed up and used 0.8MgCl2, 0.2M NaCl instead of PEG during precipitation step. Also used the wrong phage<br />
::-Infected ER2738 cells anyway…. no growth by 8/12<br />
<br />
==Week 15==<br />
<br />
'''8/10'''<br />
*Are the progeny phage from 8/6 (original progeny from first Fd-Tet application) viable/ able to reproduce?<br />
::-Used these progeny to make phage that should amplify Fd-CAT phage containing either the Fd-CAT or Litmus28ibb-J04450 phagemid<br />
<br />
'''8/11'''<br />
*Finished isolating phage<br />
{| class = "wikitable"<br />
|-<br />
! Helper Phagemid<br />
! Phagemid<br />
! A269<br />
! A320<br />
! genome size<br />
! [ ]<br />
<br />
|-<br />
| Fd-CAT<br />
| Litmus28ibb (1)<br />
| 0.029<br />
| 0.013<br />
| 4080<br />
| 2.35 x10^11<br />
<br />
|-<br />
| Fd-CAT<br />
| Ltimus28ibb (2)<br />
| 0.033<br />
| 0.015<br />
| 4080<br />
| 2.65 x10^11<br />
<br />
|-<br />
| Fd-CAT<br />
| Fd-CAT (1)<br />
| 0.326<br />
| 0.082<br />
| 7775<br />
| 1.88 x10^12<br />
<br />
|-<br />
| Fd-CAT<br />
| Fd-CAT (2)<br />
| 0.320<br />
| 0.079<br />
| 7775<br />
| 1.86 x10^12<br />
<br />
|-<br />
| M13g6A1<br />
| M13g6A1 (1)<br />
| 0.322<br />
| 0.148<br />
| 10,029<br />
| 1.04 x10^12<br />
<br />
|-<br />
| M13g6A1<br />
| M13g6A1 (2)<br />
| 0.503<br />
| 0.130<br />
| 10,029<br />
| 2.23 x10^12<br />
<br />
|-<br />
|}<br />
<br />
'''8/13'''<br />
*Infected ER2738 with Fd-CAT packaging Litmus28ibb-J04450<br />
<br />
'''8/16'''<br />
*Results from Infection on 8/11<br />
{| class = "wikitable"<br />
|-<br />
! Helper Phagemid<br />
! Phagemid<br />
! 1:1<br />
! 1:10<br />
! 1:100<br />
! 1:1000<br />
<br />
|-<br />
| M13g6A1<br />
| M13g6A1<br />
| almost lawn<br />
| 728<br />
| 287<br />
| 55<br />
<br />
|-<br />
| Fd-CAT<br />
| Fd-CAT<br />
| lawn<br />
| almost lawn<br />
| 1260<br />
| 640<br />
<br />
|-<br />
| Fd-CAT<br />
| Litmus28ibb-J04450<br />
| lawn<br />
| almost lawn<br />
| 2004<br />
| 304<br />
<br />
|-<br />
|}<br />
<br />
Results: packaging of Litmus28ibb-J04450 using Fd-CAT helper phagemid from (8/15)<br />
<br />
{| class="wikitable"<br />
|-<br />
!Antibiotic<br />
!1:10 <br />
!1:100 <br />
!1:1k <br />
!1:10k <br />
!1:100k<br />
<br />
|-<br />
|Amp <br />
|Lawn<br />
|Almost lawn <br />
|1025 <br />
|217 <br />
|25<br />
<br />
|-<br />
|Kan <br />
|9 <br />
|2 <br />
|0 <br />
|0 <br />
|0<br />
|}<br />
<br />
==Week 16==<br />
<br />
'''8/19'''<br />
<br />
Sent samples for sequencing<br />
<br />
Litmus28ibb-J04450<br />
<br />
==Week 17==<br />
<br />
'''8/27'''<br />
<br />
Transform pSB1C3-M13ori into ER2738 cells<br />
<br />
'''8/28'''<br />
<br />
Make phage with pSB1C3-M13ori as phagemid and M13g6A1 as helper phagemid<br />
<br />
'''8/29'''<br />
<br />
Finish phage isolation of M13 phage containing pSB1C3-M13ori<br />
<br />
'''9/3'''<br />
<br />
digest pSB6A1-M13g and pSB3C5 with EcoRI and PstI. Did not receive a band for M13genes so did not proceed with ligation to pSB3C5<br />
<br />
==Week 18==<br />
<br />
'''9/4'''<br />
<br />
Mini-prepped new pSB6A1-M13genes (bad quality) and pSB3C5-J04550. Digested both plasmids with EcoRI and PstI and ran products on gel. Did receive a band for the M13genes but it was too feint to gel extract<br />
<br />
'''9/5'''<br />
<br />
Mini-prep pSB6A1-M13genes again. Digest this and pSB3C5 backbone from previous day with EcoRI and PstI. M14g band was barely bright enough to extract so we extracted M13genes and pSB3C5 backbone. Pieces were ligated together overnight at 16C for 10 hours.<br />
<br />
'''9/7'''<br />
<br />
Transformed 9/5 ligation (pSB3C5-M13genes into 5alpha cells). Plated sample onto low Chlor plate (34ug/mL)<br />
<br />
'''9/9'''<br />
<br />
Still no growth for 9/7 transformation of pSB3C5-M13genes ligation PCR M13genes to attempt another ligation into pSB3C5. Diluted DNA to ~1.17ng/ul<br />
<br />
'''9/10'''<br />
<br />
Gel of 9/9 PCR of M13genes looked clean (only ran 3ul of PCR sample on gel). PCR purified the remaining PCR sample. Digested with EcoRI and PstI. Ligated to previously digested pSB3C5.<br />
<br />
==Week 18==<br />
<br />
'''9/11'''<br />
<br />
Transform 9/10 ligation (pSB3C5-M13genes) into 5alpha cells<br />
<br />
'''9/13'''<br />
<br />
Colony PCR of colonies that grew for 9/11 transformation. Looking for pSB3C5-M13genes)<br />
<br />
'''9/15'''<br />
<br />
Gel of colony PCR showed failure. Set up overnight cultures anyway.<br />
<br />
'''9/17'''<br />
<br />
Submitted Part to iGEM Registry<br />
<br />
{| class="wikitable"<br />
|-<br />
|iGEM # <br />
|Description<br />
<br />
|-<br />
|BBa_K1445000 <br />
|M13ori- the packaging signal for the M13 and fd phage<br />
|}<br />
<br />
Mini-prepped overnights from 9/15 and sent samples for sequencing<br />
<br />
Sent Litmus28ibb-J04450 sample for sequencing to sequence the remaining section that was not reached before<br />
<br />
==Week 19==<br />
<br />
'''9/21'''<br />
<br />
Up until this point, very little luck was had with pSB3C5. Retried putting M13genes onto a chloramphenicol backbone, but this time used pSB1C3. Digested M13genes and pSB1C3 with EcoRI and PstI. Gel showed incomplete digestion so did not gel extract.<br />
<br />
'''9/22'''<br />
<br />
Digested pSB1C3-J04450 with EcoRI and PstI then gel extracted the backbone. PCR purified M13genes(PCR product was clean so did not bother to gel extract). Ligated the two pieces together.<br />
<br />
'''9/23'''<br />
<br />
Transform ligations from 9/22 (pSB1C3-M13genes) into 5 alpha cells<br />
<br />
'''9/27'''<br />
<br />
Transform pSB1C3-M13genes into 5alpha cells. Plate onto low Chlor (34ug/mL)<br />
<br />
Later in day.... Set up overnight cultures of colonies<br />
<br />
'''9/28'''<br />
<br />
Mini-prepped O/N cultures from 9/27. Digested samples with EcoRI and PstI. There were no bands of the correct size on the gel.<br />
<br />
the M13 and fd phage</div>Leighlahttp://2014.igem.org/Team:CU-Boulder/Notebook/Phage_TeamTeam:CU-Boulder/Notebook/Phage Team2014-10-17T06:54:52Z<p>Leighla: /* Week 17 */</p>
<hr />
<div>{{Template:UCB-Main}}<br />
{{UCB-NavBar}}<br />
__FORCETOC__<br />
<br />
'''Phage Delivery'''<br />
==Week 1==<br />
'''Notes:''' Unless stated otherwise, all gels contain 2-log ladder<br />
<br />
'''5/9'''<br />
*Obtained BW23115 KanR cells- BW23115 cells that had their native CRISPR-Cas system knocked out by the insertion of a Kanamycin resistance gene<br />
::-Will also be called BW23115 or BW<br />
::-Conjugated BW23115 KanR cells with contain F’ notation (ex. BWF’)<br />
*Obtained ER2738 cells that contain the F’ episome (no changes from NEB sample)<br />
::-Will also be called ER. Assume that all ER samples contain the F’ episome<br />
::-Streaked sample onto LB+Tet (20ug/mL) to select for colonies containing F’ episome<br />
<br />
'''5/10'''<br />
*Did receive colonies from 5/9 selection<br />
<br />
==Week 2==<br />
'''5/12'''<br />
*Need to conjugate BW23115 KanR cells with the F’ episome<br />
::-Set up overnight cultures of ER2738 and BW23115 KanR<br />
::-When mixed, ER2738 will donate it’s F’ episome and BW23115 KanR will receive the F’ episome. F’ episome confers Tetracycline resistance<br />
<br />
'''5/13'''<br />
*Started M13 Amplification: Amplify M13 phage using the M13K07 Helper Phage<br />
::-Let precipitated in NaCl/PEG solution overnight<br />
::-Possible sources of error<br />
:::*Did not sterilize 2.5M NaCl/20% PEG-8000 solution<br />
:::*Added 4-fold PEG solution<br />
::::Compensated by adding more LB<br />
:::*During precipitation, put sample in -20C for 30 minutes before realizing mistake and moving to it to 4C. Sample partially froze<br />
*Conjugated BW23115 with F’ episome<br />
::-Added 1mL BW23115 to 1mL ER2738 overnight culture<br />
::-Incubated at 37C for 30 minutes, shaking<br />
::-Plated on LB+Kan(50ug/mL)+Tet(20ug/mL)<br />
:::*To select for BW cells that took the F’ episome (containing Tet resistance)<br />
<br />
'''5/14'''<br />
*Finished the M13 Amplification<br />
::-Visualized product on UV-vis. There was a tall spike at 269nm indicating that DNA was present. Did not test at 320nm.<br />
*Results of BW23115 Conjugation<br />
::-Many colonies indicating successful conjugation of F’ episome into BW23115<br />
::-Set up overnight to make freeze down tomorrow<br />
*Set up overnight of ER2738 to make chemically competent tomorrow<br />
<br />
'''5/15'''<br />
*Made freeze down of BW23115 KanR F’<br />
::-BW23115 E. coli strain with Kanamycin resistance gene inserted into genome and with F’ episome<br />
*Made chemically competent ER2738 cells<br />
*Transformation of Litmus28i (from NEB) into chemically competent ER2738 cells<br />
::-Added 1ul Litmus28i plasmid to 40ul competent cells<br />
::-Plated on LB + Amp(100ug/mL)<br />
::-Purpose: To make M13 phage that package Litmus28i DNA. Need phagemid (Litmus28i) DNA in infectable cells (cells containing F’ episome) to introduce M13K07 Helper Phage and make phage. <br />
<br />
'''5/16'''<br />
*Results of 5/15 transformation<br />
::-No growth for No DNA control<br />
::-Many colonies for sample<br />
<br />
==Week 3==<br />
'''5/19'''<br />
*M13 Amplification to isolate M13-Litmus28i phage<br />
::-Cells: ER2738 cells containing Litmus28i phagemid<br />
::-Helper Phage: M13K07<br />
::-Not much phage was precipitated<br />
*Set up overnight culture of ER2738 to infect tomorrow<br />
<br />
'''5/20'''<br />
*Infected ER2738 cells with M13-Litmus28i phage<br />
::-Plated only on Ampicillin(100ug/mL) (should have also plated on kanamycin)<br />
::-Infected for 4-5 hours-> should have only infected for 30 minutes maximum. This extra time gives the cells that were infected with M13-M13K07 the time to produced M13-M13K07 phage and reinfect<br />
<br />
'''5/21'''<br />
*Results from M13-Litmus28i infection of ER2738<br />
::-Solid lawn of growth for diluted and non-diluted<br />
::-Also sickly looking growth<br />
*Set up overnights<br />
::-ER2738 cells containing Litmus28i for freeze down<br />
::-BW23115 with F’ episome to make chemically competent cells <br />
::-ER2738 to redo infection<br />
<br />
'''5/22'''<br />
*Tested absorbance of phage produced through M13 amplification on 5/19<br />
::-Low absorbance of 0.018 at 269nm but no detection at wavelength 320nm<br />
::-Decided to redo M13 amplification<br />
*Made chemically competent BW23115 with f-episome<br />
*Made freeze down of ER2738 containing Litmus28i<br />
*Set up overnight of ER2738 containing Litmus28i to redo M13 amplification tomorrow<br />
<br />
'''5/23'''<br />
*Protocol switch to make phage using phagemid<br />
::-“M13 Amplification” protocol should only be used to make more M13-M13K07, not to make M13 phage containing a different phagemid<br />
::-Switched to new protocol (“Use of M13K07 Helper Phage for isolation of single stranded phagemid DNA” by NEB. Made modifications (see our protocols) to isolate phage rather than single-stranded DNA)<br />
::-Making phage….<br />
{| class = "wikitable"<br />
|-<br />
! Helper phage<br />
! Phagemid<br />
! Cells<br />
! Notes<br />
<br />
|-<br />
| M13K07<br />
| None<br />
| ER2738<br />
| Make more M13-M13K07<br />
<br />
|-<br />
| M13K07<br />
| Litmus28i<br />
| ER2738<br />
| Test packaging of Litmus28i<br />
<br />
|-<br />
|}<br />
*Made fresh antibiotics<br />
::-Chloramphenicol (34 ng/mL)<br />
:::*1.44g chloramphenicol into 42mL EtOH<br />
::-Ampicillin (50 ng/mL)<br />
:::*4g ampicillin into 80mL mili-Q H2O<br />
<br />
'''5/24'''<br />
*Isolated phage using new protocol<br />
::-Resuspended pellet in 200ul TBS and 200ul 30% glycerol<br />
::-Measured absorbance with UV-vis<br />
:::*concentration (phage/mL) = 6x10^16 x (A269-A320)/ (#of base pairs in the phage genome)<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! Abs (269nm)<br />
! Abs (320nm)<br />
! Genome size<br />
! Concentration (phage/mL)<br />
<br />
|-<br />
| M13-M13K07<br />
| 0.721<br />
| 0.060<br />
| <br />
| 4.57 x10^12<br />
<br />
|-<br />
| M13-Litmus28i<br />
| 0.250<br />
| 0.028<br />
| 2823<br />
| 4.72 x10^12<br />
<br />
|-<br />
|}<br />
*Infect ER2738 cells with M13-Litmus28i<br />
::-Wanted 1:10 phage:cell ratio. Math….<br />
:::*At 1 OD (e.coli), cell/mL = 5x10^8<br />
:::*5x10^7 phage * (1mL/4.72x10^12 phage) = 0.011ul phage<br />
*Set up overnights<br />
::-ER2738 for infection with M13-Litmus28i<br />
::-BW23115 F’ for infection with M13-Litmus28i to test infectivity of conjugated strain<br />
<br />
==Week 4==<br />
'''5/25'''<br />
*Infect ER and BWF’ cells with M13-Litmus28i<br />
::*Made 5mL culture of ER and BW that was at 1 OD<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! OD<br />
! mL sample for 1OD in 5mL<br />
! mL LB to 5mL<br />
<br />
|-<br />
| ER2738<br />
| 2.5<br />
| 2 mL<br />
| 3 mL<br />
<br />
|-<br />
| BW23115<br />
| 2.0<br />
| 2.5 mL<br />
| 2.5 mL<br />
<br />
|-<br />
|}<br />
::-Based on calculations from 5/24, we needed to add 0.011 ul phage per 1 mL of cells at 1 OD. This equates to 0.055 ul of phage into 5 mL cells; therefore we made a 1:10 dilution so we could add 0.5ul. Unfortunately, the pipet would not take up 0.5ul so we added 0.8ul of M13-Litmus28i phage<br />
::-Grew the cells for 20 minutes at 37C<br />
::-Plated 300ul onto Kanamycin (50ug/mL) and 300ul onto Ampicillin (100ug/mL) for each sample<br />
:::*Incubated overnight at 37C<br />
*'''Note:''' During the production of phage, the phagemid SHOULD be packaged preferentially over the Helper Phagemid but some Helper Phagemid will still be packaged. We plated on Amp to select for cells that were infected with phage containing Phagemid. We plated on Kan to select for cells that were infected with phage containing Helper Phagemid. This allows us to compare the packaging efficiency of Helper Phagemid: Phagemid.<br />
'''5/26'''<br />
*Results from 5/25 infection with M13-Litmus28i<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Result<br />
! Significance<br />
<br />
|-<br />
| ER2738 on Amp<br />
| Lawn<br />
| Litmus28i phagemid was successfully packaged into the M13 phage and is infectable<br />
<br />
|-<br />
| ER2738 on Kan<br />
| 100-200 colonies<br />
| Some M13 helper phage is packaged into the M13 phage but at a much lower rate than Litmus28i<br />
<br />
|-<br />
| BW23115 on Amp<br />
| Lawn<br />
| BW23115 is ‘equally’ infectable by M13 as ER2738<br />
<br />
|-<br />
| BW23115 on Kan<br />
| Lawn<br />
| BW23115 contains Kan resistance in its genome so this tells us nothing<br />
<br />
|-<br />
|}<br />
:*Conclusions:<br />
::-Cells grew on Ampicillin; therefore, Litmus28i phagemid was successfully packaged into M13 phage. <br />
::-For ER2738 samples, there was significant growth on Ampicillin compared to Kanamycin; therefore, Litmus28i phagemid is packaged preferentially over M13K07 Helper Phagemid<br />
::-M13-Litmus28i retains its infectivity of cells containing the F’ episome<br />
:*Because we received lawns, we have to redo the infection and plate less cells so we can calculate the uptake ratio between the phagemid and helper phage based on the number of colonies<br />
*Started 50 mL overnight of K12 ER2738 and BW23115<br />
<br />
'''5/27'''<br />
*Redo the infection done on 5/25<br />
::-Infectable cells: ER2738 and BW23115<br />
:::*Plated non-infected samples of each (non-diluted) to check for contaminants<br />
::-Diluted M13-Litmus28i (1) phage by a factor of 10. Added 5.5ul to each sample<br />
::-Grew samples for 20 minutes at 37C, 250rpm<br />
::-Plated 100ul of onto an Ampicillin (100ug/mL) plate and onto a Kanamycin (50ug/mL) plate. Incubated overnight at 37C.<br />
:::*Dilutions = 1:10; 1:100; and 1:1000<br />
'''5/28'''<br />
*Results from 5/27<br />
::-Controls were as expected<br />
:::*No growth for ER2738 non-infected grown on Amp, ER2738 non-infected grown on Kan, or BW23115 non-infected grown on Amp<br />
:::*Growth for BW23115 non-infected grown on Kan (BW23115 has Kan R in genome)<br />
::-Many colonies were received for all dilutions (1:10, 1:100, and 1:1000) of the following<br />
:::*ER2738 infected and plated on Amp<br />
:::*BW23115 infected and plated on Amp<br />
:::*BW23115 infected and plated on Kan<br />
::-Many (100s to 1000?) colonies grew on 1:10 and 1:100 dilutions of ER2738. 50-100 colonies grew on the 1:1000 dilution of ER2738<br />
:::*Compare this to the 100-200 colonies that grew from 2/25 infection (which was 300ul non-diluted, infected cells)<br />
:::*Reasons for increased yield<br />
::::*Added too much phage?<br />
::::*volume changed between experiment (5mL to 50mL)<br />
::::*Overnight culture may not have been saturated. If still in log phase, the cells would continue to grow<br />
*Made 50mL O/N cultures of ER2738 and BW23115 so we can repeat the infection tomorrow and plate further dilutions starting at 1:1000<br />
::-Carry out infection in 5mL and 50mL to test volume effect?<br />
<br />
'''5/29'''<br />
*Measured OD of overnights<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! OD<br />
! mL to have .1OD in 50mL<br />
! mL to have 1OD in 5mL<br />
<br />
|-<br />
| K12 ER2738<br />
| 3.0<br />
| 1.7<br />
| 1.7<br />
<br />
|-<br />
| BW23115<br />
| 2.9<br />
| 1.7<br />
| 1.7<br />
<br />
|-<br />
|}<br />
<br />
<br />
*Experiment 1: Infect cells using same method as 5/25 (in a 5mL culture)<br />
::-Started with 1OD cells in 5mL<br />
::-Added about 0.7ul (inaccuracies in pipet) of 1:10 diluted M13-Litmus28i phage<br />
::-Incubated (rotating) for 20 minutes<br />
::-Made 1:1,000 and 1:10,000 dilutions<br />
::-Plated 100ul on Ampicillin (100ug/mL) plates and on Kanamycin (50ugmL) plates<br />
:::*Included non-infected samples diluted by 1:1000<br />
::::*This negative control can be used for Experiment 2 since the non-infected parent solution is the same<br />
::-Incubate overnight at 37C<br />
*Experiment 2: Infect cells using protocol from “Eliminating helper phage from phage display”<br />
::-Diluted O/Ns to OD of 0.1 in 50 mL culture<br />
::-Grew samples until of OD of ER2728 = 0.59 and the OD of BW23115 = 0.60<br />
:::*Missed OD of 0.5 mark, but the two samples are close to each other<br />
::-Chilled samples on ice for 30 minutes<br />
::-Warmed in incubator for 35 minutes (should have been 30)<br />
::-Amount of phage. Rather than use 1:1 as mentioned in protocol, we used multiplicity of 1:10 (phage:cell)<br />
:::*Added 3.3ul of 1:10 diluted M13-Litmus28i (1) phage<br />
::::*(On 5/27 we added 5.5ul of diluted phage to 50mL of cells at OD of 1. Our cells were at OD of .6; therefore, 5.5*.6 = 3.3ul)<br />
::-Incubated for 30 minutes at 37C, not shaking<br />
:::*We later change this to shaking<br />
::-Dilutions<br />
:::*1:1,000; 1:5,000; 1:10,000; 1:50,000; 1:100,000; 1:1,000,000<br />
:::*Plated ER2738 and BW23115 on Ampicillin (100ug/mL)<br />
:::*Plated ER2738 on Kanamycin (50ug/mL)<br />
*Experiment 3: Growth Test (for growth curve)<br />
::-We were concerned by the low OD of the Overnights from the last few days. Wanted to be sure that 2.0-3.0 was not still in log phase. Cultures looked saturated but the OD seemed low.<br />
{| class = "wikitable"<br />
|-<br />
! Time<br />
! Elapsed time (min)<br />
! ER2738 (no antibiotic)<br />
! ER2738 (Tetracycline (20ug/mL))<br />
! BW23115 (with F’ episome) no antibiotic<br />
<br />
|-<br />
| 10:08<br />
| 0<br />
| 0.1<br />
| 0.1<br />
| 0.1<br />
<br />
|-<br />
| 11:10<br />
| 62<br />
| 0.24<br />
| 0.21<br />
| 0.21<br />
<br />
|-<br />
| 12:00<br />
| 112<br />
| 0.49<br />
| 0.45<br />
| 0.44<br />
<br />
|-<br />
| 13:00<br />
| 172<br />
| 1.00<br />
| 0.93<br />
| 0.98<br />
<br />
|-<br />
| 14:15<br />
| 217<br />
| 1.29<br />
| 1.21<br />
| 1.31<br />
<br />
|-<br />
| 15:40<br />
| 302<br />
| 2.1<br />
| 1.8<br />
| 2.3<br />
<br />
|-<br />
| 16:47<br />
| 369<br />
| 2.5<br />
| 1.9<br />
| 3.4<br />
<br />
|-<br />
| 18:05<br />
| 447<br />
| 2.6<br />
| 2.2<br />
| 2.5<br />
<br />
|-<br />
| 19:00<br />
| 502<br />
| 2.9<br />
| 2.3<br />
| 2.5<br />
<br />
|-<br />
| 20:00<br />
| 562<br />
| 3.0<br />
| 2.6<br />
| 2.8<br />
<br />
|-<br />
|}<br />
<br />
:::*The time point at 16:47 (369 minutes elapsed) for BW23115 conjugated (without antibiotics) is most likely an error. It has been removed from the growth plot<br />
'''[[File:UCB-Phage Delivery-140529.JPG]]'''<br />
*Other<br />
::-Made Amp and Kan plates (1 sleeve of each)<br />
::-Made 50mL O/N of ER2738 and BW23115F’ in case we need further dilutions<br />
::-Made 5mL O/N of ER2738, BW23115F’, and BW23115 (without F’ episome) to make chemically competent tomorrow<br />
:::*Did not have plate of BW23115 (without F’ episome) so used freeze down. Hoping to get O/N of a picked colony from CRISPR Team tomorrow morning<br />
<br />
'''5/30'''<br />
*Made chemically competent cells of…<br />
::-ER2738<br />
::-BW23115F’ (conjugated with F’ episome)<br />
::-BW23115 (not conjugated- without F’ episome)<br />
:::*Culture started from plate<br />
::-BW23115* not conjugated (without F’ episome)<br />
:::*Culture started from freeze down<br />
*Results from infections<br />
::-Negative Controls (cells were not infected; cells were diluted 1:1000)<br />
'''[[File:UCB-Phage Delivery-140530.JPG]]'''<br />
::-Results from Experiment 1 (5/29)<br />
'''[[File:UCB-Phage Delivery-140530-02.JPG]]'''<br />
::-Results from Experiment 2 (5/29)<br />
'''[[File:UCB-Phage Delivery-140530-03.JPG]]'''<br />
*Math<br />
::-If there are 5.00E+8 cells in 1mL of culture at OD of 1, then in 1mL of culture at OD of 0.59, there are 2.95E+8 cells. In a 50mL culture at OD of 0.59, there are 1.48E+10 cells.<br />
::-We added 3.3ul (0.0033mL) phage at concentration 4.62E+11 phage/mL which amounts to 1.52E+9 total phage<br />
::-Assuming that 1 phage infects 1 bacterium, we can assume that 1.52E+9 bacterial have the potential to be infected in the 50mL culture<br />
::-We plated 100ul of culture at various dilutions. If not diluted, the number of cells that can be potentially infected in 0.1mL equals 1.52E+9/500, or 3.05E+06 cells. We then accounted for the dilutions (for 1:1000 dilution, we divided 3.05E+06 by 1000 to receive 3.05E+03)<br />
::-The following table contains the number of cells with the potential to be infected assuming a 100% infectivity rate by M13 phage and that 1 cell is infected only once.<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Dilution<br />
! Potential infected cells<br />
! Colonies on Amp<br />
! % Potential (Amp)<br />
! Colonies on Kan<br />
! % Potential(Kan)<br />
! Kan:Amp<br />
<br />
|-<br />
| 1:1000<br />
| 1.52E+06<br />
| 476<br />
| 15.62%<br />
| 15<br />
| 0.820%<br />
| 1:19.04<br />
<br />
|-<br />
| 1:5000<br />
| 3.05E+05<br />
| 131<br />
| 21.49%<br />
| 13<br />
| 2.133%<br />
| 1:10.08<br />
<br />
|-<br />
| 1:10000<br />
| 1.52E+05<br />
| 93<br />
| 30.51%<br />
| 5<br />
| 1.640%<br />
| 1:18.60<br />
<br />
|-<br />
| 1:50000<br />
| 3.05E+04<br />
| 17<br />
| 44.29%<br />
| 0<br />
| 0.000%<br />
| <br />
<br />
|-<br />
|}<br />
<br />
*Conclusions from infections<br />
::-Results between and within the three trials are inconsistent. For example, the number of colonies received in experiments 1 and 2 from 5/29 differ greatly. Due to the differences in protocol, variation was expected but not to this extent. <br />
::-Our dilutions did not yield the expected 10 fold (or 5 fold) decrease in growth that was expected.<br />
::-Plates from 5/29 could be plated better to reduce dense areas of growth and growth around the rim.<br />
::-Though the experiment contained many errors we can say that the phagemid (Litmus 28i) is preferentially packaged compared to the helper phage (M13K07) but not to the degree we expected.<br />
::-Could receive increased occurrences of cells containing M13k07 due to infection, phage production, further infection<br />
<br />
==Week 5==<br />
<br />
'''6/2'''<br />
*Tested chemically competent cells through transformation<br />
::-Are cells contaminated?<br />
::-Are cells competent?<br />
*The samples for transformation<br />
{| class = "wikitable"<br />
|-<br />
! #<br />
! Cells (Tube label)<br />
! DNA (Tube label)<br />
! Resistance before transformation<br />
! Resistance after transformation<br />
<br />
|-<br />
| 1<br />
| K12 ER2738 5/20<br />
| p110+RBS (2) 4/23<br />
| Tet<br />
| Tet, Chlor<br />
<br />
|-<br />
| 2<br />
| BW (-f) 5/30<br />
| p110+RBS (2) 4/23<br />
| Kan<br />
| Kan, Chlor<br />
<br />
|-<br />
| 3<br />
| BW f-ep comp 5/22<br />
| p110+RBS (2) 4/23<br />
| Kan, Tet<br />
| Kan, Tet, Chlor<br />
<br />
|-<br />
| 4<br />
| BW (+f) 5/30<br />
| p110+RBS (2) 4/23<br />
| Kan, Tet<br />
| Kan, Tet, Chlor<br />
<br />
|-<br />
| 5<br />
| *BW23115 5/30<br />
| p110+RBS (2) 4/23<br />
| Kan<br />
| Kan, Chlor<br />
<br />
|-<br />
| 2B<br />
| K12 ER2738<br />
| 2B [from dis. kit]<br />
| Tet<br />
| Tet, Chlor<br />
<br />
|-<br />
| 2P<br />
| BW f-ep comp 5/22<br />
| 2P [from dis. kit]<br />
| Kan, Tet<br />
| Kan, Tet, Chlor<br />
<br />
|-<br />
|}<br />
<br />
'''6/3'''<br />
*Results from 6/2 Transformation<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Growth on Chlor<br />
! Growth on Kan<br />
! Growth on Kan+Tet<br />
! Growth on Amp<br />
<br />
|-<br />
| 1 N<br />
| X<br />
| X<br />
| X<br />
| X<br />
<br />
|-<br />
| 2 N<br />
| X<br />
| +<br />
| X<br />
| X<br />
<br />
|-<br />
| 3 N<br />
| X<br />
| +<br />
| +<br />
| X<br />
<br />
|-<br />
| 4 N<br />
| X<br />
| +<br />
| +<br />
| X<br />
<br />
|-<br />
| 5 N<br />
| X<br />
| +<br />
| X<br />
| X<br />
<br />
|-<br />
| 1<br />
| +<br />
| X<br />
| X<br />
| <br />
<br />
|-<br />
| 2<br />
| +<br />
| +<br />
| X<br />
| <br />
<br />
|-<br />
| 3<br />
| +<br />
| +<br />
| +<br />
| <br />
<br />
|-<br />
| 4<br />
| +<br />
| +<br />
| +<br />
| <br />
<br />
|-<br />
| 5<br />
| +<br />
| +<br />
| Many colonies but close to samp. 4<br />
| <br />
<br />
|-<br />
| 2P<br />
| + (~100)<br />
| +<br />
| +<br />
| <br />
<br />
|-<br />
| 1- JW<br />
| +<br />
| X<br />
| X<br />
| <br />
<br />
|-<br />
| 2- JW<br />
| +<br />
| +<br />
| X<br />
| <br />
<br />
|-<br />
| 3- JW<br />
| +<br />
| +<br />
| +<br />
| <br />
<br />
|-<br />
| 4- JW<br />
| +<br />
| +<br />
| +<br />
| <br />
<br />
|-<br />
| 5-JW<br />
| +<br />
| +<br />
| X<br />
| <br />
<br />
|-<br />
| 2B- JW<br />
| + (24)<br />
| X<br />
| X<br />
| <br />
<br />
|-<br />
|}<br />
*The transformations with DNA from the well (B2 and P2) had lower efficiencies than those with DNA from a mini-prep. Most likely this is due to the differences in DNA concentration (p110+RBS (2) 4/23 was at 254.4ng/ul)<br />
*Conclusions<br />
::-None of the competent cells were contaminated<br />
::-All of the competent cells are in fact, competent<br />
*Set up O/N of DH5-alpha cells to make competent tomorrow<br />
<br />
'''6/4'''<br />
*Isolation of single-stranded phagemid DNA using M13K07<br />
::-Added ER2738 colony to 50mL LB<br />
:::*Plate was cold. Next time warm plate before pricking<br />
::::*Best to use freshly grown plate<br />
::-After 4 hours, OD was at 0.02. Waited 45 minutes and OD was at 0.08. Therefore, we infected at OD 0.08<br />
::-Had started another culture when we did not think the first was growing. In incubator for about 1 hour. OD was 0.00. We infected anyway because last time it worked.<br />
::-Let infection proceed for 60 minutes then added 70ul of Kanamycin to be a final concentration of 70ug/mL<br />
*Primers came in to biobrick M13ori (packaging signal on Litmus28i)<br />
::-Resuspended primers and diluted 1:10<br />
<br />
'''6/5'''<br />
*Isolated single-stranded M13K07 DNA<br />
::-Final concentration = 5724 ng/ul (calculated from a 1:10 dilution)<br />
::-For second sample in pair, we resuspended it in TE but did not proceed to DNA extraction teps<br />
::-For the second culture we started 6/4, we resuspended pellet in TBS and glycerol to preserve the M13 phage. Measured absorbances (before glycerol was added)<br />
: #1<br />
::269 => 1.690A<br />
::320 => 0.103A<br />
: #2<br />
::269 => 1.453<br />
::320 => 0.059<br />
*For our first biobrick, we wanted to isolate the M13 origin, a segment ~500bp that allows for packaging into the M13 phage. We tried to achieve this by biobrick assembly and by Gibson Assembly.<br />
::-To biobrick M13 ori through biobrick assembly (the old-school way)<br />
:::PCR on Litmus 28i to amplify/biobrick M13ori<br />
::::*Used primers Gem003 F & R<br />
::::*Diluted Litmus 28i DNA 1:10<br />
:::Digestion of p11+RBS (1) to digest pSB1C3 bb with EcoRI-HF and PstI-HF<br />
:::Ran samples on gel and gel extracted pieces. We recieved very low yields (out of range for nano drop)<br />
::::*M13 ori: 4.0 ng/ul<br />
::::*pSB1C3: 1.8 ng/ul<br />
:::Digested M13 ori fragment despite poor extraction yield with EcoRI-HF and PstI-HF<br />
::::*Used 1.5x as much DNA as instructed based on inaccurate concentration<br />
'''[[File:UCB-Phage Delivery-140605.JPG]]'''<br />
::-Gel extracted red rectangles<br />
::-Ligation<br />
:::*10hr @ 16C, 10min @ 65C, 4ever @ 4C<br />
*To biobrick M13 ori through Gibson Assembly (the cool-kids way)<br />
::-PCR on Litmus 28i<br />
:::*Used primers Gem002 F & R<br />
:::*Diluted Litmus 28i DNA 1:10<br />
::-PCR on pSB1C3 (p11+RBS (1))<br />
:::*Used primers Gem001 F & R<br />
:::*Diluted pSB1C3 DNA 1:3<br />
<br />
'''6/6'''<br />
*Ran gel of PCR products from (6/5). Products will be used for Gibson Assembly<br />
::-Recieved bands for pSB1C3 around 2000bp and M13ori around 500bp<br />
::-No contamination in pSB1C3 PCR negative control<br />
::-Band in M13ori negative control that is the same size as sample. Contaminated by sample?<br />
::-Gel of PCR products #1 and #2 from 6/5<br />
'''[[File:UCB-Phage Delivery-140606.JPG]]'''<br />
:1. pSB1C3 with promoter+RBS as insert. Amplified with Gem001<br />
:2. No DNA control for Gem001<br />
:3. M13ori amplified with Gem002 from Litmus28i<br />
:4. No DNA control for Gem002<br />
*Gibson Assembly<br />
{| class = "wikitable"<br />
|-<br />
! Total Amount of Frag.<br />
! .02-.5pmol<br />
! 10 ul total<br />
<br />
|-<br />
| Gibson Assembly MM (2x)<br />
| 10 ul<br />
| 10<br />
<br />
|-<br />
| Dionized H2O<br />
| 10-x<br />
| <br />
<br />
|-<br />
|}<br />
::-Diluted pSB1C3 and M13ori PCR products 1:10<br />
::-Incubated 60min @ 50C<br />
::-Also used provided pUC16 as positive control<br />
*Transformation<br />
:1. p110+RBS Positive control<br />
:2. No DNA Negative control<br />
:3. Cas9 from distribution kit so we can have more<br />
:4. Thaw and refreeze cells Test competency of comp cells after thawed<br />
:5. Not chem comp cells Negative control for the above<br />
:6. Ligation Product<br />
:7. Gibson product<br />
::*7.2. Gibson product diluted 1:4<br />
:8. Gibson positive control<br />
::*7.2. Gibson positive control diluted 1:4<br />
::-For the Gibson product and the positive control, we transformed 2ul of product and 2ul of 1:4 diluted product. NEB recommends the first if using their competent cells and the second if using cells from other companies. Our cells are from NEB but we made them competent ourselves so we tried both ways<br />
:::*Plated on Chlor at concentrations of 170, 85, and 33 ug/mL<br />
*Primers came in<br />
::-Resuspended and made 1:10 dilutions<br />
<br />
'''6/7'''<br />
*Results from 6/6 transformation<br />
:1. Positive control<br />
::*Lots of growth, ~300-400 on 1:10 dilution<br />
:2. No DNA negative control<br />
::*No growth<br />
:3. Cas9 from distribution kit<br />
::*7 potential colonies (some are close to edges through) on non-diluted<br />
:4. Thawed then refroze cells<br />
::*Looks like (1)<br />
:5. Not chemically competent cells<br />
::*No growth<br />
:6. Ligation product<br />
::*13 potential colonies (some are close to edge)<br />
:7. Gibson Assembly Product<br />
::*170 -> No colonies<br />
::*85 -> No colonies<br />
::*33 -> 3 specks<br />
:7.2. Gibson Assembly product diluted 1:10<br />
::*170 -> 1 speck<br />
::*85 -> 3 colonies<br />
::*33 -> 13 colonies<br />
:8. Gibson positive control<br />
::*No colonies<br />
:8.2. Gibson positive control diluted 1:4<br />
::*No colonies<br />
*Made 6mL O/N cultures<br />
::-4 from (3) cas9 plate<br />
:::See Constitutive CRISPR notebook for more info on these samples<br />
::-7 from (6) Ligation product<br />
::-5 from (7.2 [85]) Diluted Gibson product on 85 ug/mL Chlor<br />
::-8 from (7.2 [33]) Diluted Gibson product on 33 ug/mL Chlor<br />
<br />
==Week 6==<br />
'''6/8'''<br />
*Check colonies for correct constructs.<br />
::-Mini-prepped all 24 O/Ns<br />
:::*Yielded low concentrations for samples 12, 15, 19, and 22<br />
::-Digested all with EcoRI and PstI (10ul reactions)<br />
::-Ran results on gel<br />
:::*All 4 cas9 samples had the expected bands of 2000 and 5000bp<br />
:::*All 7 ligation products have expected bands of 2000 and ~570bp<br />
:::*3 of 5 Gibson assemblies from 85ug/mL Chlor plate had expected bands of 2000 and ~500bp<br />
:::*3 of 8 Gibson assemblies from 33ug/mL Chlor plate had expected bands of 2000 and ~500bp<br />
'''[[File:UCB-Phage Delivery-140608-01.JPG]]'''<br />
<br />
'''[[File:UCB-Phage Delivery-140608-02.JPG]]'''<br />
::1-4: Cas9 from Stanford-Brown team<br />
::5-11: pSB1C3-M13ori cloned through ligation<br />
::12-24: pSB1C3-M13ori cloned through Gibson Assembly<br />
:::*12-16: Grown with 85 ug/mL Chlor<br />
:::*17-24: Grown with 33 ug/mL Chlor<br />
*Conclusions from gel<br />
::-We have cas9 safely in cells<br />
::-Our ligation reactions successfully yielded M13ori on pSB1C3<br />
::-Combined, we had a 46% success rate for the Gibson Assembly in yielding M13ori on pSB1C3<br />
:::*The 4 samples that had the lowest concentration after being mini-prepped (12,15, 19, and 22) correlate with samples that had the correct band pattern<br />
*We selected 4 samples from each (4 total between the two Gibson reactions) type<br />
::-For non-Gibson Assembled samples<br />
:::Plated 25ul on 170ug/mL Chlor<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Our usual method<br />
! Gibson Method<br />
<br />
|-<br />
| 1. Thaw on ice<br />
| 1. Thaw on ice <br />
<br />
|-<br />
| 2. Transfer 40ul cells to tube<br />
| 2. Transfer 50ul cell to tube<br />
<br />
|-<br />
| 3. Add DNA. 1ul for mini-prep OR up to 10ul for ligation<br />
| 3. Add 2ul to NEB cells OR 2ul of 1:4 diluted to other cells<br />
<br />
|-<br />
| 4. Mix by pipet Let sit 30min on ice<br />
| 4. Mix by pipet or flicking Let sit 30 min. on ice<br />
<br />
|-<br />
| 5. Heat shock: 42C for 45s<br />
| 5. Heat shock: 42C for 30s<br />
<br />
|-<br />
| 6. Ice for 5 minutes<br />
| 6. Ice for 2 min.<br />
<br />
|-<br />
| 7. Transfer to culture tube; Add 200ul SOC<br />
| 7. Add 950ul SOC to tube<br />
<br />
|-<br />
| 8. Shake or rotate for 60-120min at 37C<br />
| 8. Shake (250rpm) or rotate for 60 min. at 37C<br />
<br />
|-<br />
| 9.<br />
| 9. Warm plates to 37C<br />
<br />
|-<br />
| 10. Plate 100ul onto selection plate<br />
| 10. Plate 100ul onto plate<br />
<br />
|-<br />
| 11. Incubate O/N @ 37C<br />
| 11. Incubate O/N @ 37C<br />
<br />
|-<br />
|}<br />
::-Added 6mL LB and Chlor at concentration of 170ug/mL to grow O/N<br />
:*For Gibson Assembled samples<br />
::-Plated 25ul onto 170, 85, and 33ug/mL Chlor<br />
::-Samples from 85ng/mL plate<br />
:::Transferred 100ul to new tube, added media, added Chlor at 170ng/mL<br />
::-Samples from 33ng/mL plate<br />
:::Tranfered 100ul to new tubes, added media, added Chlor at 85ng/mL to one and 33ng/mL to the other<br />
:*Tomorrow, may send for sequencing and make freeze downs<br />
<br />
*Transformation<br />
:1. Positive control (p110+RBS diluted 1:10)<br />
:2. Non-diluted Gibson product<br />
:3. Gibson product diluted 1:4<br />
:4. Gibson product diluted 1:10<br />
::-Transformed each sample using our usual method and using the protocol given by Gibson<br />
<br />
::-Due to not have plates ready before transformation, in step 4, the samples sat for about 50 minutes. Then in step 8, they both recovered for about 150 minutes. Though not specified in our protocol, we did warm the plates to 37C. In step 10 for our protocol, since we only added 200ul SOC and wanted to plate on 3 selection plates (see below), we only plated 50ul (except for the positive control). <br />
::-Plated on three concentrations of Chloramphenicol (33 ug/mL, 85 ug/mL, and 170 ug/mL) to determine the differences in yield due to differences in concentration.Obvious hypothesis: more colonies will grow on plates that have a lower concentration of chlor.<br />
<br />
<br />
'''6/9'''<br />
*Made chemically competent 5alpha cells with Dan and Alex from main campus<br />
::-Waiting to hear results on competency<br />
*Will eventually make phage containing CRISPR-Cas9 that targets Kanamycin resistance. M13K07 has Kanamycin resistance so we need to switch the resistance on the M13 genes.<br />
::-PCR on pwp 2.po (plasmid that Sam gave us that contains the zeoR gene adjacent to ori) to amplify zeoR and ori. Zeo is on EM7 promoter<br />
:::-Primers: Gem008 R & R<br />
:::-Anneal temp from NEBuilder: 63.7C<br />
:::-Extension time: 90s<br />
:::-Expected band size on gel: 1300bp<br />
:::-Used phusion polymerase<br />
*PCR on M13K07 DNA to amplify M13 phage genes (also removes majority of M13 ori, all of KanR, and all of p15 ori)<br />
:::-Primers: Gem007 F & R<br />
:::-Anneal temp from NEBuilder: 60.2C<br />
:::-Extension time: 4:30<br />
:::-Expected band size on gel: about 6000bp<br />
:::-Used phusion polymerase<br />
*Freeze downs<br />
::-Note: Phagemid 1C3 was the original name for ‘pSB1C3-M13ori’<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Top Label<br />
! Side label<br />
! What?<br />
<br />
|-<br />
| Phagemid 1C3 6/9<br />
| From Lig<br />
| M13 ori inserted into 1C3 (biobricked); Done through ligation; Contains extra bases as spacer between biobrick prefrix/suffix and part for primer design<br />
<br />
|-<br />
| Phagemid 1C3 6/9<br />
| From Lig<br />
| “ “<br />
<br />
|-<br />
| Phagemid 1C3 6/9<br />
| Gibson<br />
| M13 ori inserted into 1C3 (biobricked); Done through Gibson cloning<br />
<br />
|-<br />
| Phagemid 1C3 6/9<br />
| Gibson<br />
| “ “<br />
<br />
|-<br />
|}<br />
'''6/10'''<br />
*Ran gel of PCRs from 6/9<br />
<br />
'''[[File:UCB-Phage Delivery-140610.JPG]]'''<br />
:1. Amplification of M13 genes from M13K07 (~6000bp)<br />
:2. No DNA control for (1) amplification<br />
:3. Amplification of Zeo resistance gene + plasmid ori (~1300bp)<br />
:4. No DNA control for (3) amplification<br />
*Gibson Assembly of above parts (did not gel extract)<br />
::-Diluted the PCR products 1:10 then added 3ul of M13K07 genes product and 7ul of ZeoR+ori product<br />
::-Incubated at 50C for 60 min.<br />
::-Transformed Gibson Assembly product into 5alpha cells<br />
:::*Used our usual protocol<br />
:::*Add 2ul of DNA<br />
::::-In one sample, diluted DNA 1:4 and in the other, we diluted DNA 1:10<br />
*Started Phage Amplification Protocol<br />
::-ER2738 transformed with Litmus 28i<br />
:::*Grew for ~2.5hr before reaching an OD of 0.04<br />
::-ER2738 transformed with pSB1C3-M13ori (M13ori on pSB1C3)<br />
:::*Grew for ~ 3.5hr before reaching an OD of 0.01, then in the next 1.5 hours, spiked to 0.19<br />
:::*We gave up and went home, and will restart tomorrow<br />
*Analyzed transformation results from 6/8<br />
<br />
'''6/11'''<br />
*Made chemically competent 5alpha cells<br />
*Restarted Phage Amplification Protocol<br />
::-Forgot to add phagemid antibiotic at start of growth. Added phagemid antibiotic when we added phage. Incubated for 90 minutes before adding Kanamycin (to select for cells that were infected by M13K07)<br />
::-Phage is at concentration 4.57x10^12 phage/mL<br />
:::Protocol calls for final concentration of 1 x10^8 phage/mL<br />
::::*(4.57 x10^12)*V = (1 x10^8)(50mL)<br />
::::*V = 0.00109mL<br />
:::Added 1.1ul of phage<br />
*Transformations<br />
{| class="wikitable"<br />
|-<br />
!DNA<br />
!Plate Selection<br />
|-<br />
|No DNA control<br />
|(all)<br />
|-<br />
|Positive control (p110+RBS)<br />
|(C)<br />
|-<br />
|M13 genes + ZeoR ori<br />
|(Z)<br />
|-<br />
|M13 genes +ZeoR ori diluted 1:4<br />
|(z)<br />
|} <br />
<br />
'''6/12'''<br />
*Transformation Results from 6/10 [Took ~36 hours to be clearly visible]<br />
::No DNA control<br />
:::-Amp: 0 <br />
:::-Zeo (50ug/mL): 200 colonies<br />
::M13 genes + ZeoR ori (1:4 dilution) <br />
:::-Zeo (25ug/mL): 300<br />
:::-Zeo (50ug/mL): 200<br />
:::-Zeo (100ug/mL): 100<br />
::M13 genes + ZeoR ori (1:10 dilution) <br />
:::-Zeo (25ug/mL): 300<br />
:::-Zeo (50ug/mL): 150<br />
:::-Zeo (100ug/mL): 150<br />
*Transformation Results for 6/11<br />
::-Positive (p110+RBS) on Chloramphenicol: 500 colonies<br />
::-No DNA on Amp: 0 colonies Zeo (100 ug/mL): specks<br />
::-No apparent growth on any other plate<br />
:::*Realized later that we grew our samples on the wrong plates. Will repeat transformation today<br />
*Transformation #1<br />
::This morning there were no colonies on positive (p110+RBS) coltrol from 6/11 even though we observed fast growth in the past. Without waiting for colonies to appear, we started a control transformation<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Diluted<br />
! Time at 42C<br />
<br />
|-<br />
| p110+RBS<br />
| No<br />
| 45s<br />
<br />
|-<br />
| p110+RBS<br />
| 1:10<br />
| 45s<br />
<br />
|-<br />
| p110+RBS<br />
| 1:10<br />
| 30s<br />
<br />
|-<br />
| p110+RBS<br />
| 1:10<br />
| No timer. ~43s<br />
<br />
|-<br />
| No DNA control<br />
| No<br />
| 45<br />
<br />
|-<br />
|}<br />
*Because ‘No DNA control 6/10’ yielded colonies, we researched Zeocin plates<br />
::-According to Life Technologies (Invitrogen), Zeocin requires low salt medium and a pH of 7.5<br />
::-Low Salt LB Medium (1L)<br />
:::*Ingredients<br />
::::10g Tryptone<br />
::::5g NaCl<br />
::::5g Yeast Extract<br />
:::-Mix ingredients<br />
:::-Adjust pH to 7.5 using NaOH (If go over, use HCl)<br />
:::-Add agar for plates at 15g/L. Autoclave<br />
:::-Thaw Zeocin on ice. Vortex<br />
:::-Add Zeocin to final concentration of 25ug/mL<br />
*Transformation #2<br />
::-Repeat of transformation on 6/11 but this time we will plate on the correct plates<br />
::-Also remade Zeocin plates<br />
*Finished isolation of M13 Litmus phage and M13 pSB1C3-M13ori phage<br />
::*Note: Phagemid 1C3 was the original name for ‘pSB1C3-M13ori’<br />
{| class = "wikitable"<br />
|-<br />
! Phage Sample<br />
! A269<br />
! A320<br />
! Concentration (phage/mL)<br />
<br />
|-<br />
| Litmus phage (1)<br />
| 0.181<br />
| 0.034<br />
| 3.12 x10^12<br />
<br />
|-<br />
| Litmus phage (2)<br />
| 0.227<br />
| 0.047<br />
| 3.83 x10^12<br />
<br />
|-<br />
| Phagemid 1C3 phage (1)<br />
| 0.101<br />
| 0.020<br />
| 1.87 x10^12<br />
<br />
|-<br />
| Phagemid 1C3 phage (2)<br />
| 0.126<br />
| 0.021<br />
| 2.42 x10^12<br />
<br />
|-<br />
|}<br />
::::phage/mL = 6x10^16 x (A269-A320)/ (#of base pairs in the phage genome)<br />
*Set up 50mL O/N of K12 ER2738 (containing f-episome) for infection tomorrow with Litmus phage and pSB1C3-M13ori phage<br />
<br />
<br />
'''6/13'''<br />
*Transformation Results for 6/11<br />
::-Several hundred colonies on Positive Control (p110+RBS) on Chlor<br />
::-No colonies for GA positive control on Amp<br />
::-No colonies for M13ori + ZeoR mistakenly plated on Amp<br />
::-No colonies for No DNA control on Amp<br />
::-100-ish colonies for No DNA control on Zeo<br />
::-100-ish colonies for cas9+AmpR+gRNA mistakenly plated on Zeo<br />
::-These last 3 points suggest/confirm that Zeo plates are no good<br />
*Transformation Results from 6/12 control test<br />
::-Colonies grew is about equal amounts on all plates, including No DNA control<br />
:::Either plates don’t contain Chlor or competent cells are contaminated<br />
::-Streaked competent cells onto new and old Chlor plates<br />
*Infection test of ER2738: Is Litmus preferentially packaged over M13K07 Helper phage? Is pSB1C3-M13ori preferentially packaged?<br />
::-Infect ER2738 with phage produced 6/12<br />
:::*Phage should have packaged Litmus 28i phagemid or pSB1C3-M13ori<br />
:::*Cells infected with phage packaging Litmus 28i will grow on Amp<br />
:::*Cells infected with phage packaging pSB1C3-M13ori will grow on Chlor<br />
:::*Cells infected with phage packaging M13K07 will grow on Kan<br />
::-After we plate, we can count the colonies and calculate a ratio of Litmus28i: M13K07 or pSB1C3-M13ori:M13K07 packaging<br />
<br />
'''6/14'''<br />
*Results of contamination test (streaked competent cells onto new and old Chlor plates)<br />
::-Colonies grew in low amounts on both plates. most likely the cells are contaminated<br />
*Transformation results for 6/12<br />
::-Many colonies for No DNA control on Zeo<br />
:::-Some colonies are turning pinkish-red<br />
::-Many colonies for diluted and non-diluted M13 genes+ZeoR on Zeo<br />
:::-Some colonies are turning pinkish-red<br />
*Could the white colonies be the designed colonies and can we kill the red colonies with Zeocin before killing the white colonies (aka. Use high Zeocin concentrations to select for the correct construct)<br />
::-Selected 1 red colony from No DNA Zeo control plate and 1 white colony from M13genes+ZeoR sample plate<br />
::-Added 100ul H2O then divided amongst 5 culture tubes each (with 5mL of low-salt LB, pH 7.5)<br />
::-Then added Zeocin to a final concentration of: 0, 25, 50, 75, 100ug/mL.<br />
*Results of 6/13 infection<br />
::-Litmus 28i infected cells<br />
:::*on Kan: Individual colonies for 1:10, 1:100, and 1:1000. No colonies on 1:5000<br />
:::*on Amp: lawn for 1:10, 1:100, and near lawn for 1:1000. Single colonies for 1:5000<br />
::-pSB1C3-M13ori infected cells<br />
:::*on Kan: Same as Litmus 28i samples on Kan<br />
:::*on Chlor: Lawn for all dilutions. Must discredit due to recent Chlor contamination<br />
<br />
==Week 7==<br />
<br />
'''6/15'''<br />
*Results of Zeocin experiment on 6/14<br />
::-Healthy growth for both at 0ug/mL Zeo<br />
::-No growth for white colony with any Zeo<br />
::-Good growth for red colony even at 100ug/mL Zeo<br />
::-This suggests that the red colonies are naturally resistant to Zeocin. Also, our plates must not contain active Neocin. We are sure that we are adding enough. Possibly, we add it while the media is too hot or leave the plates at room temperature (decondensing) for too long and this deactivates the antibiotic? Perhaps we are not attaining the correct pH<br />
*In light of recent contamination problems on both Chlor and Zeo, we made new competent cells<br />
::-5alpha<br />
::-BW23115<br />
::-BW23115 conjugated (contains f-episome)<br />
<br />
'''6/16'''<br />
*Made new Chlor antibiotic. Made to Chlor plates<br />
*Contamination Test: Streaked Zeo and new Chlor plates each with<br />
::-Colony from Zeo contaminated plate<br />
::-Colony from Chlor contaminated plate<br />
::-Non-transformed OLD chemically competent cells<br />
::-Non-transformed NEW chemically competent cells<br />
*Redid bacterial infection<br />
::-For both samples, we used ER2738 (not from the same ‘O/N’ (also, not a real O/N))<br />
::-Infected one sample with isolated ‘Litmus28i phage’ and one with ‘pSB1C3-M13ori’<br />
::-Alterations to protocol<br />
:::*Did not started from saturated O/N. Started each with a colony, waited several hours until at OD ~1. We then added these cells to fresh 50mL LB to have an OD of 0.1.<br />
:::*Missed the 30 minute infection mark. Infected for ~45 minutes.<br />
::-Plated on Kan and Amp (Litmus28i sample) or Chlor (pSB1C3-M13ori sample) at dilutions of<br />
:::*1:100, 1:1k, 1:10k, and 1:100k<br />
<br />
'''6/17'''<br />
*Q5 PCR to replace KanR with ZeoR in M13K07<br />
::-Used recommendations<br />
::-Unfortunately, I (Jo) don’t know the difference between tightening and loosening the thermocycler lid; therefore, our M13K07 sample to amplify the M13K07 genes evaporated. But that’s ok, because we were sick of Zeocin anyway and decided mid-PCR to not waste our time with a Gibson Assembly and transformation. Instead we will be using the Old-School method of digestion and ligation because let’s face it, it’s a classic (and Gibson sucks) =)<br />
*Results from infection Test<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Litmus 28i<br />
! 1:100<br />
! 1:1k<br />
! 1:10k<br />
! 1:100k<br />
<br />
|-<br />
| Amp<br />
| ~6000?<br />
| ~2056<br />
| 377<br />
| 36<br />
<br />
|-<br />
| Kan<br />
| 130<br />
| 8<br />
| 3<br />
| 0<br />
<br />
|-<br />
|}<br />
{| class = "wikitable"<br />
|-<br />
! pSB1C3-M13ori<br />
! 1:100<br />
! 1:1k<br />
! 1:10k<br />
! 1:100k<br />
<br />
|-<br />
| Chlor<br />
| 8<br />
| 1<br />
| 0<br />
| 0<br />
<br />
|-<br />
| Kan<br />
| 97<br />
| 9<br />
| 0<br />
| 0<br />
<br />
|-<br />
|}<br />
::-We consider the Litmus 28i to have been a success. The pSB1C3-M13ori …. not so much. Looking back at the Litmus28i and the M13K07, we noticed that the M13ori is facing the opposite direction as the plasmid ori. Ours faces the same direction as the plasmid ori. Because we are working with phagemids that are single stranded, we think that by flipping the M13ori, we may be able to recover functionality. We will also look into other reasons.<br />
*Results from contamination test (6/16)<br />
::-Old Chlor plate (6/12)<br />
:::-Non-transformed chemically competent cells<br />
::::*Old: some colonies<br />
::::*New: No colonies<br />
::-New Chlor plate (6/16)<br />
:::-Colony from<br />
::::*Zeo contaminated plate: No growth<br />
::::*Chlor contaminated plate: Much growth<br />
:::-Non-transformed chemically competent cells<br />
::::*Old: some colonies<br />
::::*New: No growth<br />
::-Zeo (25ug/mL, Low NaCl, pH 7.5) (6/12)<br />
:::-Colony from<br />
::::*Zeo contaminated plate: Much growth<br />
::::*Chlor contaminated plate: No growth<br />
:::-Non-transformed chemically competent cells<br />
::::*Old: some specks<br />
::::*New: some specks<br />
<br />
'''6/18'''<br />
*Ordered primers to…<br />
::-biobrick M13ori (in other direction)<br />
::-biobrick M13K07 genes<br />
'''6/19'''<br />
*Waited for primers<br />
*Set up O/N cultures to test last infection (6/16) for colonies containing pSB1C3-M13ori<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample #<br />
! Selection<br />
! Dilution<br />
! Presumably<br />
! Notes<br />
<br />
|-<br />
| 1-8<br />
| Chlor<br />
| 1:100<br />
| pSB1C3-M13ori<br />
| <br />
<br />
|-<br />
| 9<br />
| Chlor<br />
| 1:1k<br />
| pSB1C3-M13ori<br />
| <br />
<br />
|-<br />
| 10-14<br />
| Chlor<br />
| 1:1k<br />
| pSB1C3-M13ori<br />
| These colonies were not present on plate during initial counting on 6/17<br />
<br />
|-<br />
| 15-16<br />
| Kan<br />
| 1:1k<br />
| M13K07<br />
| <br />
<br />
|-<br />
|}<br />
:::-Selection and dilution refer to the plate. Cells were then grown under selection.<br />
:::-More colonies were seen on all Chlor plates. No new colonies appeared on Kan plates<br />
*Tomorrow, we will mini-prep, digest, and run the samples on a gel to verify gene transfer.<br />
<br />
'''6/20'''<br />
*Primers are in!<br />
*Cloning of pSB1C3-M13ori(New)<br />
::-PCR to amplify M13ori (packaging signal) from Litmus 28i to be in the other direction<br />
:::-Primers (Gem011 F&R) add cut sites to make part biobrick compatible<br />
:::-Was able to gel extract<br />
::::*Band at the same size as sample….. contaminated primers?<br />
:::-Then digested with E and P<br />
::-Digested pSB1C3 plasmid with E and P to linearize backbone<br />
::::*Was able to gel extract<br />
'''[[File:UCB-Phage Delivery-140620.JPG]]'''<br />
<br />
:1. Spill over from 2<br />
:2 and 3. pSB1C3 digested with EcoRI and PstI<br />
::-Insert <100bp so cannot be seen<br />
:4. M13ori<br />
*Ligation of M13ori (packaging signal) into pSB1C3<br />
::10h at 16C<br />
::20m at 80C<br />
::Hold at 4C<br />
<br />
*PCR to amplify M13K07 genes from M13K07 DNA isolated from phage<br />
::-Primers (Gem012 F&R) add cut sites to make part biobrick compatible<br />
::-Very light bands and not band might correlate with 6000pb but not enough resolution on gel to be certain. Bands were too light so did not extract<br />
::-Set up PCR again using Q5 to run O/N<br />
:::*Received only a feint, smudgy band that was too large. We did not bother with extracting the DNA<br />
*Test of 6/19 O/Ns (14 that are presumably pSB1C3-M13ori and 2 that are presumably M13K07)<br />
::-Mini-prepped all O/Ns<br />
::-Digested all with Pst-I (common RE between pSB1C3-M13ori and M13K07)<br />
:::-Even though these are phagemids, we assumed that because they were still in the cell, the plasmids were still double stranded so could be recognized by RE. Our assumption was valid<br />
:::-Results of digestion<br />
::::*1,2,4-14: all had bands that corresponded to pSB1C3-M13ori cut once<br />
::::*3: Slight band at expected size but very feint<br />
::::*15, 16: as expected, they contain bands of ~9000bp, correlating with the M13K07 phagemid. Interestingly, sample 15 also contained a (brighter) band that corresponds with pSB1C3-M13ori. We assume that this was an incident of double infection (chance or did this occur at high frequency?)<br />
'''[[File:UCB-Phage Delivery-140620-02.JPG]]'''<br />
::Check table from 6/19 for more details<br />
:::-1-14: pSB1C3-M13ori<br />
:::-15-16: M13K07 from 1:1k diluted plate<br />
<br />
'''6/21'''<br />
*Needed to transform our pSB1C3-M13ori(New) (6/20) into cells. First, we transformed into 5alpha cells; however, we need to infect these cells later in order to make phage. Our 5alphas are not competent so we repeated the transformation, this time using ER2738 cells which contain the F’ episome, allowing us to infect with the M13 phage.<br />
::-Also transformed sample #1 from 6/20 mini-preps (pSB1C3-M13ori(Old) into ER2738 cells. Do not see any notes about this plasmid being in infectable cells during the initial experiment. Reason for experiment failure???<br />
*Ran 6/20 O/N PCR (to amplify M13K07 genes) on gel (also re-ran the previous sample from the day with more DNA)<br />
::-Still, band looks too big. Brightest band still occurs between 8000 and 9000bp. Lighter band around 7000bp-- might be ~6000pb but too light to tell<br />
*Alternative plan for amplifying M13K07 genes<br />
::-Digest sample with PstI (cuts outside of the target region)<br />
'''[[File:UCB-Phage Delivery-140621.JPG]]'''<br />
::Used sample #16 from 6/20 b/c it is double stranded so will cut<br />
:-PCR amplified the linearized digestion product<br />
:::1. PCR of M13genes<br />
:::2. PCR of M13genes + DMSO<br />
::::Gel extracted pieces boxed in red<br />
::-Used primers Gem012 F&R<br />
::-Gel extracted piece but received very low yield<br />
*Alternative to alternative plan for amplifying M13K07 genes<br />
::-Digested with PstI and AgeI<br />
'''[[File:UCB-Phage Delivery-140621-02.JPG]]'''<br />
:1. Cut with PstI and AgeI<br />
:2. Uncut<br />
:3. Cut with PstI<br />
:4. Just PCR<br />
::-Gel extracted ~6000pb band after double digestion<br />
::-PCR (Phusion)<br />
:::-Digestion (A+P) product<br />
:::-Gel extraction product<br />
:::-No DNA control<br />
<br />
==Week 8==<br />
<br />
'''6/22'''<br />
*Did receive colonies from 6/21 transformations. Selected colonies for overnight<br />
{|class="wikitable"<br />
|-<br />
!number<br />
!colonies<br />
|-<br />
|1-7<br />
|pSB1C3-M13ori(NEW) in 5alpha<br />
|-<br />
|8-14<br />
|pSB1C3-M13ori(NEW) in ER2738<br />
|-<br />
|15-18<br />
|pSB1C3-M13ori(OLD) in ER2738<br />
|-<br />
|19<br />
|p110+RBS in 5alpha as control<br />
|-<br />
|20<br />
|p110+RBS in ER2738 as control<br />
|}<br />
<br />
*Gel of 6/21 O/N PCR<br />
::Digestion (AgeI+PstI) product ? Yielded the three bands that appear with every PCR of M13K07<br />
'''[[File:UCB-Phage Delivery-140622.JPG]]'''<br />
<br />
:1. M13K07-> digested (AgeI + PstI) -> PCR<br />
:2. M13K07-> digested (AgeI+ PstI) -> extraction-> PCR<br />
:3. No DNA control for PCR<br />
::-Gel extraction product ? No bands<br />
::-No DNA control ? No bands<br />
*PCR purified the above PCR (Digestion (AgeI+PstI) product) and PCR from 6/21<br />
*Digested PCR purified sample with EcoRI+PstI and ran gel<br />
'''[[File:UCB-Phage Delivery-140622-02.JPG]]'''<br />
:1. PCR-> PCR purified<br />
:2. Sample after DpnI digest<br />
:3. Sample after digestion with EcoRI and PstI<br />
:4. Samples after digestion with DpnI then EcoRI and PstI<br />
*Ligation of above digestion (M13K07 genes) with pSB1C3<br />
<br />
<br />
'''6/23'''<br />
*Transformed pSB1C3-M13ori(New) (6/22 ligation)<br />
*Check overnights from 6/22 for the correct insert<br />
::-Mini-prep O/Ns<br />
::-Digested with EcoRI and PstI<br />
'''[[File:UCB-Phage Delivery-140622-03.JPG]]'''<br />
:::1-7: pSB1C3-M13ori(New) in 5alpha<br />
:::8-14: pSB1C3-M13ori(New) in ER2738<br />
:::15-18: pSB1C3-M13ori(Old)<br />
::-All were the expected size (though gel wiggled)<br />
::-Sent 4 samples for sequencing (iGEM primers: VF2 and VR)<br />
:::# 4: Divergent (new) phagemid 1C3 transformed into 5alpha<br />
:::# 11, 13: Divergent (new) phagemid 1C3 transformed into ER2738<br />
:::# 16: Convergent (old) phagemid 1C3 transformed into ER2738<br />
<br />
'''6/24'''<br />
*Results of 6/23 transformation (pSB1C3-M13genes) from 6/22 ligation)<br />
::-Received ~100 colonies<br />
::-Set up O/N cultures for 8 of the colonies<br />
::-Time passed…..<br />
::-Mini-prepped the 8 O/N samples mentioned above (yes, it was a long day)<br />
:::*Digested samples (E+P)<br />
*Started phage amplification protocol<br />
::-Amount of phage added = 10.9ul of 1:10 diluted phage (M13 phage (1) from 5/24 (4.575 x10^12 phage/mL)) to a final concentration of 1x10^8 phage/mL in 50mL<br />
::-Managed to get both samples to the 14-28hr incubation<br />
<br />
'''6/25'''<br />
*Checked mini-prep samples from 6/24 (pSB1C3-M13genes)<br />
::Ran the digestion overnight (6/24 to 6/25)<br />
'''[[File:UCB-Phage Delivery-140625.JPG]]'''<br />
::-All contained a band at 2000bp. Mostly empty vector or contained small, light band. Sample 1 had prominent band at ~1250bp.<br />
::-None of samples contained M13genes<br />
*M13K07 is on P15A ori (10-12 copy number) whereas the pSB1C3 ori is on pUC19 (500-700 copies). It’s possible that this overexpression is detrimental to cell<br />
::Alternative low copy plasmids found in distribution kit<br />
:::*2013 (plate 5)<br />
::::pSB6A1 (1K)<br />
::::pSB3C5 (3C)<br />
::::pSB3K3 (5E)<br />
:::*2014 (plate 4)<br />
::::pSB3C5 (4D)<br />
::::pSB6A1 (2L)<br />
::-Suspended and transformed the above plasmids into 5alphas<br />
::-Next, we will select colonies, mini-prep, digest, gel extract, ligate with M13genes<br />
*Started phage amplification protocol <br />
::-Phagemids in ER2738<br />
:::pSB1C3-M13ori(New)<br />
:::pSB1C3-M13ori(Old) <br />
::-Set up O/N of ER2738 for infection tomorrow<br />
<br />
'''6/26'''<br />
*Results of transformation of plasmids from the distribution kit<br />
::-Only received colonies pSB6A1 (2L from 2014) and pSB3C5 (4D from 2014)<br />
::-Pricked colonies for O/N<br />
*PCR of M13 genes so we can ligate it into the above backbones tomorrow<br />
::-DNA: M13K07 digested with EcoRI+PstI (6/21)<br />
::-Primers: Gem012 F & R<br />
*Finished phage amplification protocol<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Samples<br />
! A269<br />
! A320<br />
! Concentration<br />
<br />
|-<br />
| Old phagemid 1C3 (1)<br />
| 0.832<br />
| 0.492<br />
| 7.828 x10^12<br />
<br />
|-<br />
| Old phagemid 1C3 (2)<br />
| 0.324<br />
| 0.083<br />
| 5.549 x10^12<br />
<br />
|-<br />
| New phagemid 1C3 (1)<br />
| 0.391<br />
| 0.077<br />
| 7.229 x10^12<br />
<br />
|-<br />
| New phagemid 1C3 (2)<br />
| 0.402<br />
| 0.639<br />
| -5.457 x10^12<br />
<br />
|-<br />
|}<br />
::::*While resuspending “New phagemid 1C3 (2)”, the tip fell off and we lost half of sample. Evidently, we lost most of phage so we tossed sample<br />
::-Prepped and Infected ER2738 with “Old phagemid 1C3 (1)” and “New phagemid 1C3 (2)”<br />
:::Added 1.6ul of 1:10 diluted “Old phagemid 1C3 (1) to ER2738 cells<br />
:::Added 1.8ul of 1:10 diluted “New phagemid 1C3 (1) to ER2738 cells<br />
::-Plated the infected cells at dilutions<br />
:::1:1 onto Chlor+Kan plates<br />
:::1:10 onto Chlor+Kan plates<br />
:::1:100 onto Chlor and onto Kan plates<br />
:::1:1000 onto Chlor and onto Kan plates<br />
:::1:10000 onto Chlor and onto Kan plates<br />
:::1:100000 onto Chlor and onto Kan plates<br />
<br />
'''6/27'''<br />
*To ligate M13genes onto different backbones<br />
::-Mini-prepped the O/Ns from 6/26 to get backbones with low copy number<br />
:::pSB6A1<br />
:::pSB3C5 <br />
::::*did not grow as well<br />
::-PCR purified PCR (to amplify M13 genes with Gem012 F & R) from 6/26<br />
::-Digestions (50ul)<br />
:::Digest pSB6A1 with E + P<br />
:::Digest pSB3C5 with E + P<br />
:::Digest PCR purification product with E + P<br />
'''[[File:UCB-Phage Delivery-140627.JPG]]'''<br />
:1. pSB6A1<br />
:2. pSB3C5<br />
:3. M13genes<br />
::-Gel extraction of the above digestions<br />
:::For each: Added 10ul of 6x loading dye to 50ul digestions and divided the total volume between 2 wells<br />
::-Ligation<br />
:::1) pSB6A1-M13genes<br />
:::2) pSB3C5-M13genes<br />
*Results from 6/26 infection (after 22hrs in incubator)<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Anti-Sense Phagemid 1C3<br />
! 1:100<br />
! 1:1000<br />
! 1:10000<br />
! 1:100000<br />
<br />
|-<br />
| Chlor<br />
| 85<br />
| 6<br />
| 2<br />
| 0<br />
<br />
|-<br />
| Kan<br />
| 405<br />
| 13<br />
| 2<br />
| 1<br />
<br />
|-<br />
|}<br />
{| class = "wikitable"<br />
|-<br />
! Sense Phagemid1C3<br />
! 1:100<br />
! 1:1000<br />
! 1:10000<br />
! 1:100000<br />
<br />
|-<br />
| Chlor<br />
| 121<br />
| 24<br />
| 3<br />
| 0<br />
<br />
|-<br />
| Kan<br />
| 25<br />
| 1<br />
| 0<br />
| 0<br />
<br />
|-<br />
|}<br />
-We also plated the infected cells on plates containing Chlor and Kan to test for the possibility of double infection<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Phagemid 1C3<br />
! 1:1<br />
! 1:10<br />
<br />
|-<br />
| Anti-Sense<br />
| 59<br />
| 0<br />
<br />
|-<br />
| Sense<br />
| 4<br />
| 0<br />
<br />
|-<br />
|}<br />
<br />
<br />
'''6/28'''<br />
*We noticed that there were more colonies on our infection plates from 6/26 than on 6/27; therefore, we recounted colonies<br />
::-No increase of colonies on Kan plates<br />
::-Significant increase of colonies on Chlor plates<br />
::-Sense refers to the first phagemid 1C3 where the M13ori is in the sense direction compared to the plasmid ori<br />
::-Anti-Sense refers to the new phagemid 1C3 where the M13ori is in the anti-sense direction compared to the plasmid ori<br />
'''[[File:UCB-Phage Delivery-140628.JPG]]'''<br />
::Numbers on the left are after 22 hours. Numbers on the right are after 38.75 hours<br />
*Results of transformation from 6/27 (pSB6A1-M13genes and pSB3C5-M13genes)<br />
::-We have many colonies. Unfortunately, some are red, suggesting that original insert (J04450) was not successfully separated from backbone through gel extraction<br />
::-Selected colonies to grow overnight in 5mL LB<br />
<br />
==Week 9==<br />
<br />
'''6/29'''<br />
*Check O/N cultures for correct constructs (pSB6A1-M13genes and pSB3C5-M13genes)<br />
::-Mini-prep samples<br />
::-Digested with EcoRI and PstI to check insert sizes<br />
::-Gel<br />
'''[[File:UCB-Phage Delivery-140629.JPG]]'''<br />
::lanes….<br />
:::1-11: pSB6A1-M13genes<br />
:::12-18: pSB3C5-M13genes<br />
::-Epic failure<br />
::-All pSB6A1 backbones are empty<br />
::-Half of pSB3C5 backbones were empty. The others contained random inserts (1700 OR 3500). We don’t know what these inserts are. 1700 band is likely the digestion product that appears when we digest M13genes<br />
*Question: Can we use empty vectors from these mini-preps as ligation vectors?<br />
::-Digest pSB6A1 mini-prep with…. (see if both cut sites were retained during re-ligation)<br />
:::no enzyme<br />
:::EcoRI<br />
:::PstI<br />
:::EcoRI + PstI<br />
'''[[File:UCB-Phage Delivery-140629-02.JPG]]'''<br />
::lanes....<br />
:::1-2: last two samples of pSB3C5-M13genes from above gel<br />
:::3. Uncut<br />
:::4. Cut with EcoRI<br />
:::5. Cut with PstI<br />
:::6. Cut with EcoRI and PstI<br />
:-Appears that two backbones are ligated together<br />
<br />
'''6/30'''<br />
*Made chemically competent ER2738 cells that contain Litmus28i DNA<br />
*To amplify M13genes in order to retry ligation<br />
::-PCR of M13K07 DNA (diluted 1:100) to amplify the M13 genes<br />
::-PCR purify PCR product<br />
::-Run on gel: PCR purification, PCR, noDNAcontrol<br />
:::*PCR and purification showed bands at ~9k; therefore, did not get product<br />
:::*No DNA control was clean<br />
*Made freeze downs of<br />
::pSB3C5-J04450<br />
::pSB6A1-J04450<br />
::‘empty’ pSB3C5<br />
::‘empty’ pSB6A1<br />
<br />
'''7/1'''<br />
*Digestion of PCR product from 6/30 to figure out where mistake is<br />
:-Not really useful<br />
'''[[File:UCB-Phage Delivery-140701.JPG]]'''<br />
:Lanes...<br />
:1. PCR pur -> digested with AgeI<br />
:2. PCR pur -> digested with NgoMIV<br />
:3. PCR pur -> digested with PstI<br />
:4. PCR pur -> uncut<br />
:5. PCR pur -> digested with dpnI<br />
:6. Uncut plasmid DNA<br />
*Talked to Mary: She says we were adding to much DNA<br />
::-PCR again. Used 1:100 dilution of 1:100 diluted M13K07 DNA. (aka 1:10000 dilution)<br />
'''[[File:UCB-Phage Delivery-140701-02.JPG]]'''<br />
*Received a beautiful band at 6k bp<br />
:lanes<br />
:1. 1:100 dilution<br />
:2. 1:1000 dilution<br />
:3. 1:10 000 dilution<br />
:4. No DNA control<br />
<br />
'''7/2'''<br />
*To biobrick M13 genes (pSB6A1-M13genes and pSB3C5-M13genes)<br />
::-PCR purified 7/1 PCR product (Primers = Gem012)<br />
::-Digested with EcoRI-HF and PstI-HF<br />
::-Ligation to<br />
::::pSBA61 (digested and gel extracted)<br />
::::pSB3C5 (digested and gel extracted)<br />
<br />
'''7/3'''<br />
*Transformation<br />
::-pSB6A1-M13genes into 5alpha cells and ER2738 with pSB1C3-M13ori<br />
::-pSB3C5-M13genes into 5alpha cells and ER2738 with Litmus28i<br />
::-Transformed into cells containing a phagemid in order to skip some steps<br />
<br />
'''7/3-7/7 Vacation!'''<br />
<br />
==Week 10==<br />
<br />
'''7/7'''<br />
*Made O/N cultures of 7/3 transformation colonies<br />
::-Transformation results were not recorded until 7/8 (see below)<br />
<br />
'''7/8'''<br />
*Transformation results ( CC = Chemically comp cells, ER = ER2738)<br />
::-No growth on No DNA controls<br />
:::ER-Litmus28i CC on AMP-Chlor-Tet<br />
:::ER-phagemid1C3 CC on AMP-Chlor-Tet <br />
:::5alpha CC on Amp <br />
:::5alpha CC on Chlor <br />
::-Lots of red colonies on positive controls, no white colonies<br />
:::ER-Litmus28i CC + pSB3C5 on AMP-Chlor-Tet<br />
:::ER-phagemid1C3 CC + pSB6A1 on AMP-Chlor-Tet<br />
::-Lots of white colonies on sample plates, some red colonies<br />
:::ER-Lit CC + M13genes-pSB3C5 on AMP-Chlor-Tet<br />
:::ER-phagemid1C3 CC + M13genes-pSB6A1 on AMP-Chlor-Tet<br />
:::5alpha CC + M13genes-pSB3C5 on Chlor<br />
:::5alpha CC + M13genes-pSB6A1 on Amp<br />
*Overnights from 7/7 look healthy<br />
::-Renamed O/N to have numbers instead of long names<br />
{| class = "wikitable"<br />
|-<br />
! Sample #<br />
! Cells<br />
! DNA<br />
<br />
|-<br />
| 1-5<br />
| ER-Lit<br />
| M13genes-pSB3C5<br />
<br />
|-<br />
| 6-10<br />
| ER-phagemid1C3<br />
| M13genes-pSB6A1<br />
<br />
|-<br />
| 11-15<br />
| 5alpha<br />
| M13genes-pSB3C5<br />
<br />
|-<br />
| 16-20<br />
| 5alpha<br />
| M13genes-pSB6A1<br />
<br />
|-<br />
|}<br />
::-Mini-prepped DNA<br />
::-Digest the mini-preps with EcoRI-HF and PstI-HF<br />
::-Run digestions on gel to check sizes<br />
'''[[File:UCB-Phage Delivery-140708.JPG]]'''<br />
<br />
:lanes<br />
::top-left: pSB3C5-M13genes in ER2738 with Litmus28i<br />
::top-right: pSB6A1-M13genes in ER2738 with pSB1C3-1C3<br />
::bottom-left: pSB3C5-M13genes in 5alpha<br />
::bottom-right: pSB6A1-M13genes in 5alpha<br />
:None are correct<br />
*Primers Gem013 came in. Resuspend and diluted primers<br />
::-O/N of pSB3C5 to use for PCR tomorrow<br />
<br />
'''7/9'''<br />
*Wanted to make Litmus28i biobrick compatible for use as a phagemid backbone for us and other iGEM teams<br />
::-Mini-prepped O/N of pSB3C5<br />
::-PCR of pSB3C5 to amplify J04450 with Litmus28i compatible cut sites<br />
:::Primers: Gem013<br />
::::*At the 3’ end, these primers are the same as VF2 and VR so will bind the region flanking J04450. This conserves the terminators that exist between the biobrick prefix and VF2 on one side and those between the biobrick suffix and VR on the other side. Therefore, we are amplifying, VF2 priming site, terminators, J04450, terminators, and VR priming site<br />
::::*At the 5’ end, these primers contain unique restriction sites found in the Litmus28i MCS<br />
*PCR of M13 genes…. again (did 4 samples)<br />
::-Used the 1:10000 dilutions<br />
<br />
'''7/10'''<br />
*Note: The following two projects were done in parallel when possible<br />
*To make Litmus28i biobrick compatible<br />
::-Ran gel of PCR from 7/9 (J04450 amplification with Gem013)<br />
'''[[File:UCB-Phage Delivery-140710.JPG]]'''<br />
<br />
:1-4: PCRs of M13genes<br />
:5. No DNA control for M13genes PCR<br />
:6. PCR of pSB3C5 backbone<br />
:7. No DNA control for pSB3C5 backbone<br />
::-PRC purified the PCR product<br />
::-Digestion #1<br />
:::Restriction enzymes had the same buffer conditions but different activation temperatures so we had to do a 2 part digestion<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Restriction enzymes<br />
! Notes<br />
<br />
|-<br />
| J04450<br />
| Sac1<br />
| PCR purified<br />
<br />
|-<br />
| Litmus 28i<br />
| Sac1<br />
| From NEB tube<br />
<br />
|-<br />
|}<br />
:::*Incubate 1hr at 37 C<br />
:::*Heat inactivated 20 minutes at 80C<br />
::-Digestion #2<br />
:::-Added 1ul BsmI to both samples<br />
:::-Incubated 1hr at 65C<br />
:::-Heat inactivated 20 minutes at 80C<br />
::Ran gel<br />
:::-See gel below<br />
:::-Tried to extract J04450 segment but received very low yield. Since band otherwise looked clean, we decided to redo digestion then skip straight to ligation<br />
::Repeat digestion #1 for J04450<br />
::Repeat digestion #2 for J04450<br />
::Ligation (10hr at 16C, 10 min at 80C)<br />
:::3. Litmus28i + J04450<br />
*To retry ligation to biobrick backbone<br />
::-Ran gel of PCR from 7/9 (M13genes amplified with Gem012)<br />
:::See gel below<br />
::-PCR purified the PCR product<br />
::-Digestion<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Restriction enzymes<br />
! Notes<br />
<br />
|-<br />
| M13 genes<br />
| EcoRI-HF + PstI-HF<br />
| PCR purified<br />
<br />
|-<br />
| 6A1<br />
| EcoRI-HF + PstI-HF<br />
| ‘empty’ pSB6A1<br />
<br />
|-<br />
| 3C5<br />
| EcoRI-HF + PstI-HF<br />
| contains J04450<br />
<br />
|-<br />
|}<br />
:Incubate 1hr at 37C<br />
:Heat inactivated 20 minutes at 80C<br />
::-Ran gel<br />
:::*See gel below<br />
:::*Tried to extract M13genes and pSB3C5 segments but received very low yields. Since bands otherwise looked clean, we decided to redo digestion then skip straight to ligation<br />
::-Repeat digestion for M13genes and pSB3C5<br />
::-Ligation (10h at 16C, 10m at 80C)<br />
:::1. pSB3C5 + M13genes<br />
:::2. pSB6A1 + M13genes<br />
*Gel<br />
'''[[File:UCB-Phage Delivery-140710-02.JPG]]'''<br />
:1. pSB1A3<br />
:2. Limtus28i<br />
:3. M13genes<br />
:4. pSB3C5<br />
:5. J04450<br />
<br />
'''7/11'''<br />
*Transformed ligations from 7/10 into 5alpha cells<br />
::1. pSB3C5 + M13genes<br />
::2. pSB6A1 + M13genes<br />
::3. Litmus28i + J04450<br />
<br />
<br />
'''7/12'''<br />
*Results of 7/11 transformation<br />
::-No growth on no DNA control (Amp or Chlor)<br />
::-Lots of growth on Lit-J04450 -> some red-> colonies are too close together to prick individual colonies<br />
:::-It’s possible that these red colonies are satellites =(<br />
:::-Swiped some and plated on new Amp plate (restreak)<br />
::-Lawn of positive control (Litmus 28i) on Amp<br />
::-Many colonies for L1 (pSB3C5-M13genes) and L2 (pSB6A1-M13genes)<br />
:::-Due to high number of red colonies on Lit-J04450 plate, we assumed that most of these colonies contain empty vector<br />
:::-Did not make O/N<br />
*Ligated M13 genes to pSB6A1<br />
::-Used the remained of our digested M13genes.<br />
::-Both digestions from 7/10<br />
*Infection Experiment<br />
::-had 3 cell stocks (each taken from a different colony the night before)<br />
::-Tested Litmus 28i, Tandem phagemid 1C3, and double infection<br />
:::*Therefore, there were 9 flasks total.<br />
::-negative control: Streaked parent cells (non-infected) onto Chlor, Amp, and kan<br />
::-Included double infection plates for Litmus 28i and phagemid 1C3<br />
<br />
==Week 11==<br />
<br />
'''7/13'''<br />
*Transformed 7/12 ligation (pSB6A1-M13genes)<br />
*Litmus28i-J04450<br />
::-All growth from 7/12 restreak was white<br />
::-Pricked some red colonies for liquid O/N-will hopefully see red tomorrow<br />
::-Pricked a few red colonies and put into 200ul H2O (a few colonies per tube- 2 tubes total).<br />
:::*Plated 150ul onto Amp plates<br />
::-Put original plate into incubator to hopefully get bigger colonies<br />
<br />
'''7/14'''<br />
*Finished TWIV ppt<br />
*Set up 2 liquid culture of red Litmus 28i colonies (Litmus28i-J04450)-- slow growth <br />
::-Mini-prepped red Litmus 28i ‘O/N’ from earlier in day<br />
::-Digested samples—to check for insert and correct cut sites<br />
*Made O/Ns of pSB6A1-M13genes colonies from 7/13 transformation<br />
<br />
<br />
'''7/15'''<br />
*Ran gel of Litmus28i-J04450 samples<br />
'''[[File:UCB-Phage Delivery-140715.JPG]]'''<br />
:1. J04450-Litmus28ibb #1 (EcoRI+PstI)<br />
:2. J04450-Litmus28ibb #1 (uncut)<br />
:3. J04450-Litmus28ibb #2 (EcoRI+PstI)<br />
:4. J04450-Litmus28ibb #2 (EcoRI+PstI)<br />
::*Accidentally added restriction enzymes<br />
:5. Litmus28i (EcoRI+PstI)<br />
::*Only has PstI site<br />
<br />
::-Verifies that Litmus28i-J00450 had correct cut sites<br />
::-From this point on, biobricked Litmus28i is called Litmus28ibb<br />
*Check 7/14 O/Ns for pSB6A1-M13genes<br />
::-Mini-prepped liquid cultures<br />
::-Digested with EcoRI and PstI<br />
::::Received bold bands of just under 4000bp. Some lanes had a very feint band ~330bp. None were the correct size<br />
*Transformed Litmus28ibb-J04450 into ER2738 cells<br />
*Remake phage packaging<br />
::-pSB1C3-M13ori(New)<br />
::-pSB1C3-M13ori(Old)<br />
<br />
'''7/16'''<br />
*Results of 7/15 transformation of Litmus28i into 6/30 ER2738 cells<br />
::-lawn on no DNA control-- most likely contaminated cells<br />
::::Streaked chem comp 5alpha and ER onto Amp and Amp+Tet plates to determine if problem is with cells or plates<br />
::-O/N culture of ER to make new chem comp cells<br />
<br />
'''7/17'''<br />
*Finished phage isolation<br />
::Note:<br />
:::*pSB1C3-M13ori (New): M13ori and plasmid ori are convergent<br />
:::*pSB1C3-M13ori (Old): M13ori and plasmid ori are tandem<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! 269nm<br />
! 320nm<br />
! [] phage/mL<br />
<br />
|-<br />
| pSB1C3-M13ori (New)<br />
| 0.386<br />
| 0.034<br />
| 8.126 E12<br />
<br />
|-<br />
| pSB1C3-M13ori (New)<br />
| 0.419<br />
| 0.036<br />
| 8.842 E12<br />
<br />
|-<br />
| pSB1C3-M13ori (Old)<br />
| 0.283<br />
| 0.025<br />
| 5.956 E12<br />
<br />
|-<br />
| pSB1C3-M13ori (Old)<br />
| 0.326<br />
| 0.024<br />
| 6.972 E12<br />
<br />
|-<br />
|}<br />
*Infection to compare the new and the old pSB1C3-M13ori<br />
::-Prepped cells for infection<br />
::-Infected cells with pSB1C3-M13ori (New) or pSB1C3-M13ori (New)<br />
::-and plated on dilutions of 1:100, 1:1000, and 1:10000 on Chlor and kan<br />
::-Also plated 1:1000 dilution on lowered Chlor concentration (34ug/mL)<br />
*Sent Litmus28ibb-J04450 for sequencing<br />
<br />
'''7/18'''<br />
*Results from contamination test<br />
::-5alpha on Amp = no growth<br />
::-5alpha on Amp+Tet = colonies<br />
::-ER on Amp = lawn<br />
::-ER on Amp+Tet = lawn<br />
*Made new chemically competent ER2738<br />
*Transformed Litmus28ibb-J04450 into ER2738 made on 3/30 and ER2738 made on 7/18<br />
::-Had two samples of Litmus28ibb-J04450 and two cell stocks, so 4 samples total<br />
<br />
'''7/19'''<br />
*Results of 7/17 infection<br />
::-pSB1C3-M13ori(Old) (tandem-when plasmid ori and M13ori point in the same direction) packages better than pSB1C3-M13ori(New) (when ori and M13ori are convergent), implying that directionality matters.<br />
'''[[File:UCB-phage lab7-19-141012.jpg]]'''<br />
<br />
==Week 12==<br />
<br />
'''7/20'''<br />
*Freeze downs<br />
:-Litmus28i-J04450 Litmus 28i is now biobrick compatible<br />
<br />
'''7/21'''<br />
*Send samples for sequencing<br />
::-pSB1C3-M13ori(Old)<br />
::-pSB1C3-M13ori(New)<br />
*To ligate M13ori to a kanamycin backbone (pSB1K3)<br />
::-Resuspended pSB1K3-J04450 from distribution kit (6B on plate 4)<br />
:::*Contains J04450 as insert (full RFP construct)<br />
::-Transform pSB1K3 into 5alpha cells<br />
<br />
'''7/22'''<br />
*Pricked colony from pSB1K3-J04450 transformation<br />
::-Mini-prepped DNA to get a supply of DNA<br />
*To put M13ori (M13 phackaging signal) onto Kanamycin resistance o we can test packaging ratios with M13K07 on the same antibiotic<br />
::-Digested pSB1K3 with EcoRI-HF and XbaI<br />
::-Digested pSB1C3-M13ori (Old) with EcoRI-HF and SpeI-HF<br />
:::*DNA was not sufficiently cut. Too much DNA? Problem with enzyme (SpeI-HF)?<br />
'''[[File:UCB-Phage Delivery-140722.JPG]]'''<br />
:1. pSB1K3<br />
:2. M13ori<br />
<br />
'''7/23'''<br />
*Sequencing samples from 7/21 were lost in the mail. Resent samples<br />
*Yet another attempt to biobrick M13genes<br />
::1. using primers that would amplify genes and M13 ori parts from M13K07<br />
::2. using primers that would amplify genes, M13 ori parts, and plasmid ori from M13K07<br />
'''[[File:UCB-Phage Delivery-140723.JPG]]'''<br />
:1. Amplified only the M13genes and M13ori<br />
:2. No DNA control for 1<br />
:3. Amplified M13genes, M13ori, and plasmid ori<br />
:4. No DNA control for 3<br />
::-When run on a gel, samples were clean with only one band at around 7kb. No contamination in no DNA controls<br />
*Digestions<br />
::-pSB1K3 with EcoRI-HF and PstI-HF to check for correct insert<br />
::-pSB1C3-M13ori (Old and new) with EcoRI-HF and SpeI-HF to test for efficient cutting with different stock of restriction enzyme<br />
'''[[File:UCB-Phage Delivery-140723-02.JPG]]'''<br />
:1-3: pSB1K3<br />
:4. pSB1C3-M13ori (Old)<br />
:5. pSB1C3-M13ori (New)<br />
*Still had inefficient cutting. Tested M13ori next to Litmus28i to disern the problem<br />
:*Uncut<br />
:*Cut once with (E, X, S, or P)<br />
:*Cut twice with (E+S or E+P)<br />
'''[[File:UCB-Phage Delivery-140723-03.JPG]]'''<br />
:1. M13ori uncut<br />
:2. M13ori Ecori-HF<br />
:3. M13ori SpeI-HF<br />
:4. M13ori XbaI<br />
:5. M13ori PstI-HF<br />
:6. M13ori EcoRI-HF + SpeI-HF<br />
:7. M13ori EcoRI-HF + PstI-HF<br />
:8. Litmus28i uncut<br />
:9. Litmus28i EcoRI-HF<br />
:10. Litmus28i SpeI-HF<br />
:11. Litmus28i XbaI<br />
:12. Litmus28i PstI-HF<br />
:13. Litmus28i EcoRI-HF + SpeI-HF<br />
:14. Litmus28i EcoRI-HF + PstI-HF<br />
*Only partial digest with only SpeI-HF for both. Complete digestion with all others, including E+S<br />
<br />
'''7/24'''<br />
*To Biobrick M13genes using pSB6A1<br />
::-PCR purified M13 genes (did both samples at the same time)<br />
::-Digested PCR purification and pSB6A1 with EcoRI-HF and PstI-HF<br />
'''[[File:UCB-Phage Delivery-140724.JPG]]'''<br />
*Gel extracted M13genes<br />
<br />
:1. M13genes<br />
:2. pSB6A1<br />
:-Bands of pSB6A1 were too light to gel extract<br />
*To swap the kanamycin resistance marker on M13K07 with ampicillin resistance<br />
::-PCR amplified AmpR from pSB6A1<br />
*made O/N culture of pSB6A1 from freeze down to mini-prep<br />
::-Also streaked cells onto plate<br />
<br />
'''7/25'''<br />
*To change resistance marker on M13K07<br />
:1. Biobrick method<br />
::*Tried to mini-prep O/N but pellet (after liquid culture was spun) was not red even after 16+ hours. Set up O/N from plate colonies<br />
:2. Swap only resistance method<br />
::*PCR purified 7/24 PCR<br />
::*Ran product on gel-> band of correct size<br />
'''[[File:UCB-Phage Delivery-140725.JPG]]'''<br />
<br />
:1. PCR product<br />
:2. PCR product-> PCR purified<br />
:3. No DNA control<br />
::-Digested the PCR purification products of M13genes+ori and AmpR with AgeI-HF and NotI-HF<br />
::*Bands on previous gel looked clean enough for both so did not gel extract<br />
::-Overnight ligation<br />
*Set up O/Ns of pSB1K3 colonies<br />
<br />
'''7/26'''<br />
*Transform<br />
::-pSB6A1+M13genes<br />
*To biobrick M13genes<br />
::-Digestion<br />
:::*pSB6A1 with EcoRI and PstI<br />
'''[[File:UCB-Phage Delivery-140726.JPG]]'''<br />
:Gel extracted backbone (band ~4000bp)<br />
*Ligations (10hrs 16C, 10min at 80C)<br />
::-pSB6A1+M13genes<br />
<br />
==Week 13==<br />
<br />
'''7/27'''<br />
*Transformation results from 7/26<br />
::-No growth for no DNA controls ''on Amp''<br />
::-No growth for M13 genes onto pSB6A1 ''on Amp''<br />
*Set up O/Ns of 5alpha and ER2738 to make competent cell tomorrow<br />
*Made freeze down of pSB1K3<br />
*Plated ER competent cells from 5/15 and 6/16 Onto Chlor plates to check for contamination<br />
<br />
'''7/28'''<br />
*No growth of ER2738 on Chlor (either sample)<br />
::-ER is not contaminated with ChlorR<br />
*Make new 5alpha chem comp cells <br />
*Transformations<br />
::-pSB6A1-M13genes<br />
::-positive control for Amp (pSB6A1)<br />
::-No DNA control<br />
<br />
'''7/29'''<br />
*Set up O/Ns of pSB6A1-M13genes<br />
::-Plated sample on reduced Amp and regular Tet<br />
::-Later in day…<br />
::-Mini-prepped O/Ns<br />
::-Digested and ran on a gel to check for insert sizes<br />
'''[[File:UCB-Phage Delivery-140729.JPG]]'''<br />
:*All but the 4th lane with 3 bands look correct<br />
<br />
'''7/30'''<br />
*Lawn on last night’s plating-> replate<br />
*Digest pSB3C5 and pSB6A1-M13genes with E and P to move M13genes to a Chlor backbone<br />
'''[[File:UCB-Phage Delivery-140730.JPG]]'''<br />
::-Extracted bands<br />
::-Ligations<br />
:::*pSB3C5 + M13genes<br />
*Sent samples for sequencing<br />
**M13 genes on pSB6A1 VF2<br />
**M13 genes on pSB6A1 VR<br />
<br />
'''Test experiment for high school kids coming to lab (7/29-7/31)'''<br />
*General idea: Have 2 strands of DNA, one has an EcoRI site while the other contains a SNP, abolishing the cut sight. We pretend that one is pathogenic (the one that is not cut) and tells kids to figure out which one is which <br />
*PCR with Dream-Taq<br />
::-Did not have special fastdigest enzyme<br />
::-Hypothesized that green dye will interfere with enzyme effectivity<br />
*PCR with Dream-Taq (no green dye)<br />
::-Digest with EcoRI fastdigest-> did not cut<br />
*Redid experiment several times<br />
::-Should digest with XbaI<br />
::-PCR purified PCR products<br />
<br />
'''7/31'''<br />
*Spent the morning with Heritage High School<br />
*Made phage<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Phage<br />
! A269<br />
! A320<br />
! genome size<br />
! [ ]<br />
<br />
|-<br />
| Litmus28ibb-J04450 (1)<br />
| 0.823<br />
| 0.197<br />
| <br />
| 9.35 x10^12<br />
<br />
|-<br />
| Litmus28ibb-J04450 (2)<br />
| 0.940<br />
| 0.168<br />
| <br />
| 1.14 x10^13<br />
<br />
|-<br />
|}<br />
<br />
'''8/2'''<br />
*Digestion<br />
::-pSB6A1-M13genes (E+P)<br />
::-pSB3C5 (E+P)<br />
<br />
==Week 14==<br />
<br />
'''8/3'''<br />
*Another attempt to clone M13genes onto pSB3C5<br />
::-Run 8/2 digestions on gel<br />
'''[[File:UCB-Phage Delivery-140803.JPG]]'''<br />
:1. M13genes (E+P)<br />
:2. pSB3C5 (E+P)<br />
::-Gel extracted <br />
:::*M13genes away from pSB6A1 backbone<br />
:::*pSB3C5 backbone away from J04450<br />
::-Ligations<br />
:::*pSB3C5 + M13genes<br />
:::*pSB3C5 + no insert<br />
::-Transformed ligations into ER2738<br />
<br />
'''8/5'''<br />
*Transformation of pSB3C5+M13genes failed<br />
*Made phage<br />
::-Fd-CAT DNA packaged with Fd-CAT<br />
::-phagemid 1C3 packaged with M13K07<br />
::-amilCP on pSB1C3 packaged with M13K07<br />
::-Litmus28ibb-J04450 packaged with M13K07<br />
<br />
'''8/6'''<br />
*Isolated phage<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Phage<br />
! [ ]<br />
<br />
|-<br />
| Fd CAT<br />
| 2.72 x10^12<br />
<br />
|-<br />
| Litmus28ibb-J04450<br />
| 8.37 x10^12<br />
<br />
|-<br />
| pSB1C3-amilCP<br />
| 1.42 x10^13<br />
<br />
|-<br />
| pSB1C3-M13ori<br />
| 4.32 x10^12<br />
<br />
|-<br />
|}<br />
<br />
'''8/6'''<br />
*Started making phage with Fd CAT the should contain Litmus28ibb-RFP<br />
*Infection<br />
::-Infected ER2738 cells with Litmus28ibb-RFP, pSB1C3-M13ori, or pSB1C3-amilCP<br />
::-Plated Litmus samples on 100ug/mL Amp and 50ug/mL Kan<br />
::-Plated other two samples on 34ug/mL Chlor and 25ug/mL Kan<br />
<br />
'''8/7'''<br />
*Isolated phage containing Litmus28ibb-RFP using helper phage<br />
::-M13K07 (as control)<br />
::-Fd-CAT (no phage pellet was observed-worried that there was no phage)<br />
*Went to CSU to have them test M13ori part compared to amilCP. Grew the samples in 5mL O/Ns then diluted to 0.5OD and grew 30 minutes rather than starting from a fresh colony<br />
<br />
'''8/8'''<br />
*At CSU<br />
::-Finished phage protocol (Test packaging of M13ori part)<br />
::-Only used 20mL infection samples<br />
::-grew to 0.55 OD<br />
::-When making phage (after 14 hour incubation), there was very little growth<br />
::-Plated non-diluted and diluted 1:1000 of M13ori and amilCP sample<br />
*Infected cells using phage isolated 8/7. Plated at dilutions of 1:100 and 1:100k<br />
*Want to test progeny Fd-CAT phage (made 8/7) for infectability<br />
::-Start phage isolation prodocol using Fd-CAT phage from 8/7 do deliver the helper phagemid<br />
:::*Phagemid:<br />
::::*None: will make Fd-CAT phage packaing Fd-CAT phagemid<br />
::::*Litmus28ibb-J04450: make Fd-CAT phage packaging Litmus28ibb-J04450 phagemid<br />
*Remake phage (Fd-CAT) using fresh stock from Mike<br />
<br />
<br />
'''8/9'''<br />
*Results of 8/8 infection<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Helper Phagemid<br />
! phagemid<br />
! Selection<br />
! 1:1000 dilution<br />
! 1:00k dilution<br />
<br />
|-<br />
| M13K07<br />
| Litmus28ibb-RFP<br />
| Kan+Tet<br />
| 400-500 (some red)<br />
| 61 white; 20 red<br />
<br />
|-<br />
| same<br />
| same<br />
| Amp+Tet<br />
| lawn<br />
| 2<br />
<br />
|-<br />
| Fd-CAT<br />
| Litmus28ibb-RFP<br />
| Chlor+Tet<br />
| 0<br />
| 0<br />
<br />
|-<br />
| same<br />
| same<br />
| Amp+Tet<br />
| 10<br />
| 707<br />
<br />
|-<br />
|}<br />
*NOTE: The same sample was plated on two different plates. For example, the sample using M13K07 as the helper phage was plated on Kan+Tet and Amp+Tet<br />
*Mini-prep<br />
::-pSC3C5-J04450 -- very little growth, low DNA yield<br />
::-Fd-CAT infected cells<br />
:::*Digest and run on a gel to verify presence of a band- there was a very feint band<br />
*Finished isolating phage…. messed up and used 0.8MgCl2, 0.2M NaCl instead of PEG during precipitation step. Also used the wrong phage<br />
::-Infected ER2738 cells anyway…. no growth by 8/12<br />
<br />
==Week 15==<br />
<br />
'''8/10'''<br />
*Are the progeny phage from 8/6 (original progeny from first Fd-Tet application) viable/ able to reproduce?<br />
::-Used these progeny to make phage that should amplify Fd-CAT phage containing either the Fd-CAT or Litmus28ibb-J04450 phagemid<br />
<br />
'''8/11'''<br />
*Finished isolating phage<br />
{| class = "wikitable"<br />
|-<br />
! Helper Phagemid<br />
! Phagemid<br />
! A269<br />
! A320<br />
! genome size<br />
! [ ]<br />
<br />
|-<br />
| Fd-CAT<br />
| Litmus28ibb (1)<br />
| 0.029<br />
| 0.013<br />
| 4080<br />
| 2.35 x10^11<br />
<br />
|-<br />
| Fd-CAT<br />
| Ltimus28ibb (2)<br />
| 0.033<br />
| 0.015<br />
| 4080<br />
| 2.65 x10^11<br />
<br />
|-<br />
| Fd-CAT<br />
| Fd-CAT (1)<br />
| 0.326<br />
| 0.082<br />
| 7775<br />
| 1.88 x10^12<br />
<br />
|-<br />
| Fd-CAT<br />
| Fd-CAT (2)<br />
| 0.320<br />
| 0.079<br />
| 7775<br />
| 1.86 x10^12<br />
<br />
|-<br />
| M13g6A1<br />
| M13g6A1 (1)<br />
| 0.322<br />
| 0.148<br />
| 10,029<br />
| 1.04 x10^12<br />
<br />
|-<br />
| M13g6A1<br />
| M13g6A1 (2)<br />
| 0.503<br />
| 0.130<br />
| 10,029<br />
| 2.23 x10^12<br />
<br />
|-<br />
|}<br />
<br />
'''8/13'''<br />
*Infected ER2738 with Fd-CAT packaging Litmus28ibb-J04450<br />
<br />
'''8/16'''<br />
*Results from Infection on 8/11<br />
{| class = "wikitable"<br />
|-<br />
! Helper Phagemid<br />
! Phagemid<br />
! 1:1<br />
! 1:10<br />
! 1:100<br />
! 1:1000<br />
<br />
|-<br />
| M13g6A1<br />
| M13g6A1<br />
| almost lawn<br />
| 728<br />
| 287<br />
| 55<br />
<br />
|-<br />
| Fd-CAT<br />
| Fd-CAT<br />
| lawn<br />
| almost lawn<br />
| 1260<br />
| 640<br />
<br />
|-<br />
| Fd-CAT<br />
| Litmus28ibb-J04450<br />
| lawn<br />
| almost lawn<br />
| 2004<br />
| 304<br />
<br />
|-<br />
|}<br />
<br />
Results: packaging of Litmus28ibb-J04450 using Fd-CAT helper phagemid from (8/15)<br />
<br />
{| class="wikitable"<br />
|-<br />
!Antibiotic<br />
!1:10 <br />
!1:100 <br />
!1:1k <br />
!1:10k <br />
!1:100k<br />
<br />
|-<br />
|Amp <br />
|Lawn<br />
|Almost lawn <br />
|1025 <br />
|217 <br />
|25<br />
<br />
|-<br />
|Kan <br />
|9 <br />
|2 <br />
|0 <br />
|0 <br />
|0<br />
|}<br />
<br />
==Week 16==<br />
<br />
'''8/19'''<br />
<br />
Sent samples for sequencing<br />
<br />
Litmus28ibb-J04450<br />
<br />
==Week 17==<br />
<br />
'''8/27'''<br />
<br />
Transform pSB1C3-M13ori into ER2738 cells<br />
<br />
'''8/28'''<br />
<br />
Make phage with pSB1C3-M13ori as phagemid and M13g6A1 as helper phagemid<br />
<br />
'''8/29'''<br />
<br />
Finish phage isolation of M13 phage containing pSB1C3-M13ori<br />
<br />
'''9/3'''<br />
<br />
digest pSB6A1-M13g and pSB3C5 with EcoRI and PstI. Did not receive a band for M13genes so did not proceed with ligation to pSB3C5<br />
<br />
==Week 18==<br />
<br />
'''9/4'''<br />
<br />
Mini-prepped new pSB6A1-M13genes (bad quality) and pSB3C5-J04550. Digested both plasmids with EcoRI and PstI and ran products on gel. Did receive a band for the M13genes but it was too feint to gel extract<br />
<br />
'''9/5'''<br />
<br />
Mini-prep pSB6A1-M13genes again. Digest this and pSB3C5 backbone from previous day with EcoRI and PstI. M14g band was barely bright enough to extract so we extracted M13genes and pSB3C5 backbone. Pieces were ligated together overnight at 16C for 10 hours.<br />
<br />
'''9/7'''<br />
<br />
Transformed 9/5 ligation (pSB3C5-M13genes into 5alpha cells). Plated sample onto low Chlor plate (34ug/mL)<br />
<br />
'''9/9'''<br />
<br />
Still no growth for 9/7 transformation of pSB3C5-M13genes ligation PCR M13genes to attempt another ligation into pSB3C5. Diluted DNA to ~1.17ng/ul<br />
<br />
'''9/10'''<br />
<br />
Gel of 9/9 PCR of M13genes looked clean (only ran 3ul of PCR sample on gel). PCR purified the remaining PCR sample. Digested with EcoRI and PstI. Ligated to previously digested pSB3C5.<br />
<br />
==Week 18==<br />
<br />
'''9/11'''<br />
<br />
Transform 9/10 ligation (pSB3C5-M13genes) into 5alpha cells<br />
<br />
'''9/13'''<br />
<br />
Colony PCR of colonies that grew for 9/11 transformation. Looking for pSB3C5-M13genes)<br />
<br />
'''9/15'''<br />
<br />
Gel of colony PCR showed failure. Set up overnight cultures anyway.<br />
<br />
'''9/17'''<br />
<br />
Submitted Part to iGEM Registry<br />
<br />
{| class="wikitable"<br />
|-<br />
|iGEM # <br />
|Description<br />
<br />
|-<br />
|BBa_K1445000 <br />
|M13ori- the packaging signal for the M13 and fd phage<br />
|}<br />
<br />
Mini-prepped overnights from 9/15 and sent samples for sequencing<br />
<br />
Sent Litmus28ibb-J04450 sample for sequencing to sequence the remaining section that was not reached before<br />
<br />
==Week 18==<br />
<br />
'''9/21'''<br />
<br />
Up until this point, very little luck was had with pSB3C5. Retried putting M13genes onto a chloramphenicol backbone, but this time used pSB1C3. Digested M13genes and pSB1C3 with EcoRI and PstI. Gel showed incomplete digestion so did not gel extract.<br />
<br />
'''9/22'''<br />
<br />
Digested pSB1C3-J04450 with EcoRI and PstI then gel extracted the backbone. PCR purified M13genes(PCR product was clean so did not bother to gel extract). Ligated the two pieces together.<br />
<br />
'''9/23'''<br />
<br />
Transform ligations from 9/22 (pSB1C3-M13genes) into 5 alpha cells<br />
<br />
'''9/27'''<br />
<br />
Transform pSB1C3-M13genes into 5alpha cells. Plate onto low Chlor (34ug/mL)<br />
<br />
Later in day.... Set up overnight cultures of colonies<br />
<br />
'''9/28'''<br />
<br />
Mini-prepped O/N cultures from 9/27. Digested samples with EcoRI and PstI. There were no bands of the correct size on the gel.<br />
<br />
the M13 and fd phage</div>Leighlahttp://2014.igem.org/Team:CU-Boulder/Notebook/Phage_TeamTeam:CU-Boulder/Notebook/Phage Team2014-10-17T06:54:17Z<p>Leighla: /* Week 17 */</p>
<hr />
<div>{{Template:UCB-Main}}<br />
{{UCB-NavBar}}<br />
__FORCETOC__<br />
<br />
'''Phage Delivery'''<br />
==Week 1==<br />
'''Notes:''' Unless stated otherwise, all gels contain 2-log ladder<br />
<br />
'''5/9'''<br />
*Obtained BW23115 KanR cells- BW23115 cells that had their native CRISPR-Cas system knocked out by the insertion of a Kanamycin resistance gene<br />
::-Will also be called BW23115 or BW<br />
::-Conjugated BW23115 KanR cells with contain F’ notation (ex. BWF’)<br />
*Obtained ER2738 cells that contain the F’ episome (no changes from NEB sample)<br />
::-Will also be called ER. Assume that all ER samples contain the F’ episome<br />
::-Streaked sample onto LB+Tet (20ug/mL) to select for colonies containing F’ episome<br />
<br />
'''5/10'''<br />
*Did receive colonies from 5/9 selection<br />
<br />
==Week 2==<br />
'''5/12'''<br />
*Need to conjugate BW23115 KanR cells with the F’ episome<br />
::-Set up overnight cultures of ER2738 and BW23115 KanR<br />
::-When mixed, ER2738 will donate it’s F’ episome and BW23115 KanR will receive the F’ episome. F’ episome confers Tetracycline resistance<br />
<br />
'''5/13'''<br />
*Started M13 Amplification: Amplify M13 phage using the M13K07 Helper Phage<br />
::-Let precipitated in NaCl/PEG solution overnight<br />
::-Possible sources of error<br />
:::*Did not sterilize 2.5M NaCl/20% PEG-8000 solution<br />
:::*Added 4-fold PEG solution<br />
::::Compensated by adding more LB<br />
:::*During precipitation, put sample in -20C for 30 minutes before realizing mistake and moving to it to 4C. Sample partially froze<br />
*Conjugated BW23115 with F’ episome<br />
::-Added 1mL BW23115 to 1mL ER2738 overnight culture<br />
::-Incubated at 37C for 30 minutes, shaking<br />
::-Plated on LB+Kan(50ug/mL)+Tet(20ug/mL)<br />
:::*To select for BW cells that took the F’ episome (containing Tet resistance)<br />
<br />
'''5/14'''<br />
*Finished the M13 Amplification<br />
::-Visualized product on UV-vis. There was a tall spike at 269nm indicating that DNA was present. Did not test at 320nm.<br />
*Results of BW23115 Conjugation<br />
::-Many colonies indicating successful conjugation of F’ episome into BW23115<br />
::-Set up overnight to make freeze down tomorrow<br />
*Set up overnight of ER2738 to make chemically competent tomorrow<br />
<br />
'''5/15'''<br />
*Made freeze down of BW23115 KanR F’<br />
::-BW23115 E. coli strain with Kanamycin resistance gene inserted into genome and with F’ episome<br />
*Made chemically competent ER2738 cells<br />
*Transformation of Litmus28i (from NEB) into chemically competent ER2738 cells<br />
::-Added 1ul Litmus28i plasmid to 40ul competent cells<br />
::-Plated on LB + Amp(100ug/mL)<br />
::-Purpose: To make M13 phage that package Litmus28i DNA. Need phagemid (Litmus28i) DNA in infectable cells (cells containing F’ episome) to introduce M13K07 Helper Phage and make phage. <br />
<br />
'''5/16'''<br />
*Results of 5/15 transformation<br />
::-No growth for No DNA control<br />
::-Many colonies for sample<br />
<br />
==Week 3==<br />
'''5/19'''<br />
*M13 Amplification to isolate M13-Litmus28i phage<br />
::-Cells: ER2738 cells containing Litmus28i phagemid<br />
::-Helper Phage: M13K07<br />
::-Not much phage was precipitated<br />
*Set up overnight culture of ER2738 to infect tomorrow<br />
<br />
'''5/20'''<br />
*Infected ER2738 cells with M13-Litmus28i phage<br />
::-Plated only on Ampicillin(100ug/mL) (should have also plated on kanamycin)<br />
::-Infected for 4-5 hours-> should have only infected for 30 minutes maximum. This extra time gives the cells that were infected with M13-M13K07 the time to produced M13-M13K07 phage and reinfect<br />
<br />
'''5/21'''<br />
*Results from M13-Litmus28i infection of ER2738<br />
::-Solid lawn of growth for diluted and non-diluted<br />
::-Also sickly looking growth<br />
*Set up overnights<br />
::-ER2738 cells containing Litmus28i for freeze down<br />
::-BW23115 with F’ episome to make chemically competent cells <br />
::-ER2738 to redo infection<br />
<br />
'''5/22'''<br />
*Tested absorbance of phage produced through M13 amplification on 5/19<br />
::-Low absorbance of 0.018 at 269nm but no detection at wavelength 320nm<br />
::-Decided to redo M13 amplification<br />
*Made chemically competent BW23115 with f-episome<br />
*Made freeze down of ER2738 containing Litmus28i<br />
*Set up overnight of ER2738 containing Litmus28i to redo M13 amplification tomorrow<br />
<br />
'''5/23'''<br />
*Protocol switch to make phage using phagemid<br />
::-“M13 Amplification” protocol should only be used to make more M13-M13K07, not to make M13 phage containing a different phagemid<br />
::-Switched to new protocol (“Use of M13K07 Helper Phage for isolation of single stranded phagemid DNA” by NEB. Made modifications (see our protocols) to isolate phage rather than single-stranded DNA)<br />
::-Making phage….<br />
{| class = "wikitable"<br />
|-<br />
! Helper phage<br />
! Phagemid<br />
! Cells<br />
! Notes<br />
<br />
|-<br />
| M13K07<br />
| None<br />
| ER2738<br />
| Make more M13-M13K07<br />
<br />
|-<br />
| M13K07<br />
| Litmus28i<br />
| ER2738<br />
| Test packaging of Litmus28i<br />
<br />
|-<br />
|}<br />
*Made fresh antibiotics<br />
::-Chloramphenicol (34 ng/mL)<br />
:::*1.44g chloramphenicol into 42mL EtOH<br />
::-Ampicillin (50 ng/mL)<br />
:::*4g ampicillin into 80mL mili-Q H2O<br />
<br />
'''5/24'''<br />
*Isolated phage using new protocol<br />
::-Resuspended pellet in 200ul TBS and 200ul 30% glycerol<br />
::-Measured absorbance with UV-vis<br />
:::*concentration (phage/mL) = 6x10^16 x (A269-A320)/ (#of base pairs in the phage genome)<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! Abs (269nm)<br />
! Abs (320nm)<br />
! Genome size<br />
! Concentration (phage/mL)<br />
<br />
|-<br />
| M13-M13K07<br />
| 0.721<br />
| 0.060<br />
| <br />
| 4.57 x10^12<br />
<br />
|-<br />
| M13-Litmus28i<br />
| 0.250<br />
| 0.028<br />
| 2823<br />
| 4.72 x10^12<br />
<br />
|-<br />
|}<br />
*Infect ER2738 cells with M13-Litmus28i<br />
::-Wanted 1:10 phage:cell ratio. Math….<br />
:::*At 1 OD (e.coli), cell/mL = 5x10^8<br />
:::*5x10^7 phage * (1mL/4.72x10^12 phage) = 0.011ul phage<br />
*Set up overnights<br />
::-ER2738 for infection with M13-Litmus28i<br />
::-BW23115 F’ for infection with M13-Litmus28i to test infectivity of conjugated strain<br />
<br />
==Week 4==<br />
'''5/25'''<br />
*Infect ER and BWF’ cells with M13-Litmus28i<br />
::*Made 5mL culture of ER and BW that was at 1 OD<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! OD<br />
! mL sample for 1OD in 5mL<br />
! mL LB to 5mL<br />
<br />
|-<br />
| ER2738<br />
| 2.5<br />
| 2 mL<br />
| 3 mL<br />
<br />
|-<br />
| BW23115<br />
| 2.0<br />
| 2.5 mL<br />
| 2.5 mL<br />
<br />
|-<br />
|}<br />
::-Based on calculations from 5/24, we needed to add 0.011 ul phage per 1 mL of cells at 1 OD. This equates to 0.055 ul of phage into 5 mL cells; therefore we made a 1:10 dilution so we could add 0.5ul. Unfortunately, the pipet would not take up 0.5ul so we added 0.8ul of M13-Litmus28i phage<br />
::-Grew the cells for 20 minutes at 37C<br />
::-Plated 300ul onto Kanamycin (50ug/mL) and 300ul onto Ampicillin (100ug/mL) for each sample<br />
:::*Incubated overnight at 37C<br />
*'''Note:''' During the production of phage, the phagemid SHOULD be packaged preferentially over the Helper Phagemid but some Helper Phagemid will still be packaged. We plated on Amp to select for cells that were infected with phage containing Phagemid. We plated on Kan to select for cells that were infected with phage containing Helper Phagemid. This allows us to compare the packaging efficiency of Helper Phagemid: Phagemid.<br />
'''5/26'''<br />
*Results from 5/25 infection with M13-Litmus28i<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Result<br />
! Significance<br />
<br />
|-<br />
| ER2738 on Amp<br />
| Lawn<br />
| Litmus28i phagemid was successfully packaged into the M13 phage and is infectable<br />
<br />
|-<br />
| ER2738 on Kan<br />
| 100-200 colonies<br />
| Some M13 helper phage is packaged into the M13 phage but at a much lower rate than Litmus28i<br />
<br />
|-<br />
| BW23115 on Amp<br />
| Lawn<br />
| BW23115 is ‘equally’ infectable by M13 as ER2738<br />
<br />
|-<br />
| BW23115 on Kan<br />
| Lawn<br />
| BW23115 contains Kan resistance in its genome so this tells us nothing<br />
<br />
|-<br />
|}<br />
:*Conclusions:<br />
::-Cells grew on Ampicillin; therefore, Litmus28i phagemid was successfully packaged into M13 phage. <br />
::-For ER2738 samples, there was significant growth on Ampicillin compared to Kanamycin; therefore, Litmus28i phagemid is packaged preferentially over M13K07 Helper Phagemid<br />
::-M13-Litmus28i retains its infectivity of cells containing the F’ episome<br />
:*Because we received lawns, we have to redo the infection and plate less cells so we can calculate the uptake ratio between the phagemid and helper phage based on the number of colonies<br />
*Started 50 mL overnight of K12 ER2738 and BW23115<br />
<br />
'''5/27'''<br />
*Redo the infection done on 5/25<br />
::-Infectable cells: ER2738 and BW23115<br />
:::*Plated non-infected samples of each (non-diluted) to check for contaminants<br />
::-Diluted M13-Litmus28i (1) phage by a factor of 10. Added 5.5ul to each sample<br />
::-Grew samples for 20 minutes at 37C, 250rpm<br />
::-Plated 100ul of onto an Ampicillin (100ug/mL) plate and onto a Kanamycin (50ug/mL) plate. Incubated overnight at 37C.<br />
:::*Dilutions = 1:10; 1:100; and 1:1000<br />
'''5/28'''<br />
*Results from 5/27<br />
::-Controls were as expected<br />
:::*No growth for ER2738 non-infected grown on Amp, ER2738 non-infected grown on Kan, or BW23115 non-infected grown on Amp<br />
:::*Growth for BW23115 non-infected grown on Kan (BW23115 has Kan R in genome)<br />
::-Many colonies were received for all dilutions (1:10, 1:100, and 1:1000) of the following<br />
:::*ER2738 infected and plated on Amp<br />
:::*BW23115 infected and plated on Amp<br />
:::*BW23115 infected and plated on Kan<br />
::-Many (100s to 1000?) colonies grew on 1:10 and 1:100 dilutions of ER2738. 50-100 colonies grew on the 1:1000 dilution of ER2738<br />
:::*Compare this to the 100-200 colonies that grew from 2/25 infection (which was 300ul non-diluted, infected cells)<br />
:::*Reasons for increased yield<br />
::::*Added too much phage?<br />
::::*volume changed between experiment (5mL to 50mL)<br />
::::*Overnight culture may not have been saturated. If still in log phase, the cells would continue to grow<br />
*Made 50mL O/N cultures of ER2738 and BW23115 so we can repeat the infection tomorrow and plate further dilutions starting at 1:1000<br />
::-Carry out infection in 5mL and 50mL to test volume effect?<br />
<br />
'''5/29'''<br />
*Measured OD of overnights<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! OD<br />
! mL to have .1OD in 50mL<br />
! mL to have 1OD in 5mL<br />
<br />
|-<br />
| K12 ER2738<br />
| 3.0<br />
| 1.7<br />
| 1.7<br />
<br />
|-<br />
| BW23115<br />
| 2.9<br />
| 1.7<br />
| 1.7<br />
<br />
|-<br />
|}<br />
<br />
<br />
*Experiment 1: Infect cells using same method as 5/25 (in a 5mL culture)<br />
::-Started with 1OD cells in 5mL<br />
::-Added about 0.7ul (inaccuracies in pipet) of 1:10 diluted M13-Litmus28i phage<br />
::-Incubated (rotating) for 20 minutes<br />
::-Made 1:1,000 and 1:10,000 dilutions<br />
::-Plated 100ul on Ampicillin (100ug/mL) plates and on Kanamycin (50ugmL) plates<br />
:::*Included non-infected samples diluted by 1:1000<br />
::::*This negative control can be used for Experiment 2 since the non-infected parent solution is the same<br />
::-Incubate overnight at 37C<br />
*Experiment 2: Infect cells using protocol from “Eliminating helper phage from phage display”<br />
::-Diluted O/Ns to OD of 0.1 in 50 mL culture<br />
::-Grew samples until of OD of ER2728 = 0.59 and the OD of BW23115 = 0.60<br />
:::*Missed OD of 0.5 mark, but the two samples are close to each other<br />
::-Chilled samples on ice for 30 minutes<br />
::-Warmed in incubator for 35 minutes (should have been 30)<br />
::-Amount of phage. Rather than use 1:1 as mentioned in protocol, we used multiplicity of 1:10 (phage:cell)<br />
:::*Added 3.3ul of 1:10 diluted M13-Litmus28i (1) phage<br />
::::*(On 5/27 we added 5.5ul of diluted phage to 50mL of cells at OD of 1. Our cells were at OD of .6; therefore, 5.5*.6 = 3.3ul)<br />
::-Incubated for 30 minutes at 37C, not shaking<br />
:::*We later change this to shaking<br />
::-Dilutions<br />
:::*1:1,000; 1:5,000; 1:10,000; 1:50,000; 1:100,000; 1:1,000,000<br />
:::*Plated ER2738 and BW23115 on Ampicillin (100ug/mL)<br />
:::*Plated ER2738 on Kanamycin (50ug/mL)<br />
*Experiment 3: Growth Test (for growth curve)<br />
::-We were concerned by the low OD of the Overnights from the last few days. Wanted to be sure that 2.0-3.0 was not still in log phase. Cultures looked saturated but the OD seemed low.<br />
{| class = "wikitable"<br />
|-<br />
! Time<br />
! Elapsed time (min)<br />
! ER2738 (no antibiotic)<br />
! ER2738 (Tetracycline (20ug/mL))<br />
! BW23115 (with F’ episome) no antibiotic<br />
<br />
|-<br />
| 10:08<br />
| 0<br />
| 0.1<br />
| 0.1<br />
| 0.1<br />
<br />
|-<br />
| 11:10<br />
| 62<br />
| 0.24<br />
| 0.21<br />
| 0.21<br />
<br />
|-<br />
| 12:00<br />
| 112<br />
| 0.49<br />
| 0.45<br />
| 0.44<br />
<br />
|-<br />
| 13:00<br />
| 172<br />
| 1.00<br />
| 0.93<br />
| 0.98<br />
<br />
|-<br />
| 14:15<br />
| 217<br />
| 1.29<br />
| 1.21<br />
| 1.31<br />
<br />
|-<br />
| 15:40<br />
| 302<br />
| 2.1<br />
| 1.8<br />
| 2.3<br />
<br />
|-<br />
| 16:47<br />
| 369<br />
| 2.5<br />
| 1.9<br />
| 3.4<br />
<br />
|-<br />
| 18:05<br />
| 447<br />
| 2.6<br />
| 2.2<br />
| 2.5<br />
<br />
|-<br />
| 19:00<br />
| 502<br />
| 2.9<br />
| 2.3<br />
| 2.5<br />
<br />
|-<br />
| 20:00<br />
| 562<br />
| 3.0<br />
| 2.6<br />
| 2.8<br />
<br />
|-<br />
|}<br />
<br />
:::*The time point at 16:47 (369 minutes elapsed) for BW23115 conjugated (without antibiotics) is most likely an error. It has been removed from the growth plot<br />
'''[[File:UCB-Phage Delivery-140529.JPG]]'''<br />
*Other<br />
::-Made Amp and Kan plates (1 sleeve of each)<br />
::-Made 50mL O/N of ER2738 and BW23115F’ in case we need further dilutions<br />
::-Made 5mL O/N of ER2738, BW23115F’, and BW23115 (without F’ episome) to make chemically competent tomorrow<br />
:::*Did not have plate of BW23115 (without F’ episome) so used freeze down. Hoping to get O/N of a picked colony from CRISPR Team tomorrow morning<br />
<br />
'''5/30'''<br />
*Made chemically competent cells of…<br />
::-ER2738<br />
::-BW23115F’ (conjugated with F’ episome)<br />
::-BW23115 (not conjugated- without F’ episome)<br />
:::*Culture started from plate<br />
::-BW23115* not conjugated (without F’ episome)<br />
:::*Culture started from freeze down<br />
*Results from infections<br />
::-Negative Controls (cells were not infected; cells were diluted 1:1000)<br />
'''[[File:UCB-Phage Delivery-140530.JPG]]'''<br />
::-Results from Experiment 1 (5/29)<br />
'''[[File:UCB-Phage Delivery-140530-02.JPG]]'''<br />
::-Results from Experiment 2 (5/29)<br />
'''[[File:UCB-Phage Delivery-140530-03.JPG]]'''<br />
*Math<br />
::-If there are 5.00E+8 cells in 1mL of culture at OD of 1, then in 1mL of culture at OD of 0.59, there are 2.95E+8 cells. In a 50mL culture at OD of 0.59, there are 1.48E+10 cells.<br />
::-We added 3.3ul (0.0033mL) phage at concentration 4.62E+11 phage/mL which amounts to 1.52E+9 total phage<br />
::-Assuming that 1 phage infects 1 bacterium, we can assume that 1.52E+9 bacterial have the potential to be infected in the 50mL culture<br />
::-We plated 100ul of culture at various dilutions. If not diluted, the number of cells that can be potentially infected in 0.1mL equals 1.52E+9/500, or 3.05E+06 cells. We then accounted for the dilutions (for 1:1000 dilution, we divided 3.05E+06 by 1000 to receive 3.05E+03)<br />
::-The following table contains the number of cells with the potential to be infected assuming a 100% infectivity rate by M13 phage and that 1 cell is infected only once.<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Dilution<br />
! Potential infected cells<br />
! Colonies on Amp<br />
! % Potential (Amp)<br />
! Colonies on Kan<br />
! % Potential(Kan)<br />
! Kan:Amp<br />
<br />
|-<br />
| 1:1000<br />
| 1.52E+06<br />
| 476<br />
| 15.62%<br />
| 15<br />
| 0.820%<br />
| 1:19.04<br />
<br />
|-<br />
| 1:5000<br />
| 3.05E+05<br />
| 131<br />
| 21.49%<br />
| 13<br />
| 2.133%<br />
| 1:10.08<br />
<br />
|-<br />
| 1:10000<br />
| 1.52E+05<br />
| 93<br />
| 30.51%<br />
| 5<br />
| 1.640%<br />
| 1:18.60<br />
<br />
|-<br />
| 1:50000<br />
| 3.05E+04<br />
| 17<br />
| 44.29%<br />
| 0<br />
| 0.000%<br />
| <br />
<br />
|-<br />
|}<br />
<br />
*Conclusions from infections<br />
::-Results between and within the three trials are inconsistent. For example, the number of colonies received in experiments 1 and 2 from 5/29 differ greatly. Due to the differences in protocol, variation was expected but not to this extent. <br />
::-Our dilutions did not yield the expected 10 fold (or 5 fold) decrease in growth that was expected.<br />
::-Plates from 5/29 could be plated better to reduce dense areas of growth and growth around the rim.<br />
::-Though the experiment contained many errors we can say that the phagemid (Litmus 28i) is preferentially packaged compared to the helper phage (M13K07) but not to the degree we expected.<br />
::-Could receive increased occurrences of cells containing M13k07 due to infection, phage production, further infection<br />
<br />
==Week 5==<br />
<br />
'''6/2'''<br />
*Tested chemically competent cells through transformation<br />
::-Are cells contaminated?<br />
::-Are cells competent?<br />
*The samples for transformation<br />
{| class = "wikitable"<br />
|-<br />
! #<br />
! Cells (Tube label)<br />
! DNA (Tube label)<br />
! Resistance before transformation<br />
! Resistance after transformation<br />
<br />
|-<br />
| 1<br />
| K12 ER2738 5/20<br />
| p110+RBS (2) 4/23<br />
| Tet<br />
| Tet, Chlor<br />
<br />
|-<br />
| 2<br />
| BW (-f) 5/30<br />
| p110+RBS (2) 4/23<br />
| Kan<br />
| Kan, Chlor<br />
<br />
|-<br />
| 3<br />
| BW f-ep comp 5/22<br />
| p110+RBS (2) 4/23<br />
| Kan, Tet<br />
| Kan, Tet, Chlor<br />
<br />
|-<br />
| 4<br />
| BW (+f) 5/30<br />
| p110+RBS (2) 4/23<br />
| Kan, Tet<br />
| Kan, Tet, Chlor<br />
<br />
|-<br />
| 5<br />
| *BW23115 5/30<br />
| p110+RBS (2) 4/23<br />
| Kan<br />
| Kan, Chlor<br />
<br />
|-<br />
| 2B<br />
| K12 ER2738<br />
| 2B [from dis. kit]<br />
| Tet<br />
| Tet, Chlor<br />
<br />
|-<br />
| 2P<br />
| BW f-ep comp 5/22<br />
| 2P [from dis. kit]<br />
| Kan, Tet<br />
| Kan, Tet, Chlor<br />
<br />
|-<br />
|}<br />
<br />
'''6/3'''<br />
*Results from 6/2 Transformation<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Growth on Chlor<br />
! Growth on Kan<br />
! Growth on Kan+Tet<br />
! Growth on Amp<br />
<br />
|-<br />
| 1 N<br />
| X<br />
| X<br />
| X<br />
| X<br />
<br />
|-<br />
| 2 N<br />
| X<br />
| +<br />
| X<br />
| X<br />
<br />
|-<br />
| 3 N<br />
| X<br />
| +<br />
| +<br />
| X<br />
<br />
|-<br />
| 4 N<br />
| X<br />
| +<br />
| +<br />
| X<br />
<br />
|-<br />
| 5 N<br />
| X<br />
| +<br />
| X<br />
| X<br />
<br />
|-<br />
| 1<br />
| +<br />
| X<br />
| X<br />
| <br />
<br />
|-<br />
| 2<br />
| +<br />
| +<br />
| X<br />
| <br />
<br />
|-<br />
| 3<br />
| +<br />
| +<br />
| +<br />
| <br />
<br />
|-<br />
| 4<br />
| +<br />
| +<br />
| +<br />
| <br />
<br />
|-<br />
| 5<br />
| +<br />
| +<br />
| Many colonies but close to samp. 4<br />
| <br />
<br />
|-<br />
| 2P<br />
| + (~100)<br />
| +<br />
| +<br />
| <br />
<br />
|-<br />
| 1- JW<br />
| +<br />
| X<br />
| X<br />
| <br />
<br />
|-<br />
| 2- JW<br />
| +<br />
| +<br />
| X<br />
| <br />
<br />
|-<br />
| 3- JW<br />
| +<br />
| +<br />
| +<br />
| <br />
<br />
|-<br />
| 4- JW<br />
| +<br />
| +<br />
| +<br />
| <br />
<br />
|-<br />
| 5-JW<br />
| +<br />
| +<br />
| X<br />
| <br />
<br />
|-<br />
| 2B- JW<br />
| + (24)<br />
| X<br />
| X<br />
| <br />
<br />
|-<br />
|}<br />
*The transformations with DNA from the well (B2 and P2) had lower efficiencies than those with DNA from a mini-prep. Most likely this is due to the differences in DNA concentration (p110+RBS (2) 4/23 was at 254.4ng/ul)<br />
*Conclusions<br />
::-None of the competent cells were contaminated<br />
::-All of the competent cells are in fact, competent<br />
*Set up O/N of DH5-alpha cells to make competent tomorrow<br />
<br />
'''6/4'''<br />
*Isolation of single-stranded phagemid DNA using M13K07<br />
::-Added ER2738 colony to 50mL LB<br />
:::*Plate was cold. Next time warm plate before pricking<br />
::::*Best to use freshly grown plate<br />
::-After 4 hours, OD was at 0.02. Waited 45 minutes and OD was at 0.08. Therefore, we infected at OD 0.08<br />
::-Had started another culture when we did not think the first was growing. In incubator for about 1 hour. OD was 0.00. We infected anyway because last time it worked.<br />
::-Let infection proceed for 60 minutes then added 70ul of Kanamycin to be a final concentration of 70ug/mL<br />
*Primers came in to biobrick M13ori (packaging signal on Litmus28i)<br />
::-Resuspended primers and diluted 1:10<br />
<br />
'''6/5'''<br />
*Isolated single-stranded M13K07 DNA<br />
::-Final concentration = 5724 ng/ul (calculated from a 1:10 dilution)<br />
::-For second sample in pair, we resuspended it in TE but did not proceed to DNA extraction teps<br />
::-For the second culture we started 6/4, we resuspended pellet in TBS and glycerol to preserve the M13 phage. Measured absorbances (before glycerol was added)<br />
: #1<br />
::269 => 1.690A<br />
::320 => 0.103A<br />
: #2<br />
::269 => 1.453<br />
::320 => 0.059<br />
*For our first biobrick, we wanted to isolate the M13 origin, a segment ~500bp that allows for packaging into the M13 phage. We tried to achieve this by biobrick assembly and by Gibson Assembly.<br />
::-To biobrick M13 ori through biobrick assembly (the old-school way)<br />
:::PCR on Litmus 28i to amplify/biobrick M13ori<br />
::::*Used primers Gem003 F & R<br />
::::*Diluted Litmus 28i DNA 1:10<br />
:::Digestion of p11+RBS (1) to digest pSB1C3 bb with EcoRI-HF and PstI-HF<br />
:::Ran samples on gel and gel extracted pieces. We recieved very low yields (out of range for nano drop)<br />
::::*M13 ori: 4.0 ng/ul<br />
::::*pSB1C3: 1.8 ng/ul<br />
:::Digested M13 ori fragment despite poor extraction yield with EcoRI-HF and PstI-HF<br />
::::*Used 1.5x as much DNA as instructed based on inaccurate concentration<br />
'''[[File:UCB-Phage Delivery-140605.JPG]]'''<br />
::-Gel extracted red rectangles<br />
::-Ligation<br />
:::*10hr @ 16C, 10min @ 65C, 4ever @ 4C<br />
*To biobrick M13 ori through Gibson Assembly (the cool-kids way)<br />
::-PCR on Litmus 28i<br />
:::*Used primers Gem002 F & R<br />
:::*Diluted Litmus 28i DNA 1:10<br />
::-PCR on pSB1C3 (p11+RBS (1))<br />
:::*Used primers Gem001 F & R<br />
:::*Diluted pSB1C3 DNA 1:3<br />
<br />
'''6/6'''<br />
*Ran gel of PCR products from (6/5). Products will be used for Gibson Assembly<br />
::-Recieved bands for pSB1C3 around 2000bp and M13ori around 500bp<br />
::-No contamination in pSB1C3 PCR negative control<br />
::-Band in M13ori negative control that is the same size as sample. Contaminated by sample?<br />
::-Gel of PCR products #1 and #2 from 6/5<br />
'''[[File:UCB-Phage Delivery-140606.JPG]]'''<br />
:1. pSB1C3 with promoter+RBS as insert. Amplified with Gem001<br />
:2. No DNA control for Gem001<br />
:3. M13ori amplified with Gem002 from Litmus28i<br />
:4. No DNA control for Gem002<br />
*Gibson Assembly<br />
{| class = "wikitable"<br />
|-<br />
! Total Amount of Frag.<br />
! .02-.5pmol<br />
! 10 ul total<br />
<br />
|-<br />
| Gibson Assembly MM (2x)<br />
| 10 ul<br />
| 10<br />
<br />
|-<br />
| Dionized H2O<br />
| 10-x<br />
| <br />
<br />
|-<br />
|}<br />
::-Diluted pSB1C3 and M13ori PCR products 1:10<br />
::-Incubated 60min @ 50C<br />
::-Also used provided pUC16 as positive control<br />
*Transformation<br />
:1. p110+RBS Positive control<br />
:2. No DNA Negative control<br />
:3. Cas9 from distribution kit so we can have more<br />
:4. Thaw and refreeze cells Test competency of comp cells after thawed<br />
:5. Not chem comp cells Negative control for the above<br />
:6. Ligation Product<br />
:7. Gibson product<br />
::*7.2. Gibson product diluted 1:4<br />
:8. Gibson positive control<br />
::*7.2. Gibson positive control diluted 1:4<br />
::-For the Gibson product and the positive control, we transformed 2ul of product and 2ul of 1:4 diluted product. NEB recommends the first if using their competent cells and the second if using cells from other companies. Our cells are from NEB but we made them competent ourselves so we tried both ways<br />
:::*Plated on Chlor at concentrations of 170, 85, and 33 ug/mL<br />
*Primers came in<br />
::-Resuspended and made 1:10 dilutions<br />
<br />
'''6/7'''<br />
*Results from 6/6 transformation<br />
:1. Positive control<br />
::*Lots of growth, ~300-400 on 1:10 dilution<br />
:2. No DNA negative control<br />
::*No growth<br />
:3. Cas9 from distribution kit<br />
::*7 potential colonies (some are close to edges through) on non-diluted<br />
:4. Thawed then refroze cells<br />
::*Looks like (1)<br />
:5. Not chemically competent cells<br />
::*No growth<br />
:6. Ligation product<br />
::*13 potential colonies (some are close to edge)<br />
:7. Gibson Assembly Product<br />
::*170 -> No colonies<br />
::*85 -> No colonies<br />
::*33 -> 3 specks<br />
:7.2. Gibson Assembly product diluted 1:10<br />
::*170 -> 1 speck<br />
::*85 -> 3 colonies<br />
::*33 -> 13 colonies<br />
:8. Gibson positive control<br />
::*No colonies<br />
:8.2. Gibson positive control diluted 1:4<br />
::*No colonies<br />
*Made 6mL O/N cultures<br />
::-4 from (3) cas9 plate<br />
:::See Constitutive CRISPR notebook for more info on these samples<br />
::-7 from (6) Ligation product<br />
::-5 from (7.2 [85]) Diluted Gibson product on 85 ug/mL Chlor<br />
::-8 from (7.2 [33]) Diluted Gibson product on 33 ug/mL Chlor<br />
<br />
==Week 6==<br />
'''6/8'''<br />
*Check colonies for correct constructs.<br />
::-Mini-prepped all 24 O/Ns<br />
:::*Yielded low concentrations for samples 12, 15, 19, and 22<br />
::-Digested all with EcoRI and PstI (10ul reactions)<br />
::-Ran results on gel<br />
:::*All 4 cas9 samples had the expected bands of 2000 and 5000bp<br />
:::*All 7 ligation products have expected bands of 2000 and ~570bp<br />
:::*3 of 5 Gibson assemblies from 85ug/mL Chlor plate had expected bands of 2000 and ~500bp<br />
:::*3 of 8 Gibson assemblies from 33ug/mL Chlor plate had expected bands of 2000 and ~500bp<br />
'''[[File:UCB-Phage Delivery-140608-01.JPG]]'''<br />
<br />
'''[[File:UCB-Phage Delivery-140608-02.JPG]]'''<br />
::1-4: Cas9 from Stanford-Brown team<br />
::5-11: pSB1C3-M13ori cloned through ligation<br />
::12-24: pSB1C3-M13ori cloned through Gibson Assembly<br />
:::*12-16: Grown with 85 ug/mL Chlor<br />
:::*17-24: Grown with 33 ug/mL Chlor<br />
*Conclusions from gel<br />
::-We have cas9 safely in cells<br />
::-Our ligation reactions successfully yielded M13ori on pSB1C3<br />
::-Combined, we had a 46% success rate for the Gibson Assembly in yielding M13ori on pSB1C3<br />
:::*The 4 samples that had the lowest concentration after being mini-prepped (12,15, 19, and 22) correlate with samples that had the correct band pattern<br />
*We selected 4 samples from each (4 total between the two Gibson reactions) type<br />
::-For non-Gibson Assembled samples<br />
:::Plated 25ul on 170ug/mL Chlor<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Our usual method<br />
! Gibson Method<br />
<br />
|-<br />
| 1. Thaw on ice<br />
| 1. Thaw on ice <br />
<br />
|-<br />
| 2. Transfer 40ul cells to tube<br />
| 2. Transfer 50ul cell to tube<br />
<br />
|-<br />
| 3. Add DNA. 1ul for mini-prep OR up to 10ul for ligation<br />
| 3. Add 2ul to NEB cells OR 2ul of 1:4 diluted to other cells<br />
<br />
|-<br />
| 4. Mix by pipet Let sit 30min on ice<br />
| 4. Mix by pipet or flicking Let sit 30 min. on ice<br />
<br />
|-<br />
| 5. Heat shock: 42C for 45s<br />
| 5. Heat shock: 42C for 30s<br />
<br />
|-<br />
| 6. Ice for 5 minutes<br />
| 6. Ice for 2 min.<br />
<br />
|-<br />
| 7. Transfer to culture tube; Add 200ul SOC<br />
| 7. Add 950ul SOC to tube<br />
<br />
|-<br />
| 8. Shake or rotate for 60-120min at 37C<br />
| 8. Shake (250rpm) or rotate for 60 min. at 37C<br />
<br />
|-<br />
| 9.<br />
| 9. Warm plates to 37C<br />
<br />
|-<br />
| 10. Plate 100ul onto selection plate<br />
| 10. Plate 100ul onto plate<br />
<br />
|-<br />
| 11. Incubate O/N @ 37C<br />
| 11. Incubate O/N @ 37C<br />
<br />
|-<br />
|}<br />
::-Added 6mL LB and Chlor at concentration of 170ug/mL to grow O/N<br />
:*For Gibson Assembled samples<br />
::-Plated 25ul onto 170, 85, and 33ug/mL Chlor<br />
::-Samples from 85ng/mL plate<br />
:::Transferred 100ul to new tube, added media, added Chlor at 170ng/mL<br />
::-Samples from 33ng/mL plate<br />
:::Tranfered 100ul to new tubes, added media, added Chlor at 85ng/mL to one and 33ng/mL to the other<br />
:*Tomorrow, may send for sequencing and make freeze downs<br />
<br />
*Transformation<br />
:1. Positive control (p110+RBS diluted 1:10)<br />
:2. Non-diluted Gibson product<br />
:3. Gibson product diluted 1:4<br />
:4. Gibson product diluted 1:10<br />
::-Transformed each sample using our usual method and using the protocol given by Gibson<br />
<br />
::-Due to not have plates ready before transformation, in step 4, the samples sat for about 50 minutes. Then in step 8, they both recovered for about 150 minutes. Though not specified in our protocol, we did warm the plates to 37C. In step 10 for our protocol, since we only added 200ul SOC and wanted to plate on 3 selection plates (see below), we only plated 50ul (except for the positive control). <br />
::-Plated on three concentrations of Chloramphenicol (33 ug/mL, 85 ug/mL, and 170 ug/mL) to determine the differences in yield due to differences in concentration.Obvious hypothesis: more colonies will grow on plates that have a lower concentration of chlor.<br />
<br />
<br />
'''6/9'''<br />
*Made chemically competent 5alpha cells with Dan and Alex from main campus<br />
::-Waiting to hear results on competency<br />
*Will eventually make phage containing CRISPR-Cas9 that targets Kanamycin resistance. M13K07 has Kanamycin resistance so we need to switch the resistance on the M13 genes.<br />
::-PCR on pwp 2.po (plasmid that Sam gave us that contains the zeoR gene adjacent to ori) to amplify zeoR and ori. Zeo is on EM7 promoter<br />
:::-Primers: Gem008 R & R<br />
:::-Anneal temp from NEBuilder: 63.7C<br />
:::-Extension time: 90s<br />
:::-Expected band size on gel: 1300bp<br />
:::-Used phusion polymerase<br />
*PCR on M13K07 DNA to amplify M13 phage genes (also removes majority of M13 ori, all of KanR, and all of p15 ori)<br />
:::-Primers: Gem007 F & R<br />
:::-Anneal temp from NEBuilder: 60.2C<br />
:::-Extension time: 4:30<br />
:::-Expected band size on gel: about 6000bp<br />
:::-Used phusion polymerase<br />
*Freeze downs<br />
::-Note: Phagemid 1C3 was the original name for ‘pSB1C3-M13ori’<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Top Label<br />
! Side label<br />
! What?<br />
<br />
|-<br />
| Phagemid 1C3 6/9<br />
| From Lig<br />
| M13 ori inserted into 1C3 (biobricked); Done through ligation; Contains extra bases as spacer between biobrick prefrix/suffix and part for primer design<br />
<br />
|-<br />
| Phagemid 1C3 6/9<br />
| From Lig<br />
| “ “<br />
<br />
|-<br />
| Phagemid 1C3 6/9<br />
| Gibson<br />
| M13 ori inserted into 1C3 (biobricked); Done through Gibson cloning<br />
<br />
|-<br />
| Phagemid 1C3 6/9<br />
| Gibson<br />
| “ “<br />
<br />
|-<br />
|}<br />
'''6/10'''<br />
*Ran gel of PCRs from 6/9<br />
<br />
'''[[File:UCB-Phage Delivery-140610.JPG]]'''<br />
:1. Amplification of M13 genes from M13K07 (~6000bp)<br />
:2. No DNA control for (1) amplification<br />
:3. Amplification of Zeo resistance gene + plasmid ori (~1300bp)<br />
:4. No DNA control for (3) amplification<br />
*Gibson Assembly of above parts (did not gel extract)<br />
::-Diluted the PCR products 1:10 then added 3ul of M13K07 genes product and 7ul of ZeoR+ori product<br />
::-Incubated at 50C for 60 min.<br />
::-Transformed Gibson Assembly product into 5alpha cells<br />
:::*Used our usual protocol<br />
:::*Add 2ul of DNA<br />
::::-In one sample, diluted DNA 1:4 and in the other, we diluted DNA 1:10<br />
*Started Phage Amplification Protocol<br />
::-ER2738 transformed with Litmus 28i<br />
:::*Grew for ~2.5hr before reaching an OD of 0.04<br />
::-ER2738 transformed with pSB1C3-M13ori (M13ori on pSB1C3)<br />
:::*Grew for ~ 3.5hr before reaching an OD of 0.01, then in the next 1.5 hours, spiked to 0.19<br />
:::*We gave up and went home, and will restart tomorrow<br />
*Analyzed transformation results from 6/8<br />
<br />
'''6/11'''<br />
*Made chemically competent 5alpha cells<br />
*Restarted Phage Amplification Protocol<br />
::-Forgot to add phagemid antibiotic at start of growth. Added phagemid antibiotic when we added phage. Incubated for 90 minutes before adding Kanamycin (to select for cells that were infected by M13K07)<br />
::-Phage is at concentration 4.57x10^12 phage/mL<br />
:::Protocol calls for final concentration of 1 x10^8 phage/mL<br />
::::*(4.57 x10^12)*V = (1 x10^8)(50mL)<br />
::::*V = 0.00109mL<br />
:::Added 1.1ul of phage<br />
*Transformations<br />
{| class="wikitable"<br />
|-<br />
!DNA<br />
!Plate Selection<br />
|-<br />
|No DNA control<br />
|(all)<br />
|-<br />
|Positive control (p110+RBS)<br />
|(C)<br />
|-<br />
|M13 genes + ZeoR ori<br />
|(Z)<br />
|-<br />
|M13 genes +ZeoR ori diluted 1:4<br />
|(z)<br />
|} <br />
<br />
'''6/12'''<br />
*Transformation Results from 6/10 [Took ~36 hours to be clearly visible]<br />
::No DNA control<br />
:::-Amp: 0 <br />
:::-Zeo (50ug/mL): 200 colonies<br />
::M13 genes + ZeoR ori (1:4 dilution) <br />
:::-Zeo (25ug/mL): 300<br />
:::-Zeo (50ug/mL): 200<br />
:::-Zeo (100ug/mL): 100<br />
::M13 genes + ZeoR ori (1:10 dilution) <br />
:::-Zeo (25ug/mL): 300<br />
:::-Zeo (50ug/mL): 150<br />
:::-Zeo (100ug/mL): 150<br />
*Transformation Results for 6/11<br />
::-Positive (p110+RBS) on Chloramphenicol: 500 colonies<br />
::-No DNA on Amp: 0 colonies Zeo (100 ug/mL): specks<br />
::-No apparent growth on any other plate<br />
:::*Realized later that we grew our samples on the wrong plates. Will repeat transformation today<br />
*Transformation #1<br />
::This morning there were no colonies on positive (p110+RBS) coltrol from 6/11 even though we observed fast growth in the past. Without waiting for colonies to appear, we started a control transformation<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Diluted<br />
! Time at 42C<br />
<br />
|-<br />
| p110+RBS<br />
| No<br />
| 45s<br />
<br />
|-<br />
| p110+RBS<br />
| 1:10<br />
| 45s<br />
<br />
|-<br />
| p110+RBS<br />
| 1:10<br />
| 30s<br />
<br />
|-<br />
| p110+RBS<br />
| 1:10<br />
| No timer. ~43s<br />
<br />
|-<br />
| No DNA control<br />
| No<br />
| 45<br />
<br />
|-<br />
|}<br />
*Because ‘No DNA control 6/10’ yielded colonies, we researched Zeocin plates<br />
::-According to Life Technologies (Invitrogen), Zeocin requires low salt medium and a pH of 7.5<br />
::-Low Salt LB Medium (1L)<br />
:::*Ingredients<br />
::::10g Tryptone<br />
::::5g NaCl<br />
::::5g Yeast Extract<br />
:::-Mix ingredients<br />
:::-Adjust pH to 7.5 using NaOH (If go over, use HCl)<br />
:::-Add agar for plates at 15g/L. Autoclave<br />
:::-Thaw Zeocin on ice. Vortex<br />
:::-Add Zeocin to final concentration of 25ug/mL<br />
*Transformation #2<br />
::-Repeat of transformation on 6/11 but this time we will plate on the correct plates<br />
::-Also remade Zeocin plates<br />
*Finished isolation of M13 Litmus phage and M13 pSB1C3-M13ori phage<br />
::*Note: Phagemid 1C3 was the original name for ‘pSB1C3-M13ori’<br />
{| class = "wikitable"<br />
|-<br />
! Phage Sample<br />
! A269<br />
! A320<br />
! Concentration (phage/mL)<br />
<br />
|-<br />
| Litmus phage (1)<br />
| 0.181<br />
| 0.034<br />
| 3.12 x10^12<br />
<br />
|-<br />
| Litmus phage (2)<br />
| 0.227<br />
| 0.047<br />
| 3.83 x10^12<br />
<br />
|-<br />
| Phagemid 1C3 phage (1)<br />
| 0.101<br />
| 0.020<br />
| 1.87 x10^12<br />
<br />
|-<br />
| Phagemid 1C3 phage (2)<br />
| 0.126<br />
| 0.021<br />
| 2.42 x10^12<br />
<br />
|-<br />
|}<br />
::::phage/mL = 6x10^16 x (A269-A320)/ (#of base pairs in the phage genome)<br />
*Set up 50mL O/N of K12 ER2738 (containing f-episome) for infection tomorrow with Litmus phage and pSB1C3-M13ori phage<br />
<br />
<br />
'''6/13'''<br />
*Transformation Results for 6/11<br />
::-Several hundred colonies on Positive Control (p110+RBS) on Chlor<br />
::-No colonies for GA positive control on Amp<br />
::-No colonies for M13ori + ZeoR mistakenly plated on Amp<br />
::-No colonies for No DNA control on Amp<br />
::-100-ish colonies for No DNA control on Zeo<br />
::-100-ish colonies for cas9+AmpR+gRNA mistakenly plated on Zeo<br />
::-These last 3 points suggest/confirm that Zeo plates are no good<br />
*Transformation Results from 6/12 control test<br />
::-Colonies grew is about equal amounts on all plates, including No DNA control<br />
:::Either plates don’t contain Chlor or competent cells are contaminated<br />
::-Streaked competent cells onto new and old Chlor plates<br />
*Infection test of ER2738: Is Litmus preferentially packaged over M13K07 Helper phage? Is pSB1C3-M13ori preferentially packaged?<br />
::-Infect ER2738 with phage produced 6/12<br />
:::*Phage should have packaged Litmus 28i phagemid or pSB1C3-M13ori<br />
:::*Cells infected with phage packaging Litmus 28i will grow on Amp<br />
:::*Cells infected with phage packaging pSB1C3-M13ori will grow on Chlor<br />
:::*Cells infected with phage packaging M13K07 will grow on Kan<br />
::-After we plate, we can count the colonies and calculate a ratio of Litmus28i: M13K07 or pSB1C3-M13ori:M13K07 packaging<br />
<br />
'''6/14'''<br />
*Results of contamination test (streaked competent cells onto new and old Chlor plates)<br />
::-Colonies grew in low amounts on both plates. most likely the cells are contaminated<br />
*Transformation results for 6/12<br />
::-Many colonies for No DNA control on Zeo<br />
:::-Some colonies are turning pinkish-red<br />
::-Many colonies for diluted and non-diluted M13 genes+ZeoR on Zeo<br />
:::-Some colonies are turning pinkish-red<br />
*Could the white colonies be the designed colonies and can we kill the red colonies with Zeocin before killing the white colonies (aka. Use high Zeocin concentrations to select for the correct construct)<br />
::-Selected 1 red colony from No DNA Zeo control plate and 1 white colony from M13genes+ZeoR sample plate<br />
::-Added 100ul H2O then divided amongst 5 culture tubes each (with 5mL of low-salt LB, pH 7.5)<br />
::-Then added Zeocin to a final concentration of: 0, 25, 50, 75, 100ug/mL.<br />
*Results of 6/13 infection<br />
::-Litmus 28i infected cells<br />
:::*on Kan: Individual colonies for 1:10, 1:100, and 1:1000. No colonies on 1:5000<br />
:::*on Amp: lawn for 1:10, 1:100, and near lawn for 1:1000. Single colonies for 1:5000<br />
::-pSB1C3-M13ori infected cells<br />
:::*on Kan: Same as Litmus 28i samples on Kan<br />
:::*on Chlor: Lawn for all dilutions. Must discredit due to recent Chlor contamination<br />
<br />
==Week 7==<br />
<br />
'''6/15'''<br />
*Results of Zeocin experiment on 6/14<br />
::-Healthy growth for both at 0ug/mL Zeo<br />
::-No growth for white colony with any Zeo<br />
::-Good growth for red colony even at 100ug/mL Zeo<br />
::-This suggests that the red colonies are naturally resistant to Zeocin. Also, our plates must not contain active Neocin. We are sure that we are adding enough. Possibly, we add it while the media is too hot or leave the plates at room temperature (decondensing) for too long and this deactivates the antibiotic? Perhaps we are not attaining the correct pH<br />
*In light of recent contamination problems on both Chlor and Zeo, we made new competent cells<br />
::-5alpha<br />
::-BW23115<br />
::-BW23115 conjugated (contains f-episome)<br />
<br />
'''6/16'''<br />
*Made new Chlor antibiotic. Made to Chlor plates<br />
*Contamination Test: Streaked Zeo and new Chlor plates each with<br />
::-Colony from Zeo contaminated plate<br />
::-Colony from Chlor contaminated plate<br />
::-Non-transformed OLD chemically competent cells<br />
::-Non-transformed NEW chemically competent cells<br />
*Redid bacterial infection<br />
::-For both samples, we used ER2738 (not from the same ‘O/N’ (also, not a real O/N))<br />
::-Infected one sample with isolated ‘Litmus28i phage’ and one with ‘pSB1C3-M13ori’<br />
::-Alterations to protocol<br />
:::*Did not started from saturated O/N. Started each with a colony, waited several hours until at OD ~1. We then added these cells to fresh 50mL LB to have an OD of 0.1.<br />
:::*Missed the 30 minute infection mark. Infected for ~45 minutes.<br />
::-Plated on Kan and Amp (Litmus28i sample) or Chlor (pSB1C3-M13ori sample) at dilutions of<br />
:::*1:100, 1:1k, 1:10k, and 1:100k<br />
<br />
'''6/17'''<br />
*Q5 PCR to replace KanR with ZeoR in M13K07<br />
::-Used recommendations<br />
::-Unfortunately, I (Jo) don’t know the difference between tightening and loosening the thermocycler lid; therefore, our M13K07 sample to amplify the M13K07 genes evaporated. But that’s ok, because we were sick of Zeocin anyway and decided mid-PCR to not waste our time with a Gibson Assembly and transformation. Instead we will be using the Old-School method of digestion and ligation because let’s face it, it’s a classic (and Gibson sucks) =)<br />
*Results from infection Test<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Litmus 28i<br />
! 1:100<br />
! 1:1k<br />
! 1:10k<br />
! 1:100k<br />
<br />
|-<br />
| Amp<br />
| ~6000?<br />
| ~2056<br />
| 377<br />
| 36<br />
<br />
|-<br />
| Kan<br />
| 130<br />
| 8<br />
| 3<br />
| 0<br />
<br />
|-<br />
|}<br />
{| class = "wikitable"<br />
|-<br />
! pSB1C3-M13ori<br />
! 1:100<br />
! 1:1k<br />
! 1:10k<br />
! 1:100k<br />
<br />
|-<br />
| Chlor<br />
| 8<br />
| 1<br />
| 0<br />
| 0<br />
<br />
|-<br />
| Kan<br />
| 97<br />
| 9<br />
| 0<br />
| 0<br />
<br />
|-<br />
|}<br />
::-We consider the Litmus 28i to have been a success. The pSB1C3-M13ori …. not so much. Looking back at the Litmus28i and the M13K07, we noticed that the M13ori is facing the opposite direction as the plasmid ori. Ours faces the same direction as the plasmid ori. Because we are working with phagemids that are single stranded, we think that by flipping the M13ori, we may be able to recover functionality. We will also look into other reasons.<br />
*Results from contamination test (6/16)<br />
::-Old Chlor plate (6/12)<br />
:::-Non-transformed chemically competent cells<br />
::::*Old: some colonies<br />
::::*New: No colonies<br />
::-New Chlor plate (6/16)<br />
:::-Colony from<br />
::::*Zeo contaminated plate: No growth<br />
::::*Chlor contaminated plate: Much growth<br />
:::-Non-transformed chemically competent cells<br />
::::*Old: some colonies<br />
::::*New: No growth<br />
::-Zeo (25ug/mL, Low NaCl, pH 7.5) (6/12)<br />
:::-Colony from<br />
::::*Zeo contaminated plate: Much growth<br />
::::*Chlor contaminated plate: No growth<br />
:::-Non-transformed chemically competent cells<br />
::::*Old: some specks<br />
::::*New: some specks<br />
<br />
'''6/18'''<br />
*Ordered primers to…<br />
::-biobrick M13ori (in other direction)<br />
::-biobrick M13K07 genes<br />
'''6/19'''<br />
*Waited for primers<br />
*Set up O/N cultures to test last infection (6/16) for colonies containing pSB1C3-M13ori<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample #<br />
! Selection<br />
! Dilution<br />
! Presumably<br />
! Notes<br />
<br />
|-<br />
| 1-8<br />
| Chlor<br />
| 1:100<br />
| pSB1C3-M13ori<br />
| <br />
<br />
|-<br />
| 9<br />
| Chlor<br />
| 1:1k<br />
| pSB1C3-M13ori<br />
| <br />
<br />
|-<br />
| 10-14<br />
| Chlor<br />
| 1:1k<br />
| pSB1C3-M13ori<br />
| These colonies were not present on plate during initial counting on 6/17<br />
<br />
|-<br />
| 15-16<br />
| Kan<br />
| 1:1k<br />
| M13K07<br />
| <br />
<br />
|-<br />
|}<br />
:::-Selection and dilution refer to the plate. Cells were then grown under selection.<br />
:::-More colonies were seen on all Chlor plates. No new colonies appeared on Kan plates<br />
*Tomorrow, we will mini-prep, digest, and run the samples on a gel to verify gene transfer.<br />
<br />
'''6/20'''<br />
*Primers are in!<br />
*Cloning of pSB1C3-M13ori(New)<br />
::-PCR to amplify M13ori (packaging signal) from Litmus 28i to be in the other direction<br />
:::-Primers (Gem011 F&R) add cut sites to make part biobrick compatible<br />
:::-Was able to gel extract<br />
::::*Band at the same size as sample….. contaminated primers?<br />
:::-Then digested with E and P<br />
::-Digested pSB1C3 plasmid with E and P to linearize backbone<br />
::::*Was able to gel extract<br />
'''[[File:UCB-Phage Delivery-140620.JPG]]'''<br />
<br />
:1. Spill over from 2<br />
:2 and 3. pSB1C3 digested with EcoRI and PstI<br />
::-Insert <100bp so cannot be seen<br />
:4. M13ori<br />
*Ligation of M13ori (packaging signal) into pSB1C3<br />
::10h at 16C<br />
::20m at 80C<br />
::Hold at 4C<br />
<br />
*PCR to amplify M13K07 genes from M13K07 DNA isolated from phage<br />
::-Primers (Gem012 F&R) add cut sites to make part biobrick compatible<br />
::-Very light bands and not band might correlate with 6000pb but not enough resolution on gel to be certain. Bands were too light so did not extract<br />
::-Set up PCR again using Q5 to run O/N<br />
:::*Received only a feint, smudgy band that was too large. We did not bother with extracting the DNA<br />
*Test of 6/19 O/Ns (14 that are presumably pSB1C3-M13ori and 2 that are presumably M13K07)<br />
::-Mini-prepped all O/Ns<br />
::-Digested all with Pst-I (common RE between pSB1C3-M13ori and M13K07)<br />
:::-Even though these are phagemids, we assumed that because they were still in the cell, the plasmids were still double stranded so could be recognized by RE. Our assumption was valid<br />
:::-Results of digestion<br />
::::*1,2,4-14: all had bands that corresponded to pSB1C3-M13ori cut once<br />
::::*3: Slight band at expected size but very feint<br />
::::*15, 16: as expected, they contain bands of ~9000bp, correlating with the M13K07 phagemid. Interestingly, sample 15 also contained a (brighter) band that corresponds with pSB1C3-M13ori. We assume that this was an incident of double infection (chance or did this occur at high frequency?)<br />
'''[[File:UCB-Phage Delivery-140620-02.JPG]]'''<br />
::Check table from 6/19 for more details<br />
:::-1-14: pSB1C3-M13ori<br />
:::-15-16: M13K07 from 1:1k diluted plate<br />
<br />
'''6/21'''<br />
*Needed to transform our pSB1C3-M13ori(New) (6/20) into cells. First, we transformed into 5alpha cells; however, we need to infect these cells later in order to make phage. Our 5alphas are not competent so we repeated the transformation, this time using ER2738 cells which contain the F’ episome, allowing us to infect with the M13 phage.<br />
::-Also transformed sample #1 from 6/20 mini-preps (pSB1C3-M13ori(Old) into ER2738 cells. Do not see any notes about this plasmid being in infectable cells during the initial experiment. Reason for experiment failure???<br />
*Ran 6/20 O/N PCR (to amplify M13K07 genes) on gel (also re-ran the previous sample from the day with more DNA)<br />
::-Still, band looks too big. Brightest band still occurs between 8000 and 9000bp. Lighter band around 7000bp-- might be ~6000pb but too light to tell<br />
*Alternative plan for amplifying M13K07 genes<br />
::-Digest sample with PstI (cuts outside of the target region)<br />
'''[[File:UCB-Phage Delivery-140621.JPG]]'''<br />
::Used sample #16 from 6/20 b/c it is double stranded so will cut<br />
:-PCR amplified the linearized digestion product<br />
:::1. PCR of M13genes<br />
:::2. PCR of M13genes + DMSO<br />
::::Gel extracted pieces boxed in red<br />
::-Used primers Gem012 F&R<br />
::-Gel extracted piece but received very low yield<br />
*Alternative to alternative plan for amplifying M13K07 genes<br />
::-Digested with PstI and AgeI<br />
'''[[File:UCB-Phage Delivery-140621-02.JPG]]'''<br />
:1. Cut with PstI and AgeI<br />
:2. Uncut<br />
:3. Cut with PstI<br />
:4. Just PCR<br />
::-Gel extracted ~6000pb band after double digestion<br />
::-PCR (Phusion)<br />
:::-Digestion (A+P) product<br />
:::-Gel extraction product<br />
:::-No DNA control<br />
<br />
==Week 8==<br />
<br />
'''6/22'''<br />
*Did receive colonies from 6/21 transformations. Selected colonies for overnight<br />
{|class="wikitable"<br />
|-<br />
!number<br />
!colonies<br />
|-<br />
|1-7<br />
|pSB1C3-M13ori(NEW) in 5alpha<br />
|-<br />
|8-14<br />
|pSB1C3-M13ori(NEW) in ER2738<br />
|-<br />
|15-18<br />
|pSB1C3-M13ori(OLD) in ER2738<br />
|-<br />
|19<br />
|p110+RBS in 5alpha as control<br />
|-<br />
|20<br />
|p110+RBS in ER2738 as control<br />
|}<br />
<br />
*Gel of 6/21 O/N PCR<br />
::Digestion (AgeI+PstI) product ? Yielded the three bands that appear with every PCR of M13K07<br />
'''[[File:UCB-Phage Delivery-140622.JPG]]'''<br />
<br />
:1. M13K07-> digested (AgeI + PstI) -> PCR<br />
:2. M13K07-> digested (AgeI+ PstI) -> extraction-> PCR<br />
:3. No DNA control for PCR<br />
::-Gel extraction product ? No bands<br />
::-No DNA control ? No bands<br />
*PCR purified the above PCR (Digestion (AgeI+PstI) product) and PCR from 6/21<br />
*Digested PCR purified sample with EcoRI+PstI and ran gel<br />
'''[[File:UCB-Phage Delivery-140622-02.JPG]]'''<br />
:1. PCR-> PCR purified<br />
:2. Sample after DpnI digest<br />
:3. Sample after digestion with EcoRI and PstI<br />
:4. Samples after digestion with DpnI then EcoRI and PstI<br />
*Ligation of above digestion (M13K07 genes) with pSB1C3<br />
<br />
<br />
'''6/23'''<br />
*Transformed pSB1C3-M13ori(New) (6/22 ligation)<br />
*Check overnights from 6/22 for the correct insert<br />
::-Mini-prep O/Ns<br />
::-Digested with EcoRI and PstI<br />
'''[[File:UCB-Phage Delivery-140622-03.JPG]]'''<br />
:::1-7: pSB1C3-M13ori(New) in 5alpha<br />
:::8-14: pSB1C3-M13ori(New) in ER2738<br />
:::15-18: pSB1C3-M13ori(Old)<br />
::-All were the expected size (though gel wiggled)<br />
::-Sent 4 samples for sequencing (iGEM primers: VF2 and VR)<br />
:::# 4: Divergent (new) phagemid 1C3 transformed into 5alpha<br />
:::# 11, 13: Divergent (new) phagemid 1C3 transformed into ER2738<br />
:::# 16: Convergent (old) phagemid 1C3 transformed into ER2738<br />
<br />
'''6/24'''<br />
*Results of 6/23 transformation (pSB1C3-M13genes) from 6/22 ligation)<br />
::-Received ~100 colonies<br />
::-Set up O/N cultures for 8 of the colonies<br />
::-Time passed…..<br />
::-Mini-prepped the 8 O/N samples mentioned above (yes, it was a long day)<br />
:::*Digested samples (E+P)<br />
*Started phage amplification protocol<br />
::-Amount of phage added = 10.9ul of 1:10 diluted phage (M13 phage (1) from 5/24 (4.575 x10^12 phage/mL)) to a final concentration of 1x10^8 phage/mL in 50mL<br />
::-Managed to get both samples to the 14-28hr incubation<br />
<br />
'''6/25'''<br />
*Checked mini-prep samples from 6/24 (pSB1C3-M13genes)<br />
::Ran the digestion overnight (6/24 to 6/25)<br />
'''[[File:UCB-Phage Delivery-140625.JPG]]'''<br />
::-All contained a band at 2000bp. Mostly empty vector or contained small, light band. Sample 1 had prominent band at ~1250bp.<br />
::-None of samples contained M13genes<br />
*M13K07 is on P15A ori (10-12 copy number) whereas the pSB1C3 ori is on pUC19 (500-700 copies). It’s possible that this overexpression is detrimental to cell<br />
::Alternative low copy plasmids found in distribution kit<br />
:::*2013 (plate 5)<br />
::::pSB6A1 (1K)<br />
::::pSB3C5 (3C)<br />
::::pSB3K3 (5E)<br />
:::*2014 (plate 4)<br />
::::pSB3C5 (4D)<br />
::::pSB6A1 (2L)<br />
::-Suspended and transformed the above plasmids into 5alphas<br />
::-Next, we will select colonies, mini-prep, digest, gel extract, ligate with M13genes<br />
*Started phage amplification protocol <br />
::-Phagemids in ER2738<br />
:::pSB1C3-M13ori(New)<br />
:::pSB1C3-M13ori(Old) <br />
::-Set up O/N of ER2738 for infection tomorrow<br />
<br />
'''6/26'''<br />
*Results of transformation of plasmids from the distribution kit<br />
::-Only received colonies pSB6A1 (2L from 2014) and pSB3C5 (4D from 2014)<br />
::-Pricked colonies for O/N<br />
*PCR of M13 genes so we can ligate it into the above backbones tomorrow<br />
::-DNA: M13K07 digested with EcoRI+PstI (6/21)<br />
::-Primers: Gem012 F & R<br />
*Finished phage amplification protocol<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Samples<br />
! A269<br />
! A320<br />
! Concentration<br />
<br />
|-<br />
| Old phagemid 1C3 (1)<br />
| 0.832<br />
| 0.492<br />
| 7.828 x10^12<br />
<br />
|-<br />
| Old phagemid 1C3 (2)<br />
| 0.324<br />
| 0.083<br />
| 5.549 x10^12<br />
<br />
|-<br />
| New phagemid 1C3 (1)<br />
| 0.391<br />
| 0.077<br />
| 7.229 x10^12<br />
<br />
|-<br />
| New phagemid 1C3 (2)<br />
| 0.402<br />
| 0.639<br />
| -5.457 x10^12<br />
<br />
|-<br />
|}<br />
::::*While resuspending “New phagemid 1C3 (2)”, the tip fell off and we lost half of sample. Evidently, we lost most of phage so we tossed sample<br />
::-Prepped and Infected ER2738 with “Old phagemid 1C3 (1)” and “New phagemid 1C3 (2)”<br />
:::Added 1.6ul of 1:10 diluted “Old phagemid 1C3 (1) to ER2738 cells<br />
:::Added 1.8ul of 1:10 diluted “New phagemid 1C3 (1) to ER2738 cells<br />
::-Plated the infected cells at dilutions<br />
:::1:1 onto Chlor+Kan plates<br />
:::1:10 onto Chlor+Kan plates<br />
:::1:100 onto Chlor and onto Kan plates<br />
:::1:1000 onto Chlor and onto Kan plates<br />
:::1:10000 onto Chlor and onto Kan plates<br />
:::1:100000 onto Chlor and onto Kan plates<br />
<br />
'''6/27'''<br />
*To ligate M13genes onto different backbones<br />
::-Mini-prepped the O/Ns from 6/26 to get backbones with low copy number<br />
:::pSB6A1<br />
:::pSB3C5 <br />
::::*did not grow as well<br />
::-PCR purified PCR (to amplify M13 genes with Gem012 F & R) from 6/26<br />
::-Digestions (50ul)<br />
:::Digest pSB6A1 with E + P<br />
:::Digest pSB3C5 with E + P<br />
:::Digest PCR purification product with E + P<br />
'''[[File:UCB-Phage Delivery-140627.JPG]]'''<br />
:1. pSB6A1<br />
:2. pSB3C5<br />
:3. M13genes<br />
::-Gel extraction of the above digestions<br />
:::For each: Added 10ul of 6x loading dye to 50ul digestions and divided the total volume between 2 wells<br />
::-Ligation<br />
:::1) pSB6A1-M13genes<br />
:::2) pSB3C5-M13genes<br />
*Results from 6/26 infection (after 22hrs in incubator)<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Anti-Sense Phagemid 1C3<br />
! 1:100<br />
! 1:1000<br />
! 1:10000<br />
! 1:100000<br />
<br />
|-<br />
| Chlor<br />
| 85<br />
| 6<br />
| 2<br />
| 0<br />
<br />
|-<br />
| Kan<br />
| 405<br />
| 13<br />
| 2<br />
| 1<br />
<br />
|-<br />
|}<br />
{| class = "wikitable"<br />
|-<br />
! Sense Phagemid1C3<br />
! 1:100<br />
! 1:1000<br />
! 1:10000<br />
! 1:100000<br />
<br />
|-<br />
| Chlor<br />
| 121<br />
| 24<br />
| 3<br />
| 0<br />
<br />
|-<br />
| Kan<br />
| 25<br />
| 1<br />
| 0<br />
| 0<br />
<br />
|-<br />
|}<br />
-We also plated the infected cells on plates containing Chlor and Kan to test for the possibility of double infection<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Phagemid 1C3<br />
! 1:1<br />
! 1:10<br />
<br />
|-<br />
| Anti-Sense<br />
| 59<br />
| 0<br />
<br />
|-<br />
| Sense<br />
| 4<br />
| 0<br />
<br />
|-<br />
|}<br />
<br />
<br />
'''6/28'''<br />
*We noticed that there were more colonies on our infection plates from 6/26 than on 6/27; therefore, we recounted colonies<br />
::-No increase of colonies on Kan plates<br />
::-Significant increase of colonies on Chlor plates<br />
::-Sense refers to the first phagemid 1C3 where the M13ori is in the sense direction compared to the plasmid ori<br />
::-Anti-Sense refers to the new phagemid 1C3 where the M13ori is in the anti-sense direction compared to the plasmid ori<br />
'''[[File:UCB-Phage Delivery-140628.JPG]]'''<br />
::Numbers on the left are after 22 hours. Numbers on the right are after 38.75 hours<br />
*Results of transformation from 6/27 (pSB6A1-M13genes and pSB3C5-M13genes)<br />
::-We have many colonies. Unfortunately, some are red, suggesting that original insert (J04450) was not successfully separated from backbone through gel extraction<br />
::-Selected colonies to grow overnight in 5mL LB<br />
<br />
==Week 9==<br />
<br />
'''6/29'''<br />
*Check O/N cultures for correct constructs (pSB6A1-M13genes and pSB3C5-M13genes)<br />
::-Mini-prep samples<br />
::-Digested with EcoRI and PstI to check insert sizes<br />
::-Gel<br />
'''[[File:UCB-Phage Delivery-140629.JPG]]'''<br />
::lanes….<br />
:::1-11: pSB6A1-M13genes<br />
:::12-18: pSB3C5-M13genes<br />
::-Epic failure<br />
::-All pSB6A1 backbones are empty<br />
::-Half of pSB3C5 backbones were empty. The others contained random inserts (1700 OR 3500). We don’t know what these inserts are. 1700 band is likely the digestion product that appears when we digest M13genes<br />
*Question: Can we use empty vectors from these mini-preps as ligation vectors?<br />
::-Digest pSB6A1 mini-prep with…. (see if both cut sites were retained during re-ligation)<br />
:::no enzyme<br />
:::EcoRI<br />
:::PstI<br />
:::EcoRI + PstI<br />
'''[[File:UCB-Phage Delivery-140629-02.JPG]]'''<br />
::lanes....<br />
:::1-2: last two samples of pSB3C5-M13genes from above gel<br />
:::3. Uncut<br />
:::4. Cut with EcoRI<br />
:::5. Cut with PstI<br />
:::6. Cut with EcoRI and PstI<br />
:-Appears that two backbones are ligated together<br />
<br />
'''6/30'''<br />
*Made chemically competent ER2738 cells that contain Litmus28i DNA<br />
*To amplify M13genes in order to retry ligation<br />
::-PCR of M13K07 DNA (diluted 1:100) to amplify the M13 genes<br />
::-PCR purify PCR product<br />
::-Run on gel: PCR purification, PCR, noDNAcontrol<br />
:::*PCR and purification showed bands at ~9k; therefore, did not get product<br />
:::*No DNA control was clean<br />
*Made freeze downs of<br />
::pSB3C5-J04450<br />
::pSB6A1-J04450<br />
::‘empty’ pSB3C5<br />
::‘empty’ pSB6A1<br />
<br />
'''7/1'''<br />
*Digestion of PCR product from 6/30 to figure out where mistake is<br />
:-Not really useful<br />
'''[[File:UCB-Phage Delivery-140701.JPG]]'''<br />
:Lanes...<br />
:1. PCR pur -> digested with AgeI<br />
:2. PCR pur -> digested with NgoMIV<br />
:3. PCR pur -> digested with PstI<br />
:4. PCR pur -> uncut<br />
:5. PCR pur -> digested with dpnI<br />
:6. Uncut plasmid DNA<br />
*Talked to Mary: She says we were adding to much DNA<br />
::-PCR again. Used 1:100 dilution of 1:100 diluted M13K07 DNA. (aka 1:10000 dilution)<br />
'''[[File:UCB-Phage Delivery-140701-02.JPG]]'''<br />
*Received a beautiful band at 6k bp<br />
:lanes<br />
:1. 1:100 dilution<br />
:2. 1:1000 dilution<br />
:3. 1:10 000 dilution<br />
:4. No DNA control<br />
<br />
'''7/2'''<br />
*To biobrick M13 genes (pSB6A1-M13genes and pSB3C5-M13genes)<br />
::-PCR purified 7/1 PCR product (Primers = Gem012)<br />
::-Digested with EcoRI-HF and PstI-HF<br />
::-Ligation to<br />
::::pSBA61 (digested and gel extracted)<br />
::::pSB3C5 (digested and gel extracted)<br />
<br />
'''7/3'''<br />
*Transformation<br />
::-pSB6A1-M13genes into 5alpha cells and ER2738 with pSB1C3-M13ori<br />
::-pSB3C5-M13genes into 5alpha cells and ER2738 with Litmus28i<br />
::-Transformed into cells containing a phagemid in order to skip some steps<br />
<br />
'''7/3-7/7 Vacation!'''<br />
<br />
==Week 10==<br />
<br />
'''7/7'''<br />
*Made O/N cultures of 7/3 transformation colonies<br />
::-Transformation results were not recorded until 7/8 (see below)<br />
<br />
'''7/8'''<br />
*Transformation results ( CC = Chemically comp cells, ER = ER2738)<br />
::-No growth on No DNA controls<br />
:::ER-Litmus28i CC on AMP-Chlor-Tet<br />
:::ER-phagemid1C3 CC on AMP-Chlor-Tet <br />
:::5alpha CC on Amp <br />
:::5alpha CC on Chlor <br />
::-Lots of red colonies on positive controls, no white colonies<br />
:::ER-Litmus28i CC + pSB3C5 on AMP-Chlor-Tet<br />
:::ER-phagemid1C3 CC + pSB6A1 on AMP-Chlor-Tet<br />
::-Lots of white colonies on sample plates, some red colonies<br />
:::ER-Lit CC + M13genes-pSB3C5 on AMP-Chlor-Tet<br />
:::ER-phagemid1C3 CC + M13genes-pSB6A1 on AMP-Chlor-Tet<br />
:::5alpha CC + M13genes-pSB3C5 on Chlor<br />
:::5alpha CC + M13genes-pSB6A1 on Amp<br />
*Overnights from 7/7 look healthy<br />
::-Renamed O/N to have numbers instead of long names<br />
{| class = "wikitable"<br />
|-<br />
! Sample #<br />
! Cells<br />
! DNA<br />
<br />
|-<br />
| 1-5<br />
| ER-Lit<br />
| M13genes-pSB3C5<br />
<br />
|-<br />
| 6-10<br />
| ER-phagemid1C3<br />
| M13genes-pSB6A1<br />
<br />
|-<br />
| 11-15<br />
| 5alpha<br />
| M13genes-pSB3C5<br />
<br />
|-<br />
| 16-20<br />
| 5alpha<br />
| M13genes-pSB6A1<br />
<br />
|-<br />
|}<br />
::-Mini-prepped DNA<br />
::-Digest the mini-preps with EcoRI-HF and PstI-HF<br />
::-Run digestions on gel to check sizes<br />
'''[[File:UCB-Phage Delivery-140708.JPG]]'''<br />
<br />
:lanes<br />
::top-left: pSB3C5-M13genes in ER2738 with Litmus28i<br />
::top-right: pSB6A1-M13genes in ER2738 with pSB1C3-1C3<br />
::bottom-left: pSB3C5-M13genes in 5alpha<br />
::bottom-right: pSB6A1-M13genes in 5alpha<br />
:None are correct<br />
*Primers Gem013 came in. Resuspend and diluted primers<br />
::-O/N of pSB3C5 to use for PCR tomorrow<br />
<br />
'''7/9'''<br />
*Wanted to make Litmus28i biobrick compatible for use as a phagemid backbone for us and other iGEM teams<br />
::-Mini-prepped O/N of pSB3C5<br />
::-PCR of pSB3C5 to amplify J04450 with Litmus28i compatible cut sites<br />
:::Primers: Gem013<br />
::::*At the 3’ end, these primers are the same as VF2 and VR so will bind the region flanking J04450. This conserves the terminators that exist between the biobrick prefix and VF2 on one side and those between the biobrick suffix and VR on the other side. Therefore, we are amplifying, VF2 priming site, terminators, J04450, terminators, and VR priming site<br />
::::*At the 5’ end, these primers contain unique restriction sites found in the Litmus28i MCS<br />
*PCR of M13 genes…. again (did 4 samples)<br />
::-Used the 1:10000 dilutions<br />
<br />
'''7/10'''<br />
*Note: The following two projects were done in parallel when possible<br />
*To make Litmus28i biobrick compatible<br />
::-Ran gel of PCR from 7/9 (J04450 amplification with Gem013)<br />
'''[[File:UCB-Phage Delivery-140710.JPG]]'''<br />
<br />
:1-4: PCRs of M13genes<br />
:5. No DNA control for M13genes PCR<br />
:6. PCR of pSB3C5 backbone<br />
:7. No DNA control for pSB3C5 backbone<br />
::-PRC purified the PCR product<br />
::-Digestion #1<br />
:::Restriction enzymes had the same buffer conditions but different activation temperatures so we had to do a 2 part digestion<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Restriction enzymes<br />
! Notes<br />
<br />
|-<br />
| J04450<br />
| Sac1<br />
| PCR purified<br />
<br />
|-<br />
| Litmus 28i<br />
| Sac1<br />
| From NEB tube<br />
<br />
|-<br />
|}<br />
:::*Incubate 1hr at 37 C<br />
:::*Heat inactivated 20 minutes at 80C<br />
::-Digestion #2<br />
:::-Added 1ul BsmI to both samples<br />
:::-Incubated 1hr at 65C<br />
:::-Heat inactivated 20 minutes at 80C<br />
::Ran gel<br />
:::-See gel below<br />
:::-Tried to extract J04450 segment but received very low yield. Since band otherwise looked clean, we decided to redo digestion then skip straight to ligation<br />
::Repeat digestion #1 for J04450<br />
::Repeat digestion #2 for J04450<br />
::Ligation (10hr at 16C, 10 min at 80C)<br />
:::3. Litmus28i + J04450<br />
*To retry ligation to biobrick backbone<br />
::-Ran gel of PCR from 7/9 (M13genes amplified with Gem012)<br />
:::See gel below<br />
::-PCR purified the PCR product<br />
::-Digestion<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Restriction enzymes<br />
! Notes<br />
<br />
|-<br />
| M13 genes<br />
| EcoRI-HF + PstI-HF<br />
| PCR purified<br />
<br />
|-<br />
| 6A1<br />
| EcoRI-HF + PstI-HF<br />
| ‘empty’ pSB6A1<br />
<br />
|-<br />
| 3C5<br />
| EcoRI-HF + PstI-HF<br />
| contains J04450<br />
<br />
|-<br />
|}<br />
:Incubate 1hr at 37C<br />
:Heat inactivated 20 minutes at 80C<br />
::-Ran gel<br />
:::*See gel below<br />
:::*Tried to extract M13genes and pSB3C5 segments but received very low yields. Since bands otherwise looked clean, we decided to redo digestion then skip straight to ligation<br />
::-Repeat digestion for M13genes and pSB3C5<br />
::-Ligation (10h at 16C, 10m at 80C)<br />
:::1. pSB3C5 + M13genes<br />
:::2. pSB6A1 + M13genes<br />
*Gel<br />
'''[[File:UCB-Phage Delivery-140710-02.JPG]]'''<br />
:1. pSB1A3<br />
:2. Limtus28i<br />
:3. M13genes<br />
:4. pSB3C5<br />
:5. J04450<br />
<br />
'''7/11'''<br />
*Transformed ligations from 7/10 into 5alpha cells<br />
::1. pSB3C5 + M13genes<br />
::2. pSB6A1 + M13genes<br />
::3. Litmus28i + J04450<br />
<br />
<br />
'''7/12'''<br />
*Results of 7/11 transformation<br />
::-No growth on no DNA control (Amp or Chlor)<br />
::-Lots of growth on Lit-J04450 -> some red-> colonies are too close together to prick individual colonies<br />
:::-It’s possible that these red colonies are satellites =(<br />
:::-Swiped some and plated on new Amp plate (restreak)<br />
::-Lawn of positive control (Litmus 28i) on Amp<br />
::-Many colonies for L1 (pSB3C5-M13genes) and L2 (pSB6A1-M13genes)<br />
:::-Due to high number of red colonies on Lit-J04450 plate, we assumed that most of these colonies contain empty vector<br />
:::-Did not make O/N<br />
*Ligated M13 genes to pSB6A1<br />
::-Used the remained of our digested M13genes.<br />
::-Both digestions from 7/10<br />
*Infection Experiment<br />
::-had 3 cell stocks (each taken from a different colony the night before)<br />
::-Tested Litmus 28i, Tandem phagemid 1C3, and double infection<br />
:::*Therefore, there were 9 flasks total.<br />
::-negative control: Streaked parent cells (non-infected) onto Chlor, Amp, and kan<br />
::-Included double infection plates for Litmus 28i and phagemid 1C3<br />
<br />
==Week 11==<br />
<br />
'''7/13'''<br />
*Transformed 7/12 ligation (pSB6A1-M13genes)<br />
*Litmus28i-J04450<br />
::-All growth from 7/12 restreak was white<br />
::-Pricked some red colonies for liquid O/N-will hopefully see red tomorrow<br />
::-Pricked a few red colonies and put into 200ul H2O (a few colonies per tube- 2 tubes total).<br />
:::*Plated 150ul onto Amp plates<br />
::-Put original plate into incubator to hopefully get bigger colonies<br />
<br />
'''7/14'''<br />
*Finished TWIV ppt<br />
*Set up 2 liquid culture of red Litmus 28i colonies (Litmus28i-J04450)-- slow growth <br />
::-Mini-prepped red Litmus 28i ‘O/N’ from earlier in day<br />
::-Digested samples—to check for insert and correct cut sites<br />
*Made O/Ns of pSB6A1-M13genes colonies from 7/13 transformation<br />
<br />
<br />
'''7/15'''<br />
*Ran gel of Litmus28i-J04450 samples<br />
'''[[File:UCB-Phage Delivery-140715.JPG]]'''<br />
:1. J04450-Litmus28ibb #1 (EcoRI+PstI)<br />
:2. J04450-Litmus28ibb #1 (uncut)<br />
:3. J04450-Litmus28ibb #2 (EcoRI+PstI)<br />
:4. J04450-Litmus28ibb #2 (EcoRI+PstI)<br />
::*Accidentally added restriction enzymes<br />
:5. Litmus28i (EcoRI+PstI)<br />
::*Only has PstI site<br />
<br />
::-Verifies that Litmus28i-J00450 had correct cut sites<br />
::-From this point on, biobricked Litmus28i is called Litmus28ibb<br />
*Check 7/14 O/Ns for pSB6A1-M13genes<br />
::-Mini-prepped liquid cultures<br />
::-Digested with EcoRI and PstI<br />
::::Received bold bands of just under 4000bp. Some lanes had a very feint band ~330bp. None were the correct size<br />
*Transformed Litmus28ibb-J04450 into ER2738 cells<br />
*Remake phage packaging<br />
::-pSB1C3-M13ori(New)<br />
::-pSB1C3-M13ori(Old)<br />
<br />
'''7/16'''<br />
*Results of 7/15 transformation of Litmus28i into 6/30 ER2738 cells<br />
::-lawn on no DNA control-- most likely contaminated cells<br />
::::Streaked chem comp 5alpha and ER onto Amp and Amp+Tet plates to determine if problem is with cells or plates<br />
::-O/N culture of ER to make new chem comp cells<br />
<br />
'''7/17'''<br />
*Finished phage isolation<br />
::Note:<br />
:::*pSB1C3-M13ori (New): M13ori and plasmid ori are convergent<br />
:::*pSB1C3-M13ori (Old): M13ori and plasmid ori are tandem<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! 269nm<br />
! 320nm<br />
! [] phage/mL<br />
<br />
|-<br />
| pSB1C3-M13ori (New)<br />
| 0.386<br />
| 0.034<br />
| 8.126 E12<br />
<br />
|-<br />
| pSB1C3-M13ori (New)<br />
| 0.419<br />
| 0.036<br />
| 8.842 E12<br />
<br />
|-<br />
| pSB1C3-M13ori (Old)<br />
| 0.283<br />
| 0.025<br />
| 5.956 E12<br />
<br />
|-<br />
| pSB1C3-M13ori (Old)<br />
| 0.326<br />
| 0.024<br />
| 6.972 E12<br />
<br />
|-<br />
|}<br />
*Infection to compare the new and the old pSB1C3-M13ori<br />
::-Prepped cells for infection<br />
::-Infected cells with pSB1C3-M13ori (New) or pSB1C3-M13ori (New)<br />
::-and plated on dilutions of 1:100, 1:1000, and 1:10000 on Chlor and kan<br />
::-Also plated 1:1000 dilution on lowered Chlor concentration (34ug/mL)<br />
*Sent Litmus28ibb-J04450 for sequencing<br />
<br />
'''7/18'''<br />
*Results from contamination test<br />
::-5alpha on Amp = no growth<br />
::-5alpha on Amp+Tet = colonies<br />
::-ER on Amp = lawn<br />
::-ER on Amp+Tet = lawn<br />
*Made new chemically competent ER2738<br />
*Transformed Litmus28ibb-J04450 into ER2738 made on 3/30 and ER2738 made on 7/18<br />
::-Had two samples of Litmus28ibb-J04450 and two cell stocks, so 4 samples total<br />
<br />
'''7/19'''<br />
*Results of 7/17 infection<br />
::-pSB1C3-M13ori(Old) (tandem-when plasmid ori and M13ori point in the same direction) packages better than pSB1C3-M13ori(New) (when ori and M13ori are convergent), implying that directionality matters.<br />
'''[[File:UCB-phage lab7-19-141012.jpg]]'''<br />
<br />
==Week 12==<br />
<br />
'''7/20'''<br />
*Freeze downs<br />
:-Litmus28i-J04450 Litmus 28i is now biobrick compatible<br />
<br />
'''7/21'''<br />
*Send samples for sequencing<br />
::-pSB1C3-M13ori(Old)<br />
::-pSB1C3-M13ori(New)<br />
*To ligate M13ori to a kanamycin backbone (pSB1K3)<br />
::-Resuspended pSB1K3-J04450 from distribution kit (6B on plate 4)<br />
:::*Contains J04450 as insert (full RFP construct)<br />
::-Transform pSB1K3 into 5alpha cells<br />
<br />
'''7/22'''<br />
*Pricked colony from pSB1K3-J04450 transformation<br />
::-Mini-prepped DNA to get a supply of DNA<br />
*To put M13ori (M13 phackaging signal) onto Kanamycin resistance o we can test packaging ratios with M13K07 on the same antibiotic<br />
::-Digested pSB1K3 with EcoRI-HF and XbaI<br />
::-Digested pSB1C3-M13ori (Old) with EcoRI-HF and SpeI-HF<br />
:::*DNA was not sufficiently cut. Too much DNA? Problem with enzyme (SpeI-HF)?<br />
'''[[File:UCB-Phage Delivery-140722.JPG]]'''<br />
:1. pSB1K3<br />
:2. M13ori<br />
<br />
'''7/23'''<br />
*Sequencing samples from 7/21 were lost in the mail. Resent samples<br />
*Yet another attempt to biobrick M13genes<br />
::1. using primers that would amplify genes and M13 ori parts from M13K07<br />
::2. using primers that would amplify genes, M13 ori parts, and plasmid ori from M13K07<br />
'''[[File:UCB-Phage Delivery-140723.JPG]]'''<br />
:1. Amplified only the M13genes and M13ori<br />
:2. No DNA control for 1<br />
:3. Amplified M13genes, M13ori, and plasmid ori<br />
:4. No DNA control for 3<br />
::-When run on a gel, samples were clean with only one band at around 7kb. No contamination in no DNA controls<br />
*Digestions<br />
::-pSB1K3 with EcoRI-HF and PstI-HF to check for correct insert<br />
::-pSB1C3-M13ori (Old and new) with EcoRI-HF and SpeI-HF to test for efficient cutting with different stock of restriction enzyme<br />
'''[[File:UCB-Phage Delivery-140723-02.JPG]]'''<br />
:1-3: pSB1K3<br />
:4. pSB1C3-M13ori (Old)<br />
:5. pSB1C3-M13ori (New)<br />
*Still had inefficient cutting. Tested M13ori next to Litmus28i to disern the problem<br />
:*Uncut<br />
:*Cut once with (E, X, S, or P)<br />
:*Cut twice with (E+S or E+P)<br />
'''[[File:UCB-Phage Delivery-140723-03.JPG]]'''<br />
:1. M13ori uncut<br />
:2. M13ori Ecori-HF<br />
:3. M13ori SpeI-HF<br />
:4. M13ori XbaI<br />
:5. M13ori PstI-HF<br />
:6. M13ori EcoRI-HF + SpeI-HF<br />
:7. M13ori EcoRI-HF + PstI-HF<br />
:8. Litmus28i uncut<br />
:9. Litmus28i EcoRI-HF<br />
:10. Litmus28i SpeI-HF<br />
:11. Litmus28i XbaI<br />
:12. Litmus28i PstI-HF<br />
:13. Litmus28i EcoRI-HF + SpeI-HF<br />
:14. Litmus28i EcoRI-HF + PstI-HF<br />
*Only partial digest with only SpeI-HF for both. Complete digestion with all others, including E+S<br />
<br />
'''7/24'''<br />
*To Biobrick M13genes using pSB6A1<br />
::-PCR purified M13 genes (did both samples at the same time)<br />
::-Digested PCR purification and pSB6A1 with EcoRI-HF and PstI-HF<br />
'''[[File:UCB-Phage Delivery-140724.JPG]]'''<br />
*Gel extracted M13genes<br />
<br />
:1. M13genes<br />
:2. pSB6A1<br />
:-Bands of pSB6A1 were too light to gel extract<br />
*To swap the kanamycin resistance marker on M13K07 with ampicillin resistance<br />
::-PCR amplified AmpR from pSB6A1<br />
*made O/N culture of pSB6A1 from freeze down to mini-prep<br />
::-Also streaked cells onto plate<br />
<br />
'''7/25'''<br />
*To change resistance marker on M13K07<br />
:1. Biobrick method<br />
::*Tried to mini-prep O/N but pellet (after liquid culture was spun) was not red even after 16+ hours. Set up O/N from plate colonies<br />
:2. Swap only resistance method<br />
::*PCR purified 7/24 PCR<br />
::*Ran product on gel-> band of correct size<br />
'''[[File:UCB-Phage Delivery-140725.JPG]]'''<br />
<br />
:1. PCR product<br />
:2. PCR product-> PCR purified<br />
:3. No DNA control<br />
::-Digested the PCR purification products of M13genes+ori and AmpR with AgeI-HF and NotI-HF<br />
::*Bands on previous gel looked clean enough for both so did not gel extract<br />
::-Overnight ligation<br />
*Set up O/Ns of pSB1K3 colonies<br />
<br />
'''7/26'''<br />
*Transform<br />
::-pSB6A1+M13genes<br />
*To biobrick M13genes<br />
::-Digestion<br />
:::*pSB6A1 with EcoRI and PstI<br />
'''[[File:UCB-Phage Delivery-140726.JPG]]'''<br />
:Gel extracted backbone (band ~4000bp)<br />
*Ligations (10hrs 16C, 10min at 80C)<br />
::-pSB6A1+M13genes<br />
<br />
==Week 13==<br />
<br />
'''7/27'''<br />
*Transformation results from 7/26<br />
::-No growth for no DNA controls ''on Amp''<br />
::-No growth for M13 genes onto pSB6A1 ''on Amp''<br />
*Set up O/Ns of 5alpha and ER2738 to make competent cell tomorrow<br />
*Made freeze down of pSB1K3<br />
*Plated ER competent cells from 5/15 and 6/16 Onto Chlor plates to check for contamination<br />
<br />
'''7/28'''<br />
*No growth of ER2738 on Chlor (either sample)<br />
::-ER is not contaminated with ChlorR<br />
*Make new 5alpha chem comp cells <br />
*Transformations<br />
::-pSB6A1-M13genes<br />
::-positive control for Amp (pSB6A1)<br />
::-No DNA control<br />
<br />
'''7/29'''<br />
*Set up O/Ns of pSB6A1-M13genes<br />
::-Plated sample on reduced Amp and regular Tet<br />
::-Later in day…<br />
::-Mini-prepped O/Ns<br />
::-Digested and ran on a gel to check for insert sizes<br />
'''[[File:UCB-Phage Delivery-140729.JPG]]'''<br />
:*All but the 4th lane with 3 bands look correct<br />
<br />
'''7/30'''<br />
*Lawn on last night’s plating-> replate<br />
*Digest pSB3C5 and pSB6A1-M13genes with E and P to move M13genes to a Chlor backbone<br />
'''[[File:UCB-Phage Delivery-140730.JPG]]'''<br />
::-Extracted bands<br />
::-Ligations<br />
:::*pSB3C5 + M13genes<br />
*Sent samples for sequencing<br />
**M13 genes on pSB6A1 VF2<br />
**M13 genes on pSB6A1 VR<br />
<br />
'''Test experiment for high school kids coming to lab (7/29-7/31)'''<br />
*General idea: Have 2 strands of DNA, one has an EcoRI site while the other contains a SNP, abolishing the cut sight. We pretend that one is pathogenic (the one that is not cut) and tells kids to figure out which one is which <br />
*PCR with Dream-Taq<br />
::-Did not have special fastdigest enzyme<br />
::-Hypothesized that green dye will interfere with enzyme effectivity<br />
*PCR with Dream-Taq (no green dye)<br />
::-Digest with EcoRI fastdigest-> did not cut<br />
*Redid experiment several times<br />
::-Should digest with XbaI<br />
::-PCR purified PCR products<br />
<br />
'''7/31'''<br />
*Spent the morning with Heritage High School<br />
*Made phage<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Phage<br />
! A269<br />
! A320<br />
! genome size<br />
! [ ]<br />
<br />
|-<br />
| Litmus28ibb-J04450 (1)<br />
| 0.823<br />
| 0.197<br />
| <br />
| 9.35 x10^12<br />
<br />
|-<br />
| Litmus28ibb-J04450 (2)<br />
| 0.940<br />
| 0.168<br />
| <br />
| 1.14 x10^13<br />
<br />
|-<br />
|}<br />
<br />
'''8/2'''<br />
*Digestion<br />
::-pSB6A1-M13genes (E+P)<br />
::-pSB3C5 (E+P)<br />
<br />
==Week 14==<br />
<br />
'''8/3'''<br />
*Another attempt to clone M13genes onto pSB3C5<br />
::-Run 8/2 digestions on gel<br />
'''[[File:UCB-Phage Delivery-140803.JPG]]'''<br />
:1. M13genes (E+P)<br />
:2. pSB3C5 (E+P)<br />
::-Gel extracted <br />
:::*M13genes away from pSB6A1 backbone<br />
:::*pSB3C5 backbone away from J04450<br />
::-Ligations<br />
:::*pSB3C5 + M13genes<br />
:::*pSB3C5 + no insert<br />
::-Transformed ligations into ER2738<br />
<br />
'''8/5'''<br />
*Transformation of pSB3C5+M13genes failed<br />
*Made phage<br />
::-Fd-CAT DNA packaged with Fd-CAT<br />
::-phagemid 1C3 packaged with M13K07<br />
::-amilCP on pSB1C3 packaged with M13K07<br />
::-Litmus28ibb-J04450 packaged with M13K07<br />
<br />
'''8/6'''<br />
*Isolated phage<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Phage<br />
! [ ]<br />
<br />
|-<br />
| Fd CAT<br />
| 2.72 x10^12<br />
<br />
|-<br />
| Litmus28ibb-J04450<br />
| 8.37 x10^12<br />
<br />
|-<br />
| pSB1C3-amilCP<br />
| 1.42 x10^13<br />
<br />
|-<br />
| pSB1C3-M13ori<br />
| 4.32 x10^12<br />
<br />
|-<br />
|}<br />
<br />
'''8/6'''<br />
*Started making phage with Fd CAT the should contain Litmus28ibb-RFP<br />
*Infection<br />
::-Infected ER2738 cells with Litmus28ibb-RFP, pSB1C3-M13ori, or pSB1C3-amilCP<br />
::-Plated Litmus samples on 100ug/mL Amp and 50ug/mL Kan<br />
::-Plated other two samples on 34ug/mL Chlor and 25ug/mL Kan<br />
<br />
'''8/7'''<br />
*Isolated phage containing Litmus28ibb-RFP using helper phage<br />
::-M13K07 (as control)<br />
::-Fd-CAT (no phage pellet was observed-worried that there was no phage)<br />
*Went to CSU to have them test M13ori part compared to amilCP. Grew the samples in 5mL O/Ns then diluted to 0.5OD and grew 30 minutes rather than starting from a fresh colony<br />
<br />
'''8/8'''<br />
*At CSU<br />
::-Finished phage protocol (Test packaging of M13ori part)<br />
::-Only used 20mL infection samples<br />
::-grew to 0.55 OD<br />
::-When making phage (after 14 hour incubation), there was very little growth<br />
::-Plated non-diluted and diluted 1:1000 of M13ori and amilCP sample<br />
*Infected cells using phage isolated 8/7. Plated at dilutions of 1:100 and 1:100k<br />
*Want to test progeny Fd-CAT phage (made 8/7) for infectability<br />
::-Start phage isolation prodocol using Fd-CAT phage from 8/7 do deliver the helper phagemid<br />
:::*Phagemid:<br />
::::*None: will make Fd-CAT phage packaing Fd-CAT phagemid<br />
::::*Litmus28ibb-J04450: make Fd-CAT phage packaging Litmus28ibb-J04450 phagemid<br />
*Remake phage (Fd-CAT) using fresh stock from Mike<br />
<br />
<br />
'''8/9'''<br />
*Results of 8/8 infection<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Helper Phagemid<br />
! phagemid<br />
! Selection<br />
! 1:1000 dilution<br />
! 1:00k dilution<br />
<br />
|-<br />
| M13K07<br />
| Litmus28ibb-RFP<br />
| Kan+Tet<br />
| 400-500 (some red)<br />
| 61 white; 20 red<br />
<br />
|-<br />
| same<br />
| same<br />
| Amp+Tet<br />
| lawn<br />
| 2<br />
<br />
|-<br />
| Fd-CAT<br />
| Litmus28ibb-RFP<br />
| Chlor+Tet<br />
| 0<br />
| 0<br />
<br />
|-<br />
| same<br />
| same<br />
| Amp+Tet<br />
| 10<br />
| 707<br />
<br />
|-<br />
|}<br />
*NOTE: The same sample was plated on two different plates. For example, the sample using M13K07 as the helper phage was plated on Kan+Tet and Amp+Tet<br />
*Mini-prep<br />
::-pSC3C5-J04450 -- very little growth, low DNA yield<br />
::-Fd-CAT infected cells<br />
:::*Digest and run on a gel to verify presence of a band- there was a very feint band<br />
*Finished isolating phage…. messed up and used 0.8MgCl2, 0.2M NaCl instead of PEG during precipitation step. Also used the wrong phage<br />
::-Infected ER2738 cells anyway…. no growth by 8/12<br />
<br />
==Week 15==<br />
<br />
'''8/10'''<br />
*Are the progeny phage from 8/6 (original progeny from first Fd-Tet application) viable/ able to reproduce?<br />
::-Used these progeny to make phage that should amplify Fd-CAT phage containing either the Fd-CAT or Litmus28ibb-J04450 phagemid<br />
<br />
'''8/11'''<br />
*Finished isolating phage<br />
{| class = "wikitable"<br />
|-<br />
! Helper Phagemid<br />
! Phagemid<br />
! A269<br />
! A320<br />
! genome size<br />
! [ ]<br />
<br />
|-<br />
| Fd-CAT<br />
| Litmus28ibb (1)<br />
| 0.029<br />
| 0.013<br />
| 4080<br />
| 2.35 x10^11<br />
<br />
|-<br />
| Fd-CAT<br />
| Ltimus28ibb (2)<br />
| 0.033<br />
| 0.015<br />
| 4080<br />
| 2.65 x10^11<br />
<br />
|-<br />
| Fd-CAT<br />
| Fd-CAT (1)<br />
| 0.326<br />
| 0.082<br />
| 7775<br />
| 1.88 x10^12<br />
<br />
|-<br />
| Fd-CAT<br />
| Fd-CAT (2)<br />
| 0.320<br />
| 0.079<br />
| 7775<br />
| 1.86 x10^12<br />
<br />
|-<br />
| M13g6A1<br />
| M13g6A1 (1)<br />
| 0.322<br />
| 0.148<br />
| 10,029<br />
| 1.04 x10^12<br />
<br />
|-<br />
| M13g6A1<br />
| M13g6A1 (2)<br />
| 0.503<br />
| 0.130<br />
| 10,029<br />
| 2.23 x10^12<br />
<br />
|-<br />
|}<br />
<br />
'''8/13'''<br />
*Infected ER2738 with Fd-CAT packaging Litmus28ibb-J04450<br />
<br />
'''8/16'''<br />
*Results from Infection on 8/11<br />
{| class = "wikitable"<br />
|-<br />
! Helper Phagemid<br />
! Phagemid<br />
! 1:1<br />
! 1:10<br />
! 1:100<br />
! 1:1000<br />
<br />
|-<br />
| M13g6A1<br />
| M13g6A1<br />
| almost lawn<br />
| 728<br />
| 287<br />
| 55<br />
<br />
|-<br />
| Fd-CAT<br />
| Fd-CAT<br />
| lawn<br />
| almost lawn<br />
| 1260<br />
| 640<br />
<br />
|-<br />
| Fd-CAT<br />
| Litmus28ibb-J04450<br />
| lawn<br />
| almost lawn<br />
| 2004<br />
| 304<br />
<br />
|-<br />
|}<br />
<br />
Results: packaging of Litmus28ibb-J04450 using Fd-CAT helper phagemid from (8/15)<br />
<br />
{| class="wikitable"<br />
|-<br />
!Antibiotic<br />
!1:10 <br />
!1:100 <br />
!1:1k <br />
!1:10k <br />
!1:100k<br />
<br />
|-<br />
|Amp <br />
|Lawn<br />
|Almost lawn <br />
|1025 <br />
|217 <br />
|25<br />
<br />
|-<br />
|Kan <br />
|9 <br />
|2 <br />
|0 <br />
|0 <br />
|0<br />
|}<br />
<br />
==Week 16==<br />
<br />
'''8/19'''<br />
<br />
Sent samples for sequencing<br />
<br />
Litmus28ibb-J04450<br />
<br />
==Week 17==<br />
<br />
'''8/27'''<br />
<br />
Transform pSB1C3-M13ori into ER2738 cells<br />
<br />
'''8/28'''<br />
<br />
Make phage with pSB1C3-M13ori as phagemid and M13g6A1 as helper phagemid<br />
<br />
'''8/29'''<br />
<br />
Finish phage isolation of M13 phage containing pSB1C3-M13ori<br />
<br />
'''9/3'''<br />
<br />
digest pSB6A1-M13g and pSB3C5 with EcoRI and PstI. Did not receive a band for M13genes so did not proceed with ligation to pSB3C5<br />
<br />
==Week 17==<br />
<br />
'''9/4'''<br />
<br />
Mini-prepped new pSB6A1-M13genes (bad quality) and pSB3C5-J04550. Digested both plasmids with EcoRI and PstI and ran products on gel. Did receive a band for the M13genes but it was too feint to gel extract<br />
<br />
'''9/5'''<br />
<br />
Mini-prep pSB6A1-M13genes again. Digest this and pSB3C5 backbone from previous day with EcoRI and PstI. M14g band was barely bright enough to extract so we extracted M13genes and pSB3C5 backbone. Pieces were ligated together overnight at 16C for 10 hours.<br />
<br />
'''9/7'''<br />
<br />
Transformed 9/5 ligation (pSB3C5-M13genes into 5alpha cells). Plated sample onto low Chlor plate (34ug/mL)<br />
<br />
'''9/9'''<br />
<br />
Still no growth for 9/7 transformation of pSB3C5-M13genes ligation PCR M13genes to attempt another ligation into pSB3C5. Diluted DNA to ~1.17ng/ul<br />
<br />
'''9/10'''<br />
<br />
Gel of 9/9 PCR of M13genes looked clean (only ran 3ul of PCR sample on gel). PCR purified the remaining PCR sample. Digested with EcoRI and PstI. Ligated to previously digested pSB3C5.<br />
<br />
==Week 18==<br />
<br />
'''9/11'''<br />
<br />
Transform 9/10 ligation (pSB3C5-M13genes) into 5alpha cells<br />
<br />
'''9/13'''<br />
<br />
Colony PCR of colonies that grew for 9/11 transformation. Looking for pSB3C5-M13genes)<br />
<br />
'''9/15'''<br />
<br />
Gel of colony PCR showed failure. Set up overnight cultures anyway.<br />
<br />
'''9/17'''<br />
<br />
Submitted Part to iGEM Registry<br />
<br />
{| class="wikitable"<br />
|-<br />
|iGEM # <br />
|Description<br />
<br />
|-<br />
|BBa_K1445000 <br />
|M13ori- the packaging signal for the M13 and fd phage<br />
|}<br />
<br />
Mini-prepped overnights from 9/15 and sent samples for sequencing<br />
<br />
Sent Litmus28ibb-J04450 sample for sequencing to sequence the remaining section that was not reached before<br />
<br />
==Week 18==<br />
<br />
'''9/21'''<br />
<br />
Up until this point, very little luck was had with pSB3C5. Retried putting M13genes onto a chloramphenicol backbone, but this time used pSB1C3. Digested M13genes and pSB1C3 with EcoRI and PstI. Gel showed incomplete digestion so did not gel extract.<br />
<br />
'''9/22'''<br />
<br />
Digested pSB1C3-J04450 with EcoRI and PstI then gel extracted the backbone. PCR purified M13genes(PCR product was clean so did not bother to gel extract). Ligated the two pieces together.<br />
<br />
'''9/23'''<br />
<br />
Transform ligations from 9/22 (pSB1C3-M13genes) into 5 alpha cells<br />
<br />
'''9/27'''<br />
<br />
Transform pSB1C3-M13genes into 5alpha cells. Plate onto low Chlor (34ug/mL)<br />
<br />
Later in day.... Set up overnight cultures of colonies<br />
<br />
'''9/28'''<br />
<br />
Mini-prepped O/N cultures from 9/27. Digested samples with EcoRI and PstI. There were no bands of the correct size on the gel.<br />
<br />
the M13 and fd phage</div>Leighlahttp://2014.igem.org/Team:CU-Boulder/BiobricksTeam:CU-Boulder/Biobricks2014-10-17T00:37:22Z<p>Leighla: </p>
<hr />
<div>{{:Team:CU-Boulder/Wiki}}<br />
{{Template:UCB-NavBar}}<br />
<br />
Part Summaries <br />
<br />
For detailed characterization, see the parts page.<br />
<br />
==Part: BBa_K1445000 (M13ori)==<br />
The M13 origin of replication (M13ori) has been documented as the packaging signal for the M13 phage. When single stranded, the M13ori forms unique hairpins that signal packaging into a phage capsid. It was experimentally demonstrated that this part is both necessary and sufficient for plasmid packaging into M13 phage. This part does not code for phage coat proteins; therefore, cannot produce phage.<br />
<br />
<br />
<br />
==Part: BBa_K1445001 (Endogenous Type II CRISPR-Cas9 phagemid)==<br />
This composite part consists of parts BBa_K1445000 (M13ori) and BBa_K1218011 (cas9).<br />
The cas9 construct includes the native trcrRNA and promoter region upstream of the type II Cas9 endonuclease. Downstream of the Cas9 protein is a minimal CRISPR array that contains the spacer region that determines the DNA sequence that is targeted by the Cas9 endonuclease. The spacer sequence can be easily replaced through a digestion-ligation reaction using BsaI. The addition of the M13 origin of replication (M13ori) allows for the uptake of the CRISPR-Cas9 machinery into M13 phage.<br />
<br />
<br />
'''[[File:UCB-Biobrick-01-141016.JPG]]'''<br />
<br />
<br />
==Part: BBa_K1445002 (High copy BioBricked M13 phagemid vector)==<br />
This biobrick backbone has the high copy number pUC19 origin of replication and an ampicillin resistance marker. It also contains the M13 origin of replication which allows for the phagemid to be packaged into the M13 phage. It was experimentally validated that vector retains its ability to be packaged into M13 phage.<br />
<br />
'''[[File:UCB-Biobrick-02-141016.JPG]]'''<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
{{Template:UCB-Footer}}</div>Leighlahttp://2014.igem.org/Team:CU-Boulder/BiobricksTeam:CU-Boulder/Biobricks2014-10-17T00:31:54Z<p>Leighla: </p>
<hr />
<div>{{:Team:CU-Boulder/Wiki}}<br />
{{Template:UCB-NavBar}}<br />
<br />
Part Summaries<br />
<br />
For detailed characterization, see the parts page.<br />
<br />
==Part: BBa_K1445000 (M13ori)==<br />
The M13 origin of replication (M13ori) has been documented as the packaging signal for the M13 phage. When single stranded, the M13ori forms unique hairpins that signal packaging into a phage capsid. It was experimentally demonstrated that this part is both necessary and sufficient for plasmid packaging into M13 phage. This part does not code for phage coat proteins; therefore, cannot produce phage.<br />
<br />
<br />
<br />
==Part: BBa_K1445001 (Endogenous Type II CRISPR-Cas9 phagemid)==<br />
This composite part consists of parts BBa_K1445000 (M13ori) and BBa_K1218011 (cas9).<br />
The cas9 construct includes the native trcrRNA and promoter region upstream of the type II Cas9 endonuclease. Downstream of the Cas9 protein is a minimal CRISPR array that contains the spacer region that determines the DNA sequence that is targeted by the Cas9 endonuclease. The spacer sequence can be easily replaced through a digestion-ligation reaction using BsaI. The addition of the M13 origin of replication (M13ori) allows for the uptake of the CRISPR-Cas9 machinery into M13 phage.<br />
<br />
<br />
'''[[File:UCB-Biobrick-01-141016.JPG]]'''<br />
<br />
<br />
==Part: BBa_K1445002 (High copy BioBricked M13 phagemid vector)==<br />
This biobrick backbone has the high copy number pUC19 origin of replication and an ampicillin resistance marker. It also contains the M13 origin of replication which allows for the phagemid to be packaged into the M13 phage. It was experimentally validated that vector retains its ability to be packaged into M13 phage.<br />
<br />
'''[[File:UCB-Biobrick-02-141016.JPG]]'''</div>Leighlahttp://2014.igem.org/File:UCB-Biobrick-02-141016.JPGFile:UCB-Biobrick-02-141016.JPG2014-10-17T00:27:09Z<p>Leighla: </p>
<hr />
<div></div>Leighlahttp://2014.igem.org/File:UCB-Biobrick-01-141016.JPGFile:UCB-Biobrick-01-141016.JPG2014-10-17T00:23:20Z<p>Leighla: </p>
<hr />
<div></div>Leighlahttp://2014.igem.org/Team:CU-Boulder/Notebook/MeasurementTeam:CU-Boulder/Notebook/Measurement2014-10-17T00:16:26Z<p>Leighla: /* Week 10 */</p>
<hr />
<div>{{Template:UCB-Main}}<br />
{{Template:UCB-NavBar}}<br />
__FORCETOC__<br />
'''Interlab Measurement Study'''<br />
<br />
==Study Description==<br />
<br />
Different iGEM teams from around the world were offered the opportunity to participate in this study to obtain fluorescent measurements from three separate devices. Each device consisted of a specific plasmid that was to be expressed in bacterial cells. The parts for each device were provided to each team from the iGEM registry. <br />
<br />
The first plasmid was already assembled and provided to teams with highly expressive constitutive promoter for the GFP in a low-copy plasmid, pSB3K3. The other two plasmids contained the same GFP gene, but were assembled onto the pSB1C3 plasmid. The first of these held the same constitutive promoter (J23101) as the first device. The third device contained a constitutively low-expressive promoter (J23115) to drive GFP expression (Table 1).<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Device<br />
! Constitutive Promoter<br />
! Plasmid<br />
<br />
|-<br />
| Device 1<br />
| J23101 (High Expression)<br />
| pSB3K3 (Low-Copy)<br />
<br />
|-<br />
| Device 2<br />
| J23101 (High Expression)<br />
| pSB3C1<br />
<br />
|-<br />
| Device 3<br />
| J23115 (Low Expression)<br />
| pSB3C1<br />
<br />
|-<br />
|}<br />
<br />
Table 1: Overview of the three devices constructed for this study.<br />
<br />
Device 1 plasmids were transformed into competent E. coli DH5? cells and plated under antibiotic selection for cells that had taken up the plasmid. Device 2 and device 3 were assembled using restriction digestion followed by ligation. To begin, the plasmid containing the GFP gene (E0240) was digested with XbaI and PstI. The plasmids with promoters were each digested with SpeI and PstI, resulting in a location to which the GFP fragment could be inserted. These products were ligated together and transformed into competent E. coli DH5? cells and plated under antibiotic selection. Colonies were taken for overnight cultures, and DNA from these cultures was isolated for analysis. <br />
<br />
Biological triplicates were prepared as overnight cultures for all three devices and control samples. Fluorescence was measured from all samples in triplicate. First, samples were diluted as needed to obtain the same cell concentrations (by measuring OD) and then serial dilutions of samples were placed into a 96 well plate and measurements taken using a plate reader. Fluorescence values in RFU (relative fluorescent units) were compared and reported.<br />
<br />
==Results==<br />
<br />
As shown below (Data Table 1), for each of the devices, three biological replicates were measured (labeled #1, #2, and #3 for each device). Data shown includes actual measurements and derived quantities obtained by subtracting fluorescence measured from non-GFP expressing E. coli of the same strain on the same plate. Promoter J23115 for device #3 was used as distributed (containing mismatch). In addition, each sample underwent a serial dilution (concentrations listed as fractions across top of table).<br />
<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! 1.00<br />
! 0.67<br />
! 0.44<br />
! 0.3<br />
! 0.2<br />
! 13.4<br />
! 0.09<br />
<br />
|-<br />
| Dev 1 #1 Actual<br />
| 1728<br />
| 1450<br />
| 1153<br />
| 1024<br />
| 899<br />
| 881<br />
| 838<br />
<br />
|-<br />
| Calculated<br />
| 746<br />
| 525<br />
| 267<br />
| 174<br />
| 54<br />
| 48<br />
| 31<br />
<br />
|-<br />
| Dev 1 #2 Actual<br />
| 1830<br />
| 1413<br />
| 1142<br />
| 1010<br />
| 921<br />
| 841<br />
| 861<br />
<br />
|-<br />
| Calculated<br />
| 848<br />
| 488<br />
| 256<br />
| 160<br />
| 76<br />
| 8<br />
| 54<br />
<br />
|-<br />
| Dev 1 #3 Actual<br />
| 1790<br />
| 1446<br />
| 1155<br />
| 975<br />
| 911<br />
| 861<br />
| 854<br />
<br />
|-<br />
| Calculated<br />
| 808<br />
| 521<br />
| 269<br />
| 125<br />
| 66<br />
| 28<br />
| 47<br />
<br />
|-<br />
| Dev 2 #1 Actual<br />
| 992<br />
| 925<br />
| 849<br />
| 812<br />
| 805<br />
| 779<br />
| 779<br />
<br />
|-<br />
| Calculated<br />
| -76<br />
| 22<br />
| -5<br />
| -9<br />
| -2<br />
| -15<br />
| -9<br />
<br />
|-<br />
| Dev 2 #2 Actual<br />
| 1083<br />
| 924<br />
| 820<br />
| 813<br />
| 791<br />
| 776<br />
| 761<br />
<br />
|-<br />
| Calculated<br />
| 15<br />
| 21<br />
| -34<br />
| -8<br />
| -16<br />
| -18<br />
| -27<br />
<br />
|-<br />
| Dev 2 #3 Actual<br />
| 1047<br />
| 879<br />
| 824<br />
| 801<br />
| 776<br />
| 782<br />
| 772<br />
<br />
|-<br />
| Calculated<br />
| -21<br />
| -24<br />
| -30<br />
| -20<br />
| -31<br />
| -12<br />
| -16<br />
<br />
|-<br />
| Dev 3 #1 Actual<br />
| 1142<br />
| 995<br />
| 895<br />
| 831<br />
| 804<br />
| 809<br />
| 798<br />
<br />
|-<br />
| Calculated<br />
| 74<br />
| 92<br />
| 41<br />
| 10<br />
| -3<br />
| 15<br />
| 10<br />
<br />
|-<br />
| Dev 3 #2 Actual<br />
| 1125<br />
| 940<br />
| 852<br />
| 823<br />
| 796<br />
| 805<br />
| 788<br />
<br />
|-<br />
| Calculated<br />
| 57<br />
| 37<br />
| -2<br />
| 2<br />
| -11<br />
| 11<br />
| 0<br />
<br />
|-<br />
| Dev 3 #3 Actual<br />
| 1035<br />
| 951<br />
| 888<br />
| 837<br />
| 824<br />
| 812<br />
| 797<br />
<br />
|-<br />
| Calculated<br />
| -33<br />
| 48<br />
| 34<br />
| 16<br />
| 17<br />
| 18<br />
| 9<br />
<br />
|-<br />
|}<br />
<br />
Data Table 1: Fluorescence data. For each of the devices, three biological replicates were measured (labeled #1, #2, and #3 for each device). Data shown includes actual measurements and derived quantities. Fractions across top of table represent sample concentration.<br />
<br />
==Conclusions==<br />
<br />
As shown below (Data Table 3), Device 1 showed a much higher amount of fluorescence compared to the other two devices. This is likely due to differences in the plasmid between the devices, with the low copy pSB3K3 performing much higher than the pSB1C3 plasmid. Device 2 and 3 were similar in fluorescence amounts. However, device 3 outperformed device 2, an unexpected result considering the promoter differences. It was later discovered that the J23115 promoter part used for device 3 contained two mismatched base pairs. Considering that the promoter sequence is only 35 bp in length, this could have a significant impact on expression of the GFP gene in this device. <br />
<br />
[[File:UCB-interlab study gragh-141016.JPG]]<br />
<br />
Data Table 2: Fold change in fluorescence in samples compared to non-GFP expressing bacterial cells. <br />
<br><br />
<br><br />
<br />
'''Below is the Daily Lab Notebook for this Study'''<br />
<br><br />
<br><br />
<br> <br />
==Week 1==<br />
<br />
'''7/1/14'''<br />
<br />
Dry Lab only: Planning and research into this study. From the description on iGEM’s website <br />
<br />
(https://2014.igem.org/Tracks/Measurement/Interlab_study):<br />
<br />
The first device is already built and available in the distribution kit. The second and third devices must be built using the BioBricks standard protocol by the teams participating in this study.<br />
<br />
1. Existing device: BBa_I20260 (J23101-B0032-E0040-B0015) in the pSB3K3 vector. <br />
Kit location <br />
Plate 4, Well 18A<br />
<br />
2. New device to be built by the iGEM team: BBa_J23101 + BBa_E0240 (B0032-E0040-B0015), must be built in the pSB1C3 backbone <br />
Kit locations <br />
BBa_J23101 (called BBa_K823005 when in pSB1C3): Plate 1, Well 20K <br />
BBa_E0240 (in pSB1C3): Plate 2, Well 24B<br />
<br />
3. New device to be built by the iGEM team: BBa_J23115 + BBa_E0240 (B0032-E0040-B0015), must be built in the pSB1C3 backbone <br />
Kit locations <br />
BBa_J23115 (called BBa_K823012 when in pSB1C3): Plate 1, Well 22I <br />
BBa_E0240 (in pSB1C3): Plate 2, Well 24B<br />
<br />
PLEASE NOTE: The J23115 part in this year's distribution kit has two mismatched basepairs and instead matches the K823012sequence (which was already known to have these mismatches). You can either (a) use the J23115 part as distributed or (b) re-clone J23115 and correct the mismatch. Please let us know which path you will choose at measurement at igem dot org. <br />
<br />
Thank you! (*This note was not originally on the webpage – it was added around July 12th)<br />
<br />
<br />
'''7/3/14'''<br />
<br />
Resuspended DNA from 2014 distribution kit to obtain the 4 parts/devices needed for this study<br />
Transformation of Device 1 (I20260) into BWF+ comp cells<br />
- Two reactions were performed according to transformation protocol, one is negative control<br />
- Heat shock was done for 45 sec <br />
- Cultures were plated at full strength and 1:10 dilution with 20 uL each<br />
<br />
==Week 2==<br />
<br />
'''7/7/14'''<br />
<br />
Examined plates and observed that growth occurred on both, including the negative control plate. This reveals a problem. It was later discovered that the BW comp cells used in this transformation have Kan resistance, thus explaining the growth on the negative control plate. Transformation will need to be repeated using different comp cells; DH5 alpha cells have been suggested and are available.<br />
- Notation: K will be used for Kan plates and C for Chloroamp. <br />
<br />
'''7/10/14'''<br />
<br />
Transformation of all the parts needed from the distribution kit was performed. Each well in the distribution kit from iGEM contains 2-3 ng of DNA. So each transformation contains 200-300 pg/uL. All reactions were done following standard transformation protocol including 45 sec heat shock at 42°C. DH5 alpha comp cells were obtained from Dowell Lab for the reaction. <br />
<br />
Sample Volume DNA Volume Comp Cells<br />
I20260 (Dev1) 1.5 uL 40 uL <br />
J23101 1.5 uL 40 uL<br />
J23115 1.5 uL 40 uL<br />
E0240 1.5 uL 40 uL<br />
Neg Control (1.5 uL MQ H2O) 40 uL<br />
<br />
Following the transformation, 25 uL of mixture plated full strength on u of plate, 20 uL at 1:10 dilution plated on other u of plate containing the appropriate antibiotic.<br />
<br />
==Week 3==<br />
<br />
'''7/14/14'''<br />
<br />
Examination of plates from transformation shows various single colonies in the full strength and very few, if any colonies on the 1:10 dilution side of plates. This may indicate low transformation efficiency or could be due to slow growth of comp cells, which was observed by other iGEM students. Plates images shown below. Colonies were taken from I20260, E0240, and J23101 plates for overnight cultures in the appropriate antibiotic. <br />
<br />
<br />
[[File:UCB-Interlab-01-2014.png]]<br />
<br />
[[File:UCB-Interlab-02-2014.png]]<br />
<br />
[[File:UCB-Interlab-03-2014.png]]<br />
<br />
'''7/15/14'''<br />
<br />
Minipreps were done according to manufacturer’s recommendations for each of the overnight cultures. Bacterial pellets were made with the remainder of the cultures. Nanodrop performed on the miniprepped products as shown below: <br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! ng/ uL<br />
! 260/280<br />
! 260/230<br />
<br />
|-<br />
| Dev1 (I20260)<br />
| 43.9<br />
| 1.93<br />
| 2.13<br />
<br />
|-<br />
| J23101<br />
| 54.7<br />
| 1.88<br />
| 2.15<br />
<br />
|-<br />
| E0240<br />
| 13.2<br />
| 1.92<br />
| 1.10<br />
<br />
|-<br />
|}<br />
<br />
Gel electrophoresis was performed on the products. A 1% agarose gel (0.3 g agarose) with 3.0 uL EtBr in 30 mL TAE was prepared. 5 uL of a 2-Log DNA ladder was loaded followed by 5.0 uL of each sample with 1.0 uL loading dye into the following lanes:<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Well<br />
! Loaded<br />
! Expected Size (bp)<br />
<br />
|-<br />
| 1<br />
| 2-Log DNA Ladder<br />
| N/A<br />
<br />
|-<br />
| 2<br />
| D1 (I20260)<br />
| 3669<br />
<br />
|-<br />
| 3<br />
| J23101<br />
| 2100<br />
<br />
|-<br />
| 4<br />
| E0240<br />
| 2946<br />
<br />
|-<br />
| 5<br />
| D1 (I20260)<br />
| 3669<br />
<br />
|-<br />
| 6<br />
| J23101<br />
| 2100<br />
<br />
|-<br />
| 7<br />
| E0240<br />
| 2946<br />
<br />
|-<br />
| 8<br />
| H2O Control<br />
| --<br />
<br />
|-<br />
|}<br />
[[File:UCB-Interlab-04-2014.jpg]]<br />
<br />
As seen from the gel (shown above), various bands are visible for most of the samples. Since these are uncut plasmids, it is assumed that this is from supercoiling. <br />
<br />
'''7/16/14'''<br />
<br />
Restriction Digestion was performed on the 3 samples, followed by gel electrophoresis to analyze the products isolated. The digestion was done according to protocol using a 1 hour digestion at 37 °C followed by 20 min heat inactivation at 80°C. Enzymes and buffer obtained from NEB.<br />
<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! I20260<br />
! J23101<br />
! E0240<br />
<br />
|-<br />
| CutSmart Buffer<br />
| 5.0 uL<br />
| 5.0 uL<br />
| 5.0 uL<br />
<br />
|-<br />
| PstI<br />
| 1.0 uL<br />
| 1.0 uL<br />
| 1.0 uL<br />
<br />
|-<br />
| SpeI<br />
| --<br />
| 1.0 uL<br />
| --<br />
<br />
|-<br />
| XbaI<br />
| 1.0 uL<br />
| --<br />
| 1.0 uL<br />
<br />
|-<br />
| DNA (250 ng)<br />
| 5.7 uL<br />
| 4.6 uL<br />
| 18.9 uL<br />
<br />
|-<br />
| MQ H2O<br />
| 37.3 uL<br />
| 38.4 uL<br />
| 24.1 uL<br />
<br />
|-<br />
| Total Volume<br />
| 50.0 uL<br />
| 50.0 uL<br />
| 50.0 uL<br />
<br />
|-<br />
|}<br />
'''7/17/14'''<br />
<br />
Products from reaction were run on a 1% agarose gel with EtBr. 1.0 uL of loading dye was added to 5.0 uL of each sample as well as 5.0 uL of 2-Log DNA Ladder. Lanes and expected band sizes shown below. There appears to be undigested product present in the E0240 sample. <br />
<br />
{| class = "wikitable"<br />
|-<br />
! Well<br />
! Loaded<br />
! Expected Sizes (bp)<br />
<br />
|-<br />
| 1<br />
| 2-Log DNA Ladder<br />
| N/A<br />
<br />
|-<br />
| 2<br />
| D1 (I20260)<br />
| 2724, 945<br />
<br />
|-<br />
| 3<br />
| J23101<br />
| 2261<br />
<br />
|-<br />
| 4<br />
| E0240<br />
| 2042, 900<br />
<br />
|-<br />
| 5<br />
| H2O Control<br />
| --<br />
<br />
|-<br />
|}<br />
[[File:UCB-Interlab-05-2014.jpg]]<br />
<br />
<br />
<br />
J23101 and E0240 digest products are used in a ligation reaction to build Device 2. NEBioCalculator (http://nebiocalculator.neb.com ) was used to calculate the suggested ratio of 1:3 vector to insert for DNA sizes. The reaction included a negative control and was done using the following reagents according to NEB ligation protocol:<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! Sample<br />
! Neg Control<br />
<br />
|-<br />
| T4 Ligase <br />
| 1.25 uL<br />
| 1.25 uL<br />
<br />
|-<br />
| T4 Ligase Buffer<br />
| 2.5 uL<br />
| 2.5 uL<br />
<br />
|-<br />
| J23101 Vector DNA (40 ng)<br />
| 8.0 uL<br />
| 18.0 uL<br />
<br />
|-<br />
| E0240 Insert DNA (50 ng)<br />
| 10.0 uL<br />
| --<br />
<br />
|-<br />
| MQ H2O<br />
| 3.25 uL<br />
| 3.25 uL<br />
<br />
|-<br />
| Total Volume<br />
| 25.0 uL<br />
| 25.0 uL<br />
<br />
|-<br />
|}<br />
The reaction was allowed to proceed for 15 minutes at room temperature. The resulting product was transformed into DH5alpha competent cells using standard protocol. The plates were left to incubate, but even after several days no growth was observed. <br />
<br />
==Week 4==<br />
<br />
'''7/21/14'''<br />
<br />
Ligation from last week repeated using the same protocol and volumes. However, to improve chances of getting product formation, an overnight ligation will be used. The reaction was placed in a thermocycler and set to 16 °C for 16 hours. <br />
<br />
'''7/22/14'''<br />
<br />
Ligation reaction was heat inactivated at 65°C for 10 minutes. 10 uL of the product was transformed into 40 uL competent cells and plated onto the appropriate antibiotic plate in both full strength and as a 1:10 dilution. The negative control was also transformed and plated for comparison. <br />
<br />
==Week 5==<br />
<br />
'''7/28/14'''<br />
<br />
Competent cells were made with Josephina. Overnight cultures prepared from plates including device 2 and parts to build device 3. Six cultures were prepared. Shown below is the device 2 transformation plate (left) and negative control (right). <br />
[[File:UCB-Interlab-06-2014.png]]<br />
<br />
<br />
<br />
'''7/29/14'''<br />
<br />
Miniprep was performed on overnight cultures followed by Nanodrop to assess purity and concentration of samples. <br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! ng/ uL<br />
! 260/280<br />
! 260/230<br />
<br />
|-<br />
| Dev2 #1<br />
| 124.5<br />
| 1.85<br />
| 2.16<br />
<br />
|-<br />
| Dev2 #2<br />
| 160.5<br />
| 1.91<br />
| 2.29<br />
<br />
|-<br />
| E0240 #1<br />
| 150.9<br />
| 1.82<br />
| 1.61<br />
<br />
|-<br />
| E0240 #2<br />
| 91.0<br />
| 1.87<br />
| 2.15<br />
<br />
|-<br />
| J23115 #1<br />
| 114.5<br />
| 1.83<br />
| 1.97<br />
<br />
|-<br />
| J23115 #2<br />
| 148.5<br />
| 1.79<br />
| 2.29<br />
<br />
|-<br />
|}<br />
Restriction Digestion was performed on the products using standard protocol and with the following enzymes:<br />
<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! D1<br />
! D2 #1<br />
! D2 #2<br />
! E0240 #1<br />
! E0240 #2<br />
! J23115 #1<br />
! J23115 #2<br />
! (-) Control<br />
<br />
|-<br />
| CutSmart Buffer<br />
| 5.0 uL<br />
| 5.0 uL<br />
| 5.0 uL<br />
| 5.0 uL<br />
| 5.0 uL<br />
| 5.0 uL<br />
| 5.0 uL<br />
| 5.0 uL<br />
<br />
|-<br />
| PstI<br />
| 1.0 uL<br />
| 1.0 uL<br />
| 1.0 uL<br />
| 1.0 uL<br />
| 1.0 uL<br />
| 1.0 uL<br />
| 1.0 uL<br />
| 1.0 uL<br />
<br />
|-<br />
| XbaI<br />
| 1.0 uL<br />
| 1.0 uL<br />
| 1.0 uL<br />
| 1.0 uL<br />
| 1.0 uL<br />
| <br />
| <br />
| 1.0 uL<br />
<br />
|-<br />
| SpeI<br />
| --<br />
| --<br />
| --<br />
| --<br />
| --<br />
| 1.0 uL<br />
| 1.0 uL<br />
| --<br />
<br />
|-<br />
| DNA (500 ng)<br />
| 11.4 uL<br />
| 4.0 uL<br />
| 3.1 uL<br />
| 3.3 uL<br />
| 5.5 uL<br />
| 4.4 uL<br />
| 3.4 uL<br />
| --<br />
<br />
|-<br />
| MQ H2O<br />
| 31.6 uL<br />
| 39.0 uL<br />
| 39.9 uL<br />
| 39.7 uL<br />
| 37.5 uL<br />
| 38.6 uL<br />
| 39.6 uL<br />
| 43.0 uL<br />
<br />
|-<br />
| Total Volume<br />
| 50.0 uL<br />
| 50.0 uL<br />
| 50.0 uL<br />
| 50.0 uL<br />
| 50.0 uL<br />
| 50.0 uL<br />
| 50.0 uL<br />
| 50.0 uL<br />
<br />
|-<br />
|}<br />
<br />
Ligation reaction was performed to build device 3 from the digested products. This time, several samples will be made. For each reaction, a ratio of 1:3 vector to insert will again be used. A strip of numbered PCR tubes were used for the reactions, with each reaction containing the following reagents. All were placed at 16 °C for 16 hours. <br />
<br />
{| class = "wikitable"<br />
|-<br />
! T4 Ligase <br />
! 1.0 uL<br />
<br />
|-<br />
| T4 Ligase Buffer<br />
| 2.0 uL<br />
<br />
|-<br />
| J23101 Vector DNA (50 ng)<br />
| 5.0 uL<br />
<br />
|-<br />
| E0240 Insert DNA (62.5 ng)<br />
| 6.25 uL<br />
<br />
|-<br />
| MQ H2O<br />
| 5.75 uL<br />
<br />
|-<br />
| Total Volume<br />
| 20.0 uL<br />
<br />
|-<br />
|}<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Tube #<br />
<br />
|-<br />
| J23115-1 & E0240-1<br />
| 3<br />
<br />
|-<br />
| J23115-2 & E0240-1<br />
| 4<br />
<br />
|-<br />
| J23115-1 & E0240-2<br />
| 5<br />
<br />
|-<br />
| J23115-2 & E0240-2<br />
| 6<br />
<br />
|-<br />
| Neg Control – J23115-2 and H2O<br />
| 7<br />
<br />
|-<br />
| Empty<br />
| 8<br />
<br />
|-<br />
|}<br />
<br />
==Week 6==<br />
<br />
'''7/30/14'''<br />
<br />
A heat inactivation was performed on the ligation products. Next, 10 uL of each of the four device 3 samples and the negative control from tube 7 were transformed into 40 uL DH5u competent cells (made 7/28). 80 uL of each was plated after 1.5 hour incubation. <br />
<br />
Gel electrophoresis was performed on digested products from 7/29/14. A 1% agarose gel with EtBr was used and loaded with 5 uL of ladder and 5 uL of each sample with 1 uL loading dye as follows:<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Well<br />
! Loaded<br />
! Expected Sizes (bp)<br />
<br />
|-<br />
| 1<br />
| 2-Log DNA Ladder<br />
| N/A<br />
<br />
|-<br />
| 2<br />
| D1<br />
| 2724, 945<br />
<br />
|-<br />
| 3<br />
| D2-1<br />
| 2261<br />
<br />
|-<br />
| 4<br />
| D2-2<br />
| 2042, 900<br />
<br />
|-<br />
| 5<br />
| Empty<br />
| --<br />
<br />
|-<br />
| 6<br />
| 2-Log DNA Ladder<br />
| N/A<br />
<br />
|-<br />
| 7<br />
| J23115-1<br />
| 2100<br />
<br />
|-<br />
| 8<br />
| J23115-2<br />
| 2100<br />
<br />
|-<br />
| 9<br />
| E0240-1<br />
| 2100, 875<br />
<br />
|-<br />
| 10<br />
| E0240-2<br />
| 2100, 875<br />
<br />
|-<br />
| 11<br />
| (-) Control<br />
| --<br />
<br />
|-<br />
|}<br />
[[File:UCB-Interlab-07-2014.jpg]]<br />
<br />
<br />
From these data, the device 3 parts J23115-1, J23115-2, and E0240-1 are the correct size. E0240-2 may be undigested product, but will probably be excluded from further study. Device 1 reflects some undigested product in lane 2. It is unknown why a single band is present for both device 2 samples. <br />
<br />
'''8/1/14'''<br />
<br />
A discussion took place with Sam to discuss strange gel results and lack of visual fluorescence on plates or cultures. It was suggested that I have a positive control with known fluorescence, Sam will provide this. It was also suggested that multiple digests be done on samples to verify size. Plates with ligation products for device 3 removed from incubator. As shown below, all four ligation products have growth on the plates, no growth present on negative control plate (not shown). Device 3 samples used in further studies were taken from plate with J23115-1 & E0240-1.<br />
[[File:UCB-Interlab-08-2014.png]]<br />
<br />
[[File:UCB-Interlab-09-2014.png]]<br />
<br />
<br />
<br />
<br />
'''8/4/14'''<br />
<br />
20 Chloramphenicol and 10 Kan plates were made. BL21_GFP plate obtained from Sam to use as positive control. These cells have Amp resistance and need 0.3% glucose in media. GFP expression will be induced with 0.3 mM IPTG.<br />
An overnight culture of 10 mL LB with Amp and glucose was started for the BL21 sample. Overnight cultures of D2-2 and D3 from plate J23115-1 & E0240-2 was also prepared. In order to rule out contamination of any of the parts or other devices into device 1, a streak plate was made on a chloroamp plate (D1 has Kan resistance). <br />
<br />
'''8/5/14'''<br />
<br />
Control plate with device 1 looks good – no growth. Some of the DH5u cells are slow to grow so it will be left for several more days in incubator to be sure. Unfortunately, positive control cells BL21_GFP obtained from Garcea lab did not grow. The plate they were taken from may be too old. An amp plate was prepared and streaked from glycerol stock. Colonies were taken from all four device 3 plate for overnight cultures.<br />
<br />
==Week 7==<br />
<br />
'''8/8/14'''<br />
<br />
Control plate with device 1 shows no growth. This indicates that this product has not been contaminated with any of the parts from device 2 or device 3. <br />
BL21_GFP cells from overnight culture were diluted to OD of 0.4 and induced with 0.3 mM IPTG and allowed to regrow for 30 minutes (to OD 0.525). Device 3 overnight culture was also diluted to OD of 0.4 and allowed to regrow for 30 minutes, resulting in 0.494 OD. 100 uL of each sample (with LB as negative control blank) underwent a 1:2 serial dilution and was placed into a 96 well plate. Fluorescent measurements were taken using a Synergy 2 Plate Reader (courtesy of the Cech lab, University of Colorado at Boulder) using the built-in ZsGreen protocol (excitation 485/20, emission reading 528/20), raw data shown below.<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Full<br />
! 50%<br />
! 25%<br />
! 12.5%<br />
! 6.25%<br />
<br />
|-<br />
| LB<br />
| 265<br />
| 269<br />
| 259<br />
| 267<br />
| 260<br />
<br />
|-<br />
| Dev 1<br />
| 1078<br />
| 541<br />
| 360<br />
| 288<br />
| 249<br />
<br />
|-<br />
| BL21<br />
| 780<br />
| 549<br />
| 308<br />
| 212<br />
| 218<br />
<br />
|-<br />
|}<br />
After subtracting the fluorescence readings by LB alone, both samples demonstrate clear values that decline with decreasing concentrations as expected. The purpose of using the BL21 cell line was to compare cells with known fluorescence against the devices prepared for this study. These results indicate that these cells are producing fluorescence and thus are thought to contain the GFP gene and promoter. With these results, fluorescent measurements can now be taken of all three devices in biological triplicate. After discussing these data with Sam, it was also suggested that cells lacking the GFP gene (such as one of the parts for the promoter for device 2 or 3) be used for a negative control in further studies. These cells would be used to account for fluorescence from the DH5u cells and the pSB1C3 backbone. <br />
<br />
'''8/12/14'''<br />
<br />
Overnight cultures prepared of device 1, 2, and 3. BL21 culture induced with 0.3mM IPTG.<br />
<br />
<br />
'''8/13/14'''<br />
<br />
Minipreps and nanodrops prepared from overnight cultures with data shown below. Device 1 and device 2 samples demonstrate very low concentrations. These samples were then used for restriction digestion with multiple enzymes followed by agarose gel electrophoresis (1% agarose with EtBr as previously described). <br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! ng/ uL<br />
! 260/280<br />
! 260/230<br />
<br />
|-<br />
| Dev 1<br />
| 9.6<br />
| 1.90<br />
| 1.40<br />
<br />
|-<br />
| Dev 2<br />
| 35.7<br />
| 1.87<br />
| 2.29<br />
<br />
|-<br />
| Dev 3<br />
| 230.8<br />
| 1.87<br />
| 2.37<br />
<br />
|-<br />
|}<br />
<br />
Restriction Digestion<br />
{| class = "wikitable"<br />
|-<br />
! Sample <br />
! FD Buffer<br />
! NcoI<br />
! HaeII<br />
! NotI<br />
! DNA (200 ng)<br />
! MQ H2O<br />
! Total Volume<br />
! Bands Expected (Bp)<br />
<br />
|-<br />
| D1-Double<br />
| 2.0 uL<br />
| 1.0 uL<br />
| --<br />
| --<br />
| 20.8 uL<br />
| 1.16 uL<br />
| 25.0 uL<br />
| 3296, 373<br />
<br />
|-<br />
| D2-1 Single<br />
| 2.0 uL<br />
| --<br />
| 1.0 uL<br />
| --<br />
| 5.6 uL<br />
| 16.4 uL<br />
| 25.0 uL<br />
| 2981<br />
<br />
|-<br />
| D2-1 Double<br />
| 2.0 uL<br />
| --<br />
| --<br />
| 1.0 uL<br />
| 5.6 uL<br />
| 16.4 uL<br />
| 25.0 uL<br />
| 2046, 935<br />
<br />
|-<br />
| D2-1 Triple<br />
| 2.0 uL<br />
| --<br />
| 1.0 uL<br />
| 1.0 uL<br />
| 5.6 uL<br />
| 15.4 uL<br />
| 25.0 uL<br />
| 1595, 935, 451<br />
<br />
|-<br />
| D3-1 Single<br />
| 2.0 uL<br />
| --<br />
| 1.0 uL<br />
| --<br />
| 0.86 uL<br />
| 21.14 uL<br />
| 25.0 uL<br />
| 2981<br />
<br />
|-<br />
| D3-1 Double<br />
| 2.0 uL<br />
| --<br />
| --<br />
| 1.0 uL<br />
| 0.86 uL<br />
| 21.14 uL<br />
| 25.0 uL<br />
| 2046, 935<br />
<br />
|-<br />
| D3-1 Triple<br />
| 2.0 uL<br />
| --<br />
| 1.0 uL<br />
| 1.0 uL<br />
| 0.86 uL<br />
| 20.14 uL<br />
| 25.0 uL<br />
| 1595, 935, 451<br />
<br />
|-<br />
|}<br />
{| class = "wikitable"<br />
|-<br />
! Well<br />
! Loaded<br />
! Expected Sizes (bp)<br />
<br />
|-<br />
| 1<br />
| 2-Log DNA Ladder<br />
| N/A<br />
<br />
|-<br />
| 2<br />
| D1 Double<br />
| 3296, 373<br />
<br />
|-<br />
| 3<br />
| D2 Single<br />
| 2981<br />
<br />
|-<br />
| 4<br />
| D2 Double<br />
| 2046, 935<br />
<br />
|-<br />
| 5<br />
| D2 Triple<br />
| 1595, 935, 451<br />
<br />
|-<br />
| 6<br />
| Empty<br />
| N/A<br />
<br />
|-<br />
| 7<br />
| 2-Log DNA Ladder<br />
| N/A<br />
<br />
|-<br />
| 8<br />
| D3 Single<br />
| 2981<br />
<br />
|-<br />
| 9<br />
| D3 Double<br />
| 2046, 935<br />
<br />
|-<br />
| 10<br />
| D3 Triple<br />
| 1595, 935, 451<br />
<br />
|-<br />
| 11<br />
| (-) Control<br />
| --<br />
<br />
|-<br />
|}<br />
[[File:UCB-Interlab-10-2014.png]]<br />
<br />
As seen from the image above, smaller bands ran off the bottom of the gel and could not be readily visualized. However, the approximate sizes of the bands are as expected for each sample. <br />
Another 96 well plate was loaded using the sample protocol as previously described. Samples were measured after dilution and regrowth to OD of 0.5. J23101 and BL21 samples were used as controls, raw data shown below. <br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Full<br />
! 50%<br />
! 25%<br />
! 12.5%<br />
! 6.25%<br />
<br />
|-<br />
| LB<br />
| 706<br />
| 268<br />
| 188<br />
| 137<br />
| 123<br />
<br />
|-<br />
| J23101<br />
| 935<br />
| 294<br />
| 204<br />
| 145<br />
| 125<br />
<br />
|-<br />
| BL21<br />
| 1277<br />
| 349<br />
| 226<br />
| 159<br />
| 130<br />
<br />
|-<br />
| Dev 1<br />
| 1623<br />
| 438<br />
| 258<br />
| 182<br />
| 145<br />
<br />
|-<br />
| Dev 2<br />
| 920<br />
| 303<br />
| 193<br />
| 134<br />
| 123<br />
<br />
|-<br />
| Dev 3<br />
| 1147<br />
| 367<br />
| 219<br />
| 157<br />
| 126<br />
<br />
|-<br />
|}<br />
As with the previous test, all 3 devices demonstrate fluorescence in excess of the negative control sample. Values at full and below 12.5% concentration may be beyond the optimal range for the instrument to detect. The dilution amounts should be adjusted for the full test based on these data. <br />
<br />
==Week 8==<br />
<br />
'''8/18/14'''<br />
<br />
Made LB agar plates with appropriate antibiotics. 100 uL of each sample was plated and placed in incubator for overnight growth. These will be used to pull individual colonies for the biological triplicates needed in the fluorescent measurements to be performed. A repeat miniprep was performed on the device 1 sample from 8/13/14 in an attempt to obtain a higher concentration. <br />
<br />
'''8/19/14'''<br />
<br />
Plates made yesterday grew too well and single colonies could not be isolated. New streak plates were created for each sample (plates were not imaged). <br />
<br />
'''8/20/14'''<br />
<br />
Three colonies were taken from each streak plate to be used as biological triplicates in the study. Plates are shown below (Top row from left; Dev 1, Dev 2, Dev 3. Bottom row: J23101 negative control, BL21 positive control).<br />
[[File:UCB-Interlab-11-2014.png]]<br />
<br />
==Week 9==<br />
<br />
'''8/21/14'''<br />
<br />
Samples were monitored throughout the day and diluted as necessary to keep them in log phase. Since samples did not grow at the same rate, some difficulties were apparent when trying to get each sample to the same OD at the same time as the other samples. The volume per well will be increased to 150 uL and the concentrations adjusted for the measurement.<br />
<br />
<br />
'''8/22/14'''<br />
<br />
Fluorescence measurements were taken of device 1, 2 and 3 biological triplicate samples as previously described. Samples were all in log phage growth with OD of approximately 0.5 as shown below. Due to the number of samples, two separate 96 well plates were prepared using the same controls. Raw data is shown below.<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Final OD<br />
<br />
|-<br />
| J23101<br />
| 0.508<br />
<br />
|-<br />
| Dev 1-1<br />
| 0.515<br />
<br />
|-<br />
| Dev 1-2<br />
| 0.514<br />
<br />
|-<br />
| Dev 1-3<br />
| 0.509<br />
<br />
|-<br />
| Dev 2-1<br />
| 0.490<br />
<br />
|-<br />
| Dev 2-2<br />
| 0.508<br />
<br />
|-<br />
| Dev 2-3<br />
| 0.526<br />
<br />
|-<br />
| Dev 3-1<br />
| 0.512<br />
<br />
|-<br />
| Dev 3-2<br />
| 0.501<br />
<br />
|-<br />
| Dev 3-3<br />
| 0.520<br />
<br />
|-<br />
|}<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! 100<br />
! 0.67<br />
! 0.44<br />
! 0.3<br />
! 0.2<br />
! 13.4<br />
! 0.09<br />
<br />
|-<br />
| LB Blank<br />
| 778<br />
| 785<br />
| 774<br />
| 766<br />
| 780<br />
| 775<br />
| 788<br />
<br />
|-<br />
| Neg Control (J23101)<br />
| 1068<br />
| 903<br />
| 854<br />
| 821<br />
| 807<br />
| 794<br />
| 788<br />
<br />
|-<br />
| Dev 3 #1<br />
| 1142<br />
| 995<br />
| 895<br />
| 831<br />
| 804<br />
| 809<br />
| 798<br />
<br />
|-<br />
| Dev 3 #2<br />
| 1125<br />
| 940<br />
| 852<br />
| 823<br />
| 796<br />
| 805<br />
| 788<br />
<br />
|-<br />
| Dev 3 #3<br />
| 1035<br />
| 951<br />
| 888<br />
| 837<br />
| 824<br />
| 812<br />
| 797<br />
<br />
|-<br />
| Dev 2 #1<br />
| 992<br />
| 925<br />
| 849<br />
| 812<br />
| 805<br />
| 779<br />
| 779<br />
<br />
|-<br />
| Dev 2 #2<br />
| 1083<br />
| 924<br />
| 820<br />
| 813<br />
| 791<br />
| 776<br />
| 761<br />
<br />
|-<br />
| Dev 2 #3<br />
| 1047<br />
| 879<br />
| 824<br />
| 801<br />
| 776<br />
| 782<br />
| 772<br />
<br />
|-<br />
|}<br />
Plate #1 raw data with sample concentrations shown as decimals. <br />
{| class = "wikitable"<br />
|-<br />
! <br />
! 100<br />
! 0.67<br />
! 0.44<br />
! 0.3<br />
! 0.2<br />
! 13.4<br />
! 0.09<br />
<br />
|-<br />
| LB Blank<br />
| 816<br />
| 825<br />
| 849<br />
| 816<br />
| 844<br />
| 827<br />
| 825<br />
<br />
|-<br />
| Neg Control (J23101)<br />
| 982<br />
| 925<br />
| 886<br />
| 850<br />
| 845<br />
| 833<br />
| 807<br />
<br />
|-<br />
| Dev 1 #1<br />
| 1728<br />
| 1450<br />
| 1153<br />
| 1024<br />
| 899<br />
| 881<br />
| 838<br />
<br />
|-<br />
| Dev 1 #2<br />
| 1830<br />
| 1413<br />
| 1142<br />
| 1010<br />
| 921<br />
| 841<br />
| 861<br />
<br />
|-<br />
| Dev 1 #3<br />
| 1790<br />
| 1446<br />
| 1155<br />
| 975<br />
| 911<br />
| 861<br />
| 854<br />
<br />
|-<br />
|}<br />
Plate #2 raw data with sample concentrations shown as decimals. <br />
<br />
==Week 10==<br />
<br />
'''9/4/14'''<br />
<br />
Minipreps done on triplicate samples for J, D1, D2, & D3<br />
<br />
'''9/5/14'''<br />
<br />
Nanodrop done on mini prepped samples (results shown below) followed by restriction digestion using NcoI. The products were run on a 1% agarose gel with EtBr as previously described. This was done to ensure that all of the samples used in the fluorescence measurements are of the correct size. Although the gel is slightly distorted, the size of the bands correspond with the expected products (image not shown). <br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! ng/ uL<br />
! 260/280<br />
! 260/230<br />
<br />
|-<br />
| Dev 1 #1<br />
| 13.7<br />
| 1.99<br />
| 2.24<br />
<br />
|-<br />
| Dev 1 #2<br />
| 12.0<br />
| 1.94<br />
| 2.20<br />
<br />
|-<br />
| Dev 1 #3<br />
| 14.1<br />
| 1.86<br />
| 1.56<br />
<br />
|-<br />
| Dev 2-1 #1<br />
| 79.0<br />
| 1.87<br />
| 2.24<br />
<br />
|-<br />
| Dev 2-1 #2<br />
| 68.0<br />
| 1.83<br />
| 2.14<br />
<br />
|-<br />
| Dev 2-1 #3<br />
| 88.1<br />
| 1.84<br />
| 2.24<br />
<br />
|-<br />
| Dev 3 #1<br />
| 577.3<br />
| 1.85<br />
| 2.37<br />
<br />
|-<br />
| Dev 3 #2<br />
| 272.8<br />
| 1.87<br />
| 2.33<br />
<br />
|-<br />
| Dev 3 #3<br />
| 840.2<br />
| 1.86<br />
| 2.35<br />
<br />
|-<br />
| J23115 #1<br />
| 254.3<br />
| 1.85<br />
| 1.70<br />
<br />
|-<br />
| J23115 #2<br />
| 557.1<br />
| 1.82<br />
| 2.26<br />
<br />
|-<br />
| J23115 #3<br />
| 784.5<br />
| 1.84<br />
| 2.28<br />
<br />
|-<br />
|}<br />
<br />
Restriction Digestion<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! FD Buffer<br />
! NcoI<br />
! DNA (250 ng)<br />
! MQ H2O<br />
! Total Volume<br />
! Bands Expected (Bp)<br />
<br />
|-<br />
| J1<br />
| 2.0 uL<br />
| 1.0 uL<br />
| 1.0 uL<br />
| 21.0 uL<br />
| 25.0 uL<br />
| 1335, 770<br />
<br />
|-<br />
| J2<br />
| 2.0 uL<br />
| 1.0 uL<br />
| 0.9 uL†<br />
| 21.1 uL<br />
| 25.0 uL<br />
| 1335, 770<br />
<br />
|-<br />
| J3<br />
| 2.0 uL<br />
| 1.0 uL<br />
| 0.3 uL† (0.95 of 1:3)<br />
| 21.1 uL<br />
| 25.0 uL<br />
| 1335, 770<br />
<br />
|-<br />
| D2-1*<br />
| 2.0 uL<br />
| 1.0 uL<br />
| 2.5 uL <br />
| 14.5 uL<br />
| 20.0 uL<br />
| 1991, 990<br />
<br />
|-<br />
| D2-2*<br />
| 2.0 uL<br />
| 1.0 uL<br />
| 2.9 uL<br />
| 14.1 uL<br />
| 20.0 uL<br />
| 1991, 990<br />
<br />
|-<br />
| D2-3*<br />
| 2.0 uL<br />
| 1.0 uL<br />
| 2.3 uL<br />
| 14.7 uL<br />
| 20.0 uL<br />
| 1991, 990<br />
<br />
|-<br />
| D3-1<br />
| 2.0 uL<br />
| 1.0 uL<br />
| 0.45 uL† (0.9 of 1:2)<br />
| 21.1 uL<br />
| 25.0 uL<br />
| 1991, 990<br />
<br />
|-<br />
| D3-2<br />
| 2.0 uL<br />
| 1.0 uL<br />
| 0.92 uL<br />
| 21.1 uL<br />
| 25.0 uL<br />
| 1991, 990<br />
<br />
|-<br />
| D3-3<br />
| 2.0 uL<br />
| 1.0 uL<br />
| 0.3 (0.9 of 1:3) uL†<br />
| 21.1 uL<br />
| 25.0 uL<br />
| 1991, 990<br />
<br />
|-<br />
|}<br />
*Digest done with 200 ng and 20 uL volume 2 hours prior to the other reactions<br />
†Volume after DNA was diluted<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Lane #<br />
! Sample<br />
! Bands Expected (Bp)<br />
<br />
|-<br />
| 1<br />
| 2-Log DNA Ladder<br />
| N/A<br />
<br />
|-<br />
| 2<br />
| J1 (Hole in Gel)<br />
| 1335, 770<br />
<br />
|-<br />
| 3<br />
| J1<br />
| 1335, 770<br />
<br />
|-<br />
| 4<br />
| J2<br />
| 1335, 770<br />
<br />
|-<br />
| 5<br />
| J3<br />
| 1335, 770<br />
<br />
|-<br />
| 6<br />
| 2-Log DNA Ladder<br />
| N/A<br />
<br />
|-<br />
| 7<br />
| D2-1*<br />
| 1991, 990<br />
<br />
|-<br />
| 8<br />
| D2-2*<br />
| 1991, 990<br />
<br />
|-<br />
| 9<br />
| D2-3*<br />
| 1991, 990<br />
<br />
|-<br />
| 10<br />
| 2-Log DNA Ladder<br />
| N/A<br />
<br />
|-<br />
| 11<br />
| D3-1<br />
| 1991, 990<br />
<br />
|-<br />
| 12<br />
| D3-2<br />
| 1991, 990<br />
<br />
|-<br />
| 13<br />
| D3-3<br />
| 1991, 990<br />
<br />
|-<br />
| 14<br />
| 2-Log DNA Ladder<br />
| N/A<br />
<br />
|-<br />
| 15<br />
| H2O Control<br />
| N/A<br />
<br />
|-<br />
|}<br />
DATA ANALYSIS <br />
Analysis of the fluorescence data measurements obtained is shown below. <br />
{| class = "wikitable"<br />
|-<br />
! <br />
! 1.00<br />
! 0.67<br />
! 0.44<br />
! 0.3<br />
! 0.2<br />
! 13.4<br />
! 0.09<br />
<br />
|-<br />
| Dev 1 #1 Actual<br />
| 1728<br />
| 1450<br />
| 1153<br />
| 1024<br />
| 899<br />
| 881<br />
| 838<br />
<br />
|-<br />
| Calculated<br />
| 746<br />
| 525<br />
| 267<br />
| 174<br />
| 54<br />
| 48<br />
| 31<br />
<br />
|-<br />
| Dev 1 #2 Actual<br />
| 1830<br />
| 1413<br />
| 1142<br />
| 1010<br />
| 921<br />
| 841<br />
| 861<br />
<br />
|-<br />
| Calculated<br />
| 848<br />
| 488<br />
| 256<br />
| 160<br />
| 76<br />
| 8<br />
| 54<br />
<br />
|-<br />
| Dev 1 #3 Actual<br />
| 1790<br />
| 1446<br />
| 1155<br />
| 975<br />
| 911<br />
| 861<br />
| 854<br />
<br />
|-<br />
| Calculated<br />
| 808<br />
| 521<br />
| 269<br />
| 125<br />
| 66<br />
| 28<br />
| 47<br />
<br />
|-<br />
| Dev 2 #1 Actual<br />
| 992<br />
| 925<br />
| 849<br />
| 812<br />
| 805<br />
| 779<br />
| 779<br />
<br />
|-<br />
| Calculated<br />
| -76<br />
| 22<br />
| -5<br />
| -9<br />
| -2<br />
| -15<br />
| -9<br />
<br />
|-<br />
| Dev 2 #2 Actual<br />
| 1083<br />
| 924<br />
| 820<br />
| 813<br />
| 791<br />
| 776<br />
| 761<br />
<br />
|-<br />
| Calculated<br />
| 15<br />
| 21<br />
| -34<br />
| -8<br />
| -16<br />
| -18<br />
| -27<br />
<br />
|-<br />
| Dev 2 #3 Actual<br />
| 1047<br />
| 879<br />
| 824<br />
| 801<br />
| 776<br />
| 782<br />
| 772<br />
<br />
|-<br />
| Calculated<br />
| -21<br />
| -24<br />
| -30<br />
| -20<br />
| -31<br />
| -12<br />
| -16<br />
<br />
|-<br />
| Dev 3 #1 Actual<br />
| 1142<br />
| 995<br />
| 895<br />
| 831<br />
| 804<br />
| 809<br />
| 798<br />
<br />
|-<br />
| Calculated<br />
| 74<br />
| 92<br />
| 41<br />
| 10<br />
| -3<br />
| 15<br />
| 10<br />
<br />
|-<br />
| Dev 3 #2 Actual<br />
| 1125<br />
| 940<br />
| 852<br />
| 823<br />
| 796<br />
| 805<br />
| 788<br />
<br />
|-<br />
| Calculated<br />
| 57<br />
| 37<br />
| -2<br />
| 2<br />
| -11<br />
| 11<br />
| 0<br />
<br />
|-<br />
| Dev 3 #3 Actual<br />
| 1035<br />
| 951<br />
| 888<br />
| 837<br />
| 824<br />
| 812<br />
| 797<br />
<br />
|-<br />
| Calculated<br />
| -33<br />
| 48<br />
| 34<br />
| 16<br />
| 17<br />
| 18<br />
| 9<br />
<br />
|-<br />
|}<br />
Table 1: Fluorescence data. For each of the three devices, 3 biological replicates were measured (labeled #1, #2, #3 for each device). Data shown includes actual measurements and derived quantities obtained by subtracting fluorescence measured from non-GFP expressing E. coli of the same strain on the same plate. Promoter J23115 for device #3 was used as distributed. In addition, each sample underwent a serial dilution (concentrations listed as fractions across top of table).<br />
<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! 100<br />
! 0.67<br />
! 0.44<br />
! 0.3<br />
! 0.2<br />
! 13.4<br />
! 0.09<br />
<br />
|-<br />
| Dev 1 Ave<br />
| 1.82<br />
| 1.55<br />
| 1.30<br />
| 1.18<br />
| 1.08<br />
| 1.03<br />
| 1.05<br />
<br />
|-<br />
| Dev 1 Std. Dev<br />
| 0.05<br />
| 0.02<br />
| 0.01<br />
| 0.03<br />
| 0.01<br />
| 0.02<br />
| 0.01<br />
<br />
|-<br />
| Dev 2 Ave<br />
| 0.97<br />
| 1.01<br />
| 0.97<br />
| 0.98<br />
| 0.98<br />
| 0.98<br />
| 0.98<br />
<br />
|-<br />
| Dev 2 Std. Dev<br />
| 0.04<br />
| 0.03<br />
| 0.02<br />
| 0.01<br />
| 0.02<br />
| 0.00<br />
| 0.01<br />
<br />
|-<br />
| Dev 3 Ave<br />
| 1.03<br />
| 1.07<br />
| 1.03<br />
| 1.01<br />
| 1.00<br />
| 1.02<br />
| 1.01<br />
<br />
|-<br />
| Dev 3 Std. Dev<br />
| 0.05<br />
| 0.03<br />
| 0.03<br />
| 0.01<br />
| 0.02<br />
| 0.00<br />
| 0.01<br />
<br />
|-<br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
<br />
|-<br />
| <br />
| <br />
| Total: Dev 1<br />
| <br />
| <br />
| 1.23<br />
| <br />
| <br />
<br />
|-<br />
| <br />
| <br />
| Total: Dev 2<br />
| <br />
| <br />
| 0.99<br />
| <br />
| <br />
<br />
|-<br />
| <br />
| <br />
| Total: Dev 3<br />
| <br />
| <br />
| 1.02<br />
| <br />
| <br />
<br />
|-<br />
|}<br />
Table 2: Average device fluorescence. For each of the three devices, the average and standard sample deviation are given for the triplicates at each concentration. Data is given as fold change over non-GFP expressing E. coli of same DH5u strain. Total data on lower portion of table is an overall average of each device calculated using the averages for each device at all concentrations. No samples were excluded.<br />
<br />
[[File:UCB-Interlab-12-2014.png]]<br />
<br><br />
<br><br />
<br><br />
<br />
{{Template:UCB-Footer}}</div>Leighlahttp://2014.igem.org/Team:CU-Boulder/Notebook/MeasurementTeam:CU-Boulder/Notebook/Measurement2014-10-16T23:57:39Z<p>Leighla: </p>
<hr />
<div>{{Template:UCB-Main}}<br />
{{Template:UCB-NavBar}}<br />
__FORCETOC__<br />
'''Interlab Measurement Study'''<br />
<br />
==Study Description==<br />
<br />
Different iGEM teams from around the world were offered the opportunity to participate in this study to obtain fluorescent measurements from three separate devices. Each device consisted of a specific plasmid that was to be expressed in bacterial cells. The parts for each device were provided to each team from the iGEM registry. <br />
<br />
The first plasmid was already assembled and provided to teams with highly expressive constitutive promoter for the GFP in a low-copy plasmid, pSB3K3. The other two plasmids contained the same GFP gene, but were assembled onto the pSB1C3 plasmid. The first of these held the same constitutive promoter (J23101) as the first device. The third device contained a constitutively low-expressive promoter (J23115) to drive GFP expression (Table 1).<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Device<br />
! Constitutive Promoter<br />
! Plasmid<br />
<br />
|-<br />
| Device 1<br />
| J23101 (High Expression)<br />
| pSB3K3 (Low-Copy)<br />
<br />
|-<br />
| Device 2<br />
| J23101 (High Expression)<br />
| pSB3C1<br />
<br />
|-<br />
| Device 3<br />
| J23115 (Low Expression)<br />
| pSB3C1<br />
<br />
|-<br />
|}<br />
<br />
Table 1: Overview of the three devices constructed for this study.<br />
<br />
Device 1 plasmids were transformed into competent E. coli DH5? cells and plated under antibiotic selection for cells that had taken up the plasmid. Device 2 and device 3 were assembled using restriction digestion followed by ligation. To begin, the plasmid containing the GFP gene (E0240) was digested with XbaI and PstI. The plasmids with promoters were each digested with SpeI and PstI, resulting in a location to which the GFP fragment could be inserted. These products were ligated together and transformed into competent E. coli DH5? cells and plated under antibiotic selection. Colonies were taken for overnight cultures, and DNA from these cultures was isolated for analysis. <br />
<br />
Biological triplicates were prepared as overnight cultures for all three devices and control samples. Fluorescence was measured from all samples in triplicate. First, samples were diluted as needed to obtain the same cell concentrations (by measuring OD) and then serial dilutions of samples were placed into a 96 well plate and measurements taken using a plate reader. Fluorescence values in RFU (relative fluorescent units) were compared and reported.<br />
<br />
==Results==<br />
<br />
As shown below (Data Table 1), for each of the devices, three biological replicates were measured (labeled #1, #2, and #3 for each device). Data shown includes actual measurements and derived quantities obtained by subtracting fluorescence measured from non-GFP expressing E. coli of the same strain on the same plate. Promoter J23115 for device #3 was used as distributed (containing mismatch). In addition, each sample underwent a serial dilution (concentrations listed as fractions across top of table).<br />
<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! 1.00<br />
! 0.67<br />
! 0.44<br />
! 0.3<br />
! 0.2<br />
! 13.4<br />
! 0.09<br />
<br />
|-<br />
| Dev 1 #1 Actual<br />
| 1728<br />
| 1450<br />
| 1153<br />
| 1024<br />
| 899<br />
| 881<br />
| 838<br />
<br />
|-<br />
| Calculated<br />
| 746<br />
| 525<br />
| 267<br />
| 174<br />
| 54<br />
| 48<br />
| 31<br />
<br />
|-<br />
| Dev 1 #2 Actual<br />
| 1830<br />
| 1413<br />
| 1142<br />
| 1010<br />
| 921<br />
| 841<br />
| 861<br />
<br />
|-<br />
| Calculated<br />
| 848<br />
| 488<br />
| 256<br />
| 160<br />
| 76<br />
| 8<br />
| 54<br />
<br />
|-<br />
| Dev 1 #3 Actual<br />
| 1790<br />
| 1446<br />
| 1155<br />
| 975<br />
| 911<br />
| 861<br />
| 854<br />
<br />
|-<br />
| Calculated<br />
| 808<br />
| 521<br />
| 269<br />
| 125<br />
| 66<br />
| 28<br />
| 47<br />
<br />
|-<br />
| Dev 2 #1 Actual<br />
| 992<br />
| 925<br />
| 849<br />
| 812<br />
| 805<br />
| 779<br />
| 779<br />
<br />
|-<br />
| Calculated<br />
| -76<br />
| 22<br />
| -5<br />
| -9<br />
| -2<br />
| -15<br />
| -9<br />
<br />
|-<br />
| Dev 2 #2 Actual<br />
| 1083<br />
| 924<br />
| 820<br />
| 813<br />
| 791<br />
| 776<br />
| 761<br />
<br />
|-<br />
| Calculated<br />
| 15<br />
| 21<br />
| -34<br />
| -8<br />
| -16<br />
| -18<br />
| -27<br />
<br />
|-<br />
| Dev 2 #3 Actual<br />
| 1047<br />
| 879<br />
| 824<br />
| 801<br />
| 776<br />
| 782<br />
| 772<br />
<br />
|-<br />
| Calculated<br />
| -21<br />
| -24<br />
| -30<br />
| -20<br />
| -31<br />
| -12<br />
| -16<br />
<br />
|-<br />
| Dev 3 #1 Actual<br />
| 1142<br />
| 995<br />
| 895<br />
| 831<br />
| 804<br />
| 809<br />
| 798<br />
<br />
|-<br />
| Calculated<br />
| 74<br />
| 92<br />
| 41<br />
| 10<br />
| -3<br />
| 15<br />
| 10<br />
<br />
|-<br />
| Dev 3 #2 Actual<br />
| 1125<br />
| 940<br />
| 852<br />
| 823<br />
| 796<br />
| 805<br />
| 788<br />
<br />
|-<br />
| Calculated<br />
| 57<br />
| 37<br />
| -2<br />
| 2<br />
| -11<br />
| 11<br />
| 0<br />
<br />
|-<br />
| Dev 3 #3 Actual<br />
| 1035<br />
| 951<br />
| 888<br />
| 837<br />
| 824<br />
| 812<br />
| 797<br />
<br />
|-<br />
| Calculated<br />
| -33<br />
| 48<br />
| 34<br />
| 16<br />
| 17<br />
| 18<br />
| 9<br />
<br />
|-<br />
|}<br />
<br />
Data Table 1: Fluorescence data. For each of the devices, three biological replicates were measured (labeled #1, #2, and #3 for each device). Data shown includes actual measurements and derived quantities. Fractions across top of table represent sample concentration.<br />
<br />
==Conclusions==<br />
<br />
As shown below (Data Table 3), Device 1 showed a much higher amount of fluorescence compared to the other two devices. This is likely due to differences in the plasmid between the devices, with the low copy pSB3K3 performing much higher than the pSB1C3 plasmid. Device 2 and 3 were similar in fluorescence amounts. However, device 3 outperformed device 2, an unexpected result considering the promoter differences. It was later discovered that the J23115 promoter part used for device 3 contained two mismatched base pairs. Considering that the promoter sequence is only 35 bp in length, this could have a significant impact on expression of the GFP gene in this device. <br />
<br />
[[File:UCB-interlab study gragh-141016.JPG]]<br />
<br />
Data Table 2: Fold change in fluorescence in samples compared to non-GFP expressing bacterial cells. <br />
<br><br />
<br><br />
<br />
'''Below is the Daily Lab Notebook for this Study'''<br />
<br><br />
<br><br />
<br> <br />
==Week 1==<br />
<br />
'''7/1/14'''<br />
<br />
Dry Lab only: Planning and research into this study. From the description on iGEM’s website <br />
<br />
(https://2014.igem.org/Tracks/Measurement/Interlab_study):<br />
<br />
The first device is already built and available in the distribution kit. The second and third devices must be built using the BioBricks standard protocol by the teams participating in this study.<br />
<br />
1. Existing device: BBa_I20260 (J23101-B0032-E0040-B0015) in the pSB3K3 vector. <br />
Kit location <br />
Plate 4, Well 18A<br />
<br />
2. New device to be built by the iGEM team: BBa_J23101 + BBa_E0240 (B0032-E0040-B0015), must be built in the pSB1C3 backbone <br />
Kit locations <br />
BBa_J23101 (called BBa_K823005 when in pSB1C3): Plate 1, Well 20K <br />
BBa_E0240 (in pSB1C3): Plate 2, Well 24B<br />
<br />
3. New device to be built by the iGEM team: BBa_J23115 + BBa_E0240 (B0032-E0040-B0015), must be built in the pSB1C3 backbone <br />
Kit locations <br />
BBa_J23115 (called BBa_K823012 when in pSB1C3): Plate 1, Well 22I <br />
BBa_E0240 (in pSB1C3): Plate 2, Well 24B<br />
<br />
PLEASE NOTE: The J23115 part in this year's distribution kit has two mismatched basepairs and instead matches the K823012sequence (which was already known to have these mismatches). You can either (a) use the J23115 part as distributed or (b) re-clone J23115 and correct the mismatch. Please let us know which path you will choose at measurement at igem dot org. <br />
<br />
Thank you! (*This note was not originally on the webpage – it was added around July 12th)<br />
<br />
<br />
'''7/3/14'''<br />
<br />
Resuspended DNA from 2014 distribution kit to obtain the 4 parts/devices needed for this study<br />
Transformation of Device 1 (I20260) into BWF+ comp cells<br />
- Two reactions were performed according to transformation protocol, one is negative control<br />
- Heat shock was done for 45 sec <br />
- Cultures were plated at full strength and 1:10 dilution with 20 uL each<br />
<br />
==Week 2==<br />
<br />
'''7/7/14'''<br />
<br />
Examined plates and observed that growth occurred on both, including the negative control plate. This reveals a problem. It was later discovered that the BW comp cells used in this transformation have Kan resistance, thus explaining the growth on the negative control plate. Transformation will need to be repeated using different comp cells; DH5 alpha cells have been suggested and are available.<br />
- Notation: K will be used for Kan plates and C for Chloroamp. <br />
<br />
'''7/10/14'''<br />
<br />
Transformation of all the parts needed from the distribution kit was performed. Each well in the distribution kit from iGEM contains 2-3 ng of DNA. So each transformation contains 200-300 pg/uL. All reactions were done following standard transformation protocol including 45 sec heat shock at 42°C. DH5 alpha comp cells were obtained from Dowell Lab for the reaction. <br />
<br />
Sample Volume DNA Volume Comp Cells<br />
I20260 (Dev1) 1.5 uL 40 uL <br />
J23101 1.5 uL 40 uL<br />
J23115 1.5 uL 40 uL<br />
E0240 1.5 uL 40 uL<br />
Neg Control (1.5 uL MQ H2O) 40 uL<br />
<br />
Following the transformation, 25 uL of mixture plated full strength on u of plate, 20 uL at 1:10 dilution plated on other u of plate containing the appropriate antibiotic.<br />
<br />
==Week 3==<br />
<br />
'''7/14/14'''<br />
<br />
Examination of plates from transformation shows various single colonies in the full strength and very few, if any colonies on the 1:10 dilution side of plates. This may indicate low transformation efficiency or could be due to slow growth of comp cells, which was observed by other iGEM students. Plates images shown below. Colonies were taken from I20260, E0240, and J23101 plates for overnight cultures in the appropriate antibiotic. <br />
<br />
<br />
[[File:UCB-Interlab-01-2014.png]]<br />
<br />
[[File:UCB-Interlab-02-2014.png]]<br />
<br />
[[File:UCB-Interlab-03-2014.png]]<br />
<br />
'''7/15/14'''<br />
<br />
Minipreps were done according to manufacturer’s recommendations for each of the overnight cultures. Bacterial pellets were made with the remainder of the cultures. Nanodrop performed on the miniprepped products as shown below: <br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! ng/ uL<br />
! 260/280<br />
! 260/230<br />
<br />
|-<br />
| Dev1 (I20260)<br />
| 43.9<br />
| 1.93<br />
| 2.13<br />
<br />
|-<br />
| J23101<br />
| 54.7<br />
| 1.88<br />
| 2.15<br />
<br />
|-<br />
| E0240<br />
| 13.2<br />
| 1.92<br />
| 1.10<br />
<br />
|-<br />
|}<br />
<br />
Gel electrophoresis was performed on the products. A 1% agarose gel (0.3 g agarose) with 3.0 uL EtBr in 30 mL TAE was prepared. 5 uL of a 2-Log DNA ladder was loaded followed by 5.0 uL of each sample with 1.0 uL loading dye into the following lanes:<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Well<br />
! Loaded<br />
! Expected Size (bp)<br />
<br />
|-<br />
| 1<br />
| 2-Log DNA Ladder<br />
| N/A<br />
<br />
|-<br />
| 2<br />
| D1 (I20260)<br />
| 3669<br />
<br />
|-<br />
| 3<br />
| J23101<br />
| 2100<br />
<br />
|-<br />
| 4<br />
| E0240<br />
| 2946<br />
<br />
|-<br />
| 5<br />
| D1 (I20260)<br />
| 3669<br />
<br />
|-<br />
| 6<br />
| J23101<br />
| 2100<br />
<br />
|-<br />
| 7<br />
| E0240<br />
| 2946<br />
<br />
|-<br />
| 8<br />
| H2O Control<br />
| --<br />
<br />
|-<br />
|}<br />
[[File:UCB-Interlab-04-2014.jpg]]<br />
<br />
As seen from the gel (shown above), various bands are visible for most of the samples. Since these are uncut plasmids, it is assumed that this is from supercoiling. <br />
<br />
'''7/16/14'''<br />
<br />
Restriction Digestion was performed on the 3 samples, followed by gel electrophoresis to analyze the products isolated. The digestion was done according to protocol using a 1 hour digestion at 37 °C followed by 20 min heat inactivation at 80°C. Enzymes and buffer obtained from NEB.<br />
<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! I20260<br />
! J23101<br />
! E0240<br />
<br />
|-<br />
| CutSmart Buffer<br />
| 5.0 uL<br />
| 5.0 uL<br />
| 5.0 uL<br />
<br />
|-<br />
| PstI<br />
| 1.0 uL<br />
| 1.0 uL<br />
| 1.0 uL<br />
<br />
|-<br />
| SpeI<br />
| --<br />
| 1.0 uL<br />
| --<br />
<br />
|-<br />
| XbaI<br />
| 1.0 uL<br />
| --<br />
| 1.0 uL<br />
<br />
|-<br />
| DNA (250 ng)<br />
| 5.7 uL<br />
| 4.6 uL<br />
| 18.9 uL<br />
<br />
|-<br />
| MQ H2O<br />
| 37.3 uL<br />
| 38.4 uL<br />
| 24.1 uL<br />
<br />
|-<br />
| Total Volume<br />
| 50.0 uL<br />
| 50.0 uL<br />
| 50.0 uL<br />
<br />
|-<br />
|}<br />
'''7/17/14'''<br />
<br />
Products from reaction were run on a 1% agarose gel with EtBr. 1.0 uL of loading dye was added to 5.0 uL of each sample as well as 5.0 uL of 2-Log DNA Ladder. Lanes and expected band sizes shown below. There appears to be undigested product present in the E0240 sample. <br />
<br />
{| class = "wikitable"<br />
|-<br />
! Well<br />
! Loaded<br />
! Expected Sizes (bp)<br />
<br />
|-<br />
| 1<br />
| 2-Log DNA Ladder<br />
| N/A<br />
<br />
|-<br />
| 2<br />
| D1 (I20260)<br />
| 2724, 945<br />
<br />
|-<br />
| 3<br />
| J23101<br />
| 2261<br />
<br />
|-<br />
| 4<br />
| E0240<br />
| 2042, 900<br />
<br />
|-<br />
| 5<br />
| H2O Control<br />
| --<br />
<br />
|-<br />
|}<br />
[[File:UCB-Interlab-05-2014.jpg]]<br />
<br />
<br />
<br />
J23101 and E0240 digest products are used in a ligation reaction to build Device 2. NEBioCalculator (http://nebiocalculator.neb.com ) was used to calculate the suggested ratio of 1:3 vector to insert for DNA sizes. The reaction included a negative control and was done using the following reagents according to NEB ligation protocol:<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! Sample<br />
! Neg Control<br />
<br />
|-<br />
| T4 Ligase <br />
| 1.25 uL<br />
| 1.25 uL<br />
<br />
|-<br />
| T4 Ligase Buffer<br />
| 2.5 uL<br />
| 2.5 uL<br />
<br />
|-<br />
| J23101 Vector DNA (40 ng)<br />
| 8.0 uL<br />
| 18.0 uL<br />
<br />
|-<br />
| E0240 Insert DNA (50 ng)<br />
| 10.0 uL<br />
| --<br />
<br />
|-<br />
| MQ H2O<br />
| 3.25 uL<br />
| 3.25 uL<br />
<br />
|-<br />
| Total Volume<br />
| 25.0 uL<br />
| 25.0 uL<br />
<br />
|-<br />
|}<br />
The reaction was allowed to proceed for 15 minutes at room temperature. The resulting product was transformed into DH5alpha competent cells using standard protocol. The plates were left to incubate, but even after several days no growth was observed. <br />
<br />
==Week 4==<br />
<br />
'''7/21/14'''<br />
<br />
Ligation from last week repeated using the same protocol and volumes. However, to improve chances of getting product formation, an overnight ligation will be used. The reaction was placed in a thermocycler and set to 16 °C for 16 hours. <br />
<br />
'''7/22/14'''<br />
<br />
Ligation reaction was heat inactivated at 65°C for 10 minutes. 10 uL of the product was transformed into 40 uL competent cells and plated onto the appropriate antibiotic plate in both full strength and as a 1:10 dilution. The negative control was also transformed and plated for comparison. <br />
<br />
==Week 5==<br />
<br />
'''7/28/14'''<br />
<br />
Competent cells were made with Josephina. Overnight cultures prepared from plates including device 2 and parts to build device 3. Six cultures were prepared. Shown below is the device 2 transformation plate (left) and negative control (right). <br />
[[File:UCB-Interlab-06-2014.png]]<br />
<br />
<br />
<br />
'''7/29/14'''<br />
<br />
Miniprep was performed on overnight cultures followed by Nanodrop to assess purity and concentration of samples. <br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! ng/ uL<br />
! 260/280<br />
! 260/230<br />
<br />
|-<br />
| Dev2 #1<br />
| 124.5<br />
| 1.85<br />
| 2.16<br />
<br />
|-<br />
| Dev2 #2<br />
| 160.5<br />
| 1.91<br />
| 2.29<br />
<br />
|-<br />
| E0240 #1<br />
| 150.9<br />
| 1.82<br />
| 1.61<br />
<br />
|-<br />
| E0240 #2<br />
| 91.0<br />
| 1.87<br />
| 2.15<br />
<br />
|-<br />
| J23115 #1<br />
| 114.5<br />
| 1.83<br />
| 1.97<br />
<br />
|-<br />
| J23115 #2<br />
| 148.5<br />
| 1.79<br />
| 2.29<br />
<br />
|-<br />
|}<br />
Restriction Digestion was performed on the products using standard protocol and with the following enzymes:<br />
<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! D1<br />
! D2 #1<br />
! D2 #2<br />
! E0240 #1<br />
! E0240 #2<br />
! J23115 #1<br />
! J23115 #2<br />
! (-) Control<br />
<br />
|-<br />
| CutSmart Buffer<br />
| 5.0 uL<br />
| 5.0 uL<br />
| 5.0 uL<br />
| 5.0 uL<br />
| 5.0 uL<br />
| 5.0 uL<br />
| 5.0 uL<br />
| 5.0 uL<br />
<br />
|-<br />
| PstI<br />
| 1.0 uL<br />
| 1.0 uL<br />
| 1.0 uL<br />
| 1.0 uL<br />
| 1.0 uL<br />
| 1.0 uL<br />
| 1.0 uL<br />
| 1.0 uL<br />
<br />
|-<br />
| XbaI<br />
| 1.0 uL<br />
| 1.0 uL<br />
| 1.0 uL<br />
| 1.0 uL<br />
| 1.0 uL<br />
| <br />
| <br />
| 1.0 uL<br />
<br />
|-<br />
| SpeI<br />
| --<br />
| --<br />
| --<br />
| --<br />
| --<br />
| 1.0 uL<br />
| 1.0 uL<br />
| --<br />
<br />
|-<br />
| DNA (500 ng)<br />
| 11.4 uL<br />
| 4.0 uL<br />
| 3.1 uL<br />
| 3.3 uL<br />
| 5.5 uL<br />
| 4.4 uL<br />
| 3.4 uL<br />
| --<br />
<br />
|-<br />
| MQ H2O<br />
| 31.6 uL<br />
| 39.0 uL<br />
| 39.9 uL<br />
| 39.7 uL<br />
| 37.5 uL<br />
| 38.6 uL<br />
| 39.6 uL<br />
| 43.0 uL<br />
<br />
|-<br />
| Total Volume<br />
| 50.0 uL<br />
| 50.0 uL<br />
| 50.0 uL<br />
| 50.0 uL<br />
| 50.0 uL<br />
| 50.0 uL<br />
| 50.0 uL<br />
| 50.0 uL<br />
<br />
|-<br />
|}<br />
<br />
Ligation reaction was performed to build device 3 from the digested products. This time, several samples will be made. For each reaction, a ratio of 1:3 vector to insert will again be used. A strip of numbered PCR tubes were used for the reactions, with each reaction containing the following reagents. All were placed at 16 °C for 16 hours. <br />
<br />
{| class = "wikitable"<br />
|-<br />
! T4 Ligase <br />
! 1.0 uL<br />
<br />
|-<br />
| T4 Ligase Buffer<br />
| 2.0 uL<br />
<br />
|-<br />
| J23101 Vector DNA (50 ng)<br />
| 5.0 uL<br />
<br />
|-<br />
| E0240 Insert DNA (62.5 ng)<br />
| 6.25 uL<br />
<br />
|-<br />
| MQ H2O<br />
| 5.75 uL<br />
<br />
|-<br />
| Total Volume<br />
| 20.0 uL<br />
<br />
|-<br />
|}<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Tube #<br />
<br />
|-<br />
| J23115-1 & E0240-1<br />
| 3<br />
<br />
|-<br />
| J23115-2 & E0240-1<br />
| 4<br />
<br />
|-<br />
| J23115-1 & E0240-2<br />
| 5<br />
<br />
|-<br />
| J23115-2 & E0240-2<br />
| 6<br />
<br />
|-<br />
| Neg Control – J23115-2 and H2O<br />
| 7<br />
<br />
|-<br />
| Empty<br />
| 8<br />
<br />
|-<br />
|}<br />
<br />
==Week 6==<br />
<br />
'''7/30/14'''<br />
<br />
A heat inactivation was performed on the ligation products. Next, 10 uL of each of the four device 3 samples and the negative control from tube 7 were transformed into 40 uL DH5u competent cells (made 7/28). 80 uL of each was plated after 1.5 hour incubation. <br />
<br />
Gel electrophoresis was performed on digested products from 7/29/14. A 1% agarose gel with EtBr was used and loaded with 5 uL of ladder and 5 uL of each sample with 1 uL loading dye as follows:<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Well<br />
! Loaded<br />
! Expected Sizes (bp)<br />
<br />
|-<br />
| 1<br />
| 2-Log DNA Ladder<br />
| N/A<br />
<br />
|-<br />
| 2<br />
| D1<br />
| 2724, 945<br />
<br />
|-<br />
| 3<br />
| D2-1<br />
| 2261<br />
<br />
|-<br />
| 4<br />
| D2-2<br />
| 2042, 900<br />
<br />
|-<br />
| 5<br />
| Empty<br />
| --<br />
<br />
|-<br />
| 6<br />
| 2-Log DNA Ladder<br />
| N/A<br />
<br />
|-<br />
| 7<br />
| J23115-1<br />
| 2100<br />
<br />
|-<br />
| 8<br />
| J23115-2<br />
| 2100<br />
<br />
|-<br />
| 9<br />
| E0240-1<br />
| 2100, 875<br />
<br />
|-<br />
| 10<br />
| E0240-2<br />
| 2100, 875<br />
<br />
|-<br />
| 11<br />
| (-) Control<br />
| --<br />
<br />
|-<br />
|}<br />
[[File:UCB-Interlab-07-2014.jpg]]<br />
<br />
<br />
From these data, the device 3 parts J23115-1, J23115-2, and E0240-1 are the correct size. E0240-2 may be undigested product, but will probably be excluded from further study. Device 1 reflects some undigested product in lane 2. It is unknown why a single band is present for both device 2 samples. <br />
<br />
'''8/1/14'''<br />
<br />
A discussion took place with Sam to discuss strange gel results and lack of visual fluorescence on plates or cultures. It was suggested that I have a positive control with known fluorescence, Sam will provide this. It was also suggested that multiple digests be done on samples to verify size. Plates with ligation products for device 3 removed from incubator. As shown below, all four ligation products have growth on the plates, no growth present on negative control plate (not shown). Device 3 samples used in further studies were taken from plate with J23115-1 & E0240-1.<br />
[[File:UCB-Interlab-08-2014.png]]<br />
<br />
[[File:UCB-Interlab-09-2014.png]]<br />
<br />
<br />
<br />
<br />
'''8/4/14'''<br />
<br />
20 Chloramphenicol and 10 Kan plates were made. BL21_GFP plate obtained from Sam to use as positive control. These cells have Amp resistance and need 0.3% glucose in media. GFP expression will be induced with 0.3 mM IPTG.<br />
An overnight culture of 10 mL LB with Amp and glucose was started for the BL21 sample. Overnight cultures of D2-2 and D3 from plate J23115-1 & E0240-2 was also prepared. In order to rule out contamination of any of the parts or other devices into device 1, a streak plate was made on a chloroamp plate (D1 has Kan resistance). <br />
<br />
'''8/5/14'''<br />
<br />
Control plate with device 1 looks good – no growth. Some of the DH5u cells are slow to grow so it will be left for several more days in incubator to be sure. Unfortunately, positive control cells BL21_GFP obtained from Garcea lab did not grow. The plate they were taken from may be too old. An amp plate was prepared and streaked from glycerol stock. Colonies were taken from all four device 3 plate for overnight cultures.<br />
<br />
==Week 7==<br />
<br />
'''8/8/14'''<br />
<br />
Control plate with device 1 shows no growth. This indicates that this product has not been contaminated with any of the parts from device 2 or device 3. <br />
BL21_GFP cells from overnight culture were diluted to OD of 0.4 and induced with 0.3 mM IPTG and allowed to regrow for 30 minutes (to OD 0.525). Device 3 overnight culture was also diluted to OD of 0.4 and allowed to regrow for 30 minutes, resulting in 0.494 OD. 100 uL of each sample (with LB as negative control blank) underwent a 1:2 serial dilution and was placed into a 96 well plate. Fluorescent measurements were taken using a Synergy 2 Plate Reader (courtesy of the Cech lab, University of Colorado at Boulder) using the built-in ZsGreen protocol (excitation 485/20, emission reading 528/20), raw data shown below.<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Full<br />
! 50%<br />
! 25%<br />
! 12.5%<br />
! 6.25%<br />
<br />
|-<br />
| LB<br />
| 265<br />
| 269<br />
| 259<br />
| 267<br />
| 260<br />
<br />
|-<br />
| Dev 1<br />
| 1078<br />
| 541<br />
| 360<br />
| 288<br />
| 249<br />
<br />
|-<br />
| BL21<br />
| 780<br />
| 549<br />
| 308<br />
| 212<br />
| 218<br />
<br />
|-<br />
|}<br />
After subtracting the fluorescence readings by LB alone, both samples demonstrate clear values that decline with decreasing concentrations as expected. The purpose of using the BL21 cell line was to compare cells with known fluorescence against the devices prepared for this study. These results indicate that these cells are producing fluorescence and thus are thought to contain the GFP gene and promoter. With these results, fluorescent measurements can now be taken of all three devices in biological triplicate. After discussing these data with Sam, it was also suggested that cells lacking the GFP gene (such as one of the parts for the promoter for device 2 or 3) be used for a negative control in further studies. These cells would be used to account for fluorescence from the DH5u cells and the pSB1C3 backbone. <br />
<br />
'''8/12/14'''<br />
<br />
Overnight cultures prepared of device 1, 2, and 3. BL21 culture induced with 0.3mM IPTG.<br />
<br />
<br />
'''8/13/14'''<br />
<br />
Minipreps and nanodrops prepared from overnight cultures with data shown below. Device 1 and device 2 samples demonstrate very low concentrations. These samples were then used for restriction digestion with multiple enzymes followed by agarose gel electrophoresis (1% agarose with EtBr as previously described). <br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! ng/ uL<br />
! 260/280<br />
! 260/230<br />
<br />
|-<br />
| Dev 1<br />
| 9.6<br />
| 1.90<br />
| 1.40<br />
<br />
|-<br />
| Dev 2<br />
| 35.7<br />
| 1.87<br />
| 2.29<br />
<br />
|-<br />
| Dev 3<br />
| 230.8<br />
| 1.87<br />
| 2.37<br />
<br />
|-<br />
|}<br />
<br />
Restriction Digestion<br />
{| class = "wikitable"<br />
|-<br />
! Sample <br />
! FD Buffer<br />
! NcoI<br />
! HaeII<br />
! NotI<br />
! DNA (200 ng)<br />
! MQ H2O<br />
! Total Volume<br />
! Bands Expected (Bp)<br />
<br />
|-<br />
| D1-Double<br />
| 2.0 uL<br />
| 1.0 uL<br />
| --<br />
| --<br />
| 20.8 uL<br />
| 1.16 uL<br />
| 25.0 uL<br />
| 3296, 373<br />
<br />
|-<br />
| D2-1 Single<br />
| 2.0 uL<br />
| --<br />
| 1.0 uL<br />
| --<br />
| 5.6 uL<br />
| 16.4 uL<br />
| 25.0 uL<br />
| 2981<br />
<br />
|-<br />
| D2-1 Double<br />
| 2.0 uL<br />
| --<br />
| --<br />
| 1.0 uL<br />
| 5.6 uL<br />
| 16.4 uL<br />
| 25.0 uL<br />
| 2046, 935<br />
<br />
|-<br />
| D2-1 Triple<br />
| 2.0 uL<br />
| --<br />
| 1.0 uL<br />
| 1.0 uL<br />
| 5.6 uL<br />
| 15.4 uL<br />
| 25.0 uL<br />
| 1595, 935, 451<br />
<br />
|-<br />
| D3-1 Single<br />
| 2.0 uL<br />
| --<br />
| 1.0 uL<br />
| --<br />
| 0.86 uL<br />
| 21.14 uL<br />
| 25.0 uL<br />
| 2981<br />
<br />
|-<br />
| D3-1 Double<br />
| 2.0 uL<br />
| --<br />
| --<br />
| 1.0 uL<br />
| 0.86 uL<br />
| 21.14 uL<br />
| 25.0 uL<br />
| 2046, 935<br />
<br />
|-<br />
| D3-1 Triple<br />
| 2.0 uL<br />
| --<br />
| 1.0 uL<br />
| 1.0 uL<br />
| 0.86 uL<br />
| 20.14 uL<br />
| 25.0 uL<br />
| 1595, 935, 451<br />
<br />
|-<br />
|}<br />
{| class = "wikitable"<br />
|-<br />
! Well<br />
! Loaded<br />
! Expected Sizes (bp)<br />
<br />
|-<br />
| 1<br />
| 2-Log DNA Ladder<br />
| N/A<br />
<br />
|-<br />
| 2<br />
| D1 Double<br />
| 3296, 373<br />
<br />
|-<br />
| 3<br />
| D2 Single<br />
| 2981<br />
<br />
|-<br />
| 4<br />
| D2 Double<br />
| 2046, 935<br />
<br />
|-<br />
| 5<br />
| D2 Triple<br />
| 1595, 935, 451<br />
<br />
|-<br />
| 6<br />
| Empty<br />
| N/A<br />
<br />
|-<br />
| 7<br />
| 2-Log DNA Ladder<br />
| N/A<br />
<br />
|-<br />
| 8<br />
| D3 Single<br />
| 2981<br />
<br />
|-<br />
| 9<br />
| D3 Double<br />
| 2046, 935<br />
<br />
|-<br />
| 10<br />
| D3 Triple<br />
| 1595, 935, 451<br />
<br />
|-<br />
| 11<br />
| (-) Control<br />
| --<br />
<br />
|-<br />
|}<br />
[[File:UCB-Interlab-10-2014.png]]<br />
<br />
As seen from the image above, smaller bands ran off the bottom of the gel and could not be readily visualized. However, the approximate sizes of the bands are as expected for each sample. <br />
Another 96 well plate was loaded using the sample protocol as previously described. Samples were measured after dilution and regrowth to OD of 0.5. J23101 and BL21 samples were used as controls, raw data shown below. <br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Full<br />
! 50%<br />
! 25%<br />
! 12.5%<br />
! 6.25%<br />
<br />
|-<br />
| LB<br />
| 706<br />
| 268<br />
| 188<br />
| 137<br />
| 123<br />
<br />
|-<br />
| J23101<br />
| 935<br />
| 294<br />
| 204<br />
| 145<br />
| 125<br />
<br />
|-<br />
| BL21<br />
| 1277<br />
| 349<br />
| 226<br />
| 159<br />
| 130<br />
<br />
|-<br />
| Dev 1<br />
| 1623<br />
| 438<br />
| 258<br />
| 182<br />
| 145<br />
<br />
|-<br />
| Dev 2<br />
| 920<br />
| 303<br />
| 193<br />
| 134<br />
| 123<br />
<br />
|-<br />
| Dev 3<br />
| 1147<br />
| 367<br />
| 219<br />
| 157<br />
| 126<br />
<br />
|-<br />
|}<br />
As with the previous test, all 3 devices demonstrate fluorescence in excess of the negative control sample. Values at full and below 12.5% concentration may be beyond the optimal range for the instrument to detect. The dilution amounts should be adjusted for the full test based on these data. <br />
<br />
==Week 8==<br />
<br />
'''8/18/14'''<br />
<br />
Made LB agar plates with appropriate antibiotics. 100 uL of each sample was plated and placed in incubator for overnight growth. These will be used to pull individual colonies for the biological triplicates needed in the fluorescent measurements to be performed. A repeat miniprep was performed on the device 1 sample from 8/13/14 in an attempt to obtain a higher concentration. <br />
<br />
'''8/19/14'''<br />
<br />
Plates made yesterday grew too well and single colonies could not be isolated. New streak plates were created for each sample (plates were not imaged). <br />
<br />
'''8/20/14'''<br />
<br />
Three colonies were taken from each streak plate to be used as biological triplicates in the study. Plates are shown below (Top row from left; Dev 1, Dev 2, Dev 3. Bottom row: J23101 negative control, BL21 positive control).<br />
[[File:UCB-Interlab-11-2014.png]]<br />
<br />
==Week 9==<br />
<br />
'''8/21/14'''<br />
<br />
Samples were monitored throughout the day and diluted as necessary to keep them in log phase. Since samples did not grow at the same rate, some difficulties were apparent when trying to get each sample to the same OD at the same time as the other samples. The volume per well will be increased to 150 uL and the concentrations adjusted for the measurement.<br />
<br />
<br />
'''8/22/14'''<br />
<br />
Fluorescence measurements were taken of device 1, 2 and 3 biological triplicate samples as previously described. Samples were all in log phage growth with OD of approximately 0.5 as shown below. Due to the number of samples, two separate 96 well plates were prepared using the same controls. Raw data is shown below.<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Final OD<br />
<br />
|-<br />
| J23101<br />
| 0.508<br />
<br />
|-<br />
| Dev 1-1<br />
| 0.515<br />
<br />
|-<br />
| Dev 1-2<br />
| 0.514<br />
<br />
|-<br />
| Dev 1-3<br />
| 0.509<br />
<br />
|-<br />
| Dev 2-1<br />
| 0.490<br />
<br />
|-<br />
| Dev 2-2<br />
| 0.508<br />
<br />
|-<br />
| Dev 2-3<br />
| 0.526<br />
<br />
|-<br />
| Dev 3-1<br />
| 0.512<br />
<br />
|-<br />
| Dev 3-2<br />
| 0.501<br />
<br />
|-<br />
| Dev 3-3<br />
| 0.520<br />
<br />
|-<br />
|}<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! 100<br />
! 0.67<br />
! 0.44<br />
! 0.3<br />
! 0.2<br />
! 13.4<br />
! 0.09<br />
<br />
|-<br />
| LB Blank<br />
| 778<br />
| 785<br />
| 774<br />
| 766<br />
| 780<br />
| 775<br />
| 788<br />
<br />
|-<br />
| Neg Control (J23101)<br />
| 1068<br />
| 903<br />
| 854<br />
| 821<br />
| 807<br />
| 794<br />
| 788<br />
<br />
|-<br />
| Dev 3 #1<br />
| 1142<br />
| 995<br />
| 895<br />
| 831<br />
| 804<br />
| 809<br />
| 798<br />
<br />
|-<br />
| Dev 3 #2<br />
| 1125<br />
| 940<br />
| 852<br />
| 823<br />
| 796<br />
| 805<br />
| 788<br />
<br />
|-<br />
| Dev 3 #3<br />
| 1035<br />
| 951<br />
| 888<br />
| 837<br />
| 824<br />
| 812<br />
| 797<br />
<br />
|-<br />
| Dev 2 #1<br />
| 992<br />
| 925<br />
| 849<br />
| 812<br />
| 805<br />
| 779<br />
| 779<br />
<br />
|-<br />
| Dev 2 #2<br />
| 1083<br />
| 924<br />
| 820<br />
| 813<br />
| 791<br />
| 776<br />
| 761<br />
<br />
|-<br />
| Dev 2 #3<br />
| 1047<br />
| 879<br />
| 824<br />
| 801<br />
| 776<br />
| 782<br />
| 772<br />
<br />
|-<br />
|}<br />
Plate #1 raw data with sample concentrations shown as decimals. <br />
{| class = "wikitable"<br />
|-<br />
! <br />
! 100<br />
! 0.67<br />
! 0.44<br />
! 0.3<br />
! 0.2<br />
! 13.4<br />
! 0.09<br />
<br />
|-<br />
| LB Blank<br />
| 816<br />
| 825<br />
| 849<br />
| 816<br />
| 844<br />
| 827<br />
| 825<br />
<br />
|-<br />
| Neg Control (J23101)<br />
| 982<br />
| 925<br />
| 886<br />
| 850<br />
| 845<br />
| 833<br />
| 807<br />
<br />
|-<br />
| Dev 1 #1<br />
| 1728<br />
| 1450<br />
| 1153<br />
| 1024<br />
| 899<br />
| 881<br />
| 838<br />
<br />
|-<br />
| Dev 1 #2<br />
| 1830<br />
| 1413<br />
| 1142<br />
| 1010<br />
| 921<br />
| 841<br />
| 861<br />
<br />
|-<br />
| Dev 1 #3<br />
| 1790<br />
| 1446<br />
| 1155<br />
| 975<br />
| 911<br />
| 861<br />
| 854<br />
<br />
|-<br />
|}<br />
Plate #2 raw data with sample concentrations shown as decimals. <br />
<br />
==Week 10==<br />
<br />
'''9/4/14'''<br />
<br />
Minipreps done on triplicate samples for J, D1, D2, & D3<br />
<br />
'''9/5/14'''<br />
<br />
Nanodrop done on mini prepped samples (results shown below) followed by restriction digestion using NcoI. The products were run on a 1% agarose gel with EtBr as previously described. This was done to ensure that all of the samples used in the fluorescence measurements are of the correct size. Although the gel is slightly distorted, the size of the bands correspond with the expected products (image not shown). <br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! ng/ uL<br />
! 260/280<br />
! 260/230<br />
<br />
|-<br />
| Dev 1 #1<br />
| 13.7<br />
| 1.99<br />
| 2.24<br />
<br />
|-<br />
| Dev 1 #2<br />
| 12.0<br />
| 1.94<br />
| 2.20<br />
<br />
|-<br />
| Dev 1 #3<br />
| 14.1<br />
| 1.86<br />
| 1.56<br />
<br />
|-<br />
| Dev 2-1 #1<br />
| 79.0<br />
| 1.87<br />
| 2.24<br />
<br />
|-<br />
| Dev 2-1 #2<br />
| 68.0<br />
| 1.83<br />
| 2.14<br />
<br />
|-<br />
| Dev 2-1 #3<br />
| 88.1<br />
| 1.84<br />
| 2.24<br />
<br />
|-<br />
| Dev 3 #1<br />
| 577.3<br />
| 1.85<br />
| 2.37<br />
<br />
|-<br />
| Dev 3 #2<br />
| 272.8<br />
| 1.87<br />
| 2.33<br />
<br />
|-<br />
| Dev 3 #3<br />
| 840.2<br />
| 1.86<br />
| 2.35<br />
<br />
|-<br />
| J23115 #1<br />
| 254.3<br />
| 1.85<br />
| 1.70<br />
<br />
|-<br />
| J23115 #2<br />
| 557.1<br />
| 1.82<br />
| 2.26<br />
<br />
|-<br />
| J23115 #3<br />
| 784.5<br />
| 1.84<br />
| 2.28<br />
<br />
|-<br />
|}<br />
<br />
Restriction Digestion<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! FD Buffer<br />
! NcoI<br />
! DNA (250 ng)<br />
! MQ H2O<br />
! Total Volume<br />
! Bands Expected (Bp)<br />
<br />
|-<br />
| J1<br />
| 2.0 uL<br />
| 1.0 uL<br />
| 1.0 uL<br />
| 21.0 uL<br />
| 25.0 uL<br />
| 1335, 770<br />
<br />
|-<br />
| J2<br />
| 2.0 uL<br />
| 1.0 uL<br />
| 0.9 uL†<br />
| 21.1 uL<br />
| 25.0 uL<br />
| 1335, 770<br />
<br />
|-<br />
| J3<br />
| 2.0 uL<br />
| 1.0 uL<br />
| 0.3 uL† (0.95 of 1:3)<br />
| 21.1 uL<br />
| 25.0 uL<br />
| 1335, 770<br />
<br />
|-<br />
| D2-1*<br />
| 2.0 uL<br />
| 1.0 uL<br />
| 2.5 uL <br />
| 14.5 uL<br />
| 20.0 uL<br />
| 1991, 990<br />
<br />
|-<br />
| D2-2*<br />
| 2.0 uL<br />
| 1.0 uL<br />
| 2.9 uL<br />
| 14.1 uL<br />
| 20.0 uL<br />
| 1991, 990<br />
<br />
|-<br />
| D2-3*<br />
| 2.0 uL<br />
| 1.0 uL<br />
| 2.3 uL<br />
| 14.7 uL<br />
| 20.0 uL<br />
| 1991, 990<br />
<br />
|-<br />
| D3-1<br />
| 2.0 uL<br />
| 1.0 uL<br />
| 0.45 uL† (0.9 of 1:2)<br />
| 21.1 uL<br />
| 25.0 uL<br />
| 1991, 990<br />
<br />
|-<br />
| D3-2<br />
| 2.0 uL<br />
| 1.0 uL<br />
| 0.92 uL<br />
| 21.1 uL<br />
| 25.0 uL<br />
| 1991, 990<br />
<br />
|-<br />
| D3-3<br />
| 2.0 uL<br />
| 1.0 uL<br />
| 0.3 (0.9 of 1:3) uL†<br />
| 21.1 uL<br />
| 25.0 uL<br />
| 1991, 990<br />
<br />
|-<br />
|}<br />
*Digest done with 200 ng and 20 uL volume 2 hours prior to the other reactions<br />
†Volume after DNA was diluted<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Lane #<br />
! Sample<br />
! Bands Expected (Bp)<br />
<br />
|-<br />
| 1<br />
| 2-Log DNA Ladder<br />
| N/A<br />
<br />
|-<br />
| 2<br />
| J1 (Hole in Gel)<br />
| 1335, 770<br />
<br />
|-<br />
| 3<br />
| J1<br />
| 1335, 770<br />
<br />
|-<br />
| 4<br />
| J2<br />
| 1335, 770<br />
<br />
|-<br />
| 5<br />
| J3<br />
| 1335, 770<br />
<br />
|-<br />
| 6<br />
| 2-Log DNA Ladder<br />
| N/A<br />
<br />
|-<br />
| 7<br />
| D2-1*<br />
| 1991, 990<br />
<br />
|-<br />
| 8<br />
| D2-2*<br />
| 1991, 990<br />
<br />
|-<br />
| 9<br />
| D2-3*<br />
| 1991, 990<br />
<br />
|-<br />
| 10<br />
| 2-Log DNA Ladder<br />
| N/A<br />
<br />
|-<br />
| 11<br />
| D3-1<br />
| 1991, 990<br />
<br />
|-<br />
| 12<br />
| D3-2<br />
| 1991, 990<br />
<br />
|-<br />
| 13<br />
| D3-3<br />
| 1991, 990<br />
<br />
|-<br />
| 14<br />
| 2-Log DNA Ladder<br />
| N/A<br />
<br />
|-<br />
| 15<br />
| H2O Control<br />
| N/A<br />
<br />
|-<br />
|}<br />
DATA ANALYSIS <br />
Analysis of the fluorescence data measurements obtained is shown below. <br />
{| class = "wikitable"<br />
|-<br />
! <br />
! 1.00<br />
! 0.67<br />
! 0.44<br />
! 0.3<br />
! 0.2<br />
! 13.4<br />
! 0.09<br />
<br />
|-<br />
| Dev 1 #1 Actual<br />
| 1728<br />
| 1450<br />
| 1153<br />
| 1024<br />
| 899<br />
| 881<br />
| 838<br />
<br />
|-<br />
| Calculated<br />
| 746<br />
| 525<br />
| 267<br />
| 174<br />
| 54<br />
| 48<br />
| 31<br />
<br />
|-<br />
| Dev 1 #2 Actual<br />
| 1830<br />
| 1413<br />
| 1142<br />
| 1010<br />
| 921<br />
| 841<br />
| 861<br />
<br />
|-<br />
| Calculated<br />
| 848<br />
| 488<br />
| 256<br />
| 160<br />
| 76<br />
| 8<br />
| 54<br />
<br />
|-<br />
| Dev 1 #3 Actual<br />
| 1790<br />
| 1446<br />
| 1155<br />
| 975<br />
| 911<br />
| 861<br />
| 854<br />
<br />
|-<br />
| Calculated<br />
| 808<br />
| 521<br />
| 269<br />
| 125<br />
| 66<br />
| 28<br />
| 47<br />
<br />
|-<br />
| Dev 2 #1 Actual<br />
| 992<br />
| 925<br />
| 849<br />
| 812<br />
| 805<br />
| 779<br />
| 779<br />
<br />
|-<br />
| Calculated<br />
| -76<br />
| 22<br />
| -5<br />
| -9<br />
| -2<br />
| -15<br />
| -9<br />
<br />
|-<br />
| Dev 2 #2 Actual<br />
| 1083<br />
| 924<br />
| 820<br />
| 813<br />
| 791<br />
| 776<br />
| 761<br />
<br />
|-<br />
| Calculated<br />
| 15<br />
| 21<br />
| -34<br />
| -8<br />
| -16<br />
| -18<br />
| -27<br />
<br />
|-<br />
| Dev 2 #3 Actual<br />
| 1047<br />
| 879<br />
| 824<br />
| 801<br />
| 776<br />
| 782<br />
| 772<br />
<br />
|-<br />
| Calculated<br />
| -21<br />
| -24<br />
| -30<br />
| -20<br />
| -31<br />
| -12<br />
| -16<br />
<br />
|-<br />
| Dev 3 #1 Actual<br />
| 1142<br />
| 995<br />
| 895<br />
| 831<br />
| 804<br />
| 809<br />
| 798<br />
<br />
|-<br />
| Calculated<br />
| 74<br />
| 92<br />
| 41<br />
| 10<br />
| -3<br />
| 15<br />
| 10<br />
<br />
|-<br />
| Dev 3 #2 Actual<br />
| 1125<br />
| 940<br />
| 852<br />
| 823<br />
| 796<br />
| 805<br />
| 788<br />
<br />
|-<br />
| Calculated<br />
| 57<br />
| 37<br />
| -2<br />
| 2<br />
| -11<br />
| 11<br />
| 0<br />
<br />
|-<br />
| Dev 3 #3 Actual<br />
| 1035<br />
| 951<br />
| 888<br />
| 837<br />
| 824<br />
| 812<br />
| 797<br />
<br />
|-<br />
| Calculated<br />
| -33<br />
| 48<br />
| 34<br />
| 16<br />
| 17<br />
| 18<br />
| 9<br />
<br />
|-<br />
|}<br />
Table 1: Fluorescence data. For each of the three devices, 3 biological replicates were measured (labeled #1, #2, #3 for each device). Data shown includes actual measurements and derived quantities obtained by subtracting fluorescence measured from non-GFP expressing E. coli of the same strain on the same plate. Promoter J23115 for device #3 was used as distributed. In addition, each sample underwent a serial dilution (concentrations listed as fractions across top of table).<br />
<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! 100<br />
! 0.67<br />
! 0.44<br />
! 0.3<br />
! 0.2<br />
! 13.4<br />
! 0.09<br />
<br />
|-<br />
| Dev 1 Ave<br />
| 1.82<br />
| 1.55<br />
| 1.30<br />
| 1.18<br />
| 1.08<br />
| 1.03<br />
| 1.05<br />
<br />
|-<br />
| Dev 1 Std. Dev<br />
| 0.05<br />
| 0.02<br />
| 0.01<br />
| 0.03<br />
| 0.01<br />
| 0.02<br />
| 0.01<br />
<br />
|-<br />
| Dev 2 Ave<br />
| 0.97<br />
| 1.01<br />
| 0.97<br />
| 0.98<br />
| 0.98<br />
| 0.98<br />
| 0.98<br />
<br />
|-<br />
| Dev 2 Std. Dev<br />
| 0.04<br />
| 0.03<br />
| 0.02<br />
| 0.01<br />
| 0.02<br />
| 0.00<br />
| 0.01<br />
<br />
|-<br />
| Dev 3 Ave<br />
| 1.03<br />
| 1.07<br />
| 1.03<br />
| 1.01<br />
| 1.00<br />
| 1.02<br />
| 1.01<br />
<br />
|-<br />
| Dev 3 Std. Dev<br />
| 0.05<br />
| 0.03<br />
| 0.03<br />
| 0.01<br />
| 0.02<br />
| 0.00<br />
| 0.01<br />
<br />
|-<br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
<br />
|-<br />
| <br />
| <br />
| Total: Dev 1<br />
| <br />
| <br />
| 1.23<br />
| <br />
| <br />
<br />
|-<br />
| <br />
| <br />
| Total: Dev 2<br />
| <br />
| <br />
| 0.99<br />
| <br />
| <br />
<br />
|-<br />
| <br />
| <br />
| Total: Dev 3<br />
| <br />
| <br />
| 1.02<br />
| <br />
| <br />
<br />
|-<br />
|}<br />
Table 2: Average device fluorescence. For each of the three devices, the average and standard sample deviation are given for the triplicates at each concentration. Data is given as fold change over non-GFP expressing E. coli of same DH5u strain. Total data on lower portion of table is an overall average of each device calculated using the averages for each device at all concentrations. No samples were excluded.<br />
<br />
[[File:UCB-Interlab-12-2014.png]]<br />
<br />
{{Template:UCB-Footer}}</div>Leighlahttp://2014.igem.org/Team:CU-Boulder/CollaborationTeam:CU-Boulder/Collaboration2014-10-16T23:47:48Z<p>Leighla: </p>
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<br><br />
<br><br />
Members from our team reached out to another local iGEM team, CSU, to meet and talk about our projects at the beginning of the summer. We were able to discuss our projects and our experimental design over some appetizers and drinks at Oskar Blues Brewery. It was here we decided to work together August. <br />
<p><br />
Representatives of our lab traveled to CSU for a two-day experiment to validate our packaging signal part (BBa_1445000). We explained the theory behind our project and shared our protocol for phage amplification. The CSU team then produced phage using our packaging signal part and compared its delivery to a similar plasmid containing an insert with no known phagemid packaging ability. In order to accommodate the time constraints, we shortened the protocol by removing the lengthy incubation. This, along with other errors along the way likely led to the unexpected results.<br />
<p> <br />
During our stay, we related to their current problems with Gibson Assembly and offered solutions that had worked for us. Additionally, we cultivated ideas for a combined outreach program. These plans were not realized this year but set the groundwork for future and more extensive collaboration between the schools next summer.<br />
<p><br />
<p><br />
We also participated in the following surveys for other iGEM teams:<br />
<li>Complexity – Zurich<br />
<li>Policy and Practices – Warwick<br />
<li>On the concept of living and Bioart – Paris-Saclay<br />
<li>Bioethics and Biotechnology in XXI century – Brasil-SP<br />
<li>Modeling and Survey – AMU-Poznan<br />
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{{Template:UCB-Footer}}</div>Leighlahttp://2014.igem.org/Team:CU-Boulder/CollaborationTeam:CU-Boulder/Collaboration2014-10-16T23:46:45Z<p>Leighla: </p>
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<img src="https://static.igem.org/mediawiki/2014/thumb/3/3b/UCB-collaboration_CSU-141012.jpg/800px-UCB-collaboration_CSU-141012.jpg"width:800px; margin-left: 10px; margin-right: auto;"><br />
<br><br />
<br><br />
Members from our team reached out to another local iGEM team, CSU, to meet and talk about our projects at the beginning of the summer. We were able to discuss our projects and our experimental design over some appetizers and drinks at Oskar Blues Brewery. It was here we decided to work together August. <br />
<p><br />
Representatives of our lab traveled to CSU for a two-day experiment to validate our packaging signal part (BBa_1445000). We explained the theory behind our project and shared our protocol for phage amplification. The CSU team then produced phage using our packaging signal part and compared its delivery to a similar plasmid containing an insert with no known phagemid packaging ability. In order to accommodate the time constraints, we shortened the protocol by removing the lengthy incubation. This, along with other errors along the way likely led to the unexpected results.<br />
<p> <br />
During our stay, we related to their current problems with Gibson Assembly and offered solutions that had worked for us. Additionally, we cultivated ideas for a combined outreach program. These plans were not realized this year but set the groundwork for future and more extensive collaboration between the schools next summer.<br />
<p><br />
<p><br />
<li>We also participated in the following surveys for other iGEM teams:<br />
<li>Complexity – Zurich<br />
Policy and Practices – Warwick<br />
On the concept of living and Bioart – Paris-Saclay<br />
Bioethics and Biotechnology in XXI century – Brasil-SP<br />
Modeling and Survey – AMU-Poznan<br />
<br><br />
<br><br />
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{{Template:UCB-Footer}}</div>Leighlahttp://2014.igem.org/Team:CU-Boulder/CollaborationTeam:CU-Boulder/Collaboration2014-10-16T23:44:08Z<p>Leighla: </p>
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<br><br />
<br><br />
Members from our team reached out to another local iGEM team, CSU, to meet and talk about our projects at the beginning of the summer. We were able to discuss our projects and our experimental design over some appetizers and drinks at Oskar Blues Brewery. It was here we decided to work together August. <br />
<p><br />
Representatives of our lab traveled to CSU for a two-day experiment to validate our packaging signal part (BBa_1445000). We explained the theory behind our project and shared our protocol for phage amplification. The CSU team then produced phage using our packaging signal part and compared its delivery to a similar plasmid containing an insert with no known phagemid packaging ability. In order to accommodate the time constraints, we shortened the protocol by removing the lengthy incubation. This, along with other errors along the way likely led to the unexpected results.<br />
<p> <br />
During our stay, we related to their current problems with Gibson Assembly and offered solutions that had worked for us. Additionally, we cultivated ideas for a combined outreach program. These plans were not realized this year but set the groundwork for future and more extensive collaboration between the schools next summer.<br />
<p><br />
<p><br />
<ln>We also participated in the following surveys for other iGEM teams:<br />
<ln>Complexity – Zurich<br />
Policy and Practices – Warwick<br />
On the concept of living and Bioart – Paris-Saclay<br />
Bioethics and Biotechnology in XXI century – Brasil-SP<br />
Modeling and Survey – AMU-Poznan<br />
<br><br />
<br><br />
</body><br />
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<br />
{{Template:UCB-Footer}}</div>Leighlahttp://2014.igem.org/Team:CU-Boulder/CollaborationTeam:CU-Boulder/Collaboration2014-10-16T23:42:30Z<p>Leighla: </p>
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<br><br />
<br><br />
Members from our team reached out to another local iGEM team, CSU, to meet and talk about our projects at the beginning of the summer. We were able to discuss our projects and our experimental design over some appetizers and drinks at Oskar Blues Brewery. It was here we decided to work together August. <br />
<p><br />
Representatives of our lab traveled to CSU for a two-day experiment to validate our packaging signal part (BBa_1445000). We explained the theory behind our project and shared our protocol for phage amplification. The CSU team then produced phage using our packaging signal part and compared its delivery to a similar plasmid containing an insert with no known phagemid packaging ability. In order to accommodate the time constraints, we shortened the protocol by removing the lengthy incubation. This, along with other errors along the way likely led to the unexpected results.<br />
<p> <br />
During our stay, we related to their current problems with Gibson Assembly and offered solutions that had worked for us. Additionally, we cultivated ideas for a combined outreach program. These plans were not realized this year but set the groundwork for future and more extensive collaboration between the schools next summer.<br />
<p><br />
<p><br />
We also participated in the following surveys for other iGEM teams:<br />
Complexity – Zurich<br />
Policy and Practices – Warwick<br />
On the concept of living and Bioart – Paris-Saclay<br />
Bioethics and Biotechnology in XXI century – Brasil-SP<br />
Modeling and Survey – AMU-Poznan<br />
<br><br />
<br><br />
</body><br />
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<br />
{{Template:UCB-Footer}}</div>Leighlahttp://2014.igem.org/Team:CU-Boulder/CollaborationTeam:CU-Boulder/Collaboration2014-10-16T23:41:25Z<p>Leighla: </p>
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<br><br />
<br><br />
Members from our team reached out to another local iGEM team, CSU, to meet and talk about our projects at the beginning of the summer. We were able to discuss our projects and our experimental design over some appetizers and drinks at Oskar Blues Brewery. It was here we decided to work together August. <br />
<p><br />
Representatives of our lab traveled to CSU for a two-day experiment to validate our packaging signal part (BBa_1445000). We explained the theory behind our project and shared our protocol for phage amplification. The CSU team then produced phage using our packaging signal part and compared its delivery to a similar plasmid containing an insert with no known phagemid packaging ability. In order to accommodate the time constraints, we shortened the protocol by removing the lengthy incubation. This, along with other errors along the way likely led to the unexpected results.<br />
<p> <br />
During our stay, we related to their current problems with Gibson Assembly and offered solutions that had worked for us. Additionally, we cultivated ideas for a combined outreach program. These plans were not realized this year but set the groundwork for future and more extensive collaboration between the schools next summer.<br />
<p><br />
<p><br />
We also participated in the following surveys for other iGEM teams:<br />
Complexity – Zurich<br />
Policy and Practices – Warwick<br />
On the concept of living and Bioart – Paris-Saclay<br />
Bioethics and Biotechnology in XXI century – Brasil-SP<br />
Modeling and Survey – AMU-Poznan<br />
<br><br />
<br><br />
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<body><br />
{{Template:UCB-Footer}}</div>Leighlahttp://2014.igem.org/Team:CU-Boulder/CollaborationTeam:CU-Boulder/Collaboration2014-10-16T23:40:43Z<p>Leighla: </p>
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<br><br />
<br><br />
Members from our team reached out to another local iGEM team, CSU, to meet and talk about our projects at the beginning of the summer. We were able to discuss our projects and our experimental design over some appetizers and drinks at Oskar Blues Brewery. It was here we decided to work together August. <br />
<p><br />
Representatives of our lab traveled to CSU for a two-day experiment to validate our packaging signal part (BBa_1445000). We explained the theory behind our project and shared our protocol for phage amplification. The CSU team then produced phage using our packaging signal part and compared its delivery to a similar plasmid containing an insert with no known phagemid packaging ability. In order to accommodate the time constraints, we shortened the protocol by removing the lengthy incubation. This, along with other errors along the way likely led to the unexpected results.<br />
<p> <br />
During our stay, we related to their current problems with Gibson Assembly and offered solutions that had worked for us. Additionally, we cultivated ideas for a combined outreach program. These plans were not realized this year but set the groundwork for future and more extensive collaboration between the schools next summer.<br />
<p><br />
<p><br />
We also participated in the following surveys for other iGEM teams:<br />
Complexity – Zurich<br />
Policy and Practices – Warwick<br />
On the concept of living and Bioart – Paris-Saclay<br />
Bioethics and Biotechnology in XXI century – Brasil-SP<br />
Modeling and Survey – AMU-Poznan<br />
<br />
<br />
{{Template:UCB-Footer}}</div>Leighlahttp://2014.igem.org/Team:CU-Boulder/CollaborationTeam:CU-Boulder/Collaboration2014-10-16T23:36:25Z<p>Leighla: </p>
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<br><br />
<br><br />
Members from our team reached out to another local iGEM team, CSU, to meet and talk about our projects at the beginning of the summer. We were able to discuss our projects and our experimental design over some appetizers and drinks at Oskar Blues Brewery. It was here we decided to work together August. <br />
<br><br />
Representatives of our lab traveled to CSU for a two-day experiment to validate our packaging signal part (BBa_1445000). We explained the theory behind our project and shared our protocol for phage amplification. The CSU team then produced phage using our packaging signal part and compared its delivery to a similar plasmid containing an insert with no known phagemid packaging ability. In order to accommodate the time constraints, we shortened the protocol by removing the lengthy incubation. This, along with other errors along the way likely led to the unexpected results.<br />
<br> <br />
During our stay, we related to their current problems with Gibson Assembly and offered solutions that had worked for us. Additionally, we cultivated ideas for a combined outreach program. These plans were not realized this year but set the groundwork for future and more extensive collaboration between the schools next summer.<br />
<br><br />
<br><br />
We also participated in the following surveys for other iGEM teams:<br />
Complexity – Zurich<br />
Policy and Practices – Warwick<br />
On the concept of living and Bioart – Paris-Saclay<br />
Bioethics and Biotechnology in XXI century – Brasil-SP<br />
Modeling and Survey – AMU-Poznan</div>Leighlahttp://2014.igem.org/Team:CU-Boulder/CollaborationTeam:CU-Boulder/Collaboration2014-10-16T23:35:12Z<p>Leighla: </p>
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<img src="https://static.igem.org/mediawiki/2014/thumb/3/3b/UCB-collaboration_CSU-141012.jpg/600px-UCB-collaboration_CSU-141012.jpg"width:800px; margin-left: 10px; margin-right: auto;"><br />
<br><br />
<br><br />
Members from our team reached out to another local iGEM team, CSU, to meet and talk about our projects at the beginning of the summer. We were able to discuss our projects and our experimental design over some appetizers and drinks at Oskar Blues Brewery. It was here we decided to work together August. <br />
<br><br />
Representatives of our lab traveled to CSU for a two-day experiment to validate our packaging signal part (BBa_1445000). We explained the theory behind our project and shared our protocol for phage amplification. The CSU team then produced phage using our packaging signal part and compared its delivery to a similar plasmid containing an insert with no known phagemid packaging ability. In order to accommodate the time constraints, we shortened the protocol by removing the lengthy incubation. This, along with other errors along the way likely led to the unexpected results.<br />
<br> <br />
During our stay, we related to their current problems with Gibson Assembly and offered solutions that had worked for us. Additionally, we cultivated ideas for a combined outreach program. These plans were not realized this year but set the groundwork for future and more extensive collaboration between the schools next summer.<br />
<br><br />
<br><br />
We also participated in the following surveys for other iGEM teams:<br />
Complexity – Zurich<br />
Policy and Practices – Warwick<br />
On the concept of living and Bioart – Paris-Saclay<br />
Bioethics and Biotechnology in XXI century – Brasil-SP<br />
Modeling and Survey – AMU-Poznan</div>Leighlahttp://2014.igem.org/Team:CU-Boulder/Human_PracticesTeam:CU-Boulder/Human Practices2014-10-16T22:50:36Z<p>Leighla: </p>
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<p><br />
The summer STEAM camp is a program designed to engage high school students in hands-on learning opportunities. The program emphasizes STEAM curriculum areas of science, technology, engineering, art, and mathematics. This program gives young people the opportunity to explore a wide range of real world sciences. <br />
<p><br />
CU-Boulder iGEM supported the efforts of STEAM by teaching a group of adolescents from Heritage High School about the basics of DNA. These students received an introductory lesson about DNA, followed by a hands-on laboratory experience on the Boulder campus. Students first extracted DNA from their own saliva and stored the precipitate in souvenir necklaces. For most, DNA was an abstract concept but through this project they could visualize the molecular components of their cells. To introduce students to the diagnostic techniques used in research and medicine, they were given a sample containing one of two unknown DNA fragments. One fragment was reportedly harmless while the other contained a SNP (Single Nucleotide Polymorphism) making it pathogenic. Coincidentally, this mutation erased a restriction cut site, allowing the two samples to be distinguished through a restriction digest. The students digested their own samples and visualized the products on an agarose gel to determine whether they had a harmless or ‘pathogenic’ sample. The implications of this simple diagnostic technique were discussed in context of the current rise in antibiotic resistant bacteria.<br />
<p><br />
We had a great time educating the next generation of inventors, entrepreneurs, and scientists.<br />
<br />
<img src="https://static.igem.org/mediawiki/2014/thumb/2/2a/UCB-outreach-02-141012.jpg/800px-UCB-outreach-02-141012.jpg"width: 969px; margin-left: -80px; margin-right: auto;"><br />
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{{Template:UCB-Footer}}</div>Leighlahttp://2014.igem.org/Team:CU-Boulder/Human_PracticesTeam:CU-Boulder/Human Practices2014-10-16T22:50:04Z<p>Leighla: </p>
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<br><br />
<br><br />
<br><br />
<br><br />
<p><br />
The summer STEAM camp is a program designed to engage high school students in hands-on learning opportunities. The program emphasizes STEAM curriculum areas of science, technology, engineering, art, and mathematics. This program gives young people the opportunity to explore a wide range of real world sciences. <br />
,p><br />
CU-Boulder iGEM supported the efforts of STEAM by teaching a group of adolescents from Heritage High School about the basics of DNA. These students received an introductory lesson about DNA, followed by a hands-on laboratory experience on the Boulder campus. Students first extracted DNA from their own saliva and stored the precipitate in souvenir necklaces. For most, DNA was an abstract concept but through this project they could visualize the molecular components of their cells. To introduce students to the diagnostic techniques used in research and medicine, they were given a sample containing one of two unknown DNA fragments. One fragment was reportedly harmless while the other contained a SNP (Single Nucleotide Polymorphism) making it pathogenic. Coincidentally, this mutation erased a restriction cut site, allowing the two samples to be distinguished through a restriction digest. The students digested their own samples and visualized the products on an agarose gel to determine whether they had a harmless or ‘pathogenic’ sample. The implications of this simple diagnostic technique were discussed in context of the current rise in antibiotic resistant bacteria.<br />
<p><br />
We had a great time educating the next generation of inventors, entrepreneurs, and scientists.<br />
<br />
<img src="https://static.igem.org/mediawiki/2014/thumb/2/2a/UCB-outreach-02-141012.jpg/800px-UCB-outreach-02-141012.jpg"width: 969px; margin-left: -80px; margin-right: auto;"><br />
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{{Template:UCB-Footer}}</div>Leighlahttp://2014.igem.org/Team:CU-Boulder/Human_PracticesTeam:CU-Boulder/Human Practices2014-10-16T22:43:47Z<p>Leighla: </p>
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<img src="https://static.igem.org/mediawiki/2014/thumb/9/98/UCB-outreach-141010-01.jpg/800px-UCB-outreach-141010-01.jpg"width: 969px; margin-left: -80px; margin-right: auto;"><br />
<br><br />
<br><br />
<br><br />
<br><br />
The summer STEAM camp is a program designed to engage high school students in hands-on learning opportunities. The program emphasizes STEAM curriculum areas of science, technology, engineering, art, and mathematics. This program gives young people the opportunity to explore a wide range of real world sciences. <br />
CU-Boulder iGEM supported the efforts of STEAM by teaching a group of adolescents from Heritage High School about the basics of DNA. These students received an introductory lesson about DNA, followed by a hands-on laboratory experience on the Boulder campus. Students first extracted DNA from their own saliva and stored the precipitate in souvenir necklaces. For most, DNA was an abstract concept but through this project they could visualize the molecular components of their cells. To introduce students to the diagnostic techniques used in research and medicine, they were given a sample containing one of two unknown DNA fragments. One fragment was reportedly harmless while the other contained a SNP (Single Nucleotide Polymorphism) making it pathogenic. Coincidentally, this mutation erased a restriction cut site, allowing the two samples to be distinguished through a restriction digest. The students digested their own samples and visualized the products on an agarose gel to determine whether they had a harmless or ‘pathogenic’ sample. The implications of this simple diagnostic technique were discussed in context of the current rise in antibiotic resistant bacteria.<br />
We had a great time educating the next generation of inventors, entrepreneurs, and scientists.<br />
<br />
<img src="https://static.igem.org/mediawiki/2014/thumb/2/2a/UCB-outreach-02-141012.jpg/800px-UCB-outreach-02-141012.jpg"width: 969px; margin-left: -80px; margin-right: auto;"><br />
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{{Template:UCB-Footer}}</div>Leighlahttp://2014.igem.org/Team:CU-Boulder/Notebook/Phage_TeamTeam:CU-Boulder/Notebook/Phage Team2014-10-16T17:29:09Z<p>Leighla: </p>
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__FORCETOC__<br />
<br />
'''Phage Delivery'''<br />
==Week 1==<br />
'''Notes:''' Unless stated otherwise, all gels contain 2-log ladder<br />
<br />
'''5/9'''<br />
*Obtained BW23115 KanR cells- BW23115 cells that had their native CRISPR-Cas system knocked out by the insertion of a Kanamycin resistance gene<br />
::-Will also be called BW23115 or BW<br />
::-Conjugated BW23115 KanR cells with contain F’ notation (ex. BWF’)<br />
*Obtained ER2738 cells that contain the F’ episome (no changes from NEB sample)<br />
::-Will also be called ER. Assume that all ER samples contain the F’ episome<br />
::-Streaked sample onto LB+Tet (20ug/mL) to select for colonies containing F’ episome<br />
<br />
'''5/10'''<br />
*Did receive colonies from 5/9 selection<br />
<br />
==Week 2==<br />
'''5/12'''<br />
*Need to conjugate BW23115 KanR cells with the F’ episome<br />
::-Set up overnight cultures of ER2738 and BW23115 KanR<br />
::-When mixed, ER2738 will donate it’s F’ episome and BW23115 KanR will receive the F’ episome. F’ episome confers Tetracycline resistance<br />
<br />
'''5/13'''<br />
*Started M13 Amplification: Amplify M13 phage using the M13K07 Helper Phage<br />
::-Let precipitated in NaCl/PEG solution overnight<br />
::-Possible sources of error<br />
:::*Did not sterilize 2.5M NaCl/20% PEG-8000 solution<br />
:::*Added 4-fold PEG solution<br />
::::Compensated by adding more LB<br />
:::*During precipitation, put sample in -20C for 30 minutes before realizing mistake and moving to it to 4C. Sample partially froze<br />
*Conjugated BW23115 with F’ episome<br />
::-Added 1mL BW23115 to 1mL ER2738 overnight culture<br />
::-Incubated at 37C for 30 minutes, shaking<br />
::-Plated on LB+Kan(50ug/mL)+Tet(20ug/mL)<br />
:::*To select for BW cells that took the F’ episome (containing Tet resistance)<br />
<br />
'''5/14'''<br />
*Finished the M13 Amplification<br />
::-Visualized product on UV-vis. There was a tall spike at 269nm indicating that DNA was present. Did not test at 320nm.<br />
*Results of BW23115 Conjugation<br />
::-Many colonies indicating successful conjugation of F’ episome into BW23115<br />
::-Set up overnight to make freeze down tomorrow<br />
*Set up overnight of ER2738 to make chemically competent tomorrow<br />
<br />
'''5/15'''<br />
*Made freeze down of BW23115 KanR F’<br />
::-BW23115 E. coli strain with Kanamycin resistance gene inserted into genome and with F’ episome<br />
*Made chemically competent ER2738 cells<br />
*Transformation of Litmus28i (from NEB) into chemically competent ER2738 cells<br />
::-Added 1ul Litmus28i plasmid to 40ul competent cells<br />
::-Plated on LB + Amp(100ug/mL)<br />
::-Purpose: To make M13 phage that package Litmus28i DNA. Need phagemid (Litmus28i) DNA in infectable cells (cells containing F’ episome) to introduce M13K07 Helper Phage and make phage. <br />
<br />
'''5/16'''<br />
*Results of 5/15 transformation<br />
::-No growth for No DNA control<br />
::-Many colonies for sample<br />
<br />
==Week 3==<br />
'''5/19'''<br />
*M13 Amplification to isolate M13-Litmus28i phage<br />
::-Cells: ER2738 cells containing Litmus28i phagemid<br />
::-Helper Phage: M13K07<br />
::-Not much phage was precipitated<br />
*Set up overnight culture of ER2738 to infect tomorrow<br />
<br />
'''5/20'''<br />
*Infected ER2738 cells with M13-Litmus28i phage<br />
::-Plated only on Ampicillin(100ug/mL) (should have also plated on kanamycin)<br />
::-Infected for 4-5 hours-> should have only infected for 30 minutes maximum. This extra time gives the cells that were infected with M13-M13K07 the time to produced M13-M13K07 phage and reinfect<br />
<br />
'''5/21'''<br />
*Results from M13-Litmus28i infection of ER2738<br />
::-Solid lawn of growth for diluted and non-diluted<br />
::-Also sickly looking growth<br />
*Set up overnights<br />
::-ER2738 cells containing Litmus28i for freeze down<br />
::-BW23115 with F’ episome to make chemically competent cells <br />
::-ER2738 to redo infection<br />
<br />
'''5/22'''<br />
*Tested absorbance of phage produced through M13 amplification on 5/19<br />
::-Low absorbance of 0.018 at 269nm but no detection at wavelength 320nm<br />
::-Decided to redo M13 amplification<br />
*Made chemically competent BW23115 with f-episome<br />
*Made freeze down of ER2738 containing Litmus28i<br />
*Set up overnight of ER2738 containing Litmus28i to redo M13 amplification tomorrow<br />
<br />
'''5/23'''<br />
*Protocol switch to make phage using phagemid<br />
::-“M13 Amplification” protocol should only be used to make more M13-M13K07, not to make M13 phage containing a different phagemid<br />
::-Switched to new protocol (“Use of M13K07 Helper Phage for isolation of single stranded phagemid DNA” by NEB. Made modifications (see our protocols) to isolate phage rather than single-stranded DNA)<br />
::-Making phage….<br />
{| class = "wikitable"<br />
|-<br />
! Helper phage<br />
! Phagemid<br />
! Cells<br />
! Notes<br />
<br />
|-<br />
| M13K07<br />
| None<br />
| ER2738<br />
| Make more M13-M13K07<br />
<br />
|-<br />
| M13K07<br />
| Litmus28i<br />
| ER2738<br />
| Test packaging of Litmus28i<br />
<br />
|-<br />
|}<br />
*Made fresh antibiotics<br />
::-Chloramphenicol (34 ng/mL)<br />
:::*1.44g chloramphenicol into 42mL EtOH<br />
::-Ampicillin (50 ng/mL)<br />
:::*4g ampicillin into 80mL mili-Q H2O<br />
<br />
'''5/24'''<br />
*Isolated phage using new protocol<br />
::-Resuspended pellet in 200ul TBS and 200ul 30% glycerol<br />
::-Measured absorbance with UV-vis<br />
:::*concentration (phage/mL) = 6x10^16 x (A269-A320)/ (#of base pairs in the phage genome)<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! Abs (269nm)<br />
! Abs (320nm)<br />
! Genome size<br />
! Concentration (phage/mL)<br />
<br />
|-<br />
| M13-M13K07<br />
| 0.721<br />
| 0.060<br />
| <br />
| 4.57 x10^12<br />
<br />
|-<br />
| M13-Litmus28i<br />
| 0.250<br />
| 0.028<br />
| 2823<br />
| 4.72 x10^12<br />
<br />
|-<br />
|}<br />
*Infect ER2738 cells with M13-Litmus28i<br />
::-Wanted 1:10 phage:cell ratio. Math….<br />
:::*At 1 OD (e.coli), cell/mL = 5x10^8<br />
:::*5x10^7 phage * (1mL/4.72x10^12 phage) = 0.011ul phage<br />
*Set up overnights<br />
::-ER2738 for infection with M13-Litmus28i<br />
::-BW23115 F’ for infection with M13-Litmus28i to test infectivity of conjugated strain<br />
<br />
==Week 4==<br />
'''5/25'''<br />
*Infect ER and BWF’ cells with M13-Litmus28i<br />
::*Made 5mL culture of ER and BW that was at 1 OD<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! OD<br />
! mL sample for 1OD in 5mL<br />
! mL LB to 5mL<br />
<br />
|-<br />
| ER2738<br />
| 2.5<br />
| 2 mL<br />
| 3 mL<br />
<br />
|-<br />
| BW23115<br />
| 2.0<br />
| 2.5 mL<br />
| 2.5 mL<br />
<br />
|-<br />
|}<br />
::-Based on calculations from 5/24, we needed to add 0.011 ul phage per 1 mL of cells at 1 OD. This equates to 0.055 ul of phage into 5 mL cells; therefore we made a 1:10 dilution so we could add 0.5ul. Unfortunately, the pipet would not take up 0.5ul so we added 0.8ul of M13-Litmus28i phage<br />
::-Grew the cells for 20 minutes at 37C<br />
::-Plated 300ul onto Kanamycin (50ug/mL) and 300ul onto Ampicillin (100ug/mL) for each sample<br />
:::*Incubated overnight at 37C<br />
*'''Note:''' During the production of phage, the phagemid SHOULD be packaged preferentially over the Helper Phagemid but some Helper Phagemid will still be packaged. We plated on Amp to select for cells that were infected with phage containing Phagemid. We plated on Kan to select for cells that were infected with phage containing Helper Phagemid. This allows us to compare the packaging efficiency of Helper Phagemid: Phagemid.<br />
'''5/26'''<br />
*Results from 5/25 infection with M13-Litmus28i<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Result<br />
! Significance<br />
<br />
|-<br />
| ER2738 on Amp<br />
| Lawn<br />
| Litmus28i phagemid was successfully packaged into the M13 phage and is infectable<br />
<br />
|-<br />
| ER2738 on Kan<br />
| 100-200 colonies<br />
| Some M13 helper phage is packaged into the M13 phage but at a much lower rate than Litmus28i<br />
<br />
|-<br />
| BW23115 on Amp<br />
| Lawn<br />
| BW23115 is ‘equally’ infectable by M13 as ER2738<br />
<br />
|-<br />
| BW23115 on Kan<br />
| Lawn<br />
| BW23115 contains Kan resistance in its genome so this tells us nothing<br />
<br />
|-<br />
|}<br />
:*Conclusions:<br />
::-Cells grew on Ampicillin; therefore, Litmus28i phagemid was successfully packaged into M13 phage. <br />
::-For ER2738 samples, there was significant growth on Ampicillin compared to Kanamycin; therefore, Litmus28i phagemid is packaged preferentially over M13K07 Helper Phagemid<br />
::-M13-Litmus28i retains its infectivity of cells containing the F’ episome<br />
:*Because we received lawns, we have to redo the infection and plate less cells so we can calculate the uptake ratio between the phagemid and helper phage based on the number of colonies<br />
*Started 50 mL overnight of K12 ER2738 and BW23115<br />
<br />
'''5/27'''<br />
*Redo the infection done on 5/25<br />
::-Infectable cells: ER2738 and BW23115<br />
:::*Plated non-infected samples of each (non-diluted) to check for contaminants<br />
::-Diluted M13-Litmus28i (1) phage by a factor of 10. Added 5.5ul to each sample<br />
::-Grew samples for 20 minutes at 37C, 250rpm<br />
::-Plated 100ul of onto an Ampicillin (100ug/mL) plate and onto a Kanamycin (50ug/mL) plate. Incubated overnight at 37C.<br />
:::*Dilutions = 1:10; 1:100; and 1:1000<br />
'''5/28'''<br />
*Results from 5/27<br />
::-Controls were as expected<br />
:::*No growth for ER2738 non-infected grown on Amp, ER2738 non-infected grown on Kan, or BW23115 non-infected grown on Amp<br />
:::*Growth for BW23115 non-infected grown on Kan (BW23115 has Kan R in genome)<br />
::-Many colonies were received for all dilutions (1:10, 1:100, and 1:1000) of the following<br />
:::*ER2738 infected and plated on Amp<br />
:::*BW23115 infected and plated on Amp<br />
:::*BW23115 infected and plated on Kan<br />
::-Many (100s to 1000?) colonies grew on 1:10 and 1:100 dilutions of ER2738. 50-100 colonies grew on the 1:1000 dilution of ER2738<br />
:::*Compare this to the 100-200 colonies that grew from 2/25 infection (which was 300ul non-diluted, infected cells)<br />
:::*Reasons for increased yield<br />
::::*Added too much phage?<br />
::::*volume changed between experiment (5mL to 50mL)<br />
::::*Overnight culture may not have been saturated. If still in log phase, the cells would continue to grow<br />
*Made 50mL O/N cultures of ER2738 and BW23115 so we can repeat the infection tomorrow and plate further dilutions starting at 1:1000<br />
::-Carry out infection in 5mL and 50mL to test volume effect?<br />
<br />
'''5/29'''<br />
*Measured OD of overnights<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! OD<br />
! mL to have .1OD in 50mL<br />
! mL to have 1OD in 5mL<br />
<br />
|-<br />
| K12 ER2738<br />
| 3.0<br />
| 1.7<br />
| 1.7<br />
<br />
|-<br />
| BW23115<br />
| 2.9<br />
| 1.7<br />
| 1.7<br />
<br />
|-<br />
|}<br />
<br />
<br />
*Experiment 1: Infect cells using same method as 5/25 (in a 5mL culture)<br />
::-Started with 1OD cells in 5mL<br />
::-Added about 0.7ul (inaccuracies in pipet) of 1:10 diluted M13-Litmus28i phage<br />
::-Incubated (rotating) for 20 minutes<br />
::-Made 1:1,000 and 1:10,000 dilutions<br />
::-Plated 100ul on Ampicillin (100ug/mL) plates and on Kanamycin (50ugmL) plates<br />
:::*Included non-infected samples diluted by 1:1000<br />
::::*This negative control can be used for Experiment 2 since the non-infected parent solution is the same<br />
::-Incubate overnight at 37C<br />
*Experiment 2: Infect cells using protocol from “Eliminating helper phage from phage display”<br />
::-Diluted O/Ns to OD of 0.1 in 50 mL culture<br />
::-Grew samples until of OD of ER2728 = 0.59 and the OD of BW23115 = 0.60<br />
:::*Missed OD of 0.5 mark, but the two samples are close to each other<br />
::-Chilled samples on ice for 30 minutes<br />
::-Warmed in incubator for 35 minutes (should have been 30)<br />
::-Amount of phage. Rather than use 1:1 as mentioned in protocol, we used multiplicity of 1:10 (phage:cell)<br />
:::*Added 3.3ul of 1:10 diluted M13-Litmus28i (1) phage<br />
::::*(On 5/27 we added 5.5ul of diluted phage to 50mL of cells at OD of 1. Our cells were at OD of .6; therefore, 5.5*.6 = 3.3ul)<br />
::-Incubated for 30 minutes at 37C, not shaking<br />
:::*We later change this to shaking<br />
::-Dilutions<br />
:::*1:1,000; 1:5,000; 1:10,000; 1:50,000; 1:100,000; 1:1,000,000<br />
:::*Plated ER2738 and BW23115 on Ampicillin (100ug/mL)<br />
:::*Plated ER2738 on Kanamycin (50ug/mL)<br />
*Experiment 3: Growth Test (for growth curve)<br />
::-We were concerned by the low OD of the Overnights from the last few days. Wanted to be sure that 2.0-3.0 was not still in log phase. Cultures looked saturated but the OD seemed low.<br />
{| class = "wikitable"<br />
|-<br />
! Time<br />
! Elapsed time (min)<br />
! ER2738 (no antibiotic)<br />
! ER2738 (Tetracycline (20ug/mL))<br />
! BW23115 (with F’ episome) no antibiotic<br />
<br />
|-<br />
| 10:08<br />
| 0<br />
| 0.1<br />
| 0.1<br />
| 0.1<br />
<br />
|-<br />
| 11:10<br />
| 62<br />
| 0.24<br />
| 0.21<br />
| 0.21<br />
<br />
|-<br />
| 12:00<br />
| 112<br />
| 0.49<br />
| 0.45<br />
| 0.44<br />
<br />
|-<br />
| 13:00<br />
| 172<br />
| 1.00<br />
| 0.93<br />
| 0.98<br />
<br />
|-<br />
| 14:15<br />
| 217<br />
| 1.29<br />
| 1.21<br />
| 1.31<br />
<br />
|-<br />
| 15:40<br />
| 302<br />
| 2.1<br />
| 1.8<br />
| 2.3<br />
<br />
|-<br />
| 16:47<br />
| 369<br />
| 2.5<br />
| 1.9<br />
| 3.4<br />
<br />
|-<br />
| 18:05<br />
| 447<br />
| 2.6<br />
| 2.2<br />
| 2.5<br />
<br />
|-<br />
| 19:00<br />
| 502<br />
| 2.9<br />
| 2.3<br />
| 2.5<br />
<br />
|-<br />
| 20:00<br />
| 562<br />
| 3.0<br />
| 2.6<br />
| 2.8<br />
<br />
|-<br />
|}<br />
<br />
:::*The time point at 16:47 (369 minutes elapsed) for BW23115 conjugated (without antibiotics) is most likely an error. It has been removed from the growth plot<br />
'''[[File:UCB-Phage Delivery-140529.JPG]]'''<br />
*Other<br />
::-Made Amp and Kan plates (1 sleeve of each)<br />
::-Made 50mL O/N of ER2738 and BW23115F’ in case we need further dilutions<br />
::-Made 5mL O/N of ER2738, BW23115F’, and BW23115 (without F’ episome) to make chemically competent tomorrow<br />
:::*Did not have plate of BW23115 (without F’ episome) so used freeze down. Hoping to get O/N of a picked colony from CRISPR Team tomorrow morning<br />
<br />
'''5/30'''<br />
*Made chemically competent cells of…<br />
::-ER2738<br />
::-BW23115F’ (conjugated with F’ episome)<br />
::-BW23115 (not conjugated- without F’ episome)<br />
:::*Culture started from plate<br />
::-BW23115* not conjugated (without F’ episome)<br />
:::*Culture started from freeze down<br />
*Results from infections<br />
::-Negative Controls (cells were not infected; cells were diluted 1:1000)<br />
'''[[File:UCB-Phage Delivery-140530.JPG]]'''<br />
::-Results from Experiment 1 (5/29)<br />
'''[[File:UCB-Phage Delivery-140530-02.JPG]]'''<br />
::-Results from Experiment 2 (5/29)<br />
'''[[File:UCB-Phage Delivery-140530-03.JPG]]'''<br />
*Math<br />
::-If there are 5.00E+8 cells in 1mL of culture at OD of 1, then in 1mL of culture at OD of 0.59, there are 2.95E+8 cells. In a 50mL culture at OD of 0.59, there are 1.48E+10 cells.<br />
::-We added 3.3ul (0.0033mL) phage at concentration 4.62E+11 phage/mL which amounts to 1.52E+9 total phage<br />
::-Assuming that 1 phage infects 1 bacterium, we can assume that 1.52E+9 bacterial have the potential to be infected in the 50mL culture<br />
::-We plated 100ul of culture at various dilutions. If not diluted, the number of cells that can be potentially infected in 0.1mL equals 1.52E+9/500, or 3.05E+06 cells. We then accounted for the dilutions (for 1:1000 dilution, we divided 3.05E+06 by 1000 to receive 3.05E+03)<br />
::-The following table contains the number of cells with the potential to be infected assuming a 100% infectivity rate by M13 phage and that 1 cell is infected only once.<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Dilution<br />
! Potential infected cells<br />
! Colonies on Amp<br />
! % Potential (Amp)<br />
! Colonies on Kan<br />
! % Potential(Kan)<br />
! Kan:Amp<br />
<br />
|-<br />
| 1:1000<br />
| 1.52E+06<br />
| 476<br />
| 15.62%<br />
| 15<br />
| 0.820%<br />
| 1:19.04<br />
<br />
|-<br />
| 1:5000<br />
| 3.05E+05<br />
| 131<br />
| 21.49%<br />
| 13<br />
| 2.133%<br />
| 1:10.08<br />
<br />
|-<br />
| 1:10000<br />
| 1.52E+05<br />
| 93<br />
| 30.51%<br />
| 5<br />
| 1.640%<br />
| 1:18.60<br />
<br />
|-<br />
| 1:50000<br />
| 3.05E+04<br />
| 17<br />
| 44.29%<br />
| 0<br />
| 0.000%<br />
| <br />
<br />
|-<br />
|}<br />
<br />
*Conclusions from infections<br />
::-Results between and within the three trials are inconsistent. For example, the number of colonies received in experiments 1 and 2 from 5/29 differ greatly. Due to the differences in protocol, variation was expected but not to this extent. <br />
::-Our dilutions did not yield the expected 10 fold (or 5 fold) decrease in growth that was expected.<br />
::-Plates from 5/29 could be plated better to reduce dense areas of growth and growth around the rim.<br />
::-Though the experiment contained many errors we can say that the phagemid (Litmus 28i) is preferentially packaged compared to the helper phage (M13K07) but not to the degree we expected.<br />
::-Could receive increased occurrences of cells containing M13k07 due to infection, phage production, further infection<br />
<br />
==Week 5==<br />
<br />
'''6/2'''<br />
*Tested chemically competent cells through transformation<br />
::-Are cells contaminated?<br />
::-Are cells competent?<br />
*The samples for transformation<br />
{| class = "wikitable"<br />
|-<br />
! #<br />
! Cells (Tube label)<br />
! DNA (Tube label)<br />
! Resistance before transformation<br />
! Resistance after transformation<br />
<br />
|-<br />
| 1<br />
| K12 ER2738 5/20<br />
| p110+RBS (2) 4/23<br />
| Tet<br />
| Tet, Chlor<br />
<br />
|-<br />
| 2<br />
| BW (-f) 5/30<br />
| p110+RBS (2) 4/23<br />
| Kan<br />
| Kan, Chlor<br />
<br />
|-<br />
| 3<br />
| BW f-ep comp 5/22<br />
| p110+RBS (2) 4/23<br />
| Kan, Tet<br />
| Kan, Tet, Chlor<br />
<br />
|-<br />
| 4<br />
| BW (+f) 5/30<br />
| p110+RBS (2) 4/23<br />
| Kan, Tet<br />
| Kan, Tet, Chlor<br />
<br />
|-<br />
| 5<br />
| *BW23115 5/30<br />
| p110+RBS (2) 4/23<br />
| Kan<br />
| Kan, Chlor<br />
<br />
|-<br />
| 2B<br />
| K12 ER2738<br />
| 2B [from dis. kit]<br />
| Tet<br />
| Tet, Chlor<br />
<br />
|-<br />
| 2P<br />
| BW f-ep comp 5/22<br />
| 2P [from dis. kit]<br />
| Kan, Tet<br />
| Kan, Tet, Chlor<br />
<br />
|-<br />
|}<br />
<br />
'''6/3'''<br />
*Results from 6/2 Transformation<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Growth on Chlor<br />
! Growth on Kan<br />
! Growth on Kan+Tet<br />
! Growth on Amp<br />
<br />
|-<br />
| 1 N<br />
| X<br />
| X<br />
| X<br />
| X<br />
<br />
|-<br />
| 2 N<br />
| X<br />
| +<br />
| X<br />
| X<br />
<br />
|-<br />
| 3 N<br />
| X<br />
| +<br />
| +<br />
| X<br />
<br />
|-<br />
| 4 N<br />
| X<br />
| +<br />
| +<br />
| X<br />
<br />
|-<br />
| 5 N<br />
| X<br />
| +<br />
| X<br />
| X<br />
<br />
|-<br />
| 1<br />
| +<br />
| X<br />
| X<br />
| <br />
<br />
|-<br />
| 2<br />
| +<br />
| +<br />
| X<br />
| <br />
<br />
|-<br />
| 3<br />
| +<br />
| +<br />
| +<br />
| <br />
<br />
|-<br />
| 4<br />
| +<br />
| +<br />
| +<br />
| <br />
<br />
|-<br />
| 5<br />
| +<br />
| +<br />
| Many colonies but close to samp. 4<br />
| <br />
<br />
|-<br />
| 2P<br />
| + (~100)<br />
| +<br />
| +<br />
| <br />
<br />
|-<br />
| 1- JW<br />
| +<br />
| X<br />
| X<br />
| <br />
<br />
|-<br />
| 2- JW<br />
| +<br />
| +<br />
| X<br />
| <br />
<br />
|-<br />
| 3- JW<br />
| +<br />
| +<br />
| +<br />
| <br />
<br />
|-<br />
| 4- JW<br />
| +<br />
| +<br />
| +<br />
| <br />
<br />
|-<br />
| 5-JW<br />
| +<br />
| +<br />
| X<br />
| <br />
<br />
|-<br />
| 2B- JW<br />
| + (24)<br />
| X<br />
| X<br />
| <br />
<br />
|-<br />
|}<br />
*The transformations with DNA from the well (B2 and P2) had lower efficiencies than those with DNA from a mini-prep. Most likely this is due to the differences in DNA concentration (p110+RBS (2) 4/23 was at 254.4ng/ul)<br />
*Conclusions<br />
::-None of the competent cells were contaminated<br />
::-All of the competent cells are in fact, competent<br />
*Set up O/N of DH5-alpha cells to make competent tomorrow<br />
<br />
'''6/4'''<br />
*Isolation of single-stranded phagemid DNA using M13K07<br />
::-Added ER2738 colony to 50mL LB<br />
:::*Plate was cold. Next time warm plate before pricking<br />
::::*Best to use freshly grown plate<br />
::-After 4 hours, OD was at 0.02. Waited 45 minutes and OD was at 0.08. Therefore, we infected at OD 0.08<br />
::-Had started another culture when we did not think the first was growing. In incubator for about 1 hour. OD was 0.00. We infected anyway because last time it worked.<br />
::-Let infection proceed for 60 minutes then added 70ul of Kanamycin to be a final concentration of 70ug/mL<br />
*Primers came in to biobrick M13ori (packaging signal on Litmus28i)<br />
::-Resuspended primers and diluted 1:10<br />
<br />
'''6/5'''<br />
*Isolated single-stranded M13K07 DNA<br />
::-Final concentration = 5724 ng/ul (calculated from a 1:10 dilution)<br />
::-For second sample in pair, we resuspended it in TE but did not proceed to DNA extraction teps<br />
::-For the second culture we started 6/4, we resuspended pellet in TBS and glycerol to preserve the M13 phage. Measured absorbances (before glycerol was added)<br />
: #1<br />
::269 => 1.690A<br />
::320 => 0.103A<br />
: #2<br />
::269 => 1.453<br />
::320 => 0.059<br />
*For our first biobrick, we wanted to isolate the M13 origin, a segment ~500bp that allows for packaging into the M13 phage. We tried to achieve this by biobrick assembly and by Gibson Assembly.<br />
::-To biobrick M13 ori through biobrick assembly (the old-school way)<br />
:::PCR on Litmus 28i to amplify/biobrick M13ori<br />
::::*Used primers Gem003 F & R<br />
::::*Diluted Litmus 28i DNA 1:10<br />
:::Digestion of p11+RBS (1) to digest pSB1C3 bb with EcoRI-HF and PstI-HF<br />
:::Ran samples on gel and gel extracted pieces. We recieved very low yields (out of range for nano drop)<br />
::::*M13 ori: 4.0 ng/ul<br />
::::*pSB1C3: 1.8 ng/ul<br />
:::Digested M13 ori fragment despite poor extraction yield with EcoRI-HF and PstI-HF<br />
::::*Used 1.5x as much DNA as instructed based on inaccurate concentration<br />
'''[[File:UCB-Phage Delivery-140605.JPG]]'''<br />
::-Gel extracted red rectangles<br />
::-Ligation<br />
:::*10hr @ 16C, 10min @ 65C, 4ever @ 4C<br />
*To biobrick M13 ori through Gibson Assembly (the cool-kids way)<br />
::-PCR on Litmus 28i<br />
:::*Used primers Gem002 F & R<br />
:::*Diluted Litmus 28i DNA 1:10<br />
::-PCR on pSB1C3 (p11+RBS (1))<br />
:::*Used primers Gem001 F & R<br />
:::*Diluted pSB1C3 DNA 1:3<br />
<br />
'''6/6'''<br />
*Ran gel of PCR products from (6/5). Products will be used for Gibson Assembly<br />
::-Recieved bands for pSB1C3 around 2000bp and M13ori around 500bp<br />
::-No contamination in pSB1C3 PCR negative control<br />
::-Band in M13ori negative control that is the same size as sample. Contaminated by sample?<br />
::-Gel of PCR products #1 and #2 from 6/5<br />
'''[[File:UCB-Phage Delivery-140606.JPG]]'''<br />
:1. pSB1C3 with promoter+RBS as insert. Amplified with Gem001<br />
:2. No DNA control for Gem001<br />
:3. M13ori amplified with Gem002 from Litmus28i<br />
:4. No DNA control for Gem002<br />
*Gibson Assembly<br />
{| class = "wikitable"<br />
|-<br />
! Total Amount of Frag.<br />
! .02-.5pmol<br />
! 10 ul total<br />
<br />
|-<br />
| Gibson Assembly MM (2x)<br />
| 10 ul<br />
| 10<br />
<br />
|-<br />
| Dionized H2O<br />
| 10-x<br />
| <br />
<br />
|-<br />
|}<br />
::-Diluted pSB1C3 and M13ori PCR products 1:10<br />
::-Incubated 60min @ 50C<br />
::-Also used provided pUC16 as positive control<br />
*Transformation<br />
:1. p110+RBS Positive control<br />
:2. No DNA Negative control<br />
:3. Cas9 from distribution kit so we can have more<br />
:4. Thaw and refreeze cells Test competency of comp cells after thawed<br />
:5. Not chem comp cells Negative control for the above<br />
:6. Ligation Product<br />
:7. Gibson product<br />
::*7.2. Gibson product diluted 1:4<br />
:8. Gibson positive control<br />
::*7.2. Gibson positive control diluted 1:4<br />
::-For the Gibson product and the positive control, we transformed 2ul of product and 2ul of 1:4 diluted product. NEB recommends the first if using their competent cells and the second if using cells from other companies. Our cells are from NEB but we made them competent ourselves so we tried both ways<br />
:::*Plated on Chlor at concentrations of 170, 85, and 33 ug/mL<br />
*Primers came in<br />
::-Resuspended and made 1:10 dilutions<br />
<br />
'''6/7'''<br />
*Results from 6/6 transformation<br />
:1. Positive control<br />
::*Lots of growth, ~300-400 on 1:10 dilution<br />
:2. No DNA negative control<br />
::*No growth<br />
:3. Cas9 from distribution kit<br />
::*7 potential colonies (some are close to edges through) on non-diluted<br />
:4. Thawed then refroze cells<br />
::*Looks like (1)<br />
:5. Not chemically competent cells<br />
::*No growth<br />
:6. Ligation product<br />
::*13 potential colonies (some are close to edge)<br />
:7. Gibson Assembly Product<br />
::*170 -> No colonies<br />
::*85 -> No colonies<br />
::*33 -> 3 specks<br />
:7.2. Gibson Assembly product diluted 1:10<br />
::*170 -> 1 speck<br />
::*85 -> 3 colonies<br />
::*33 -> 13 colonies<br />
:8. Gibson positive control<br />
::*No colonies<br />
:8.2. Gibson positive control diluted 1:4<br />
::*No colonies<br />
*Made 6mL O/N cultures<br />
::-4 from (3) cas9 plate<br />
:::See Constitutive CRISPR notebook for more info on these samples<br />
::-7 from (6) Ligation product<br />
::-5 from (7.2 [85]) Diluted Gibson product on 85 ug/mL Chlor<br />
::-8 from (7.2 [33]) Diluted Gibson product on 33 ug/mL Chlor<br />
<br />
==Week 6==<br />
'''6/8'''<br />
*Check colonies for correct constructs.<br />
::-Mini-prepped all 24 O/Ns<br />
:::*Yielded low concentrations for samples 12, 15, 19, and 22<br />
::-Digested all with EcoRI and PstI (10ul reactions)<br />
::-Ran results on gel<br />
:::*All 4 cas9 samples had the expected bands of 2000 and 5000bp<br />
:::*All 7 ligation products have expected bands of 2000 and ~570bp<br />
:::*3 of 5 Gibson assemblies from 85ug/mL Chlor plate had expected bands of 2000 and ~500bp<br />
:::*3 of 8 Gibson assemblies from 33ug/mL Chlor plate had expected bands of 2000 and ~500bp<br />
'''[[File:UCB-Phage Delivery-140608-01.JPG]]'''<br />
<br />
'''[[File:UCB-Phage Delivery-140608-02.JPG]]'''<br />
::1-4: Cas9 from Stanford-Brown team<br />
::5-11: pSB1C3-M13ori cloned through ligation<br />
::12-24: pSB1C3-M13ori cloned through Gibson Assembly<br />
:::*12-16: Grown with 85 ug/mL Chlor<br />
:::*17-24: Grown with 33 ug/mL Chlor<br />
*Conclusions from gel<br />
::-We have cas9 safely in cells<br />
::-Our ligation reactions successfully yielded M13ori on pSB1C3<br />
::-Combined, we had a 46% success rate for the Gibson Assembly in yielding M13ori on pSB1C3<br />
:::*The 4 samples that had the lowest concentration after being mini-prepped (12,15, 19, and 22) correlate with samples that had the correct band pattern<br />
*We selected 4 samples from each (4 total between the two Gibson reactions) type<br />
::-For non-Gibson Assembled samples<br />
:::Plated 25ul on 170ug/mL Chlor<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Our usual method<br />
! Gibson Method<br />
<br />
|-<br />
| 1. Thaw on ice<br />
| 1. Thaw on ice <br />
<br />
|-<br />
| 2. Transfer 40ul cells to tube<br />
| 2. Transfer 50ul cell to tube<br />
<br />
|-<br />
| 3. Add DNA. 1ul for mini-prep OR up to 10ul for ligation<br />
| 3. Add 2ul to NEB cells OR 2ul of 1:4 diluted to other cells<br />
<br />
|-<br />
| 4. Mix by pipet Let sit 30min on ice<br />
| 4. Mix by pipet or flicking Let sit 30 min. on ice<br />
<br />
|-<br />
| 5. Heat shock: 42C for 45s<br />
| 5. Heat shock: 42C for 30s<br />
<br />
|-<br />
| 6. Ice for 5 minutes<br />
| 6. Ice for 2 min.<br />
<br />
|-<br />
| 7. Transfer to culture tube; Add 200ul SOC<br />
| 7. Add 950ul SOC to tube<br />
<br />
|-<br />
| 8. Shake or rotate for 60-120min at 37C<br />
| 8. Shake (250rpm) or rotate for 60 min. at 37C<br />
<br />
|-<br />
| 9.<br />
| 9. Warm plates to 37C<br />
<br />
|-<br />
| 10. Plate 100ul onto selection plate<br />
| 10. Plate 100ul onto plate<br />
<br />
|-<br />
| 11. Incubate O/N @ 37C<br />
| 11. Incubate O/N @ 37C<br />
<br />
|-<br />
|}<br />
::-Added 6mL LB and Chlor at concentration of 170ug/mL to grow O/N<br />
:*For Gibson Assembled samples<br />
::-Plated 25ul onto 170, 85, and 33ug/mL Chlor<br />
::-Samples from 85ng/mL plate<br />
:::Transferred 100ul to new tube, added media, added Chlor at 170ng/mL<br />
::-Samples from 33ng/mL plate<br />
:::Tranfered 100ul to new tubes, added media, added Chlor at 85ng/mL to one and 33ng/mL to the other<br />
:*Tomorrow, may send for sequencing and make freeze downs<br />
<br />
*Transformation<br />
:1. Positive control (p110+RBS diluted 1:10)<br />
:2. Non-diluted Gibson product<br />
:3. Gibson product diluted 1:4<br />
:4. Gibson product diluted 1:10<br />
::-Transformed each sample using our usual method and using the protocol given by Gibson<br />
<br />
::-Due to not have plates ready before transformation, in step 4, the samples sat for about 50 minutes. Then in step 8, they both recovered for about 150 minutes. Though not specified in our protocol, we did warm the plates to 37C. In step 10 for our protocol, since we only added 200ul SOC and wanted to plate on 3 selection plates (see below), we only plated 50ul (except for the positive control). <br />
::-Plated on three concentrations of Chloramphenicol (33 ug/mL, 85 ug/mL, and 170 ug/mL) to determine the differences in yield due to differences in concentration.Obvious hypothesis: more colonies will grow on plates that have a lower concentration of chlor.<br />
<br />
<br />
'''6/9'''<br />
*Made chemically competent 5alpha cells with Dan and Alex from main campus<br />
::-Waiting to hear results on competency<br />
*Will eventually make phage containing CRISPR-Cas9 that targets Kanamycin resistance. M13K07 has Kanamycin resistance so we need to switch the resistance on the M13 genes.<br />
::-PCR on pwp 2.po (plasmid that Sam gave us that contains the zeoR gene adjacent to ori) to amplify zeoR and ori. Zeo is on EM7 promoter<br />
:::-Primers: Gem008 R & R<br />
:::-Anneal temp from NEBuilder: 63.7C<br />
:::-Extension time: 90s<br />
:::-Expected band size on gel: 1300bp<br />
:::-Used phusion polymerase<br />
*PCR on M13K07 DNA to amplify M13 phage genes (also removes majority of M13 ori, all of KanR, and all of p15 ori)<br />
:::-Primers: Gem007 F & R<br />
:::-Anneal temp from NEBuilder: 60.2C<br />
:::-Extension time: 4:30<br />
:::-Expected band size on gel: about 6000bp<br />
:::-Used phusion polymerase<br />
*Freeze downs<br />
::-Note: Phagemid 1C3 was the original name for ‘pSB1C3-M13ori’<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Top Label<br />
! Side label<br />
! What?<br />
<br />
|-<br />
| Phagemid 1C3 6/9<br />
| From Lig<br />
| M13 ori inserted into 1C3 (biobricked); Done through ligation; Contains extra bases as spacer between biobrick prefrix/suffix and part for primer design<br />
<br />
|-<br />
| Phagemid 1C3 6/9<br />
| From Lig<br />
| “ “<br />
<br />
|-<br />
| Phagemid 1C3 6/9<br />
| Gibson<br />
| M13 ori inserted into 1C3 (biobricked); Done through Gibson cloning<br />
<br />
|-<br />
| Phagemid 1C3 6/9<br />
| Gibson<br />
| “ “<br />
<br />
|-<br />
|}<br />
'''6/10'''<br />
*Ran gel of PCRs from 6/9<br />
<br />
'''[[File:UCB-Phage Delivery-140610.JPG]]'''<br />
:1. Amplification of M13 genes from M13K07 (~6000bp)<br />
:2. No DNA control for (1) amplification<br />
:3. Amplification of Zeo resistance gene + plasmid ori (~1300bp)<br />
:4. No DNA control for (3) amplification<br />
*Gibson Assembly of above parts (did not gel extract)<br />
::-Diluted the PCR products 1:10 then added 3ul of M13K07 genes product and 7ul of ZeoR+ori product<br />
::-Incubated at 50C for 60 min.<br />
::-Transformed Gibson Assembly product into 5alpha cells<br />
:::*Used our usual protocol<br />
:::*Add 2ul of DNA<br />
::::-In one sample, diluted DNA 1:4 and in the other, we diluted DNA 1:10<br />
*Started Phage Amplification Protocol<br />
::-ER2738 transformed with Litmus 28i<br />
:::*Grew for ~2.5hr before reaching an OD of 0.04<br />
::-ER2738 transformed with pSB1C3-M13ori (M13ori on pSB1C3)<br />
:::*Grew for ~ 3.5hr before reaching an OD of 0.01, then in the next 1.5 hours, spiked to 0.19<br />
:::*We gave up and went home, and will restart tomorrow<br />
*Analyzed transformation results from 6/8<br />
<br />
'''6/11'''<br />
*Made chemically competent 5alpha cells<br />
*Restarted Phage Amplification Protocol<br />
::-Forgot to add phagemid antibiotic at start of growth. Added phagemid antibiotic when we added phage. Incubated for 90 minutes before adding Kanamycin (to select for cells that were infected by M13K07)<br />
::-Phage is at concentration 4.57x10^12 phage/mL<br />
:::Protocol calls for final concentration of 1 x10^8 phage/mL<br />
::::*(4.57 x10^12)*V = (1 x10^8)(50mL)<br />
::::*V = 0.00109mL<br />
:::Added 1.1ul of phage<br />
*Transformations<br />
{| class="wikitable"<br />
|-<br />
!DNA<br />
!Plate Selection<br />
|-<br />
|No DNA control<br />
|(all)<br />
|-<br />
|Positive control (p110+RBS)<br />
|(C)<br />
|-<br />
|M13 genes + ZeoR ori<br />
|(Z)<br />
|-<br />
|M13 genes +ZeoR ori diluted 1:4<br />
|(z)<br />
|} <br />
<br />
'''6/12'''<br />
*Transformation Results from 6/10 [Took ~36 hours to be clearly visible]<br />
::No DNA control<br />
:::-Amp: 0 <br />
:::-Zeo (50ug/mL): 200 colonies<br />
::M13 genes + ZeoR ori (1:4 dilution) <br />
:::-Zeo (25ug/mL): 300<br />
:::-Zeo (50ug/mL): 200<br />
:::-Zeo (100ug/mL): 100<br />
::M13 genes + ZeoR ori (1:10 dilution) <br />
:::-Zeo (25ug/mL): 300<br />
:::-Zeo (50ug/mL): 150<br />
:::-Zeo (100ug/mL): 150<br />
*Transformation Results for 6/11<br />
::-Positive (p110+RBS) on Chloramphenicol: 500 colonies<br />
::-No DNA on Amp: 0 colonies Zeo (100 ug/mL): specks<br />
::-No apparent growth on any other plate<br />
:::*Realized later that we grew our samples on the wrong plates. Will repeat transformation today<br />
*Transformation #1<br />
::This morning there were no colonies on positive (p110+RBS) coltrol from 6/11 even though we observed fast growth in the past. Without waiting for colonies to appear, we started a control transformation<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Diluted<br />
! Time at 42C<br />
<br />
|-<br />
| p110+RBS<br />
| No<br />
| 45s<br />
<br />
|-<br />
| p110+RBS<br />
| 1:10<br />
| 45s<br />
<br />
|-<br />
| p110+RBS<br />
| 1:10<br />
| 30s<br />
<br />
|-<br />
| p110+RBS<br />
| 1:10<br />
| No timer. ~43s<br />
<br />
|-<br />
| No DNA control<br />
| No<br />
| 45<br />
<br />
|-<br />
|}<br />
*Because ‘No DNA control 6/10’ yielded colonies, we researched Zeocin plates<br />
::-According to Life Technologies (Invitrogen), Zeocin requires low salt medium and a pH of 7.5<br />
::-Low Salt LB Medium (1L)<br />
:::*Ingredients<br />
::::10g Tryptone<br />
::::5g NaCl<br />
::::5g Yeast Extract<br />
:::-Mix ingredients<br />
:::-Adjust pH to 7.5 using NaOH (If go over, use HCl)<br />
:::-Add agar for plates at 15g/L. Autoclave<br />
:::-Thaw Zeocin on ice. Vortex<br />
:::-Add Zeocin to final concentration of 25ug/mL<br />
*Transformation #2<br />
::-Repeat of transformation on 6/11 but this time we will plate on the correct plates<br />
::-Also remade Zeocin plates<br />
*Finished isolation of M13 Litmus phage and M13 pSB1C3-M13ori phage<br />
::*Note: Phagemid 1C3 was the original name for ‘pSB1C3-M13ori’<br />
{| class = "wikitable"<br />
|-<br />
! Phage Sample<br />
! A269<br />
! A320<br />
! Concentration (phage/mL)<br />
<br />
|-<br />
| Litmus phage (1)<br />
| 0.181<br />
| 0.034<br />
| 3.12 x10^12<br />
<br />
|-<br />
| Litmus phage (2)<br />
| 0.227<br />
| 0.047<br />
| 3.83 x10^12<br />
<br />
|-<br />
| Phagemid 1C3 phage (1)<br />
| 0.101<br />
| 0.020<br />
| 1.87 x10^12<br />
<br />
|-<br />
| Phagemid 1C3 phage (2)<br />
| 0.126<br />
| 0.021<br />
| 2.42 x10^12<br />
<br />
|-<br />
|}<br />
::::phage/mL = 6x10^16 x (A269-A320)/ (#of base pairs in the phage genome)<br />
*Set up 50mL O/N of K12 ER2738 (containing f-episome) for infection tomorrow with Litmus phage and pSB1C3-M13ori phage<br />
<br />
<br />
'''6/13'''<br />
*Transformation Results for 6/11<br />
::-Several hundred colonies on Positive Control (p110+RBS) on Chlor<br />
::-No colonies for GA positive control on Amp<br />
::-No colonies for M13ori + ZeoR mistakenly plated on Amp<br />
::-No colonies for No DNA control on Amp<br />
::-100-ish colonies for No DNA control on Zeo<br />
::-100-ish colonies for cas9+AmpR+gRNA mistakenly plated on Zeo<br />
::-These last 3 points suggest/confirm that Zeo plates are no good<br />
*Transformation Results from 6/12 control test<br />
::-Colonies grew is about equal amounts on all plates, including No DNA control<br />
:::Either plates don’t contain Chlor or competent cells are contaminated<br />
::-Streaked competent cells onto new and old Chlor plates<br />
*Infection test of ER2738: Is Litmus preferentially packaged over M13K07 Helper phage? Is pSB1C3-M13ori preferentially packaged?<br />
::-Infect ER2738 with phage produced 6/12<br />
:::*Phage should have packaged Litmus 28i phagemid or pSB1C3-M13ori<br />
:::*Cells infected with phage packaging Litmus 28i will grow on Amp<br />
:::*Cells infected with phage packaging pSB1C3-M13ori will grow on Chlor<br />
:::*Cells infected with phage packaging M13K07 will grow on Kan<br />
::-After we plate, we can count the colonies and calculate a ratio of Litmus28i: M13K07 or pSB1C3-M13ori:M13K07 packaging<br />
<br />
'''6/14'''<br />
*Results of contamination test (streaked competent cells onto new and old Chlor plates)<br />
::-Colonies grew in low amounts on both plates. most likely the cells are contaminated<br />
*Transformation results for 6/12<br />
::-Many colonies for No DNA control on Zeo<br />
:::-Some colonies are turning pinkish-red<br />
::-Many colonies for diluted and non-diluted M13 genes+ZeoR on Zeo<br />
:::-Some colonies are turning pinkish-red<br />
*Could the white colonies be the designed colonies and can we kill the red colonies with Zeocin before killing the white colonies (aka. Use high Zeocin concentrations to select for the correct construct)<br />
::-Selected 1 red colony from No DNA Zeo control plate and 1 white colony from M13genes+ZeoR sample plate<br />
::-Added 100ul H2O then divided amongst 5 culture tubes each (with 5mL of low-salt LB, pH 7.5)<br />
::-Then added Zeocin to a final concentration of: 0, 25, 50, 75, 100ug/mL.<br />
*Results of 6/13 infection<br />
::-Litmus 28i infected cells<br />
:::*on Kan: Individual colonies for 1:10, 1:100, and 1:1000. No colonies on 1:5000<br />
:::*on Amp: lawn for 1:10, 1:100, and near lawn for 1:1000. Single colonies for 1:5000<br />
::-pSB1C3-M13ori infected cells<br />
:::*on Kan: Same as Litmus 28i samples on Kan<br />
:::*on Chlor: Lawn for all dilutions. Must discredit due to recent Chlor contamination<br />
<br />
==Week 7==<br />
<br />
'''6/15'''<br />
*Results of Zeocin experiment on 6/14<br />
::-Healthy growth for both at 0ug/mL Zeo<br />
::-No growth for white colony with any Zeo<br />
::-Good growth for red colony even at 100ug/mL Zeo<br />
::-This suggests that the red colonies are naturally resistant to Zeocin. Also, our plates must not contain active Neocin. We are sure that we are adding enough. Possibly, we add it while the media is too hot or leave the plates at room temperature (decondensing) for too long and this deactivates the antibiotic? Perhaps we are not attaining the correct pH<br />
*In light of recent contamination problems on both Chlor and Zeo, we made new competent cells<br />
::-5alpha<br />
::-BW23115<br />
::-BW23115 conjugated (contains f-episome)<br />
<br />
'''6/16'''<br />
*Made new Chlor antibiotic. Made to Chlor plates<br />
*Contamination Test: Streaked Zeo and new Chlor plates each with<br />
::-Colony from Zeo contaminated plate<br />
::-Colony from Chlor contaminated plate<br />
::-Non-transformed OLD chemically competent cells<br />
::-Non-transformed NEW chemically competent cells<br />
*Redid bacterial infection<br />
::-For both samples, we used ER2738 (not from the same ‘O/N’ (also, not a real O/N))<br />
::-Infected one sample with isolated ‘Litmus28i phage’ and one with ‘pSB1C3-M13ori’<br />
::-Alterations to protocol<br />
:::*Did not started from saturated O/N. Started each with a colony, waited several hours until at OD ~1. We then added these cells to fresh 50mL LB to have an OD of 0.1.<br />
:::*Missed the 30 minute infection mark. Infected for ~45 minutes.<br />
::-Plated on Kan and Amp (Litmus28i sample) or Chlor (pSB1C3-M13ori sample) at dilutions of<br />
:::*1:100, 1:1k, 1:10k, and 1:100k<br />
<br />
'''6/17'''<br />
*Q5 PCR to replace KanR with ZeoR in M13K07<br />
::-Used recommendations<br />
::-Unfortunately, I (Jo) don’t know the difference between tightening and loosening the thermocycler lid; therefore, our M13K07 sample to amplify the M13K07 genes evaporated. But that’s ok, because we were sick of Zeocin anyway and decided mid-PCR to not waste our time with a Gibson Assembly and transformation. Instead we will be using the Old-School method of digestion and ligation because let’s face it, it’s a classic (and Gibson sucks) =)<br />
*Results from infection Test<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Litmus 28i<br />
! 1:100<br />
! 1:1k<br />
! 1:10k<br />
! 1:100k<br />
<br />
|-<br />
| Amp<br />
| ~6000?<br />
| ~2056<br />
| 377<br />
| 36<br />
<br />
|-<br />
| Kan<br />
| 130<br />
| 8<br />
| 3<br />
| 0<br />
<br />
|-<br />
|}<br />
{| class = "wikitable"<br />
|-<br />
! pSB1C3-M13ori<br />
! 1:100<br />
! 1:1k<br />
! 1:10k<br />
! 1:100k<br />
<br />
|-<br />
| Chlor<br />
| 8<br />
| 1<br />
| 0<br />
| 0<br />
<br />
|-<br />
| Kan<br />
| 97<br />
| 9<br />
| 0<br />
| 0<br />
<br />
|-<br />
|}<br />
::-We consider the Litmus 28i to have been a success. The pSB1C3-M13ori …. not so much. Looking back at the Litmus28i and the M13K07, we noticed that the M13ori is facing the opposite direction as the plasmid ori. Ours faces the same direction as the plasmid ori. Because we are working with phagemids that are single stranded, we think that by flipping the M13ori, we may be able to recover functionality. We will also look into other reasons.<br />
*Results from contamination test (6/16)<br />
::-Old Chlor plate (6/12)<br />
:::-Non-transformed chemically competent cells<br />
::::*Old: some colonies<br />
::::*New: No colonies<br />
::-New Chlor plate (6/16)<br />
:::-Colony from<br />
::::*Zeo contaminated plate: No growth<br />
::::*Chlor contaminated plate: Much growth<br />
:::-Non-transformed chemically competent cells<br />
::::*Old: some colonies<br />
::::*New: No growth<br />
::-Zeo (25ug/mL, Low NaCl, pH 7.5) (6/12)<br />
:::-Colony from<br />
::::*Zeo contaminated plate: Much growth<br />
::::*Chlor contaminated plate: No growth<br />
:::-Non-transformed chemically competent cells<br />
::::*Old: some specks<br />
::::*New: some specks<br />
<br />
'''6/18'''<br />
*Ordered primers to…<br />
::-biobrick M13ori (in other direction)<br />
::-biobrick M13K07 genes<br />
'''6/19'''<br />
*Waited for primers<br />
*Set up O/N cultures to test last infection (6/16) for colonies containing pSB1C3-M13ori<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample #<br />
! Selection<br />
! Dilution<br />
! Presumably<br />
! Notes<br />
<br />
|-<br />
| 1-8<br />
| Chlor<br />
| 1:100<br />
| pSB1C3-M13ori<br />
| <br />
<br />
|-<br />
| 9<br />
| Chlor<br />
| 1:1k<br />
| pSB1C3-M13ori<br />
| <br />
<br />
|-<br />
| 10-14<br />
| Chlor<br />
| 1:1k<br />
| pSB1C3-M13ori<br />
| These colonies were not present on plate during initial counting on 6/17<br />
<br />
|-<br />
| 15-16<br />
| Kan<br />
| 1:1k<br />
| M13K07<br />
| <br />
<br />
|-<br />
|}<br />
:::-Selection and dilution refer to the plate. Cells were then grown under selection.<br />
:::-More colonies were seen on all Chlor plates. No new colonies appeared on Kan plates<br />
*Tomorrow, we will mini-prep, digest, and run the samples on a gel to verify gene transfer.<br />
<br />
'''6/20'''<br />
*Primers are in!<br />
*Cloning of pSB1C3-M13ori(New)<br />
::-PCR to amplify M13ori (packaging signal) from Litmus 28i to be in the other direction<br />
:::-Primers (Gem011 F&R) add cut sites to make part biobrick compatible<br />
:::-Was able to gel extract<br />
::::*Band at the same size as sample….. contaminated primers?<br />
:::-Then digested with E and P<br />
::-Digested pSB1C3 plasmid with E and P to linearize backbone<br />
::::*Was able to gel extract<br />
'''[[File:UCB-Phage Delivery-140620.JPG]]'''<br />
<br />
:1. Spill over from 2<br />
:2 and 3. pSB1C3 digested with EcoRI and PstI<br />
::-Insert <100bp so cannot be seen<br />
:4. M13ori<br />
*Ligation of M13ori (packaging signal) into pSB1C3<br />
::10h at 16C<br />
::20m at 80C<br />
::Hold at 4C<br />
<br />
*PCR to amplify M13K07 genes from M13K07 DNA isolated from phage<br />
::-Primers (Gem012 F&R) add cut sites to make part biobrick compatible<br />
::-Very light bands and not band might correlate with 6000pb but not enough resolution on gel to be certain. Bands were too light so did not extract<br />
::-Set up PCR again using Q5 to run O/N<br />
:::*Received only a feint, smudgy band that was too large. We did not bother with extracting the DNA<br />
*Test of 6/19 O/Ns (14 that are presumably pSB1C3-M13ori and 2 that are presumably M13K07)<br />
::-Mini-prepped all O/Ns<br />
::-Digested all with Pst-I (common RE between pSB1C3-M13ori and M13K07)<br />
:::-Even though these are phagemids, we assumed that because they were still in the cell, the plasmids were still double stranded so could be recognized by RE. Our assumption was valid<br />
:::-Results of digestion<br />
::::*1,2,4-14: all had bands that corresponded to pSB1C3-M13ori cut once<br />
::::*3: Slight band at expected size but very feint<br />
::::*15, 16: as expected, they contain bands of ~9000bp, correlating with the M13K07 phagemid. Interestingly, sample 15 also contained a (brighter) band that corresponds with pSB1C3-M13ori. We assume that this was an incident of double infection (chance or did this occur at high frequency?)<br />
'''[[File:UCB-Phage Delivery-140620-02.JPG]]'''<br />
::Check table from 6/19 for more details<br />
:::-1-14: pSB1C3-M13ori<br />
:::-15-16: M13K07 from 1:1k diluted plate<br />
<br />
'''6/21'''<br />
*Needed to transform our pSB1C3-M13ori(New) (6/20) into cells. First, we transformed into 5alpha cells; however, we need to infect these cells later in order to make phage. Our 5alphas are not competent so we repeated the transformation, this time using ER2738 cells which contain the F’ episome, allowing us to infect with the M13 phage.<br />
::-Also transformed sample #1 from 6/20 mini-preps (pSB1C3-M13ori(Old) into ER2738 cells. Do not see any notes about this plasmid being in infectable cells during the initial experiment. Reason for experiment failure???<br />
*Ran 6/20 O/N PCR (to amplify M13K07 genes) on gel (also re-ran the previous sample from the day with more DNA)<br />
::-Still, band looks too big. Brightest band still occurs between 8000 and 9000bp. Lighter band around 7000bp-- might be ~6000pb but too light to tell<br />
*Alternative plan for amplifying M13K07 genes<br />
::-Digest sample with PstI (cuts outside of the target region)<br />
'''[[File:UCB-Phage Delivery-140621.JPG]]'''<br />
::Used sample #16 from 6/20 b/c it is double stranded so will cut<br />
:-PCR amplified the linearized digestion product<br />
:::1. PCR of M13genes<br />
:::2. PCR of M13genes + DMSO<br />
::::Gel extracted pieces boxed in red<br />
::-Used primers Gem012 F&R<br />
::-Gel extracted piece but received very low yield<br />
*Alternative to alternative plan for amplifying M13K07 genes<br />
::-Digested with PstI and AgeI<br />
'''[[File:UCB-Phage Delivery-140621-02.JPG]]'''<br />
:1. Cut with PstI and AgeI<br />
:2. Uncut<br />
:3. Cut with PstI<br />
:4. Just PCR<br />
::-Gel extracted ~6000pb band after double digestion<br />
::-PCR (Phusion)<br />
:::-Digestion (A+P) product<br />
:::-Gel extraction product<br />
:::-No DNA control<br />
<br />
==Week 8==<br />
<br />
'''6/22'''<br />
*Did receive colonies from 6/21 transformations. Selected colonies for overnight<br />
{|class="wikitable"<br />
|-<br />
!number<br />
!colonies<br />
|-<br />
|1-7<br />
|pSB1C3-M13ori(NEW) in 5alpha<br />
|-<br />
|8-14<br />
|pSB1C3-M13ori(NEW) in ER2738<br />
|-<br />
|15-18<br />
|pSB1C3-M13ori(OLD) in ER2738<br />
|-<br />
|19<br />
|p110+RBS in 5alpha as control<br />
|-<br />
|20<br />
|p110+RBS in ER2738 as control<br />
|}<br />
<br />
*Gel of 6/21 O/N PCR<br />
::Digestion (AgeI+PstI) product ? Yielded the three bands that appear with every PCR of M13K07<br />
'''[[File:UCB-Phage Delivery-140622.JPG]]'''<br />
<br />
:1. M13K07-> digested (AgeI + PstI) -> PCR<br />
:2. M13K07-> digested (AgeI+ PstI) -> extraction-> PCR<br />
:3. No DNA control for PCR<br />
::-Gel extraction product ? No bands<br />
::-No DNA control ? No bands<br />
*PCR purified the above PCR (Digestion (AgeI+PstI) product) and PCR from 6/21<br />
*Digested PCR purified sample with EcoRI+PstI and ran gel<br />
'''[[File:UCB-Phage Delivery-140622-02.JPG]]'''<br />
:1. PCR-> PCR purified<br />
:2. Sample after DpnI digest<br />
:3. Sample after digestion with EcoRI and PstI<br />
:4. Samples after digestion with DpnI then EcoRI and PstI<br />
*Ligation of above digestion (M13K07 genes) with pSB1C3<br />
<br />
<br />
'''6/23'''<br />
*Transformed pSB1C3-M13ori(New) (6/22 ligation)<br />
*Check overnights from 6/22 for the correct insert<br />
::-Mini-prep O/Ns<br />
::-Digested with EcoRI and PstI<br />
'''[[File:UCB-Phage Delivery-140622-03.JPG]]'''<br />
:::1-7: pSB1C3-M13ori(New) in 5alpha<br />
:::8-14: pSB1C3-M13ori(New) in ER2738<br />
:::15-18: pSB1C3-M13ori(Old)<br />
::-All were the expected size (though gel wiggled)<br />
::-Sent 4 samples for sequencing (iGEM primers: VF2 and VR)<br />
:::# 4: Divergent (new) phagemid 1C3 transformed into 5alpha<br />
:::# 11, 13: Divergent (new) phagemid 1C3 transformed into ER2738<br />
:::# 16: Convergent (old) phagemid 1C3 transformed into ER2738<br />
<br />
'''6/24'''<br />
*Results of 6/23 transformation (pSB1C3-M13genes) from 6/22 ligation)<br />
::-Received ~100 colonies<br />
::-Set up O/N cultures for 8 of the colonies<br />
::-Time passed…..<br />
::-Mini-prepped the 8 O/N samples mentioned above (yes, it was a long day)<br />
:::*Digested samples (E+P)<br />
*Started phage amplification protocol<br />
::-Amount of phage added = 10.9ul of 1:10 diluted phage (M13 phage (1) from 5/24 (4.575 x10^12 phage/mL)) to a final concentration of 1x10^8 phage/mL in 50mL<br />
::-Managed to get both samples to the 14-28hr incubation<br />
<br />
'''6/25'''<br />
*Checked mini-prep samples from 6/24 (pSB1C3-M13genes)<br />
::Ran the digestion overnight (6/24 to 6/25)<br />
'''[[File:UCB-Phage Delivery-140625.JPG]]'''<br />
::-All contained a band at 2000bp. Mostly empty vector or contained small, light band. Sample 1 had prominent band at ~1250bp.<br />
::-None of samples contained M13genes<br />
*M13K07 is on P15A ori (10-12 copy number) whereas the pSB1C3 ori is on pUC19 (500-700 copies). It’s possible that this overexpression is detrimental to cell<br />
::Alternative low copy plasmids found in distribution kit<br />
:::*2013 (plate 5)<br />
::::pSB6A1 (1K)<br />
::::pSB3C5 (3C)<br />
::::pSB3K3 (5E)<br />
:::*2014 (plate 4)<br />
::::pSB3C5 (4D)<br />
::::pSB6A1 (2L)<br />
::-Suspended and transformed the above plasmids into 5alphas<br />
::-Next, we will select colonies, mini-prep, digest, gel extract, ligate with M13genes<br />
*Started phage amplification protocol <br />
::-Phagemids in ER2738<br />
:::pSB1C3-M13ori(New)<br />
:::pSB1C3-M13ori(Old) <br />
::-Set up O/N of ER2738 for infection tomorrow<br />
<br />
'''6/26'''<br />
*Results of transformation of plasmids from the distribution kit<br />
::-Only received colonies pSB6A1 (2L from 2014) and pSB3C5 (4D from 2014)<br />
::-Pricked colonies for O/N<br />
*PCR of M13 genes so we can ligate it into the above backbones tomorrow<br />
::-DNA: M13K07 digested with EcoRI+PstI (6/21)<br />
::-Primers: Gem012 F & R<br />
*Finished phage amplification protocol<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Samples<br />
! A269<br />
! A320<br />
! Concentration<br />
<br />
|-<br />
| Old phagemid 1C3 (1)<br />
| 0.832<br />
| 0.492<br />
| 7.828 x10^12<br />
<br />
|-<br />
| Old phagemid 1C3 (2)<br />
| 0.324<br />
| 0.083<br />
| 5.549 x10^12<br />
<br />
|-<br />
| New phagemid 1C3 (1)<br />
| 0.391<br />
| 0.077<br />
| 7.229 x10^12<br />
<br />
|-<br />
| New phagemid 1C3 (2)<br />
| 0.402<br />
| 0.639<br />
| -5.457 x10^12<br />
<br />
|-<br />
|}<br />
::::*While resuspending “New phagemid 1C3 (2)”, the tip fell off and we lost half of sample. Evidently, we lost most of phage so we tossed sample<br />
::-Prepped and Infected ER2738 with “Old phagemid 1C3 (1)” and “New phagemid 1C3 (2)”<br />
:::Added 1.6ul of 1:10 diluted “Old phagemid 1C3 (1) to ER2738 cells<br />
:::Added 1.8ul of 1:10 diluted “New phagemid 1C3 (1) to ER2738 cells<br />
::-Plated the infected cells at dilutions<br />
:::1:1 onto Chlor+Kan plates<br />
:::1:10 onto Chlor+Kan plates<br />
:::1:100 onto Chlor and onto Kan plates<br />
:::1:1000 onto Chlor and onto Kan plates<br />
:::1:10000 onto Chlor and onto Kan plates<br />
:::1:100000 onto Chlor and onto Kan plates<br />
<br />
'''6/27'''<br />
*To ligate M13genes onto different backbones<br />
::-Mini-prepped the O/Ns from 6/26 to get backbones with low copy number<br />
:::pSB6A1<br />
:::pSB3C5 <br />
::::*did not grow as well<br />
::-PCR purified PCR (to amplify M13 genes with Gem012 F & R) from 6/26<br />
::-Digestions (50ul)<br />
:::Digest pSB6A1 with E + P<br />
:::Digest pSB3C5 with E + P<br />
:::Digest PCR purification product with E + P<br />
'''[[File:UCB-Phage Delivery-140627.JPG]]'''<br />
:1. pSB6A1<br />
:2. pSB3C5<br />
:3. M13genes<br />
::-Gel extraction of the above digestions<br />
:::For each: Added 10ul of 6x loading dye to 50ul digestions and divided the total volume between 2 wells<br />
::-Ligation<br />
:::1) pSB6A1-M13genes<br />
:::2) pSB3C5-M13genes<br />
*Results from 6/26 infection (after 22hrs in incubator)<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Anti-Sense Phagemid 1C3<br />
! 1:100<br />
! 1:1000<br />
! 1:10000<br />
! 1:100000<br />
<br />
|-<br />
| Chlor<br />
| 85<br />
| 6<br />
| 2<br />
| 0<br />
<br />
|-<br />
| Kan<br />
| 405<br />
| 13<br />
| 2<br />
| 1<br />
<br />
|-<br />
|}<br />
{| class = "wikitable"<br />
|-<br />
! Sense Phagemid1C3<br />
! 1:100<br />
! 1:1000<br />
! 1:10000<br />
! 1:100000<br />
<br />
|-<br />
| Chlor<br />
| 121<br />
| 24<br />
| 3<br />
| 0<br />
<br />
|-<br />
| Kan<br />
| 25<br />
| 1<br />
| 0<br />
| 0<br />
<br />
|-<br />
|}<br />
-We also plated the infected cells on plates containing Chlor and Kan to test for the possibility of double infection<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Phagemid 1C3<br />
! 1:1<br />
! 1:10<br />
<br />
|-<br />
| Anti-Sense<br />
| 59<br />
| 0<br />
<br />
|-<br />
| Sense<br />
| 4<br />
| 0<br />
<br />
|-<br />
|}<br />
<br />
<br />
'''6/28'''<br />
*We noticed that there were more colonies on our infection plates from 6/26 than on 6/27; therefore, we recounted colonies<br />
::-No increase of colonies on Kan plates<br />
::-Significant increase of colonies on Chlor plates<br />
::-Sense refers to the first phagemid 1C3 where the M13ori is in the sense direction compared to the plasmid ori<br />
::-Anti-Sense refers to the new phagemid 1C3 where the M13ori is in the anti-sense direction compared to the plasmid ori<br />
'''[[File:UCB-Phage Delivery-140628.JPG]]'''<br />
::Numbers on the left are after 22 hours. Numbers on the right are after 38.75 hours<br />
*Results of transformation from 6/27 (pSB6A1-M13genes and pSB3C5-M13genes)<br />
::-We have many colonies. Unfortunately, some are red, suggesting that original insert (J04450) was not successfully separated from backbone through gel extraction<br />
::-Selected colonies to grow overnight in 5mL LB<br />
<br />
==Week 9==<br />
<br />
'''6/29'''<br />
*Check O/N cultures for correct constructs (pSB6A1-M13genes and pSB3C5-M13genes)<br />
::-Mini-prep samples<br />
::-Digested with EcoRI and PstI to check insert sizes<br />
::-Gel<br />
'''[[File:UCB-Phage Delivery-140629.JPG]]'''<br />
::lanes….<br />
:::1-11: pSB6A1-M13genes<br />
:::12-18: pSB3C5-M13genes<br />
::-Epic failure<br />
::-All pSB6A1 backbones are empty<br />
::-Half of pSB3C5 backbones were empty. The others contained random inserts (1700 OR 3500). We don’t know what these inserts are. 1700 band is likely the digestion product that appears when we digest M13genes<br />
*Question: Can we use empty vectors from these mini-preps as ligation vectors?<br />
::-Digest pSB6A1 mini-prep with…. (see if both cut sites were retained during re-ligation)<br />
:::no enzyme<br />
:::EcoRI<br />
:::PstI<br />
:::EcoRI + PstI<br />
'''[[File:UCB-Phage Delivery-140629-02.JPG]]'''<br />
::lanes....<br />
:::1-2: last two samples of pSB3C5-M13genes from above gel<br />
:::3. Uncut<br />
:::4. Cut with EcoRI<br />
:::5. Cut with PstI<br />
:::6. Cut with EcoRI and PstI<br />
:-Appears that two backbones are ligated together<br />
<br />
'''6/30'''<br />
*Made chemically competent ER2738 cells that contain Litmus28i DNA<br />
*To amplify M13genes in order to retry ligation<br />
::-PCR of M13K07 DNA (diluted 1:100) to amplify the M13 genes<br />
::-PCR purify PCR product<br />
::-Run on gel: PCR purification, PCR, noDNAcontrol<br />
:::*PCR and purification showed bands at ~9k; therefore, did not get product<br />
:::*No DNA control was clean<br />
*Made freeze downs of<br />
::pSB3C5-J04450<br />
::pSB6A1-J04450<br />
::‘empty’ pSB3C5<br />
::‘empty’ pSB6A1<br />
<br />
'''7/1'''<br />
*Digestion of PCR product from 6/30 to figure out where mistake is<br />
:-Not really useful<br />
'''[[File:UCB-Phage Delivery-140701.JPG]]'''<br />
:Lanes...<br />
:1. PCR pur -> digested with AgeI<br />
:2. PCR pur -> digested with NgoMIV<br />
:3. PCR pur -> digested with PstI<br />
:4. PCR pur -> uncut<br />
:5. PCR pur -> digested with dpnI<br />
:6. Uncut plasmid DNA<br />
*Talked to Mary: She says we were adding to much DNA<br />
::-PCR again. Used 1:100 dilution of 1:100 diluted M13K07 DNA. (aka 1:10000 dilution)<br />
'''[[File:UCB-Phage Delivery-140701-02.JPG]]'''<br />
*Received a beautiful band at 6k bp<br />
:lanes<br />
:1. 1:100 dilution<br />
:2. 1:1000 dilution<br />
:3. 1:10 000 dilution<br />
:4. No DNA control<br />
<br />
'''7/2'''<br />
*To biobrick M13 genes (pSB6A1-M13genes and pSB3C5-M13genes)<br />
::-PCR purified 7/1 PCR product (Primers = Gem012)<br />
::-Digested with EcoRI-HF and PstI-HF<br />
::-Ligation to<br />
::::pSBA61 (digested and gel extracted)<br />
::::pSB3C5 (digested and gel extracted)<br />
<br />
'''7/3'''<br />
*Transformation<br />
::-pSB6A1-M13genes into 5alpha cells and ER2738 with pSB1C3-M13ori<br />
::-pSB3C5-M13genes into 5alpha cells and ER2738 with Litmus28i<br />
::-Transformed into cells containing a phagemid in order to skip some steps<br />
<br />
'''7/3-7/7 Vacation!'''<br />
<br />
==Week 10==<br />
<br />
'''7/7'''<br />
*Made O/N cultures of 7/3 transformation colonies<br />
::-Transformation results were not recorded until 7/8 (see below)<br />
<br />
'''7/8'''<br />
*Transformation results ( CC = Chemically comp cells, ER = ER2738)<br />
::-No growth on No DNA controls<br />
:::ER-Litmus28i CC on AMP-Chlor-Tet<br />
:::ER-phagemid1C3 CC on AMP-Chlor-Tet <br />
:::5alpha CC on Amp <br />
:::5alpha CC on Chlor <br />
::-Lots of red colonies on positive controls, no white colonies<br />
:::ER-Litmus28i CC + pSB3C5 on AMP-Chlor-Tet<br />
:::ER-phagemid1C3 CC + pSB6A1 on AMP-Chlor-Tet<br />
::-Lots of white colonies on sample plates, some red colonies<br />
:::ER-Lit CC + M13genes-pSB3C5 on AMP-Chlor-Tet<br />
:::ER-phagemid1C3 CC + M13genes-pSB6A1 on AMP-Chlor-Tet<br />
:::5alpha CC + M13genes-pSB3C5 on Chlor<br />
:::5alpha CC + M13genes-pSB6A1 on Amp<br />
*Overnights from 7/7 look healthy<br />
::-Renamed O/N to have numbers instead of long names<br />
{| class = "wikitable"<br />
|-<br />
! Sample #<br />
! Cells<br />
! DNA<br />
<br />
|-<br />
| 1-5<br />
| ER-Lit<br />
| M13genes-pSB3C5<br />
<br />
|-<br />
| 6-10<br />
| ER-phagemid1C3<br />
| M13genes-pSB6A1<br />
<br />
|-<br />
| 11-15<br />
| 5alpha<br />
| M13genes-pSB3C5<br />
<br />
|-<br />
| 16-20<br />
| 5alpha<br />
| M13genes-pSB6A1<br />
<br />
|-<br />
|}<br />
::-Mini-prepped DNA<br />
::-Digest the mini-preps with EcoRI-HF and PstI-HF<br />
::-Run digestions on gel to check sizes<br />
'''[[File:UCB-Phage Delivery-140708.JPG]]'''<br />
<br />
:lanes<br />
::top-left: pSB3C5-M13genes in ER2738 with Litmus28i<br />
::top-right: pSB6A1-M13genes in ER2738 with pSB1C3-1C3<br />
::bottom-left: pSB3C5-M13genes in 5alpha<br />
::bottom-right: pSB6A1-M13genes in 5alpha<br />
:None are correct<br />
*Primers Gem013 came in. Resuspend and diluted primers<br />
::-O/N of pSB3C5 to use for PCR tomorrow<br />
<br />
'''7/9'''<br />
*Wanted to make Litmus28i biobrick compatible for use as a phagemid backbone for us and other iGEM teams<br />
::-Mini-prepped O/N of pSB3C5<br />
::-PCR of pSB3C5 to amplify J04450 with Litmus28i compatible cut sites<br />
:::Primers: Gem013<br />
::::*At the 3’ end, these primers are the same as VF2 and VR so will bind the region flanking J04450. This conserves the terminators that exist between the biobrick prefix and VF2 on one side and those between the biobrick suffix and VR on the other side. Therefore, we are amplifying, VF2 priming site, terminators, J04450, terminators, and VR priming site<br />
::::*At the 5’ end, these primers contain unique restriction sites found in the Litmus28i MCS<br />
*PCR of M13 genes…. again (did 4 samples)<br />
::-Used the 1:10000 dilutions<br />
<br />
'''7/10'''<br />
*Note: The following two projects were done in parallel when possible<br />
*To make Litmus28i biobrick compatible<br />
::-Ran gel of PCR from 7/9 (J04450 amplification with Gem013)<br />
'''[[File:UCB-Phage Delivery-140710.JPG]]'''<br />
<br />
:1-4: PCRs of M13genes<br />
:5. No DNA control for M13genes PCR<br />
:6. PCR of pSB3C5 backbone<br />
:7. No DNA control for pSB3C5 backbone<br />
::-PRC purified the PCR product<br />
::-Digestion #1<br />
:::Restriction enzymes had the same buffer conditions but different activation temperatures so we had to do a 2 part digestion<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Restriction enzymes<br />
! Notes<br />
<br />
|-<br />
| J04450<br />
| Sac1<br />
| PCR purified<br />
<br />
|-<br />
| Litmus 28i<br />
| Sac1<br />
| From NEB tube<br />
<br />
|-<br />
|}<br />
:::*Incubate 1hr at 37 C<br />
:::*Heat inactivated 20 minutes at 80C<br />
::-Digestion #2<br />
:::-Added 1ul BsmI to both samples<br />
:::-Incubated 1hr at 65C<br />
:::-Heat inactivated 20 minutes at 80C<br />
::Ran gel<br />
:::-See gel below<br />
:::-Tried to extract J04450 segment but received very low yield. Since band otherwise looked clean, we decided to redo digestion then skip straight to ligation<br />
::Repeat digestion #1 for J04450<br />
::Repeat digestion #2 for J04450<br />
::Ligation (10hr at 16C, 10 min at 80C)<br />
:::3. Litmus28i + J04450<br />
*To retry ligation to biobrick backbone<br />
::-Ran gel of PCR from 7/9 (M13genes amplified with Gem012)<br />
:::See gel below<br />
::-PCR purified the PCR product<br />
::-Digestion<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Restriction enzymes<br />
! Notes<br />
<br />
|-<br />
| M13 genes<br />
| EcoRI-HF + PstI-HF<br />
| PCR purified<br />
<br />
|-<br />
| 6A1<br />
| EcoRI-HF + PstI-HF<br />
| ‘empty’ pSB6A1<br />
<br />
|-<br />
| 3C5<br />
| EcoRI-HF + PstI-HF<br />
| contains J04450<br />
<br />
|-<br />
|}<br />
:Incubate 1hr at 37C<br />
:Heat inactivated 20 minutes at 80C<br />
::-Ran gel<br />
:::*See gel below<br />
:::*Tried to extract M13genes and pSB3C5 segments but received very low yields. Since bands otherwise looked clean, we decided to redo digestion then skip straight to ligation<br />
::-Repeat digestion for M13genes and pSB3C5<br />
::-Ligation (10h at 16C, 10m at 80C)<br />
:::1. pSB3C5 + M13genes<br />
:::2. pSB6A1 + M13genes<br />
*Gel<br />
'''[[File:UCB-Phage Delivery-140710-02.JPG]]'''<br />
:1. pSB1A3<br />
:2. Limtus28i<br />
:3. M13genes<br />
:4. pSB3C5<br />
:5. J04450<br />
<br />
'''7/11'''<br />
*Transformed ligations from 7/10 into 5alpha cells<br />
::1. pSB3C5 + M13genes<br />
::2. pSB6A1 + M13genes<br />
::3. Litmus28i + J04450<br />
<br />
<br />
'''7/12'''<br />
*Results of 7/11 transformation<br />
::-No growth on no DNA control (Amp or Chlor)<br />
::-Lots of growth on Lit-J04450 -> some red-> colonies are too close together to prick individual colonies<br />
:::-It’s possible that these red colonies are satellites =(<br />
:::-Swiped some and plated on new Amp plate (restreak)<br />
::-Lawn of positive control (Litmus 28i) on Amp<br />
::-Many colonies for L1 (pSB3C5-M13genes) and L2 (pSB6A1-M13genes)<br />
:::-Due to high number of red colonies on Lit-J04450 plate, we assumed that most of these colonies contain empty vector<br />
:::-Did not make O/N<br />
*Ligated M13 genes to pSB6A1<br />
::-Used the remained of our digested M13genes.<br />
::-Both digestions from 7/10<br />
*Infection Experiment<br />
::-had 3 cell stocks (each taken from a different colony the night before)<br />
::-Tested Litmus 28i, Tandem phagemid 1C3, and double infection<br />
:::*Therefore, there were 9 flasks total.<br />
::-negative control: Streaked parent cells (non-infected) onto Chlor, Amp, and kan<br />
::-Included double infection plates for Litmus 28i and phagemid 1C3<br />
<br />
==Week 11==<br />
<br />
'''7/13'''<br />
*Transformed 7/12 ligation (pSB6A1-M13genes)<br />
*Litmus28i-J04450<br />
::-All growth from 7/12 restreak was white<br />
::-Pricked some red colonies for liquid O/N-will hopefully see red tomorrow<br />
::-Pricked a few red colonies and put into 200ul H2O (a few colonies per tube- 2 tubes total).<br />
:::*Plated 150ul onto Amp plates<br />
::-Put original plate into incubator to hopefully get bigger colonies<br />
<br />
'''7/14'''<br />
*Finished TWIV ppt<br />
*Set up 2 liquid culture of red Litmus 28i colonies (Litmus28i-J04450)-- slow growth <br />
::-Mini-prepped red Litmus 28i ‘O/N’ from earlier in day<br />
::-Digested samples—to check for insert and correct cut sites<br />
*Made O/Ns of pSB6A1-M13genes colonies from 7/13 transformation<br />
<br />
<br />
'''7/15'''<br />
*Ran gel of Litmus28i-J04450 samples<br />
'''[[File:UCB-Phage Delivery-140715.JPG]]'''<br />
:1. J04450-Litmus28ibb #1 (EcoRI+PstI)<br />
:2. J04450-Litmus28ibb #1 (uncut)<br />
:3. J04450-Litmus28ibb #2 (EcoRI+PstI)<br />
:4. J04450-Litmus28ibb #2 (EcoRI+PstI)<br />
::*Accidentally added restriction enzymes<br />
:5. Litmus28i (EcoRI+PstI)<br />
::*Only has PstI site<br />
<br />
::-Verifies that Litmus28i-J00450 had correct cut sites<br />
::-From this point on, biobricked Litmus28i is called Litmus28ibb<br />
*Check 7/14 O/Ns for pSB6A1-M13genes<br />
::-Mini-prepped liquid cultures<br />
::-Digested with EcoRI and PstI<br />
::::Received bold bands of just under 4000bp. Some lanes had a very feint band ~330bp. None were the correct size<br />
*Transformed Litmus28ibb-J04450 into ER2738 cells<br />
*Remake phage packaging<br />
::-pSB1C3-M13ori(New)<br />
::-pSB1C3-M13ori(Old)<br />
<br />
'''7/16'''<br />
*Results of 7/15 transformation of Litmus28i into 6/30 ER2738 cells<br />
::-lawn on no DNA control-- most likely contaminated cells<br />
::::Streaked chem comp 5alpha and ER onto Amp and Amp+Tet plates to determine if problem is with cells or plates<br />
::-O/N culture of ER to make new chem comp cells<br />
<br />
'''7/17'''<br />
*Finished phage isolation<br />
::Note:<br />
:::*pSB1C3-M13ori (New): M13ori and plasmid ori are convergent<br />
:::*pSB1C3-M13ori (Old): M13ori and plasmid ori are tandem<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! 269nm<br />
! 320nm<br />
! [] phage/mL<br />
<br />
|-<br />
| pSB1C3-M13ori (New)<br />
| 0.386<br />
| 0.034<br />
| 8.126 E12<br />
<br />
|-<br />
| pSB1C3-M13ori (New)<br />
| 0.419<br />
| 0.036<br />
| 8.842 E12<br />
<br />
|-<br />
| pSB1C3-M13ori (Old)<br />
| 0.283<br />
| 0.025<br />
| 5.956 E12<br />
<br />
|-<br />
| pSB1C3-M13ori (Old)<br />
| 0.326<br />
| 0.024<br />
| 6.972 E12<br />
<br />
|-<br />
|}<br />
*Infection to compare the new and the old pSB1C3-M13ori<br />
::-Prepped cells for infection<br />
::-Infected cells with pSB1C3-M13ori (New) or pSB1C3-M13ori (New)<br />
::-and plated on dilutions of 1:100, 1:1000, and 1:10000 on Chlor and kan<br />
::-Also plated 1:1000 dilution on lowered Chlor concentration (34ug/mL)<br />
*Sent Litmus28ibb-J04450 for sequencing<br />
<br />
'''7/18'''<br />
*Results from contamination test<br />
::-5alpha on Amp = no growth<br />
::-5alpha on Amp+Tet = colonies<br />
::-ER on Amp = lawn<br />
::-ER on Amp+Tet = lawn<br />
*Made new chemically competent ER2738<br />
*Transformed Litmus28ibb-J04450 into ER2738 made on 3/30 and ER2738 made on 7/18<br />
::-Had two samples of Litmus28ibb-J04450 and two cell stocks, so 4 samples total<br />
<br />
'''7/19'''<br />
*Results of 7/17 infection<br />
::-pSB1C3-M13ori(Old) (tandem-when plasmid ori and M13ori point in the same direction) packages better than pSB1C3-M13ori(New) (when ori and M13ori are convergent), implying that directionality matters.<br />
'''[[File:UCB-phage lab7-19-141012.jpg]]'''<br />
<br />
==Week 12==<br />
<br />
'''7/20'''<br />
*Freeze downs<br />
:-Litmus28i-J04450 Litmus 28i is now biobrick compatible<br />
<br />
'''7/21'''<br />
*Send samples for sequencing<br />
::-pSB1C3-M13ori(Old)<br />
::-pSB1C3-M13ori(New)<br />
*To ligate M13ori to a kanamycin backbone (pSB1K3)<br />
::-Resuspended pSB1K3-J04450 from distribution kit (6B on plate 4)<br />
:::*Contains J04450 as insert (full RFP construct)<br />
::-Transform pSB1K3 into 5alpha cells<br />
<br />
'''7/22'''<br />
*Pricked colony from pSB1K3-J04450 transformation<br />
::-Mini-prepped DNA to get a supply of DNA<br />
*To put M13ori (M13 phackaging signal) onto Kanamycin resistance o we can test packaging ratios with M13K07 on the same antibiotic<br />
::-Digested pSB1K3 with EcoRI-HF and XbaI<br />
::-Digested pSB1C3-M13ori (Old) with EcoRI-HF and SpeI-HF<br />
:::*DNA was not sufficiently cut. Too much DNA? Problem with enzyme (SpeI-HF)?<br />
'''[[File:UCB-Phage Delivery-140722.JPG]]'''<br />
:1. pSB1K3<br />
:2. M13ori<br />
<br />
'''7/23'''<br />
*Sequencing samples from 7/21 were lost in the mail. Resent samples<br />
*Yet another attempt to biobrick M13genes<br />
::1. using primers that would amplify genes and M13 ori parts from M13K07<br />
::2. using primers that would amplify genes, M13 ori parts, and plasmid ori from M13K07<br />
'''[[File:UCB-Phage Delivery-140723.JPG]]'''<br />
:1. Amplified only the M13genes and M13ori<br />
:2. No DNA control for 1<br />
:3. Amplified M13genes, M13ori, and plasmid ori<br />
:4. No DNA control for 3<br />
::-When run on a gel, samples were clean with only one band at around 7kb. No contamination in no DNA controls<br />
*Digestions<br />
::-pSB1K3 with EcoRI-HF and PstI-HF to check for correct insert<br />
::-pSB1C3-M13ori (Old and new) with EcoRI-HF and SpeI-HF to test for efficient cutting with different stock of restriction enzyme<br />
'''[[File:UCB-Phage Delivery-140723-02.JPG]]'''<br />
:1-3: pSB1K3<br />
:4. pSB1C3-M13ori (Old)<br />
:5. pSB1C3-M13ori (New)<br />
*Still had inefficient cutting. Tested M13ori next to Litmus28i to disern the problem<br />
:*Uncut<br />
:*Cut once with (E, X, S, or P)<br />
:*Cut twice with (E+S or E+P)<br />
'''[[File:UCB-Phage Delivery-140723-03.JPG]]'''<br />
:1. M13ori uncut<br />
:2. M13ori Ecori-HF<br />
:3. M13ori SpeI-HF<br />
:4. M13ori XbaI<br />
:5. M13ori PstI-HF<br />
:6. M13ori EcoRI-HF + SpeI-HF<br />
:7. M13ori EcoRI-HF + PstI-HF<br />
:8. Litmus28i uncut<br />
:9. Litmus28i EcoRI-HF<br />
:10. Litmus28i SpeI-HF<br />
:11. Litmus28i XbaI<br />
:12. Litmus28i PstI-HF<br />
:13. Litmus28i EcoRI-HF + SpeI-HF<br />
:14. Litmus28i EcoRI-HF + PstI-HF<br />
*Only partial digest with only SpeI-HF for both. Complete digestion with all others, including E+S<br />
<br />
'''7/24'''<br />
*To Biobrick M13genes using pSB6A1<br />
::-PCR purified M13 genes (did both samples at the same time)<br />
::-Digested PCR purification and pSB6A1 with EcoRI-HF and PstI-HF<br />
'''[[File:UCB-Phage Delivery-140724.JPG]]'''<br />
*Gel extracted M13genes<br />
<br />
:1. M13genes<br />
:2. pSB6A1<br />
:-Bands of pSB6A1 were too light to gel extract<br />
*To swap the kanamycin resistance marker on M13K07 with ampicillin resistance<br />
::-PCR amplified AmpR from pSB6A1<br />
*made O/N culture of pSB6A1 from freeze down to mini-prep<br />
::-Also streaked cells onto plate<br />
<br />
'''7/25'''<br />
*To change resistance marker on M13K07<br />
:1. Biobrick method<br />
::*Tried to mini-prep O/N but pellet (after liquid culture was spun) was not red even after 16+ hours. Set up O/N from plate colonies<br />
:2. Swap only resistance method<br />
::*PCR purified 7/24 PCR<br />
::*Ran product on gel-> band of correct size<br />
'''[[File:UCB-Phage Delivery-140725.JPG]]'''<br />
<br />
:1. PCR product<br />
:2. PCR product-> PCR purified<br />
:3. No DNA control<br />
::-Digested the PCR purification products of M13genes+ori and AmpR with AgeI-HF and NotI-HF<br />
::*Bands on previous gel looked clean enough for both so did not gel extract<br />
::-Overnight ligation<br />
*Set up O/Ns of pSB1K3 colonies<br />
<br />
'''7/26'''<br />
*Transform<br />
::-pSB6A1+M13genes<br />
*To biobrick M13genes<br />
::-Digestion<br />
:::*pSB6A1 with EcoRI and PstI<br />
'''[[File:UCB-Phage Delivery-140726.JPG]]'''<br />
:Gel extracted backbone (band ~4000bp)<br />
*Ligations (10hrs 16C, 10min at 80C)<br />
::-pSB6A1+M13genes<br />
<br />
==Week 13==<br />
<br />
'''7/27'''<br />
*Transformation results from 7/26<br />
::-No growth for no DNA controls ''on Amp''<br />
::-No growth for M13 genes onto pSB6A1 ''on Amp''<br />
*Set up O/Ns of 5alpha and ER2738 to make competent cell tomorrow<br />
*Made freeze down of pSB1K3<br />
*Plated ER competent cells from 5/15 and 6/16 Onto Chlor plates to check for contamination<br />
<br />
'''7/28'''<br />
*No growth of ER2738 on Chlor (either sample)<br />
::-ER is not contaminated with ChlorR<br />
*Make new 5alpha chem comp cells <br />
*Transformations<br />
::-pSB6A1-M13genes<br />
::-positive control for Amp (pSB6A1)<br />
::-No DNA control<br />
<br />
'''7/29'''<br />
*Set up O/Ns of pSB6A1-M13genes<br />
::-Plated sample on reduced Amp and regular Tet<br />
::-Later in day…<br />
::-Mini-prepped O/Ns<br />
::-Digested and ran on a gel to check for insert sizes<br />
'''[[File:UCB-Phage Delivery-140729.JPG]]'''<br />
:*All but the 4th lane with 3 bands look correct<br />
<br />
'''7/30'''<br />
*Lawn on last night’s plating-> replate<br />
*Digest pSB3C5 and pSB6A1-M13genes with E and P to move M13genes to a Chlor backbone<br />
'''[[File:UCB-Phage Delivery-140730.JPG]]'''<br />
::-Extracted bands<br />
::-Ligations<br />
:::*pSB3C5 + M13genes<br />
*Sent samples for sequencing<br />
**M13 genes on pSB6A1 VF2<br />
**M13 genes on pSB6A1 VR<br />
<br />
'''Test experiment for high school kids coming to lab (7/29-7/31)'''<br />
*General idea: Have 2 strands of DNA, one has an EcoRI site while the other contains a SNP, abolishing the cut sight. We pretend that one is pathogenic (the one that is not cut) and tells kids to figure out which one is which <br />
*PCR with Dream-Taq<br />
::-Did not have special fastdigest enzyme<br />
::-Hypothesized that green dye will interfere with enzyme effectivity<br />
*PCR with Dream-Taq (no green dye)<br />
::-Digest with EcoRI fastdigest-> did not cut<br />
*Redid experiment several times<br />
::-Should digest with XbaI<br />
::-PCR purified PCR products<br />
<br />
'''7/31'''<br />
*Spent the morning with Heritage High School<br />
*Made phage<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Phage<br />
! A269<br />
! A320<br />
! genome size<br />
! [ ]<br />
<br />
|-<br />
| Litmus28ibb-J04450 (1)<br />
| 0.823<br />
| 0.197<br />
| <br />
| 9.35 x10^12<br />
<br />
|-<br />
| Litmus28ibb-J04450 (2)<br />
| 0.940<br />
| 0.168<br />
| <br />
| 1.14 x10^13<br />
<br />
|-<br />
|}<br />
<br />
'''8/2'''<br />
*Digestion<br />
::-pSB6A1-M13genes (E+P)<br />
::-pSB3C5 (E+P)<br />
<br />
==Week 14==<br />
<br />
'''8/3'''<br />
*Another attempt to clone M13genes onto pSB3C5<br />
::-Run 8/2 digestions on gel<br />
'''[[File:UCB-Phage Delivery-140803.JPG]]'''<br />
:1. M13genes (E+P)<br />
:2. pSB3C5 (E+P)<br />
::-Gel extracted <br />
:::*M13genes away from pSB6A1 backbone<br />
:::*pSB3C5 backbone away from J04450<br />
::-Ligations<br />
:::*pSB3C5 + M13genes<br />
:::*pSB3C5 + no insert<br />
::-Transformed ligations into ER2738<br />
<br />
'''8/5'''<br />
*Transformation of pSB3C5+M13genes failed<br />
*Made phage<br />
::-Fd-CAT DNA packaged with Fd-CAT<br />
::-phagemid 1C3 packaged with M13K07<br />
::-amilCP on pSB1C3 packaged with M13K07<br />
::-Litmus28ibb-J04450 packaged with M13K07<br />
<br />
'''8/6'''<br />
*Isolated phage<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Phage<br />
! [ ]<br />
<br />
|-<br />
| Fd CAT<br />
| 2.72 x10^12<br />
<br />
|-<br />
| Litmus28ibb-J04450<br />
| 8.37 x10^12<br />
<br />
|-<br />
| pSB1C3-amilCP<br />
| 1.42 x10^13<br />
<br />
|-<br />
| pSB1C3-M13ori<br />
| 4.32 x10^12<br />
<br />
|-<br />
|}<br />
<br />
'''8/6'''<br />
*Started making phage with Fd CAT the should contain Litmus28ibb-RFP<br />
*Infection<br />
::-Infected ER2738 cells with Litmus28ibb-RFP, pSB1C3-M13ori, or pSB1C3-amilCP<br />
::-Plated Litmus samples on 100ug/mL Amp and 50ug/mL Kan<br />
::-Plated other two samples on 34ug/mL Chlor and 25ug/mL Kan<br />
<br />
'''8/7'''<br />
*Isolated phage containing Litmus28ibb-RFP using helper phage<br />
::-M13K07 (as control)<br />
::-Fd-CAT (no phage pellet was observed-worried that there was no phage)<br />
*Went to CSU to have them test M13ori part compared to amilCP. Grew the samples in 5mL O/Ns then diluted to 0.5OD and grew 30 minutes rather than starting from a fresh colony<br />
<br />
'''8/8'''<br />
*At CSU<br />
::-Finished phage protocol (Test packaging of M13ori part)<br />
::-Only used 20mL infection samples<br />
::-grew to 0.55 OD<br />
::-When making phage (after 14 hour incubation), there was very little growth<br />
::-Plated non-diluted and diluted 1:1000 of M13ori and amilCP sample<br />
*Infected cells using phage isolated 8/7. Plated at dilutions of 1:100 and 1:100k<br />
*Want to test progeny Fd-CAT phage (made 8/7) for infectability<br />
::-Start phage isolation prodocol using Fd-CAT phage from 8/7 do deliver the helper phagemid<br />
:::*Phagemid:<br />
::::*None: will make Fd-CAT phage packaing Fd-CAT phagemid<br />
::::*Litmus28ibb-J04450: make Fd-CAT phage packaging Litmus28ibb-J04450 phagemid<br />
*Remake phage (Fd-CAT) using fresh stock from Mike<br />
<br />
<br />
'''8/9'''<br />
*Results of 8/8 infection<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Helper Phagemid<br />
! phagemid<br />
! Selection<br />
! 1:1000 dilution<br />
! 1:00k dilution<br />
<br />
|-<br />
| M13K07<br />
| Litmus28ibb-RFP<br />
| Kan+Tet<br />
| 400-500 (some red)<br />
| 61 white; 20 red<br />
<br />
|-<br />
| same<br />
| same<br />
| Amp+Tet<br />
| lawn<br />
| 2<br />
<br />
|-<br />
| Fd-CAT<br />
| Litmus28ibb-RFP<br />
| Chlor+Tet<br />
| 0<br />
| 0<br />
<br />
|-<br />
| same<br />
| same<br />
| Amp+Tet<br />
| 10<br />
| 707<br />
<br />
|-<br />
|}<br />
*NOTE: The same sample was plated on two different plates. For example, the sample using M13K07 as the helper phage was plated on Kan+Tet and Amp+Tet<br />
*Mini-prep<br />
::-pSC3C5-J04450 -- very little growth, low DNA yield<br />
::-Fd-CAT infected cells<br />
:::*Digest and run on a gel to verify presence of a band- there was a very feint band<br />
*Finished isolating phage…. messed up and used 0.8MgCl2, 0.2M NaCl instead of PEG during precipitation step. Also used the wrong phage<br />
::-Infected ER2738 cells anyway…. no growth by 8/12<br />
<br />
==Week 15==<br />
<br />
'''8/10'''<br />
*Are the progeny phage from 8/6 (original progeny from first Fd-Tet application) viable/ able to reproduce?<br />
::-Used these progeny to make phage that should amplify Fd-CAT phage containing either the Fd-CAT or Litmus28ibb-J04450 phagemid<br />
<br />
'''8/11'''<br />
*Finished isolating phage<br />
{| class = "wikitable"<br />
|-<br />
! Helper Phagemid<br />
! Phagemid<br />
! A269<br />
! A320<br />
! genome size<br />
! [ ]<br />
<br />
|-<br />
| Fd-CAT<br />
| Litmus28ibb (1)<br />
| 0.029<br />
| 0.013<br />
| 4080<br />
| 2.35 x10^11<br />
<br />
|-<br />
| Fd-CAT<br />
| Ltimus28ibb (2)<br />
| 0.033<br />
| 0.015<br />
| 4080<br />
| 2.65 x10^11<br />
<br />
|-<br />
| Fd-CAT<br />
| Fd-CAT (1)<br />
| 0.326<br />
| 0.082<br />
| 7775<br />
| 1.88 x10^12<br />
<br />
|-<br />
| Fd-CAT<br />
| Fd-CAT (2)<br />
| 0.320<br />
| 0.079<br />
| 7775<br />
| 1.86 x10^12<br />
<br />
|-<br />
| M13g6A1<br />
| M13g6A1 (1)<br />
| 0.322<br />
| 0.148<br />
| 10,029<br />
| 1.04 x10^12<br />
<br />
|-<br />
| M13g6A1<br />
| M13g6A1 (2)<br />
| 0.503<br />
| 0.130<br />
| 10,029<br />
| 2.23 x10^12<br />
<br />
|-<br />
|}<br />
<br />
'''8/13'''<br />
*Infected ER2738 with Fd-CAT packaging Litmus28ibb-J04450<br />
<br />
'''8/16'''<br />
*Results from Infection on 8/11<br />
{| class = "wikitable"<br />
|-<br />
! Helper Phagemid<br />
! Phagemid<br />
! 1:1<br />
! 1:10<br />
! 1:100<br />
! 1:1000<br />
<br />
|-<br />
| M13g6A1<br />
| M13g6A1<br />
| almost lawn<br />
| 728<br />
| 287<br />
| 55<br />
<br />
|-<br />
| Fd-CAT<br />
| Fd-CAT<br />
| lawn<br />
| almost lawn<br />
| 1260<br />
| 640<br />
<br />
|-<br />
| Fd-CAT<br />
| Litmus28ibb-J04450<br />
| lawn<br />
| almost lawn<br />
| 2004<br />
| 304<br />
<br />
|-<br />
|}</div>Leighlahttp://2014.igem.org/File:UCB-interlab_study_gragh-141016.JPGFile:UCB-interlab study gragh-141016.JPG2014-10-16T17:22:56Z<p>Leighla: </p>
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==Our Final System: CRISPR-Cas9 phage==<br />
<br />
The final System is comprised of two parts: the delivery of the CRISPR-Cas9 machinery and the targeting of the endonuclease. <br />
<br />
[[File:UCB-Results01.jpg]]<br />
<br />
M13 phage were used as the delivery mode in this system. Depicted above is a single bacterium containing a helper phagemid and a phagemid. The phage coat and assembly proteins are expressed from the helper phagemid but this packaging signal was disrupted by the insertion of a selectable marker (either kanamycin in M13K07 or ampicillin in M13g6A1, see below) and a plasmid origin of replication so its ability to be uptaken into a phage capsid is hindered. The phagemid contains the construct to be delivered (in this case, the CRISPR-Cas9 construct) and a fully functional packaging signal so it will be packaged into the assembling phage. The produced phage containing the phagemid are isolated and can be used to infect a target population of bactium.<br />
<br />
<br />
[[File:UCB-Results02.jpg]]<br />
<br />
Once the CRISPR-Cas9 phagemid enters the cell, the bacterium will express the Cas9 endonuclease and a guide RNA (gRNA, target sequence is determined by spacer). These two parts come together and search the bacterial genome for PAM sites (Protospacer Adjacent Motifs, shown here is orange). The Cas9 will bind to the PAM site allowing the gRNA to anneal to the target sequence (shown here in green). If binding is successful, the Cas9 endonuclease cleaves the DNA resulting in a double stranded break. If the cell is unable to repair itself or does so incorrectly, the cell will die.<br />
<br />
<br />
New BioBricked Parts<br />
*BBa_K1445000: The M13 origin of replication (M13ori) is a noncoding sequence that when cloned onto a plasmid, facilitates the uptake of that plasmid into an M13 phage capsid.<br />
*BBa_K1445001: M13ori (BBa_K1445000) cloned onto a CRISPR-Cas9 construct (BBa_K1218011). The CRISPR-Cas9 part codes for a Cas9 endonuclease that is guided to a DNA sequence specified by a spacer located within the minimal CRISPR array. Inclusion of the M13ori allows for its uptake into M13 phage.<br />
*BBa_K1445002: BioBricked phagemid vector allows for the easy cloning of BioBrick parts into a backbone that can be packaged into a phage without the addition of the M13ori to the insert region.<br />
*Additionally, the genes for M13 phage coat proteins were BioBricked and cloned onto a pSB6A1 vector. This allows the phage producing genes to be cloned onto vectors with a different resistance marker if the resistance interferes with the marker on the phagemid. This part was not submitted to the registry.<br />
<br />
<br />
Phage delivery system (M13ori)<br />
The M13 origin of replication (M13ori) has been documented as the packaging signal for the M13 phage. When single stranded, the M13ori forms unique hairpins that signal packaging into a phage capsid. To verify its functionality, its packaging efficiency was compared to that of amilCP. The amilCP sequence is roughly equal in length to the M13ori and both parts were cloned onto the pSB1C3 backbone; therefore, the nucleotide sequence is the only distinguishing factor. The helper phagemid M13K07 (a plasmid containing M13 phage structural proteins and a weakened packaging signal) made phage in host cells containing pSB1C3-M13ori or pSB1C3-amilCP phagemids. ER2738 E. coli were infected by phage from the M13ori or amilCP sample to assess the respective packaging efficiencies.<br />
<br />
<br />
<br />
<br />
<br />
[[File:UCB-Results03.jpg]]<br />
<br />
Figure 1: Conjugated BW23115 E. coli infected with recombinant phage A) pSB1C3-M13ori or B) pSB1C3-amilCP phagemids on LB agar with 170 ug/mL chloramphenicol.<br />
<br />
The growth in the M13ori sample demonstrates that this phagemid was packaged and delivered by the M13 phage. The absence of growth in the amilCP sample proves that without the presence of a packaging signal, a plasmid cannot be taken up by a phage capsid. These results show that the M13ori is necessary and sufficient for phagemid packaging.<br />
<br />
Making phage with M13g6A1<br />
For the previously mentioned experiment, M13K07 was used as the helper phagemid. However, this part contains a kanamycin resistance gene, which may interfere with the analysis of Cas9 targeting of the neomycin phosphotransferase gene, which also confers kanamycin resistance.<br />
The genes that code for the phage coat proteins were amplified from M13K07 and cloned onto the pSB6A1 vector.<br />
Phage amplification using M13g6A1 as the helper phagemid (and no additional phagemid) produced M13 phage packaging M13g6A1. ER2738 cells were infected with the isolated phage and were plated onto ampicillin to verify the viability of phage produced by this helper phagemid.<br />
<br />
<br />
<br />
[[File:UCB-Results04.jpg]]<br />
<br />
Figure 2.<br />
The presence of colonies in Figure 2 confirm that M13g6A1 is capable of producing viable phage.<br />
<br />
Program to design a Spacer Sequence<br />
For the following Cas9 experiments, a spacer sequence was designed to target the neomycin phosphotransferase gene present in the targeting strain.<br />
This program inputs FASTA files in two sets. In one are the sequences that should be targeted for Cas9 binding while the other set consists of sequences that should not be targeted. It then searches for possible targeting sites that are present in the targeting sequences and absent from the sequences that should not be targeted. The best spacer sequences are then presented to the user.<br />
<br />
For this project, the sequence for the neomycin phosphotransferase gene was given to the program as the targeting sequence. The E. coli K-12 and E. coli MG1655 genomes were given to the program as sequences to no target.<br />
One of the output spacers was chosen for use in the following CRISPR-Cas9 experiments.<br />
<br />
Targeted killing of BW through Transformation (characterization of BBa_K1218011)<br />
The spacer designed to target the neomycin phosphotransferase gene was cloned into the CRISPR-Cas9 construct (BBa_K1218011). The unmodified spacer does not target the production or target strain so was used as a non-targeting control.<br />
The targeting and non-targeting constructs were transformed into the BW23115 E. coli containing the neomycin phosphotransferase through the heat shock method and selected for on chloramphenicol.<br />
<br />
<br />
<br />
[[File:UCB-Results05.jpg]]<br />
<br />
Figure 3: Transformation results of neomycin resistant E. coli with Cas9 part having either A) non-targeting or B) targeting spacer sequence on LB agar with 170 ug/mL chloramphenicol.<br />
<br />
The decrease in growth between the non-targeting (1920 colonies) and the targeting (8 colonies) samples is accredited to the difference in spacer sequence. When the spacer sequence exists in the genome of cell, the Cas9 endonuclease is able to bind and cleave the DNA, resulting in cell death, making it possible to intentionally kill bacteria by sequence specific Cas9 targeting through transformation.<br />
<br />
Interestingly, some targeted colonies survived as can be seen in Figure 1B. Sequencing showed that all eight colonies had deleted the spacer region and one or both of the adjacent repeats.<br />
<br />
Cas9 delivery through phage<br />
The M13 origin of replication, or M13ori (BBa_K1445000) was cloned upstream of the CRISPR-Cas9 construct (BBa_ K1218011) in the targeting and the non-targeting varieties to allow for their uptake into M13 phage. This composite part was packaged into phage using the M13g6A1 helper phagemid mentioned above. Progeny phage infected conjugated versions of the BW23115 E. coli containing the neomycin phosphotransferase gene and cells were plated onto chloramphenicol.<br />
<br />
<br />
<br />
[[File:UCB-Results06.jpg]]<br />
<br />
Figure 4: Infection results of M13 phage delivering A) non-targeting and B) targeting varieties of part BBa_K1445001 on LB agar with 170 ug/mL chloramphenicol.<br />
<br />
The presence of growth in both samples demonstrates that the addition of the M13ori does allow for packaging and delivery by the M13 phage.<br />
The decrease in growth from the non-targeting (143 colonies) to the targeting (11 colonies) is accredited to the differences in spacer sequence. With a spacer sequence that targets within the genome of the cell, the Cas9 endonuclease is able to bind and cleave the DNA, successfully killing the bacterium.<br />
<br />
Unmodified spacer may have off-target binding to FҠepisome, causing cell death in the production strain or targeting strain. (I tried but could NOT find the sequence. Any idea how we can say we donӴ know without sounding lazy?)<br />
<br />
<br />
Additional Work and Characterization (BBa_K1445002)<br />
Part BBa_J04450 and flanking regions to the sequencing primer sites was cloned into the Litmus28i vector to make a biobricked phagemid backbone that can be packaged into M13 phage.<br />
To verify functionality, the vector was packaged into phage using the M13K07 helper phagemid. Progeny phage were infected ER2738 e. coli and cells were selected for on ampicillin.<br />
<br />
[[File:UCB-Results07.jpg]]<br />
<br />
Figure 5: Packaging efficiency of recombinant phagemid to helper phagemid packaging. A) Sample diluted 1:1000 and grown on 100 ug/mL Ampicillin to select for BBa_K1445002. B) Sample diluted 1:1000 and grown on 50 ug/mL Kanamycin to select for M13K07 helper phagemid. BBa_K1445002 was packaged over M13K07 at a ratio of 136:1.<br />
<br />
The growth on ampicillin (1496 colonies) shows that the biobricked version of Litmus28i retains its ability to be packaged and delivered by M13 phage. Additionally, the same sample was plated on kanamycin to determine the packaging ratio of phagemid to helper phagemid. As shown in Figure 1B), very little growth was present on kanamycin (11 colonies) confirming that BBa_K1445002 was preferentially packaged over the M13K07 helper phagemid at a ratio of 136:1.<br />
<br><br />
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<h2 style="font-size: 2em;">Meet the CU Boulder iGem Team</h2><br />
<img style="width: 100%" src="http://dowell.colorado.edu/assets/images/igem2014.jpg" title="iGEM CU-Boulder 2014" /><br />
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<br />
<!--Students--><br />
<h1 style="font-size: 2em;">Students</h1><br />
<!-- Julissa Duran-Malle --><br />
<div class="row flush images"><br />
<div class="6u"><a href="https://static.igem.org/mediawiki/2014/b/be/Julissa.jpg" class="image full l"><img src="https://static.igem.org/mediawiki/2014/b/be/Julissa.jpg" title="Julissa Malle" alt="" /></a></div><br />
<div class="6u"><div class="image full r" style="padding-top: 10px;"><h1 style="font-size: 2em;">Julissa Duran-Malle</h1><p style="text-align: left; padding-left: 15px; padding-right: 15px; font-size: 1em;">I am majoring in MCDB and Neuroscience. I am involved in all the experiments in the GOLD lab. I enjoy learning about other cultures, I plan on going to Tibet next summer <br />
</p></div></div><br />
</div><br />
<!-- Daniel Garey --><br />
<div class="row flush images"><br />
<div class="6u"><div class="image full l" style="padding-top: 10px;"><h1 style="font-size: 2em;">Daniel Garey</h1><p style="text-align: left; padding-left: 15px; padding-right: 15px; font-size: 1em;">I am a junior student majoring in MCDB and Neuroscience. For the iGem I am doing dual infection project as well as the wiki. I am involved in designing and making two separate plasmids, each containing a part of the CRISPR Cas9 system, to be delivered individually by separate phage. <br />
I enjoy backpacking and scuba diving and am fascinated by other cultures</p></div><br />
</div><br />
<div class="6u"><a href="https://static.igem.org/mediawiki/2014/f/ff/ThumbGarey.png" class="image full r"><img src="https://static.igem.org/mediawiki/2014/f/ff/ThumbGarey.png" title="Daniel Garey" alt="" /></a></div><br />
</div><br />
<br />
<!-- Josephina Hendrix --><br />
<div class="row flush images"><br />
<div class="6u"><a href="https://static.igem.org/mediawiki/2014/9/9c/ThumbHendrix.jpg" class="image full l"><img src="https://static.igem.org/mediawiki/2014/9/9c/ThumbHendrix.jpg" title="Josephine Hendrix" alt=""></a></div><br />
<div class="6u"><div class="image full r" style="padding-top: 10px;"><h1 style="font-size: 2em;">Josephina Hendrix</h1><p style="text-align: left; padding-left: 15px; padding-right: 15px; font-size: 1em;">I am majoring in Molecular Cellular and Developmental Biology and Computer Science. I am focusing on phage delivery system for the iGEM project. Outside the project, I am working the Dowell lab, focusing on gene regulation and cloning in yeast. This fall I resume my project: to determine the extent of pre- and post-transcriptional gene regulation in trisomy 21 individuals.</p></div></div><br />
<br />
</div> <br />
<!-- Joshua Ivie --><br />
<div class="row flush images"><br />
<div class="6u"><div class="image full l" style="padding-top: 10px;"><h1 style="font-size: 2em;">Joshua Ivie</h1><p style="text-align: left; padding-left: 15px; padding-right: 15px; font-size: 1em;">I am a senior majoring in MCDB. I am working with the Garcea group<br />
</p></div></div> <br />
<div class="6u"><a href="https://static.igem.org/mediawiki/2014/c/c1/ThumbCustom.png" class="image full r"><img src="https://static.igem.org/mediawiki/2014/1/18/UCB-profile_Josh-141016.JPG" title="Joshua Ivie" alt=""></a></div><br />
</div><br />
<!-- Daren Kraft --><br />
<div class="row flush images"><br />
<div class="6u"><a href="https://static.igem.org/mediawiki/2014/b/b1/ThumbDaren.jpg" class="image full l"><img src="https://static.igem.org/mediawiki/2014/b/b1/ThumbDaren.jpg" title="Daren Kraft" alt=""></a></div> <br />
<div class="6u"><div class="image full r"/><h1 style="font-size: 2em;">Daren Kraft</h1><p style="text-align: left; padding-left: 15px; padding-right: 15px; font-size: 1em;">I am a junior double majoring in Molecular, Cellular, and Developmental Biology and Biochemistry. My contributions to the iGEM project include concept and experiment design, CRISPR-Cas constructs, and drafting of cloning protocol. When not in the lab, I enjoy hiking, climbing, and camping. <br />
</p></div></div><br />
</div><br />
<!-- Andrea Mariani --><br />
<div class="row flush images"><br />
<div class="6u"><div class="image full l" style="padding-top: 10px;"><h1 style="font-size: 2em;">Andrea Mariani</h1><p style="text-align: left; padding-left: 15px; padding-right: 15px; font-size: 1em;">I am junior majoring in MCDB. I am focusing on dual infection as well as putting Cas9 and CRISPR on separate plasmids. <br />
</p></div></div> <br />
<div class="6u"><a href="https://static.igem.org/mediawiki/2014/c/c1/ThumbCustom.png" class="image full r"><img src="https://static.igem.org/mediawiki/2014/c/c1/ThumbCustom.png" title="Andrea Mariani" alt=""></a></div><br />
</div><br />
<!-- Alex Martinez --><br />
<div class="row flush images"><br />
<div class="6u"><a href="https://static.igem.org/mediawiki/2014/a/af/ThumbMartinez.png" class="image full l"><img src="https://static.igem.org/mediawiki/2014/a/af/ThumbMartinez.png" title="Alexander Martinez" alt=""></a></div> <br />
<div class="6u"><div class="image full r" style="padding-top: 10px;"><h1 style="font-size: 2em;">Alexander Martinez</h1><p style="text-align: left; padding-left: 15px; padding-right: 15px; font-size: 1em;">I am a senior majoring in MCDB. Among my contributions to the projects are dual-infection experiments, Standford-Brown <br />
</p></div></div> <br />
<!-- Kirill Novik --><br />
<div class="row flush images"> <br />
<div class="6u"><div class="image full l" style="padding-top: 10px;"><h1 style="font-size: 2em;">Kirill Novik</h1><p style="text-align: left; padding-left: 15px; padding-right: 15px; font-size: 1em;">I am a senior majoring in MCDB and minoring in Computer Science. My contribution to the project is the wiki page. Outside of the lab, I am working in the Lowry lab studying genome of Mycobacterium vaccae utilizing various bioinformatics methods. <br />
</p></div></div> <br />
<div class="6u"><a href="https://static.igem.org/mediawiki/2014/4/4a/ThumbNovik.png" class="image full r"><img src="https://static.igem.org/mediawiki/2014/4/4a/ThumbNovik.png" title="Kirill Novik" alt=""></a></div> <br />
</div><br />
<br />
<br />
<!-- Kendra Shattuck --><br />
<div class="row flush images"><br />
<div class="6u"><a href="https://static.igem.org/mediawiki/2014/5/56/ThumbShattuck.png" class="image full l"><img src="https://static.igem.org/mediawiki/2014/5/56/ThumbShattuck.png" title="Kendra Shattuck" alt=""></a></div><br />
<div class="6u"><div class="image full r" style="padding-top: 10px;"><h1 style="font-size: 2em;">Kendra Shattuck</h1><p style="text-align: left; padding-left: 15px; padding-right: 15px; font-size: 1em;">I am a senior majoring in Molecular, Cellular, and Developmental Biology. My part on the iGEM project includes Interlab Measurement Study, Outreach, and the CRISPR-Cas9 plasmid engineering. <br />
</p></div></div><br />
</div><br />
<!-- Leighla Tayefeh --><br />
<div class="row flush images"><br />
<div class="6u"><div class="image full l" style="padding-top: 10px;"><h1 style="font-size: 2em;">Leighla Tayefeh</h1><p style="text-align: left; padding-left: 15px; padding-right: 15px; font-size: 1em;">I am a senior double majoring in Molecular, Cellular, and Developmental Biology and Neuroscience. Sythetic biology's vast potential to benefit society attracted me to join iGEM. I am involved in engineering a plasmid devoted to the CRISPR/Cas9 system. Outside of lab, I enjoy reading, hiking, and spending time with family and friends. <br />
</p></div></div> <br />
<div class="6u"><a href="https://static.igem.org/mediawiki/2014/a/ac/ThumbTayefeh.png" class="image full r"><img src="https://static.igem.org/mediawiki/2014/a/ac/ThumbTayefeh.png" title="Leighla Tayefeh" alt=""></a></div><br />
</div><br />
<!-- Justine Wagner --><br />
<div class="row flush images"><br />
<div class="6u"><a href="https://static.igem.org/mediawiki/2014/4/44/ThumbWagner.png" class="image full l"><img src="https://static.igem.org/mediawiki/2014/4/44/ThumbWagner.png" title="Justine Wagner" alt=""></a></div> <br />
<div class="6u"><div class="image full r" style="padding-top: 10px;"><h1 style="font-size: 2em;">Justine Wagner</h1><p style="text-align: left; padding-left: 15px; padding-right: 15px; font-size: 1em;">I am majoring MCDB and Biochemistry. I work in the Dowell lab alongside Jo (working with sythetic phagemid product). In addition to iGEM, I also conduct research in the Bowman research group with joint collaboration with Musgrave research group in which my major work consists of synthesizing various APO derived photo-initiators. When I graduate next year, I hope to be accepted into a top bioengineering graduate program where my research will be focused on either tissue engineering and/or bioartificial organ synthesis.<br />
</p></div></div><br />
</div><br />
<!-- Rishabh Yadav --><br />
<div class="row flush images"><br />
<div class="6u"><div class="image full l" style="padding-top: 10px;"><h1 style="font-size: 2em;">Rishabh Yadav</h1><p style="text-align: left; padding-left: 15px; padding-right: 15px; font-size: 1em;">I am a junior in highschool. I am developing a computer program that searches the target sites for Cas9<br />
</p></div></div> <br />
<div class="6u"><img src="https://static.igem.org/mediawiki/2014/thumb/c/c9/UCB-profile_Rishabh-141013.jpg/800px-UCB-profile_Rishabh-141013.jpg" title="Rishabh Yadav" alt="" class="image full r"></a></div> <br />
</div><br />
<!-- Sarah Zimmerman --><br />
<div class="row flush images"><br />
<div class="6u"><a href="https://static.igem.org/mediawiki/2014/5/5b/UCB-profile_Sarah-101413.JPG" class="image full l"><img src="https://static.igem.org/mediawiki/2014/5/5b/UCB-profile_Sarah-101413.JPG" title="Sarah Zimmer" alt=""></a></div><br />
<div class="6u"><div class="image full r" style="padding-top: 10px;"><h1 style="font-size: 2em;">Sarah Zimmermann</h1><p style="text-align: left; padding-left: 15px; padding-right: 15px; font-size: 1em;">I am a senior majoring in MCDB. I am working on the dual infection project.<br />
</p></div></div> <br />
</div><br />
<br><br />
</div><br />
</section><br />
<section id="work" class="main style3 primary"><br />
<div class="container small gallery"><br />
<!-- Advisors --><br />
<h1 style="font-size: 2em;">Advisors</h1><br />
<!-- Robin Dowell --><br />
<div class="row flush images"><br />
<div class="6u"><div class="image full l" style="padding-top: 10px;"><h1 style="font-size: 2em;">Robin Dowell</h1><p style="text-align: left; padding-left: 15px; padding-right: 15px; font-size: 1em;">The Dowell Lab focuses on developing informative, mechanistic models that unravel biologically meaningful information from a few individuals and limited reference information. We leverage comparative information to better understand both the diverse roles that non-coding transcripts play within the cell and the impact of variation on phenotype between closely related individuals.<br />
</p></div></div><br />
<div class="6u"><a href="https://static.igem.org/mediawiki/2014/b/b2/ThumbDowell.jpg" class="image full r"><img src="https://static.igem.org/mediawiki/2014/b/b2/ThumbDowell.jpg" title="Robin Dowell" alt=""></a></div><br />
</div><br />
<!-- Micheal Brasino --><br />
<div class="row flush images"><br />
<div class="6u"><a href="https://static.igem.org/mediawiki/2014/c/c7/Brasiano.jpg" class="image full l"><img src="https://static.igem.org/mediawiki/2014/c/c7/Brasiano.jpg" title="Michael Brasino" alt=""></a></div><br />
<div class="6u"><div class="image full r" style="padding-top: 10px;"><h1 style="font-size: 2em;">Michael Brasino</h1><p style="text-align: left; padding-left: 15px; padding-right: 15px; font-size: 1em;">I am a graduate student advisor in the materials science PhD program <br />
</p></div></div><br />
</div><br />
<!-- Samantha O'Hara --><br />
<div class="row flush images"><br />
<div class="6u"><div class="image full l" style="padding-top: 10px;"><h1 style="font-size: 2em;">Samantha O'Hara</h1><p style="text-align: left; padding-left: 15px; padding-right: 15px; font-size: 1em;">I am a graduate student advisor in MCDB. I study polyomavirus-receptor interactions and I have a wonderful Labrador retriever named, Lucy.<br />
</p></div></div><br />
<div class="6u"><a href="https://static.igem.org/mediawiki/2014/f/f5/ThumbSam.jpg" class="image full r"><img src="https://static.igem.org/mediawiki/2014/f/f5/ThumbSam.jpg" title="Samantha O'Hara" alt=""></a></div><br />
</div><br />
<!-- Cloe Simmone Pogoda --><br />
<div class="row flush images"><br />
<div class="6u"><a href="https://static.igem.org/mediawiki/2014/e/e5/ThumbPogoda.png" class="image full l"><img src="https://static.igem.org/mediawiki/2014/e/e5/ThumbPogoda.png" title="Cloe Simmone Pogoda" alt=""></a></div><br />
<div class="6u"><div class="image full r" style="padding-top: 10px;"><h1 style="font-size: 2em;">Cloe Simmone Pogoda</h1><p style="text-align: left; padding-left: 15px; padding-right: 15px; font-size: 1em;">I am a graduate student advisor in the molecular biology (MCDB) PhD program<br />
</p></div></div><br />
</div><br />
<!-- Tim Read --><br />
<div class="row flush images"><br />
<div class="6u"><div class="image full l" style="padding-top: 10px;"><h1 style="font-size: 2em;">Tim Read</h1><p style="text-align: left; padding-left: 15px; padding-right: 15px; font-size: 1em;">I am a graduate student advisor in MCDB. I study transcriptional Evolution in the Dowell Lab<br />
</p></div></div><br />
<div class="6u"><a href="https://static.igem.org/mediawiki/2014/3/3e/ThumbTim.jpg" class="image full r"><img src="https://static.igem.org/mediawiki/2014/3/3e/ThumbTim.jpg" title="Tim Read" alt="" /></a></div><br />
</div><br />
<!-- Alex Stemm-Wolf --><br />
<div class="row flush images"><br />
<div class="6u"><a href="https://static.igem.org/mediawiki/2014/a/a8/ThumbStemmwolf.png" class="image full l"><img src="https://static.igem.org/mediawiki/2014/a/a8/ThumbStemmwolf.png" title="Alex Stemm-Wolf" alt="" /></a></div><br />
<div class="6u"><div class="image full l" style="padding-top: 10px;"><h1 style="font-size: 2em;">Alexander Stemm-Wolf</h1><p style="text-align: left; padding-left: 15px; padding-right: 15px; font-size: 1em;">I am in the advising group for Gold group. I have 25 years of experience as a research technician, the last 15 in MCD Biology. <br />
</p></div></div><br />
</div><br />
</div><br />
</div><br />
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{{Template:UCB-Footer}}</div>Leighlahttp://2014.igem.org/File:UCB-profile_Josh-141016.JPGFile:UCB-profile Josh-141016.JPG2014-10-16T17:10:58Z<p>Leighla: </p>
<hr />
<div></div>Leighlahttp://2014.igem.org/Team:CU-Boulder/Notebook/CC9Phage_TeamTeam:CU-Boulder/Notebook/CC9Phage Team2014-10-16T04:48:41Z<p>Leighla: /* Week 4 */</p>
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__FORCETOC__<br />
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<br />
'''Note:'''<br />
'''Litmus28ibb is a biobricked version of the Litmus28i (from NEB) vector which contains the M13ori (packaging signal) which allows for uptake into M13 or Fd phage. While making this part biobrick compatible, we added J04450 as the insert.'''<br />
'''pCas9 (BBa_K1218011) contains and tracrRNA and Cas9 gene under native promoters, and a minimal CRISPR arrays. The guide RNA (referred here as gRNA) is inserted into the CRISPR array.'''<br />
<br><br />
<br><br />
<br><br />
<br />
==Week 1==<br />
<br />
'''7/21'''<br />
*To ligate pCas9 to Litmus28ibb<br />
::-Digestion<br />
:::-Litmus28ibb-J04450 with E+P<br />
:::-pCas9 with E+P<br />
'''[[File:UCB-Cas9Phage-140721.JPG]]'''<br />
:1. pCas9<br />
:2. Litmus28ibb<br />
::-Overnight ligations (10hrs at 37C, 10min at 80C)<br />
<br />
'''7/22'''<br />
*Transformed ligation from 7/21 (Litmus28ibb + pCas9) into ER2738 cells<br />
::-Plated on Amp + Tet<br />
::-Later in day, pricked colonies from morning transformation (Litmus28ibb and pCas9 in ER)<br />
<br />
'''7/23'''<br />
*Miniprepped O/N from 7/22 of Litmus28ibb-pCas9<br />
::-Digested<br />
:::*None were the correct size. Smaller bands looked like pSB1C3. Plated cells onto Chlor and there was significant growth. pSB1C3 from the pCas9?<br />
<br />
'''7/24'''<br />
*To put pCas9 onto Litmus28i<br />
::-Digested<br />
:::-Litmus28ibb with EcoRI+PstI<br />
:::-pSB1C3-pCas9 with EcoRI+SpeI<br />
:::-PSB3C5-J04450 with XbaI + PstI<br />
'''[[File:UCB-Cas9Phage-140724.JPG]]'''<br />
:1. Litmus28ibb<br />
:2. pCas9<br />
:3. J04450<br />
::-Overnight ligation (10hr @ 16C)<br />
<br />
'''7/25'''<br />
*Transformed ligation from 7/24 (Litmus28ibb+pCas9) into 5alpha cells<br />
<br />
'''7/26'''<br />
*No growth from 7/25 transformation<br />
*Tranform<br />
::-Litmus28ibb + pCas9 + J04450<br />
*Retry making Litmus28i with pCas9<br />
::-Ligation (10hrs at 16C, 10min at 80C)<br />
:::*Litmus28ibb + pCas9 + J04450<br />
<br />
==Week 2==<br />
<br />
'''7/27'''<br />
*Transformation results<br />
::-No growth for no DNA controls (on Chlor)<br />
::-4 colonies for Litmus28bb-JO4450-Cas9 (on Amp-> none are red)<br />
*Picked all 4 colonies for O/Ns<br />
<br />
'''7/28'''<br />
*3 new red colonies on Litmus28bb-Cas9-J04450 plate<br />
::-Set up cultures<br />
*O/Ns grew but did not bother<br />
*Transformations<br />
::-Litmus28bb-J04450-Cas9<br />
::-No DNA control<br />
<br />
'''7/29'''<br />
*Litmus28bb-Cas9-J04450 O/Ns<br />
::-Incubator shut off during night. Samplers were at RT by morning<br />
::-Add 1mL to fresh 4mL LB and Amp<br />
::-Mini-prep DNA<br />
::-Digest with EcoRI and PstI to check insert for correct size<br />
'''[[File:UCB-Cas9Phage-140729.JPG]]'''<br />
::-Lane 2 contains the expected 3000bp band for the Litmus28ibb backbone and the ~7000bp band for the pCas9-J04450 insert. Unfortunately, this sample also contains a 4000bp band.<br />
*Plated the above sample<br />
<br />
'''7/30'''<br />
*Digested Litmus28ibb-Cas9-RFP with EcoRI-HF<br />
'''[[File:UCB-Cas9Phage-140730.JPG]]'''<br />
::-Extracted large band ~9000<br />
::-Re-ligated piece<br />
*Sent samples for sequencing<br />
::-Litmus28bb with Cas9 and RFP (LCR) gRNA sequence<br />
<br />
'''8/1'''<br />
*Mini-prepped O/Ns of<br />
::-Litmus28ibb-Cas9-J04450 (LCR)<br />
:::*Digested with EcoRI-HF and PstI-HF => all samples had band ~3700<br />
*Digestions of Litmus and Cas9 to retry ligation<br />
'''[[File:UCB-Cas9Phage-140801.JPG]] '''<br />
:1. Litmus28ibb (E+P)<br />
:2. pCas9(E+P)<br />
:3. Litmus28ibb (E+P)<br />
:4. pCas9(E+S)<br />
:5. pSB3C5 (X+P)<br />
::-Gel extractions<br />
::-Ligations<br />
:::*Litmus28ibb (E+P) to pCas9 (E+P)<br />
:::*Litmus28ibb(E+P) to pCas9(E+S) and J04450(E+P)<br />
<br />
'''8/2'''<br />
*Transformation into BW cells (5/30)<br />
::-Hundreds of colonies for LCR Miniprep<br />
*Digestion to get a better idea of what this DNA is<br />
<br />
'''8/3'''<br />
*Isolate the LCR from the extra band in the gel<br />
::-Run 8/2 digestions on gel<br />
'''[[File:UCB-Cas9Phage-140803.JPG]]'''<br />
:1. LCR (PstI)<br />
:2. LCR (EcoRI)<br />
::-Gel extracted <br />
:::-LCR digested with PstI-HF (top band<br />
::-Re-ligate extracted LCR with no insert (should re-ligate to itself)<br />
::-Transformed ligation into ER2738<br />
*Made O/Ns<br />
::-colonies from 8/2 transformation<br />
::-restreak of LCR from freeze down<br />
<br />
==Week 3==<br />
<br />
'''8/4'''<br />
*Check overnights from 8/3 (LCR = Litmus28ibb+pCas9+RFP) for correct inserts<br />
::-Mini-prep samples<br />
::-Digest with EcoRI and PstI<br />
:::*All had sizes ~3700bp<br />
:::*No correct inserts<br />
*Attempt to ligate Litmus28ibb and pCas9(gRNA3)<br />
::-Digestions<br />
:::*Litmus28ibb-RFP E+P extracted Lit back bone<br />
:::*pCas9 gRNA3 E+P extracted pCas9 insert<br />
'''[[File:UCB-Cas9Phage-140804.JPG]]'''<br />
:1. Litmus28ibb<br />
:2. pCas9(gRNA3)<br />
:3. pCas9(unmod.)<br />
::-Ligations (10 hrs at 16C, 10 min at 80C)<br />
:::*Litmus28ibb + pCas9(gRNA3)<br />
<br />
'''8/5'''<br />
*Transformation into ER2738 (7/18), 5ul of ligation products<br />
::1. Litmus28ibb-Cas9gRNA3<br />
<br />
'''8/6'''<br />
*Another attempt at ligating pCas9 and Litmus28ibb<br />
::-Digest unmodified pCas9 with EcoRI-HF and PstI-HF<br />
::-Digest pCas9 containing pRNA1 with EcoRI-HF and PstI-HF<br />
'''[[File:UCB-Cas9Phage-140806.JPG]]'''<br />
:1. pCas9(unmod.)<br />
:2. pCas9(gRNA1)<br />
::-Gel extract pieces<br />
::-Ligate separately to Litmus28ibb<br />
*Results of 8/5 transformation<br />
::-6 colonies for Litmus28ibb+pCas9(gRNA3) on Tet+reduced Amp<br />
:::*Picked colonies for overnights<br />
::-No growth for no DNA control<br />
*Transformation of ligations from earlier today<br />
::-Litmus28ibb + pSB1C3-pCas9(unmod.)<br />
::-Litmus28ibb + pSB1C3-pCas9(gRNA1)<br />
*Mini-prep Litmus28ibb+pCas9(gRNA3) O/N samples from earlier in day<br />
::-Digested with EcoRI-HF and PstI-HF<br />
:::*All samples had bands of 3000 and 2000bp. None were correct<br />
<br />
'''8/8'''<br />
*M13ori into SB Cas9<br />
::-Digestions<br />
:::-unmodified pCas9 with EcoRI-HF and XbaI<br />
:::-M13ori with EcoRI-HF and SpeI-HF<br />
:::-pCas9(gRNA1) with EcoRI-HF and XbaI<br />
:::-pCas9(gRNA3) with EcoRI-HF and XbaI<br />
<br />
'''8/9'''<br />
*To clone M13ori onto pSB1C3-pCas9<br />
::-Run 8/8 digestion on a gel<br />
'''[[File:UCB-Cas9Phage-140809.JPG]]'''<br />
:1. M13ori<br />
:2. pCas9(gRNA3)<br />
:3. pCas9(gRNA1)<br />
:-Gel extracted<br />
::-Ligation Ligated M13ori to….<br />
:::-pSB1C3-pCas9(gRNA1)<br />
:::-pSB1C3-pCas9(gRNA3)<br />
:::-pSB1C3-pCas9(unmod.)<br />
::-Transform and plate onto low Chlor 934 ug/mL)<br />
<br />
==Week 4==<br />
<br />
'''8/10'''<br />
*Digested ligation products from 8/6 and 8/4. Saw many large bands that may have been undigested<br />
*O/Ns of 8/9 transformation<br />
::-1 colony for M13ori-pCas9(gRNA3) (only 1 colony grew)<br />
::-11 colonies for M13ori-pCas9(gRNA1)<br />
::-No colonies grew for M13ori-pCas9(unmodified)<br />
<br />
'''8/11'''<br />
*Test 8/10 O/Ns for correct inserts<br />
::-Digest 8/10 O/Ns with EcoRI-HF and PstI-HF<br />
'''[[File:UCB-Cas9Phage-140811.JPG]]'''<br />
:1. M13ori-pCas9(gRNA3)<br />
:2-12. M13ori-pCas9(gRNA1)<br />
:*Lanes 1, 8, and 9 are about the right size. Sent these samples for sequencing<br />
*To ligate M13ori to pSB1C3-pCas9(unmod.)<br />
::-Digest<br />
:::*pSB1C3-pCas9(unmod.) (E+P)<br />
:::*M13 digested 8/8<br />
::-Ligation: See L5 below<br />
*To ligate pCas9 and M13ori to pSB1A3<br />
::-Digest<br />
:::*pSB1A3 (E+P)<br />
:::*M13ori (E+S)<br />
:::*Unmodified pCas9 (X+P)<br />
:::*pCas9(gRNA1)-M13ori (E+P)<br />
:::*pCas9(gRNA3)-M13ori (E+P)<br />
::-Ligation: See L2-4 below<br />
*Another try to put pCas9 onto Litmus28ibb<br />
::-Digested<br />
:::*Unmodified Litmus28i (E+P)<br />
:::*pCas9(unmod.) (E+P)<br />
:::*pCas9(gRNA1)-M13ori (E+P)<br />
:::*pCas9(gRNA3)-M13ori (E+P)<br />
::-Ligation: See L1 and L6 below<br />
*Gel for all<br />
'''[[File:UCB-Cas9Phage-140811-02.JPG]]'''<br />
<br />
:1. Unmodified Litmus28i<br />
:2. pSB1C3-pCas9(unmodified)<br />
:3. pSB1A3<br />
:4. pSB1C3-M13ori<br />
'''[[File:UCB-Cas9Phage-140811-03.JPG]]'''<br />
:5. pSB1C3-pCas9(unmod.)<br />
:6. pSB1C3-M13ori-pCas9(gRNA1)<br />
:7. pSB1C3-M13ori-pCas9(gRNA3)<br />
<br />
*Ligations for both projects<br />
<br />
{| class = "wikitable"<br />
|-<br />
! #<br />
! Backbone<br />
! Insert 1<br />
! Insert 2<br />
<br />
|-<br />
| 1<br />
| Unmod. Litmus28i (E+P)<br />
| pCas9(unmod.) (E+P)<br />
| N/A<br />
<br />
|-<br />
| 2<br />
| pSB1A3 (E+P)<br />
| pCas9(unmod.) (X+P)<br />
| M13ori (E+S)<br />
<br />
|-<br />
| 3<br />
| pSB1A3 (E+P)<br />
| pCas9(gRNA1)-M13ori (E+P)<br />
| N/A<br />
<br />
|-<br />
| 4<br />
| pSB1A3 (E+P)<br />
| pCas9(gRNA3)-M13ori (E+P)<br />
| N/A<br />
<br />
|-<br />
| 5<br />
| pSB1C3-pCas9(unmod.) (E+X)<br />
| M13ori (E+S)<br />
| N/A<br />
<br />
|-<br />
| 6<br />
| Litmus28ibb (E+P)<br />
| pCas9(gRNA3) (E+P)<br />
| N/A<br />
<br />
|-<br />
| 7<br />
| Litmus28ibb (E+P)<br />
| pCas9(gRNA1) (E+P)<br />
| N/A<br />
<br />
|-<br />
| 8<br />
| Litmus28ibb (E+P)<br />
| pCas9(unmod.) (E+P)<br />
| N/A<br />
<br />
|-<br />
|}<br />
<br />
'''8/12'''<br />
*Transform ligation from 8/11 into ER2738 cells<br />
*Make phage using M13g6A1 as Helper Phage<br />
::-used pCas9(gRNA1)-M13ori or pCas9(gRNA3)-M13ori as phagemid<br />
*Freeze downs<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Top<br />
! Side<br />
<br />
|-<br />
| M. gRNA3 Cas9 (8/12)<br />
| M13ori Cas9 gRNA3 pSB1C3<br />
<br />
|-<br />
| M. gRNA1 Cas9 (8/12)<br />
| M13ori(8) Cas9 gRNA1 pSB1C3<br />
<br />
|-<br />
| M. gRNA1 Cas9 (8/12)<br />
| M13ori (9) Cas9 gRNA1 pSB1C3<br />
<br />
|-<br />
|}<br />
<br />
'''8/13'''<br />
*Finish phage isolation<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Helper phage<br />
! Phage<br />
! A269<br />
! A320<br />
! genome size<br />
! [ ]<br />
<br />
|-<br />
| Fd-CAT<br />
| M. gRNA1<br />
| 0.353<br />
| 0.025<br />
| 7687<br />
| 2.56 x10^12<br />
<br />
|-<br />
| Fd-CAT<br />
| M. gRNA3<br />
| 0.554<br />
| 0.037<br />
| 7687<br />
| 4.40 x10^12<br />
<br />
|-<br />
|}<br />
<br />
*Infection<br />
::-Infecting ER2738 and BWF’<br />
::-Phage<br />
:::*pSB1C3-pCas9(gRNA1)<br />
:::*pSB1C3-pCas9(gRNA3)<br />
*Transformation Results from 8/12<br />
::-Growth for the following… (made O/Ns)<br />
:::*pSB1A3-M13ori-pCas9(gRNA3)<br />
:::*pSB1C3-M13ori-pCas9(unmod)-M13ori<br />
:::*Litmus28ibb-pCas9(gRNA3)<br />
:::*Litmus28ibb-pCas9(gRNA1)<br />
:::*Litmus28ibb-pCas9(unmod.)<br />
::-Hundreds of colonies for pCas9(unmod.) on Unmod. Litmus28i. Didn’t trust result so didn’t pick coloneis. EcoRI and PstI sites touch so most likely, Litmus28i was only cut once<br />
<br />
'''8/15'''<br />
*Check O/Ns from 8/14 for correct inserts<br />
::-Mini-prepped samples<br />
::-Digested with E and P<br />
::-Results<br />
:::*No bands of the right size for<br />
:::-Litmus28ibb-pCas9(gRNA3)<br />
:::-Litmus28ibb-pCas9(gRNA1)<br />
:::-Litmus28ibb-pCas9(unmod.)<br />
:::-Expected band sizes for… (see below)<br />
*pCas9(gRNA3)-M13ori on pSB1A3<br />
*pCas9(unmod)-M13ori on pSB1C3<br />
'''[[File:UCB-Cas9Phage-140815.JPG]]'''<br />
<br />
*Transform into BWF’ to test for functionality of Cas9 when associated with m13ori, independent of infectivity<br />
::-M13ori-pCas9(gRNA1)<br />
::-M13ori-pCas9(gRNA3)<br />
<br />
'''8/16'''<br />
*PCR on LCR (Litmus28ibb with pCas9 and RFP to maybe get a good product to religate<br />
::-Didn’t work<br />
<br />
Results of 8/13 infection of BWF’ with M13ori-gRNA(1 or 3) to test pCas9 killing<br />
:No growth on DNA controls on Chlor(34ug/mL) or Kan(50ug/mL)+Chlor(34ug/mL)<br />
:ON Kan(50ug/mL) + Chlor (170ug/mL) in BWF'<br />
:::gRNA1--> 22 colonies<br />
:::gRNA3--> 2 colonies<br />
<br />
On Kan(25ug/mL)+Chlor(34ug/mL) in BWF’<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! 1:100<br />
! 1:1000<br />
<br />
|-<br />
| gRNA1<br />
| 617<br />
| 84<br />
<br />
|-<br />
| gRNA3<br />
| 469<br />
| 42<br />
<br />
|-<br />
|}<br />
On Chlor (34ug/mL) in BWF’<br />
<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! 1:100<br />
! 1:1000<br />
<br />
|-<br />
| gRNA1<br />
| 508<br />
| 67<br />
<br />
|-<br />
| gRNA3<br />
| 447<br />
| 41<br />
<br />
|-<br />
|}<br />
On Chlor (34ug/mL) in ER<br />
<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! 1:100<br />
! 1:1000<br />
<br />
|-<br />
| gRNA1<br />
| 702<br />
| 93<br />
<br />
|-<br />
| gRNA3<br />
| 518<br />
| 58<br />
<br />
|-<br />
|}<br />
<br />
Results of 8/15 transformation of BWF’ with M13ori—gRNA(1 or 3) to test killing<br />
<br />
Chlor (34ug/mL) + Kan (50ug/mL)<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! 1:1<br />
! 1:10<br />
<br />
|-<br />
| gRNA1<br />
| Lawn<br />
| 1904<br />
<br />
|-<br />
| gNRA3<br />
| Slightly thinner lanw<br />
| 1616<br />
<br />
|-<br />
|}<br />
<br />
Chlor (34 ug/mL)<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! Trial 1<br />
! Trial 2<br />
<br />
|-<br />
| gRNA1<br />
| 1788<br />
| 1548<br />
<br />
|-<br />
| gRNA3<br />
| 1564<br />
| 1416<br />
<br />
|-<br />
|}<br />
Samples were diluted 1:10<br />
<br />
==Week 5==<br />
<br />
'''8/17'''<br />
Make phage<br />
::pSB1C3-M13ori-pCas9(unmod)<br />
::pSB1A3-M13ori-pCas9(gRNA3)<br />
Ran uncut LCR on a gel and gel extracted the three bands<br />
<br />
'''8/18'''<br />
While making phage…. We threw away the samples….<br />
Remake phage. Same as 8/17<br />
Transform LCR gel extractions into 5alpha cells<br />
<br />
8/19<br />
Sent samples for sequencing<br />
::pSB1C3-M13ori-pCas9(unmodified)<br />
::pSB1A3-M13ori-pCas9(gRNA3)<br />
<br />
Finish making phage<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Name<br />
! A269<br />
! A320<br />
! Genome Size<br />
! [ ]<br />
<br />
|-<br />
| pSB1A3-M13ori-pCas9(gRNA3)<br />
| 0.697<br />
| 0.162<br />
| <br />
| Note: We later found that this sample did not actually contain the M13ori<br />
<br />
|-<br />
| pSB1C3-M13ori-pCas9(unmod)<br />
| 0.764<br />
| 0.146<br />
| 7687<br />
| 3.044 x10^13<br />
<br />
|-<br />
|}<br />
Infect cells (ER and BWF’)<br />
::Grew a single 75mL sample. When OD ~0.5, divided samples into 3<br />
::Added 20mL of sample to 125mL flask (3 for each strain)<br />
::Added phage to a final concentration of 1 x10^8 phage/mL<br />
::Incubated at 250rpm, 37C, for 30 minutes<br />
::Plated at dilutions (1:10, 1:100, and 1:1k)<br />
:::On Chlor(170ug/mL), Kan(50ug/mL)+Chlor(85ug/mL), Kan(50ug/mL)+Chlor(170ug/mL)<br />
<br />
<br />
'''8/21'''<br />
Finished phage amplification protocol from 8/20 but did not receive any phage. <br />
Infect BWF’ cells with the phage having pSB1C3-M13ori-pCas9 containing the scramble, gRNA1, and gRNA3.<br />
<br />
'''8/22''<br />
Present project to department<br />
<br />
==Week 6==<br />
<br />
'''8/25'''<br />
Results of 8/21 infection experiment<br />
<br />
Results on Kan and Chlor<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! Scramble<br />
! gRNA1<br />
! gRNA3<br />
<br />
|-<br />
| 1:1<br />
| 149<br />
| 143<br />
| 13<br />
<br />
|-<br />
| 1:10<br />
| 14<br />
| 16<br />
| 1<br />
<br />
|-<br />
|}<br />
<br />
Results on Chlor<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! Scramble<br />
! gRNA1<br />
! gRNA3<br />
<br />
|-<br />
| 1:1<br />
| 147<br />
| 117<br />
| 14<br />
<br />
|-<br />
| 1:10<br />
| 14<br />
| NA<br />
| 3<br />
<br />
|-<br />
|}<br />
<br />
'''8/28'''<br />
Retry ligation of pCas9 to litmus28ibb<br />
Digest Litmus28ibb-J04450, pCas9, and amilCP with EcoRI and PstI. Gel extract pieces<br />
Ligate pCas9 into Litmus28ibb and amilCP into Limus28ibb<br />
<br />
'''8/29'''<br />
Transform 8/28 ligation products into 5alpha cells<br />
<br />
'''8/29'''<br />
Transform pSB1C3-pCas9(unmodified) into BW and BWF’ to test targeting of f’ episome. Due to poor infection results we wanted to test the guide RNAs for targeting of the F’ episome. We have searched the vast webs for the sequence of the F’ episome but have yet to (and never will) find it.<br />
<br />
'''8/30'''<br />
Transformation results of pCas9 into BW and BWF’. Results were unexpected, we expected more growth for BW sample or at least equal growth. Differences in competence or concentration could explain results in initial count<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Initial count<br />
! After another 24 hours<br />
<br />
|-<br />
| No DNA controls (for BW and BWF’)<br />
| No growth<br />
| No growth<br />
<br />
|-<br />
| BW cells<br />
| 9 colonies<br />
| 222<br />
<br />
|-<br />
| BWF’ cells<br />
| ~100 colonies<br />
| 158<br />
<br />
|-<br />
|}<br />
<br />
Transformation results for ligation of pCas9 to Litmus28ibb<br />
<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! Amp (50ug/mL)<br />
! Amp (100ug/mL)<br />
! Notes<br />
<br />
|-<br />
| No DNA control<br />
| 3<br />
| 10<br />
| All bright white<br />
<br />
|-<br />
| Litmus28ibb-pCas9<br />
| ~50 (many are red)<br />
| 25-30<br />
| High proportion are red. Some are shadowy white. A few are bright white<br />
<br />
|-<br />
| Litmus28ibb-amilCP<br />
| >300<br />
| >300<br />
| Mostly shadowy, some red, some bright white<br />
<br />
|-<br />
|}<br />
<br />
Made overnight cultures of shadowy and bright coloneis from Litmus28ibb-pCas9 samples (from both plates) and of shadowy colonies from Litmus28ibb-amilCP. Since growth on no DAN control was bright white, we assumed that bright colonies on sample plates were more likely to be contamination, but didn’t really know. Since there were fewer colonies on no DNA control plates, we figures we could randomly select colonies from the sample plates that were not a result of contamination.<br />
<br />
==Week 7==<br />
<br />
'''8/31'''<br />
Mini-prep overnight cultures from 8/30 and digest samples with EcoRI and PstI. When run on a gel, none of the Litmus28ibb-pCas9 samples yielded bands of the correct size. All Litmus28ibb-amilCP samples did yield the expected band sizes. <br />
<br />
'''9/2'''<br />
Sent some of mini-prep samples from 8/31 for sequencing<br />
<br />
'''9/3'''<br />
Digestion of pCas9 with EcoRI and speI and the unmodified NEB version of Litmus28i with EcoRI and XbaI. Gel extracted pieces and ligated together (No, this part would not have the BioBrick prefix/suffix)<br />
<br />
'''9/4'''<br />
Transform 9/3 Litmus28i-pCas9 ligation into 5alpha cells. Selected on low ampicillin (50ug/mL) and high ampicillin (100ug/mL)<br />
<br />
'''9/5'''<br />
Did receive colonies from 9/4 transformation. Selected 24 colonies for colony PCR with a primer pair that would span the junction of pCas9 and Litmus28i. (forward primer bound to CRISPR region of pCas9 while reverse primer bound in the middle of the Litmus28i backbone)<br />
Colony PCR failed to reveal any promising samples. Set up overnight cultures in a hopeless effort to yield different results.<br />
<br />
==Week 8==<br />
<br />
'''9/7'''<br />
Mini-prepped overnight cultures from 9/5. Digested each with EcoRI and PstI and ran reults on gel. Results were consistent with colony PCR: failure. A few samples had very feint bands that were approximately the correct size<br />
<br />
'''9/8'''<br />
for the samples on 9/7 with light bands of approximately the correct size, we sent samples for sequencing (another hopeless attempt to get different results)<br />
<br />
'''9/9'''<br />
Sequencing results showed that Limtus28i religated<br />
<br />
==Week 9==<br />
<br />
'''9/17'''<br />
New method to clone pCas9 onto Litmus28ibb vector: PCR->Digestion->ligation method<br />
:::PCR amplified pCas9 construct from pSB1C3-pCas9. PCR amplified Litmus28ibb backbone from Litmus28ibb-J04550<br />
:::PCR purified both samples. Digested both samples with EcoRI and PstI and gel extracted the pieces<br />
:::Ligated pCas9 to Litmus28ibb vector<br />
<br />
Sent pSB1C3-M13ori-pCas9 sample for sequencing to sequence the entire plasmid<br />
<br />
'''9/18'''<br />
Transform 9/17 ligation (Litmus28ibb-pCas9) into 10beta cells<br />
<br />
'''9/19'''<br />
There was growth on no DNA control. Some colonies for sample.<br />
<br />
'''9/20''' <br />
Make chemically competent ER and 5alpha cells using DMSO from bottle instead of fancy tubes. We think this is the reason for later transformation failures<br />
<br />
Digested pCas9 and Litmus28ibb PCR purifications from 9/17 with EcoRI, PstI, and DpnI. PCR purified digestion samples then ligated pieces together.<br />
<br />
==week 10==<br />
<br />
'''9/21'''<br />
New idea: Cloning pCas9 onto Litmus28i has not worked up until this point. What if we constructed a vector that looked similar to litmus28i. pSB1A3 has the same origin of replication and almost the same ampicillin resistance marker (with a few SNPs). Need to add M13ori (which was taken from Litmus28i) and pCas9. M13ori would not be in the correct position on plasmid but this is as close as we can get with the remaining time<br />
::Digestions: M13ori-pCas9(unmod), pCas9(unmod), pSB1A3 with EcoRI and PstI. Gel showed incomplete digestion so did not gel extract<br />
<br />
Transformation into 5alpha cells: no DNA control, 9/20 ligation (Litmus28ibb-pCas9), pSB1C3-amilCP, and pSB1C3-M13ori-pCas9<br />
<br />
'''9/22'''<br />
Re-digested M13ori-pCas9(unmod) and pSB1A3-J04450 with EcoRI and PstI. pCas9(unmod) was PCR purified (product looked clean on gel so did not bother to gel extract). <br />
Ligated M13ori-pCas9(unmod) to pSB1A3 and pCas9(unmod) to pSB1A3<br />
Sent pSB1A3 to M13ori-pCas9 for sequencing to cover region not covered by previous sequencing<br />
<br />
'''9/23'''<br />
Sequencing results came back<br />
:::Litmus28ibb-J04450 contains two nucleotides that are flipped. They are located within the ampicillin resistance gene and do change two amino acids; however, sample grows on ampicillin so these are not loss of function mutation. <br />
:::pSB1C3-M13ori-pCas9 was perfect - the above two samples are ready to submit to the registry<br />
<br />
Transform ligations from 9/22 (pSB1A3- M13ori-pCas9(unmod) and pCas9(unmod)) into 5 alpha cells<br />
Colony PCR 18 samples from 9/20 Limtus28ibb-pCas9(unmod) plate.<br />
<br />
'''9/24'''<br />
Run gel of 9/23 colony PCR to check for desired Litmus28ibb-pCas9(unmod). There was a band of approximately the correct size in each samples (including the no DNA control). Set up overnight cultures of first 5 samples for further testing<br />
Results of 9/23 transformation<br />
::No growth for no DNA control on chlor (170ug/mL), Amp (50ug/mL) or Amp (100ug/mL)<br />
::7 colonies for pSB1C3-M13genes on chlor (170ug/mL)<br />
::27 colonies for pSB1A3-pCas9 on Amp (100ug/mL) and 41 colonies on Amp (50ug/mL)<br />
::0 colonies for pSB1A3-M13ori-pCas9 on Amp(50 and 100ug/mL)<br />
Set up overnight cultures of some of the samples.<br />
<br />
<br />
'''9/25'''<br />
Mini-prep overnights from 9/24<br />
Digest samples to check for insert. When run on a gel, 3 samples had large bands of ~5000bp and one had an additional band at ~6000. We made overnights of these 3 colonies<br />
<br />
'''9/26'''<br />
Digested mini-prepped DNA from the three samples that looked promising from 9/25. Each sample was digested with only EcoRI, only PstI, or EcoRI and PstI. When run on a gel, it can be seen that each samples, when cut once, yielded one band 8000bp while when cut twice, yielded 2 bands of ~2000 and 5000 as expected.<br />
Sent parts to iGEM registry: pSB1C3-M13ori-pCas9 and Litmus28ibb-J04450<br />
To add the M13ori to pSB1A3-Cas9<br />
::Digested pSB1A3-pCas9 with EcoRI and XbaL<br />
::Digested pSB1C3-M13ori with EcoRI and SpeI<br />
::Gel extracted bands of the correct sizes and combined these into a ligation reaction.<br />
<br />
'''9/27'''<br />
Transform pSB1A3-M13ori-pCas9 into Amp(50ug/mL) and Amp(100ug/mL)<br />
Later in the day…. Set up overnight cultures of colonies<br />
<br />
==Week 11==<br />
<br />
'''9/28'''<br />
Mini-prepped overnights made 9/27<br />
Digested overnight samples with EcoRI and PstI<br />
Did receive pSB1A3-M13ori-pCas9 samples with bands of the correct size (as compared to pSB1A3-pCas9)<br />
Set up overnight cultures of the samples that looked promising<br />
Transformed pSB1A3-M13ori-pCas9(unmod) and pSB1A3-M13ori-pCas9(gRNA3) into ER2738 cells<br />
<br />
'''9/29'''<br />
Did receive colonies from 9/28 transformation. Set up overnight cultures for both.<br />
Also set up overnight cultures of ER2738 and BWF’ for upcoming infection.<br />
Start phage amplification protocol using pSB1A3-M13ori-pCas9(unmod) and pSB1A3-M13ori-pCas9(gRNA3) as the phagemid. Used Fd-Cat as the helper phagemid<br />
<br />
'''9/30'''<br />
Finished phage amplification/isolation protocol for Fd phage packaging pSB1A3-M13ori-pCas9(unmod) and pSB1A3-M13ori-pCas9(gRNA3)<br />
<br />
'''10/1'''<br />
Measured concentration of phage isolated on 9/30. There were no phage for the gRNA3 sample. The pSB1A3-M13ori-pCas9(unmod) sample contained some phage but had a high level of contamination/impurity.<br />
Send samples pSB1A3-M13ori-pCas9(unmod) and pSB1A3-M13ori-pCas9(gRNA3) samples for sequencing to verify the presence of the M13ori<br />
<br />
'''10/2'''<br />
Sequencing results came in<br />
::pSB1A3-M13ori-pCas9(unmod) looks good<br />
::pSB1A3-M13ori-pCas9(gRNA3) doesn’t have the M13ori<br />
Plan<br />
:1.SDM on pSB-M13ori-pCas9(unmod) to remove BsaI cut site in pSB1A3 backbone<br />
:2.Change spacer from unmodified to the gRNA3<br />
:3.Redo the infection experiment<br />
SDM<br />
:PCR to amplify pSB1A3-M13ori-pCas9(unmod)<br />
:Treat with kinase, Ligase, and DpnI<br />
:Transform into NEB 5alpha cells<br />
<br />
<br />
'''10/3'''<br />
Set up O/N cultures of 10/2 transformation<br />
<br />
'''10/4'''<br />
Mini-prep overnights. Samples are called pSB1A3(delta BasI)-M13ori-pCas9(unmod) to indicate each part of the construct and the removed BsaI site in the pSB1A3 backbone.<br />
Changing the gRNA spacer in pCas9<br />
::Vector digest: digested pSB1A3(delta BasI)-M13ori-pCas9(unmod) with BsaI to remove unmodified spacer. Saw only one band on the gel (cut around spacer) which shows that the additional BsaI site in the pSB1A3 backbone was removed. Gel extracted the digested vector<br />
::Ligation: Used the annealed oligos for gRNA3 (our targeting sample) from the last time we changed the spacer. Ligated these oligos to the digested pSB1A3(delta BasI)--M13ori-pCas9<br />
<br />
==Week 12==<br />
<br />
'''10/5'''<br />
Transform pSB1A3(delta BasI)-M13ori-pCas9(unmod) and pSB1A3(delta BasI)-M13ori-pCas9(gRNA3) in ER2738 cells<br />
<br />
'''10/6'''<br />
Transformation results from 10/5<br />
:No growth for no DNA control<br />
:pSB1A3(delta BasI)-M13ori-pCas9(unmod) = many colonies<br />
:pSB1A3(delta BasI)-M13ori-pCas9(gRNA3) = no colonies<br />
<br />
Re-anneal primers then relegate<br />
::Phosphorylation of oligos for gRNA3. Incubate for 1 hour at 37C then 20 minutes at 65C<br />
::Add 2.5ul 1M NaCl<br />
::Anneal in thermocycler. 5 minutes at 95C then a slow cool-down for ~2 hours<br />
::Dilute product 1:10<br />
::Ligation of newly annealed primers to digest and gel extracted pSB1A3(delta BasI)-M13ori-pCas9(unmod) from 10/5<br />
<br />
'''10/7'''<br />
Transform 10/6 ligation into ER2738 cells<br />
<br />
'''10/8'''<br />
No growth for 10/7 transformation<br />
Transform ligation from 10/6 into 5alpha cells<br />
Last try to make pSB1A3-M13ori-pCas9(gRNA3)<br />
:Know from 10/1 sequencing that we have pSB1A3-pCas9(gRNA3). Therefore, only need to add M13ori upstream of pCas9 part<br />
:Digest pSB1A3-pCas9(gRNA3) with EcoRI and XbaI and pSB1C3-M13ori with ExoRI and SpeI<br />
:Gel extract digested pieces<br />
:Ligate gel extracted pieces together<br />
<br />
'''10/9'''<br />
No growth for 10/8 transformation<br />
Transform 10/8 ligation into 5alpha cells<br />
<br />
'''10/10'''<br />
Transform <br />
:pSB1C3-M13ori-pCas9(unmod) from 10/9 into ER2738 cells<br />
:pSB1A3 (delta BsaI)-M13ori-pCas9(gRNA3) from 10/6 into 5 alpha cells<br />
:pSB1A3-M13ori-pCas9(gRNA3) from 10/8 into 5 alpha cells<br />
<br />
'''10/11'''<br />
Transformation results of 10/10<br />
:1 colony for pSB1C3-M13ori-pCas9(unmod) in ER2738 (does not seem trustworthy)<br />
:No colonies for no DNA control<br />
:No colonies for the 2 other transformations<br />
<br />
Transform pSB1C3-M13ori-pCas9(unmod) into ER2738 cells again<br />
Start phage amplification protocol to make the following phage<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Phagemid<br />
! Helper Phagemid<br />
<br />
|-<br />
| pSB1C3-M13ori-pCas9(unmod)<br />
| M13g6A1<br />
<br />
|-<br />
| pSB1C3-M13ori-pCas9(unmod)<br />
| M13K07<br />
<br />
|-<br />
| pSB1C3-M13ori-pCas9(gRNA3)<br />
| M13g6A1<br />
<br />
|-<br />
| pSB1C3-M13ori-pCas9(gRNA3)<br />
| M13K07<br />
<br />
|-<br />
|}<br />
<br />
pSB1C3-M13ori-pCas9(unmod) grew unexpectedly fast so we missed the OD mark. Growth rate compared to pSB1C3-M13ori-pCas9(gRNA3) sample suggests contamination. Did not proceed with making these phage for today.<br />
<br />
'''10/12'''<br />
No growth for 10/11 transformation – conclude that ER2738 cells are not competent (these are the cells made 9/20 with DMSO from a room temp bottle instead of the alliquotes from NEB.<br />
Remake phage<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Phagemid<br />
! Helper Phagemid<br />
<br />
|-<br />
| pSB1C3-M13ori-pCas9(unmod)<br />
| M13g6A1<br />
<br />
|-<br />
| pSB1C3-M13ori-pCas9(unmod)<br />
| M13K07<br />
<br />
|-<br />
|}<br />
<br />
'''10/13'''<br />
Finish phage isolation protocol and measure<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Phagemid<br />
! Helper Phagemid<br />
! Dilution<br />
! A269<br />
! A320<br />
! Genome size<br />
! Diluted [ ]<br />
! Actual [ ]<br />
<br />
|-<br />
| pSB1C3-M13ori-pCas9(gRNA3)<br />
| M13g6A1<br />
| 1:40<br />
| 0.090<br />
| 0.015<br />
| 7687<br />
| 5.85 E11<br />
| 2.34 E13<br />
<br />
|-<br />
| pSB1C3-M13ori-pCas9(gRNA3)<br />
| M13K07<br />
| 1:40<br />
| 0.085<br />
| 0.013<br />
| 7687<br />
| 5.62 E11<br />
| 2.25 E13<br />
<br />
|-<br />
| pSB1C3-M13ori-pCas9(unmod)<br />
| M13g6A1<br />
| 1:40<br />
| 0.104<br />
| 0.022<br />
| 7687<br />
| 6.40 E11<br />
| 2.56 E13<br />
<br />
|-<br />
| pSB1C3-M13ori-pCas9(unmod)<br />
| M13K07<br />
| 1:20<br />
| 0.091<br />
| 0.016<br />
| 7687<br />
| 5.85 E11<br />
| 1.17 E13<br />
<br />
|-<br />
|}<br />
<br />
Infection with phage of BW23115F’ cells<br />
:Grew a single stock of cells then dived into 4 samples each with 50mL cells. Added the above phage<br />
:Incubated for 30 minutes at 37C, shaking<br />
:Plated onto Chlor<br />
<br />
==Week 13==<br />
<br />
'''10/14'''<br />
Infection results<br />
:No growth on Chlor (170ul/mL)<br />
:Very little to no growth on pCas9(unmod) samples. We think this is b/c the colony picked on 10/11 did not actually contain pSB1C3-M13ori-pCas9(unmod) so nothing was packaged into phage<br />
:~100 colonies for pCas9(gRNA3) samples.<br />
:The samples made with M13g6A1 were also plated on ampicillin. These plates show considerable growth. There is more growth on <br />
pCas9(unmod) than pCas9(gRNA3) sample plate. We believe this further supports our hypothesis that our pCas9(unmod) sample did not contain pSB1C3-M13ori-pCas9(unmod) so packaged M13g6A1 in the absence of a better phagemid. Also, for the pCas9(gRNA3) sample, there was considerably more growth on the ampicillin plate than on the chlor plate. This could be because pSB1C3-M13ori-pCas9(gRNA3) successfully killed the BW23115F’ cells as desired or the phagemid was not effectively packaged<br />
<br />
Transformed pSB1C3-M13ori-pCas9(unmod) into chemically competent ER2738 cells that we know work from July.<br />
<br><br />
<br><br />
<br><br />
<br><br />
{{Template:UCB-Footer}}</div>Leighlahttp://2014.igem.org/Team:CU-Boulder/Notebook/CC9Phage_TeamTeam:CU-Boulder/Notebook/CC9Phage Team2014-10-16T04:44:37Z<p>Leighla: /* Week 4 */</p>
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<div>{{Template:UCB-Main}}<br />
{{UCB-NavBar}}<br />
__FORCETOC__<br />
<br><br />
<br><br />
<br><br />
<br />
'''Note:'''<br />
'''Litmus28ibb is a biobricked version of the Litmus28i (from NEB) vector which contains the M13ori (packaging signal) which allows for uptake into M13 or Fd phage. While making this part biobrick compatible, we added J04450 as the insert.'''<br />
'''pCas9 (BBa_K1218011) contains and tracrRNA and Cas9 gene under native promoters, and a minimal CRISPR arrays. The guide RNA (referred here as gRNA) is inserted into the CRISPR array.'''<br />
<br><br />
<br><br />
<br><br />
<br />
==Week 1==<br />
<br />
'''7/21'''<br />
*To ligate pCas9 to Litmus28ibb<br />
::-Digestion<br />
:::-Litmus28ibb-J04450 with E+P<br />
:::-pCas9 with E+P<br />
'''[[File:UCB-Cas9Phage-140721.JPG]]'''<br />
:1. pCas9<br />
:2. Litmus28ibb<br />
::-Overnight ligations (10hrs at 37C, 10min at 80C)<br />
<br />
'''7/22'''<br />
*Transformed ligation from 7/21 (Litmus28ibb + pCas9) into ER2738 cells<br />
::-Plated on Amp + Tet<br />
::-Later in day, pricked colonies from morning transformation (Litmus28ibb and pCas9 in ER)<br />
<br />
'''7/23'''<br />
*Miniprepped O/N from 7/22 of Litmus28ibb-pCas9<br />
::-Digested<br />
:::*None were the correct size. Smaller bands looked like pSB1C3. Plated cells onto Chlor and there was significant growth. pSB1C3 from the pCas9?<br />
<br />
'''7/24'''<br />
*To put pCas9 onto Litmus28i<br />
::-Digested<br />
:::-Litmus28ibb with EcoRI+PstI<br />
:::-pSB1C3-pCas9 with EcoRI+SpeI<br />
:::-PSB3C5-J04450 with XbaI + PstI<br />
'''[[File:UCB-Cas9Phage-140724.JPG]]'''<br />
:1. Litmus28ibb<br />
:2. pCas9<br />
:3. J04450<br />
::-Overnight ligation (10hr @ 16C)<br />
<br />
'''7/25'''<br />
*Transformed ligation from 7/24 (Litmus28ibb+pCas9) into 5alpha cells<br />
<br />
'''7/26'''<br />
*No growth from 7/25 transformation<br />
*Tranform<br />
::-Litmus28ibb + pCas9 + J04450<br />
*Retry making Litmus28i with pCas9<br />
::-Ligation (10hrs at 16C, 10min at 80C)<br />
:::*Litmus28ibb + pCas9 + J04450<br />
<br />
==Week 2==<br />
<br />
'''7/27'''<br />
*Transformation results<br />
::-No growth for no DNA controls (on Chlor)<br />
::-4 colonies for Litmus28bb-JO4450-Cas9 (on Amp-> none are red)<br />
*Picked all 4 colonies for O/Ns<br />
<br />
'''7/28'''<br />
*3 new red colonies on Litmus28bb-Cas9-J04450 plate<br />
::-Set up cultures<br />
*O/Ns grew but did not bother<br />
*Transformations<br />
::-Litmus28bb-J04450-Cas9<br />
::-No DNA control<br />
<br />
'''7/29'''<br />
*Litmus28bb-Cas9-J04450 O/Ns<br />
::-Incubator shut off during night. Samplers were at RT by morning<br />
::-Add 1mL to fresh 4mL LB and Amp<br />
::-Mini-prep DNA<br />
::-Digest with EcoRI and PstI to check insert for correct size<br />
'''[[File:UCB-Cas9Phage-140729.JPG]]'''<br />
::-Lane 2 contains the expected 3000bp band for the Litmus28ibb backbone and the ~7000bp band for the pCas9-J04450 insert. Unfortunately, this sample also contains a 4000bp band.<br />
*Plated the above sample<br />
<br />
'''7/30'''<br />
*Digested Litmus28ibb-Cas9-RFP with EcoRI-HF<br />
'''[[File:UCB-Cas9Phage-140730.JPG]]'''<br />
::-Extracted large band ~9000<br />
::-Re-ligated piece<br />
*Sent samples for sequencing<br />
::-Litmus28bb with Cas9 and RFP (LCR) gRNA sequence<br />
<br />
'''8/1'''<br />
*Mini-prepped O/Ns of<br />
::-Litmus28ibb-Cas9-J04450 (LCR)<br />
:::*Digested with EcoRI-HF and PstI-HF => all samples had band ~3700<br />
*Digestions of Litmus and Cas9 to retry ligation<br />
'''[[File:UCB-Cas9Phage-140801.JPG]] '''<br />
:1. Litmus28ibb (E+P)<br />
:2. pCas9(E+P)<br />
:3. Litmus28ibb (E+P)<br />
:4. pCas9(E+S)<br />
:5. pSB3C5 (X+P)<br />
::-Gel extractions<br />
::-Ligations<br />
:::*Litmus28ibb (E+P) to pCas9 (E+P)<br />
:::*Litmus28ibb(E+P) to pCas9(E+S) and J04450(E+P)<br />
<br />
'''8/2'''<br />
*Transformation into BW cells (5/30)<br />
::-Hundreds of colonies for LCR Miniprep<br />
*Digestion to get a better idea of what this DNA is<br />
<br />
'''8/3'''<br />
*Isolate the LCR from the extra band in the gel<br />
::-Run 8/2 digestions on gel<br />
'''[[File:UCB-Cas9Phage-140803.JPG]]'''<br />
:1. LCR (PstI)<br />
:2. LCR (EcoRI)<br />
::-Gel extracted <br />
:::-LCR digested with PstI-HF (top band<br />
::-Re-ligate extracted LCR with no insert (should re-ligate to itself)<br />
::-Transformed ligation into ER2738<br />
*Made O/Ns<br />
::-colonies from 8/2 transformation<br />
::-restreak of LCR from freeze down<br />
<br />
==Week 3==<br />
<br />
'''8/4'''<br />
*Check overnights from 8/3 (LCR = Litmus28ibb+pCas9+RFP) for correct inserts<br />
::-Mini-prep samples<br />
::-Digest with EcoRI and PstI<br />
:::*All had sizes ~3700bp<br />
:::*No correct inserts<br />
*Attempt to ligate Litmus28ibb and pCas9(gRNA3)<br />
::-Digestions<br />
:::*Litmus28ibb-RFP E+P extracted Lit back bone<br />
:::*pCas9 gRNA3 E+P extracted pCas9 insert<br />
'''[[File:UCB-Cas9Phage-140804.JPG]]'''<br />
:1. Litmus28ibb<br />
:2. pCas9(gRNA3)<br />
:3. pCas9(unmod.)<br />
::-Ligations (10 hrs at 16C, 10 min at 80C)<br />
:::*Litmus28ibb + pCas9(gRNA3)<br />
<br />
'''8/5'''<br />
*Transformation into ER2738 (7/18), 5ul of ligation products<br />
::1. Litmus28ibb-Cas9gRNA3<br />
<br />
'''8/6'''<br />
*Another attempt at ligating pCas9 and Litmus28ibb<br />
::-Digest unmodified pCas9 with EcoRI-HF and PstI-HF<br />
::-Digest pCas9 containing pRNA1 with EcoRI-HF and PstI-HF<br />
'''[[File:UCB-Cas9Phage-140806.JPG]]'''<br />
:1. pCas9(unmod.)<br />
:2. pCas9(gRNA1)<br />
::-Gel extract pieces<br />
::-Ligate separately to Litmus28ibb<br />
*Results of 8/5 transformation<br />
::-6 colonies for Litmus28ibb+pCas9(gRNA3) on Tet+reduced Amp<br />
:::*Picked colonies for overnights<br />
::-No growth for no DNA control<br />
*Transformation of ligations from earlier today<br />
::-Litmus28ibb + pSB1C3-pCas9(unmod.)<br />
::-Litmus28ibb + pSB1C3-pCas9(gRNA1)<br />
*Mini-prep Litmus28ibb+pCas9(gRNA3) O/N samples from earlier in day<br />
::-Digested with EcoRI-HF and PstI-HF<br />
:::*All samples had bands of 3000 and 2000bp. None were correct<br />
<br />
'''8/8'''<br />
*M13ori into SB Cas9<br />
::-Digestions<br />
:::-unmodified pCas9 with EcoRI-HF and XbaI<br />
:::-M13ori with EcoRI-HF and SpeI-HF<br />
:::-pCas9(gRNA1) with EcoRI-HF and XbaI<br />
:::-pCas9(gRNA3) with EcoRI-HF and XbaI<br />
<br />
'''8/9'''<br />
*To clone M13ori onto pSB1C3-pCas9<br />
::-Run 8/8 digestion on a gel<br />
'''[[File:UCB-Cas9Phage-140809.JPG]]'''<br />
:1. M13ori<br />
:2. pCas9(gRNA3)<br />
:3. pCas9(gRNA1)<br />
:-Gel extracted<br />
::-Ligation Ligated M13ori to….<br />
:::-pSB1C3-pCas9(gRNA1)<br />
:::-pSB1C3-pCas9(gRNA3)<br />
:::-pSB1C3-pCas9(unmod.)<br />
::-Transform and plate onto low Chlor 934 ug/mL)<br />
<br />
==Week 4==<br />
<br />
'''8/10'''<br />
*Digested ligation products from 8/6 and 8/4. Saw many large bands that may have been undigested<br />
*O/Ns of 8/9 transformation<br />
::-1 colony for M13ori-pCas9(gRNA3) (only 1 colony grew)<br />
::-11 colonies for M13ori-pCas9(gRNA1)<br />
::-No colonies grew for M13ori-pCas9(unmodified)<br />
<br />
'''8/11'''<br />
*Test 8/10 O/Ns for correct inserts<br />
::-Digest 8/10 O/Ns with EcoRI-HF and PstI-HF<br />
'''[[File:UCB-Cas9Phage-140811.JPG]]'''<br />
:1. M13ori-pCas9(gRNA3)<br />
:2-12. M13ori-pCas9(gRNA1)<br />
:*Lanes 1, 8, and 9 are about the right size. Sent these samples for sequencing<br />
*To ligate M13ori to pSB1C3-pCas9(unmod.)<br />
::-Digest<br />
:::*pSB1C3-pCas9(unmod.) (E+P)<br />
:::*M13 digested 8/8<br />
::-Ligation: See L5 below<br />
*To ligate pCas9 and M13ori to pSB1A3<br />
::-Digest<br />
:::*pSB1A3 (E+P)<br />
:::*M13ori (E+S)<br />
:::*Unmodified pCas9 (X+P)<br />
:::*pCas9(gRNA1)-M13ori (E+P)<br />
:::*pCas9(gRNA3)-M13ori (E+P)<br />
::-Ligation: See L2-4 below<br />
*Another try to put pCas9 onto Litmus28ibb<br />
::-Digested<br />
:::*Unmodified Litmus28i (E+P)<br />
:::*pCas9(unmod.) (E+P)<br />
:::*pCas9(gRNA1)-M13ori (E+P)<br />
:::*pCas9(gRNA3)-M13ori (E+P)<br />
::-Ligation: See L1 and L6 below<br />
*Gel for all<br />
'''[[File:UCB-Cas9Phage-140811-02.JPG]]'''<br />
<br />
:1. Unmodified Litmus28i<br />
:2. pSB1C3-pCas9(unmodified)<br />
:3. pSB1A3<br />
:4. pSB1C3-M13ori<br />
'''[[File:UCB-Cas9Phage-140811-03.JPG]]'''<br />
:5. pSB1C3-pCas9(unmod.)<br />
:6. pSB1C3-M13ori-pCas9(gRNA1)<br />
:7. pSB1C3-M13ori-pCas9(gRNA3)<br />
<br />
*Ligations for both projects<br />
<br />
{| class = "wikitable"<br />
|-<br />
! #<br />
! Backbone<br />
! Insert 1<br />
! Insert 2<br />
<br />
|-<br />
| 1<br />
| Unmod. Litmus28i (E+P)<br />
| pCas9(unmod.) (E+P)<br />
| N/A<br />
<br />
|-<br />
| 2<br />
| pSB1A3 (E+P)<br />
| pCas9(unmod.) (X+P)<br />
| M13ori (E+S)<br />
<br />
|-<br />
| 3<br />
| pSB1A3 (E+P)<br />
| pCas9(gRNA1)-M13ori (E+P)<br />
| N/A<br />
<br />
|-<br />
| 4<br />
| pSB1A3 (E+P)<br />
| pCas9(gRNA3)-M13ori (E+P)<br />
| N/A<br />
<br />
|-<br />
| 5<br />
| pSB1C3-pCas9(unmod.) (E+X)<br />
| M13ori (E+S)<br />
| N/A<br />
<br />
|-<br />
| 6<br />
| Litmus28ibb (E+P)<br />
| pCas9(gRNA3) (E+P)<br />
| N/A<br />
<br />
|-<br />
| 7<br />
| Litmus28ibb (E+P)<br />
| pCas9(gRNA1) (E+P)<br />
| N/A<br />
<br />
|-<br />
| 8<br />
| Litmus28ibb (E+P)<br />
| pCas9(unmod.) (E+P)<br />
| N/A<br />
<br />
|-<br />
|}<br />
<br />
'''8/12'''<br />
*Transform ligation from 8/11 into ER2738 cells<br />
*Make phage using M13g6A1 as Helper Phage<br />
::-used pCas9(gRNA1)-M13ori or pCas9(gRNA3)-M13ori as phagemid<br />
*Freeze downs<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Top<br />
! Side<br />
<br />
|-<br />
| M. gRNA3 Cas9 (8/12)<br />
| M13ori Cas9 gRNA3 pSB1C3<br />
<br />
|-<br />
| M. gRNA1 Cas9 (8/12)<br />
| M13ori(8) Cas9 gRNA1 pSB1C3<br />
<br />
|-<br />
| M. gRNA1 Cas9 (8/12)<br />
| M13ori (9) Cas9 gRNA1 pSB1C3<br />
<br />
|-<br />
|}<br />
<br />
'''8/13'''<br />
*Finish phage isolation<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Helper phage<br />
! Phage<br />
! A269<br />
! A320<br />
! genome size<br />
! [ ]<br />
<br />
|-<br />
| Fd-CAT<br />
| M. gRNA1<br />
| 0.353<br />
| 0.025<br />
| 7687<br />
| 2.56 x10^12<br />
<br />
|-<br />
| Fd-CAT<br />
| M. gRNA3<br />
| 0.554<br />
| 0.037<br />
| 7687<br />
| 4.40 x10^12<br />
<br />
|-<br />
|}<br />
<br />
*Infection<br />
::-Infecting ER2738 and BWF’<br />
::-Phage<br />
:::*pSB1C3-pCas9(gRNA1)<br />
:::*pSB1C3-pCas9(gRNA3)<br />
*Transformation Results from 8/12<br />
::-Growth for the following… (made O/Ns)<br />
:::*pSB1A3-M13ori-pCas9(gRNA3)<br />
:::*pSB1C3-M13ori-pCas9(unmod)-M13ori<br />
:::*Litmus28ibb-pCas9(gRNA3)<br />
:::*Litmus28ibb-pCas9(gRNA1)<br />
:::*Litmus28ibb-pCas9(unmod.)<br />
::-Hundreds of colonies for pCas9(unmod.) on Unmod. Litmus28i. Didn’t trust result so didn’t pick coloneis. EcoRI and PstI sites touch so most likely, Litmus28i was only cut once<br />
<br />
'''8/15'''<br />
*Check O/Ns from 8/14 for correct inserts<br />
::-Mini-prepped samples<br />
::-Digested with E and P<br />
::-Results<br />
:::*No bands of the right size for<br />
:::-Litmus28ibb-pCas9(gRNA3)<br />
:::-Litmus28ibb-pCas9(gRNA1)<br />
:::-Litmus28ibb-pCas9(unmod.)<br />
:::-Expected band sizes for… (see below)<br />
*pCas9(gRNA3)-M13ori on pSB1A3<br />
*pCas9(unmod)-M13ori on pSB1C3<br />
'''[[File:UCB-Cas9Phage-140815.JPG]]'''<br />
<br />
*Transform into BWF’ to test for functionality of Cas9 when associated with m13ori, independent of infectivity<br />
::-M13ori-pCas9(gRNA1)<br />
::-M13ori-pCas9(gRNA3)<br />
<br />
'''8/16'''<br />
*PCR on LCR (Litmus28ibb with pCas9 and RFP to maybe get a good product to religate<br />
::-Didn’t work<br />
<br />
Results of 8/13 infection of BWF’ with M13ori-gRNA(1 or 3) to test pCas9 killing<br />
:No growth on DNA controls on Chlor(34ug/mL) or Kan(50ug/mL)+Chlor(34ug/mL)<br />
:ON Kan(50ug/mL) + Chlor (170ug/mL) in BWF'<br />
:::gRNA1--> 22 colonies<br />
:::gRNA3--> 2 colonies<br />
<br />
On Kan(25ug/mL)+Chlor(34ug/mL) in BWF’<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! 1:100<br />
! 1:1000<br />
<br />
|-<br />
| gRNA1<br />
| 617<br />
| 84<br />
<br />
|-<br />
| gRNA3<br />
| 469<br />
| 42<br />
<br />
|-<br />
|}<br />
On Chlor (34ug/mL) in BWF’<br />
<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! 1:100<br />
! 1:1000<br />
<br />
|-<br />
| gRNA1<br />
| 508<br />
| 67<br />
<br />
|-<br />
| gRNA3<br />
| 447<br />
| 41<br />
<br />
|-<br />
|}<br />
On Chlor (34ug/mL) in ER<br />
<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! 1:100<br />
! 1:1000<br />
<br />
|-<br />
| gRNA1<br />
| 702<br />
| 93<br />
<br />
|-<br />
| gRNA3<br />
| 518<br />
| 58<br />
<br />
|-<br />
|}<br />
<br />
Results of 8/15 transformation of BWF’ with M13ori—gRNA(1 or 3) to test killing<br />
<br />
Chlor (34ug/mL) + Kan (50ug/mL)<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! 1:1<br />
! 1:10<br />
<br />
|-<br />
| gRNA1<br />
| Lawn<br />
| 1904<br />
<br />
|-<br />
| gNRA3<br />
| Slightly thinner lanw<br />
| 1616<br />
<br />
|-<br />
|}<br />
<br />
Chlor (34 ug/mL)<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! Trial 1<br />
! Trial 2<br />
<br />
|-<br />
| gRNA1<br />
| 1788<br />
| 1548<br />
<br />
|-<br />
| gRNA3<br />
| 1564<br />
| 1416<br />
<br />
|-<br />
|}<br />
Samples were diluted 1:10<br />
<br />
'''8/17'''<br />
Make phage<br />
::pSB1C3-M13ori-pCas9(unmod)<br />
::pSB1A3-M13ori-pCas9(gRNA3)<br />
Ran uncut LCR on a gel and gel extracted the three bands<br />
<br />
'''8/18'''<br />
While making phage…. We threw away the samples….<br />
Remake phage. Same as 8/17<br />
Transform LCR gel extractions into 5alpha cells<br />
<br />
8/19<br />
Sent samples for sequencing<br />
::pSB1C3-M13ori-pCas9(unmodified)<br />
::pSB1A3-M13ori-pCas9(gRNA3)<br />
<br />
Finish making phage<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Name<br />
! A269<br />
! A320<br />
! Genome Size<br />
! [ ]<br />
<br />
|-<br />
| pSB1A3-M13ori-pCas9(gRNA3)<br />
| 0.697<br />
| 0.162<br />
| <br />
| Note: We later found that this sample did not actually contain the M13ori<br />
<br />
|-<br />
| pSB1C3-M13ori-pCas9(unmod)<br />
| 0.764<br />
| 0.146<br />
| 7687<br />
| 3.044 x10^13<br />
<br />
|-<br />
|}<br />
Infect cells (ER and BWF’)<br />
::Grew a single 75mL sample. When OD ~0.5, divided samples into 3<br />
::Added 20mL of sample to 125mL flask (3 for each strain)<br />
::Added phage to a final concentration of 1 x10^8 phage/mL<br />
::Incubated at 250rpm, 37C, for 30 minutes<br />
::Plated at dilutions (1:10, 1:100, and 1:1k)<br />
:::On Chlor(170ug/mL), Kan(50ug/mL)+Chlor(85ug/mL), Kan(50ug/mL)+Chlor(170ug/mL)<br />
<br />
<br />
'''8/21'''<br />
Finished phage amplification protocol from 8/20 but did not receive any phage. <br />
Infect BWF’ cells with the phage having pSB1C3-M13ori-pCas9 containing the scramble, gRNA1, and gRNA3.<br />
<br />
'''8/22''<br />
Present project to department<br />
<br />
'''8/25'''<br />
Results of 8/21 infection experiment<br />
<br />
Results on Kan and Chlor<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! Scramble<br />
! gRNA1<br />
! gRNA3<br />
<br />
|-<br />
| 1:1<br />
| 149<br />
| 143<br />
| 13<br />
<br />
|-<br />
| 1:10<br />
| 14<br />
| 16<br />
| 1<br />
<br />
|-<br />
|}<br />
<br />
Results on Chlor<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! Scramble<br />
! gRNA1<br />
! gRNA3<br />
<br />
|-<br />
| 1:1<br />
| 147<br />
| 117<br />
| 14<br />
<br />
|-<br />
| 1:10<br />
| 14<br />
| NA<br />
| 3<br />
<br />
|-<br />
|}<br />
<br />
'''8/28'''<br />
Retry ligation of pCas9 to litmus28ibb<br />
Digest Litmus28ibb-J04450, pCas9, and amilCP with EcoRI and PstI. Gel extract pieces<br />
Ligate pCas9 into Litmus28ibb and amilCP into Limus28ibb<br />
<br />
'''8/29'''<br />
Transform 8/28 ligation products into 5alpha cells<br />
<br />
'''8/29'''<br />
Transform pSB1C3-pCas9(unmodified) into BW and BWF’ to test targeting of f’ episome. Due to poor infection results we wanted to test the guide RNAs for targeting of the F’ episome. We have searched the vast webs for the sequence of the F’ episome but have yet to (and never will) find it.<br />
<br />
'''8/30'''<br />
Transformation results of pCas9 into BW and BWF’. Results were unexpected, we expected more growth for BW sample or at least equal growth. Differences in competence or concentration could explain results in initial count<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Initial count<br />
! After another 24 hours<br />
<br />
|-<br />
| No DNA controls (for BW and BWF’)<br />
| No growth<br />
| No growth<br />
<br />
|-<br />
| BW cells<br />
| 9 colonies<br />
| 222<br />
<br />
|-<br />
| BWF’ cells<br />
| ~100 colonies<br />
| 158<br />
<br />
|-<br />
|}<br />
<br />
Transformation results for ligation of pCas9 to Litmus28ibb<br />
<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! Amp (50ug/mL)<br />
! Amp (100ug/mL)<br />
! Notes<br />
<br />
|-<br />
| No DNA control<br />
| 3<br />
| 10<br />
| All bright white<br />
<br />
|-<br />
| Litmus28ibb-pCas9<br />
| ~50 (many are red)<br />
| 25-30<br />
| High proportion are red. Some are shadowy white. A few are bright white<br />
<br />
|-<br />
| Litmus28ibb-amilCP<br />
| >300<br />
| >300<br />
| Mostly shadowy, some red, some bright white<br />
<br />
|-<br />
|}<br />
<br />
Made overnight cultures of shadowy and bright coloneis from Litmus28ibb-pCas9 samples (from both plates) and of shadowy colonies from Litmus28ibb-amilCP. Since growth on no DAN control was bright white, we assumed that bright colonies on sample plates were more likely to be contamination, but didn’t really know. Since there were fewer colonies on no DNA control plates, we figures we could randomly select colonies from the sample plates that were not a result of contamination.<br />
<br />
'''8/31'''<br />
Mini-prep overnight cultures from 8/30 and digest samples with EcoRI and PstI. When run on a gel, none of the Litmus28ibb-pCas9 samples yielded bands of the correct size. All Litmus28ibb-amilCP samples did yield the expected band sizes. <br />
<br />
'''9/2'''<br />
Sent some of mini-prep samples from 8/31 for sequencing<br />
<br />
'''9/3'''<br />
Digestion of pCas9 with EcoRI and speI and the unmodified NEB version of Litmus28i with EcoRI and XbaI. Gel extracted pieces and ligated together (No, this part would not have the BioBrick prefix/suffix)<br />
<br />
'''9/4'''<br />
Transform 9/3 Litmus28i-pCas9 ligation into 5alpha cells. Selected on low ampicillin (50ug/mL) and high ampicillin (100ug/mL)<br />
<br />
'''9/5'''<br />
Did receive colonies from 9/4 transformation. Selected 24 colonies for colony PCR with a primer pair that would span the junction of pCas9 and Litmus28i. (forward primer bound to CRISPR region of pCas9 while reverse primer bound in the middle of the Litmus28i backbone)<br />
Colony PCR failed to reveal any promising samples. Set up overnight cultures in a hopeless effort to yield different results.<br />
<br />
'''9/7'''<br />
Mini-prepped overnight cultures from 9/5. Digested each with EcoRI and PstI and ran reults on gel. Results were consistent with colony PCR: failure. A few samples had very feint bands that were approximately the correct size<br />
<br />
'''9/8'''<br />
for the samples on 9/7 with light bands of approximately the correct size, we sent samples for sequencing (another hopeless attempt to get different results)<br />
<br />
'''9/9'''<br />
Sequencing results showed that Limtus28i religated<br />
<br />
'''9/17'''<br />
New method to clone pCas9 onto Litmus28ibb vector: PCR->Digestion->ligation method<br />
:::PCR amplified pCas9 construct from pSB1C3-pCas9. PCR amplified Litmus28ibb backbone from Litmus28ibb-J04550<br />
:::PCR purified both samples. Digested both samples with EcoRI and PstI and gel extracted the pieces<br />
:::Ligated pCas9 to Litmus28ibb vector<br />
<br />
Sent pSB1C3-M13ori-pCas9 sample for sequencing to sequence the entire plasmid<br />
<br />
'''9/18'''<br />
Transform 9/17 ligation (Litmus28ibb-pCas9) into 10beta cells<br />
<br />
'''9/19'''<br />
There was growth on no DNA control. Some colonies for sample.<br />
<br />
'''9/20''' <br />
Make chemically competent ER and 5alpha cells using DMSO from bottle instead of fancy tubes. We think this is the reason for later transformation failures<br />
<br />
Digested pCas9 and Litmus28ibb PCR purifications from 9/17 with EcoRI, PstI, and DpnI. PCR purified digestion samples then ligated pieces together.<br />
<br />
'''9/21'''<br />
New idea: Cloning pCas9 onto Litmus28i has not worked up until this point. What if we constructed a vector that looked similar to litmus28i. pSB1A3 has the same origin of replication and almost the same ampicillin resistance marker (with a few SNPs). Need to add M13ori (which was taken from Litmus28i) and pCas9. M13ori would not be in the correct position on plasmid but this is as close as we can get with the remaining time<br />
::Digestions: M13ori-pCas9(unmod), pCas9(unmod), pSB1A3 with EcoRI and PstI. Gel showed incomplete digestion so did not gel extract<br />
<br />
Transformation into 5alpha cells: no DNA control, 9/20 ligation (Litmus28ibb-pCas9), pSB1C3-amilCP, and pSB1C3-M13ori-pCas9<br />
<br />
'''9/22'''<br />
Re-digested M13ori-pCas9(unmod) and pSB1A3-J04450 with EcoRI and PstI. pCas9(unmod) was PCR purified (product looked clean on gel so did not bother to gel extract). <br />
Ligated M13ori-pCas9(unmod) to pSB1A3 and pCas9(unmod) to pSB1A3<br />
Sent pSB1A3 to M13ori-pCas9 for sequencing to cover region not covered by previous sequencing<br />
<br />
'''9/23'''<br />
Sequencing results came back<br />
:::Litmus28ibb-J04450 contains two nucleotides that are flipped. They are located within the ampicillin resistance gene and do change two amino acids; however, sample grows on ampicillin so these are not loss of function mutation. <br />
:::pSB1C3-M13ori-pCas9 was perfect - the above two samples are ready to submit to the registry<br />
<br />
Transform ligations from 9/22 (pSB1A3- M13ori-pCas9(unmod) and pCas9(unmod)) into 5 alpha cells<br />
Colony PCR 18 samples from 9/20 Limtus28ibb-pCas9(unmod) plate.<br />
<br />
'''9/24'''<br />
Run gel of 9/23 colony PCR to check for desired Litmus28ibb-pCas9(unmod). There was a band of approximately the correct size in each samples (including the no DNA control). Set up overnight cultures of first 5 samples for further testing<br />
Results of 9/23 transformation<br />
::No growth for no DNA control on chlor (170ug/mL), Amp (50ug/mL) or Amp (100ug/mL)<br />
::7 colonies for pSB1C3-M13genes on chlor (170ug/mL)<br />
::27 colonies for pSB1A3-pCas9 on Amp (100ug/mL) and 41 colonies on Amp (50ug/mL)<br />
::0 colonies for pSB1A3-M13ori-pCas9 on Amp(50 and 100ug/mL)<br />
Set up overnight cultures of some of the samples.<br />
<br />
<br />
'''9/25'''<br />
Mini-prep overnights from 9/24<br />
Digest samples to check for insert. When run on a gel, 3 samples had large bands of ~5000bp and one had an additional band at ~6000. We made overnights of these 3 colonies<br />
<br />
'''9/26'''<br />
Digested mini-prepped DNA from the three samples that looked promising from 9/25. Each sample was digested with only EcoRI, only PstI, or EcoRI and PstI. When run on a gel, it can be seen that each samples, when cut once, yielded one band 8000bp while when cut twice, yielded 2 bands of ~2000 and 5000 as expected.<br />
Sent parts to iGEM registry: pSB1C3-M13ori-pCas9 and Litmus28ibb-J04450<br />
To add the M13ori to pSB1A3-Cas9<br />
::Digested pSB1A3-pCas9 with EcoRI and XbaL<br />
::Digested pSB1C3-M13ori with EcoRI and SpeI<br />
::Gel extracted bands of the correct sizes and combined these into a ligation reaction.<br />
<br />
'''9/27'''<br />
Transform pSB1A3-M13ori-pCas9 into Amp(50ug/mL) and Amp(100ug/mL)<br />
Later in the day…. Set up overnight cultures of colonies<br />
<br />
<br />
'''9/28'''<br />
Mini-prepped overnights made 9/27<br />
Digested overnight samples with EcoRI and PstI<br />
Did receive pSB1A3-M13ori-pCas9 samples with bands of the correct size (as compared to pSB1A3-pCas9)<br />
Set up overnight cultures of the samples that looked promising<br />
Transformed pSB1A3-M13ori-pCas9(unmod) and pSB1A3-M13ori-pCas9(gRNA3) into ER2738 cells<br />
<br />
'''9/29'''<br />
Did receive colonies from 9/28 transformation. Set up overnight cultures for both.<br />
Also set up overnight cultures of ER2738 and BWF’ for upcoming infection.<br />
Start phage amplification protocol using pSB1A3-M13ori-pCas9(unmod) and pSB1A3-M13ori-pCas9(gRNA3) as the phagemid. Used Fd-Cat as the helper phagemid<br />
<br />
'''9/30'''<br />
Finished phage amplification/isolation protocol for Fd phage packaging pSB1A3-M13ori-pCas9(unmod) and pSB1A3-M13ori-pCas9(gRNA3)<br />
<br />
'''10/1'''<br />
Measured concentration of phage isolated on 9/30. There were no phage for the gRNA3 sample. The pSB1A3-M13ori-pCas9(unmod) sample contained some phage but had a high level of contamination/impurity.<br />
Send samples pSB1A3-M13ori-pCas9(unmod) and pSB1A3-M13ori-pCas9(gRNA3) samples for sequencing to verify the presence of the M13ori<br />
<br />
'''10/2'''<br />
Sequencing results came in<br />
::pSB1A3-M13ori-pCas9(unmod) looks good<br />
::pSB1A3-M13ori-pCas9(gRNA3) doesn’t have the M13ori<br />
Plan<br />
:1.SDM on pSB-M13ori-pCas9(unmod) to remove BsaI cut site in pSB1A3 backbone<br />
:2.Change spacer from unmodified to the gRNA3<br />
:3.Redo the infection experiment<br />
SDM<br />
:PCR to amplify pSB1A3-M13ori-pCas9(unmod)<br />
:Treat with kinase, Ligase, and DpnI<br />
:Transform into NEB 5alpha cells<br />
<br />
<br />
'''10/3'''<br />
Set up O/N cultures of 10/2 transformation<br />
<br />
'''10/4'''<br />
Mini-prep overnights. Samples are called pSB1A3(delta BasI)-M13ori-pCas9(unmod) to indicate each part of the construct and the removed BsaI site in the pSB1A3 backbone.<br />
Changing the gRNA spacer in pCas9<br />
::Vector digest: digested pSB1A3(delta BasI)-M13ori-pCas9(unmod) with BsaI to remove unmodified spacer. Saw only one band on the gel (cut around spacer) which shows that the additional BsaI site in the pSB1A3 backbone was removed. Gel extracted the digested vector<br />
::Ligation: Used the annealed oligos for gRNA3 (our targeting sample) from the last time we changed the spacer. Ligated these oligos to the digested pSB1A3(delta BasI)--M13ori-pCas9<br />
<br />
'''10/5'''<br />
Transform pSB1A3(delta BasI)-M13ori-pCas9(unmod) and pSB1A3(delta BasI)-M13ori-pCas9(gRNA3) in ER2738 cells<br />
<br />
'''10/6'''<br />
Transformation results from 10/5<br />
:No growth for no DNA control<br />
:pSB1A3(delta BasI)-M13ori-pCas9(unmod) = many colonies<br />
:pSB1A3(delta BasI)-M13ori-pCas9(gRNA3) = no colonies<br />
<br />
Re-anneal primers then relegate<br />
::Phosphorylation of oligos for gRNA3. Incubate for 1 hour at 37C then 20 minutes at 65C<br />
::Add 2.5ul 1M NaCl<br />
::Anneal in thermocycler. 5 minutes at 95C then a slow cool-down for ~2 hours<br />
::Dilute product 1:10<br />
::Ligation of newly annealed primers to digest and gel extracted pSB1A3(delta BasI)-M13ori-pCas9(unmod) from 10/5<br />
<br />
'''10/7'''<br />
Transform 10/6 ligation into ER2738 cells<br />
<br />
'''10/8'''<br />
No growth for 10/7 transformation<br />
Transform ligation from 10/6 into 5alpha cells<br />
Last try to make pSB1A3-M13ori-pCas9(gRNA3)<br />
:Know from 10/1 sequencing that we have pSB1A3-pCas9(gRNA3). Therefore, only need to add M13ori upstream of pCas9 part<br />
:Digest pSB1A3-pCas9(gRNA3) with EcoRI and XbaI and pSB1C3-M13ori with ExoRI and SpeI<br />
:Gel extract digested pieces<br />
:Ligate gel extracted pieces together<br />
<br />
'''10/9'''<br />
No growth for 10/8 transformation<br />
Transform 10/8 ligation into 5alpha cells<br />
<br />
'''10/10'''<br />
Transform <br />
:pSB1C3-M13ori-pCas9(unmod) from 10/9 into ER2738 cells<br />
:pSB1A3 (delta BsaI)-M13ori-pCas9(gRNA3) from 10/6 into 5 alpha cells<br />
:pSB1A3-M13ori-pCas9(gRNA3) from 10/8 into 5 alpha cells<br />
<br />
'''10/11'''<br />
Transformation results of 10/10<br />
:1 colony for pSB1C3-M13ori-pCas9(unmod) in ER2738 (does not seem trustworthy)<br />
:No colonies for no DNA control<br />
:No colonies for the 2 other transformations<br />
<br />
Transform pSB1C3-M13ori-pCas9(unmod) into ER2738 cells again<br />
Start phage amplification protocol to make the following phage<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Phagemid<br />
! Helper Phagemid<br />
<br />
|-<br />
| pSB1C3-M13ori-pCas9(unmod)<br />
| M13g6A1<br />
<br />
|-<br />
| pSB1C3-M13ori-pCas9(unmod)<br />
| M13K07<br />
<br />
|-<br />
| pSB1C3-M13ori-pCas9(gRNA3)<br />
| M13g6A1<br />
<br />
|-<br />
| pSB1C3-M13ori-pCas9(gRNA3)<br />
| M13K07<br />
<br />
|-<br />
|}<br />
<br />
pSB1C3-M13ori-pCas9(unmod) grew unexpectedly fast so we missed the OD mark. Growth rate compared to pSB1C3-M13ori-pCas9(gRNA3) sample suggests contamination. Did not proceed with making these phage for today.<br />
<br />
'''10/12'''<br />
No growth for 10/11 transformation – conclude that ER2738 cells are not competent (these are the cells made 9/20 with DMSO from a room temp bottle instead of the alliquotes from NEB.<br />
Remake phage<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Phagemid<br />
! Helper Phagemid<br />
<br />
|-<br />
| pSB1C3-M13ori-pCas9(unmod)<br />
| M13g6A1<br />
<br />
|-<br />
| pSB1C3-M13ori-pCas9(unmod)<br />
| M13K07<br />
<br />
|-<br />
|}<br />
<br />
'''10/13'''<br />
Finish phage isolation protocol and measure<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Phagemid<br />
! Helper Phagemid<br />
! Dilution<br />
! A269<br />
! A320<br />
! Genome size<br />
! Diluted [ ]<br />
! Actual [ ]<br />
<br />
|-<br />
| pSB1C3-M13ori-pCas9(gRNA3)<br />
| M13g6A1<br />
| 1:40<br />
| 0.090<br />
| 0.015<br />
| 7687<br />
| 5.85 E11<br />
| 2.34 E13<br />
<br />
|-<br />
| pSB1C3-M13ori-pCas9(gRNA3)<br />
| M13K07<br />
| 1:40<br />
| 0.085<br />
| 0.013<br />
| 7687<br />
| 5.62 E11<br />
| 2.25 E13<br />
<br />
|-<br />
| pSB1C3-M13ori-pCas9(unmod)<br />
| M13g6A1<br />
| 1:40<br />
| 0.104<br />
| 0.022<br />
| 7687<br />
| 6.40 E11<br />
| 2.56 E13<br />
<br />
|-<br />
| pSB1C3-M13ori-pCas9(unmod)<br />
| M13K07<br />
| 1:20<br />
| 0.091<br />
| 0.016<br />
| 7687<br />
| 5.85 E11<br />
| 1.17 E13<br />
<br />
|-<br />
|}<br />
<br />
Infection with phage of BW23115F’ cells<br />
:Grew a single stock of cells then dived into 4 samples each with 50mL cells. Added the above phage<br />
:Incubated for 30 minutes at 37C, shaking<br />
:Plated onto Chlor<br />
<br />
'''10/14'''<br />
Infection results<br />
:No growth on Chlor (170ul/mL)<br />
:Very little to no growth on pCas9(unmod) samples. We think this is b/c the colony picked on 10/11 did not actually contain pSB1C3-M13ori-pCas9(unmod) so nothing was packaged into phage<br />
:~100 colonies for pCas9(gRNA3) samples.<br />
:The samples made with M13g6A1 were also plated on ampicillin. These plates show considerable growth. There is more growth on <br />
pCas9(unmod) than pCas9(gRNA3) sample plate. We believe this further supports our hypothesis that our pCas9(unmod) sample did not contain pSB1C3-M13ori-pCas9(unmod) so packaged M13g6A1 in the absence of a better phagemid. Also, for the pCas9(gRNA3) sample, there was considerably more growth on the ampicillin plate than on the chlor plate. This could be because pSB1C3-M13ori-pCas9(gRNA3) successfully killed the BW23115F’ cells as desired or the phagemid was not effectively packaged<br />
<br />
Transformed pSB1C3-M13ori-pCas9(unmod) into chemically competent ER2738 cells that we know work from July.<br />
<br><br />
<br><br />
<br><br />
<br><br />
{{Template:UCB-Footer}}</div>Leighlahttp://2014.igem.org/Team:CU-Boulder/Notebook/Phage_TeamTeam:CU-Boulder/Notebook/Phage Team2014-10-16T04:25:05Z<p>Leighla: </p>
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<div>{{Template:UCB-Main}}<br />
{{UCB-NavBar}}<br />
__FORCETOC__<br />
<br />
'''Phage Delivery'''<br />
==Week 1==<br />
'''Notes:''' Unless stated otherwise, all gels contain 2-log ladder<br />
<br />
'''5/9'''<br />
*Obtained BW23115 KanR cells- BW23115 cells that had their native CRISPR-Cas system knocked out by the insertion of a Kanamycin resistance gene<br />
::-Will also be called BW23115 or BW<br />
::-Conjugated BW23115 KanR cells with contain F’ notation (ex. BWF’)<br />
*Obtained ER2738 cells that contain the F’ episome (no changes from NEB sample)<br />
::-Will also be called ER. Assume that all ER samples contain the F’ episome<br />
::-Streaked sample onto LB+Tet (20ug/mL) to select for colonies containing F’ episome<br />
<br />
'''5/10'''<br />
*Did receive colonies from 5/9 selection<br />
<br />
==Week2==<br />
'''5/12'''<br />
*Need to conjugate BW23115 KanR cells with the F’ episome<br />
::-Set up overnight cultures of ER2738 and BW23115 KanR<br />
::-When mixed, ER2738 will donate it’s F’ episome and BW23115 KanR will receive the F’ episome. F’ episome confers Tetracycline resistance<br />
<br />
'''5/13'''<br />
*Started M13 Amplification: Amplify M13 phage using the M13K07 Helper Phage<br />
::-Let precipitated in NaCl/PEG solution overnight<br />
::-Possible sources of error<br />
:::*Did not sterilize 2.5M NaCl/20% PEG-8000 solution<br />
:::*Added 4-fold PEG solution<br />
::::Compensated by adding more LB<br />
:::*During precipitation, put sample in -20C for 30 minutes before realizing mistake and moving to it to 4C. Sample partially froze<br />
*Conjugated BW23115 with F’ episome<br />
::-Added 1mL BW23115 to 1mL ER2738 overnight culture<br />
::-Incubated at 37C for 30 minutes, shaking<br />
::-Plated on LB+Kan(50ug/mL)+Tet(20ug/mL)<br />
:::*To select for BW cells that took the F’ episome (containing Tet resistance)<br />
<br />
'''5/14'''<br />
*Finished the M13 Amplification<br />
::-Visualized product on UV-vis. There was a tall spike at 269nm indicating that DNA was present. Did not test at 320nm.<br />
*Results of BW23115 Conjugation<br />
::-Many colonies indicating successful conjugation of F’ episome into BW23115<br />
::-Set up overnight to make freeze down tomorrow<br />
*Set up overnight of ER2738 to make chemically competent tomorrow<br />
<br />
'''5/15'''<br />
*Made freeze down of BW23115 KanR F’<br />
::-BW23115 E. coli strain with Kanamycin resistance gene inserted into genome and with F’ episome<br />
*Made chemically competent ER2738 cells<br />
*Transformation of Litmus28i (from NEB) into chemically competent ER2738 cells<br />
::-Added 1ul Litmus28i plasmid to 40ul competent cells<br />
::-Plated on LB + Amp(100ug/mL)<br />
::-Purpose: To make M13 phage that package Litmus28i DNA. Need phagemid (Litmus28i) DNA in infectable cells (cells containing F’ episome) to introduce M13K07 Helper Phage and make phage. <br />
<br />
'''5/16'''<br />
*Results of 5/15 transformation<br />
::-No growth for No DNA control<br />
::-Many colonies for sample<br />
<br />
==Week 3==<br />
'''5/19'''<br />
*M13 Amplification to isolate M13-Litmus28i phage<br />
::-Cells: ER2738 cells containing Litmus28i phagemid<br />
::-Helper Phage: M13K07<br />
::-Not much phage was precipitated<br />
*Set up overnight culture of ER2738 to infect tomorrow<br />
<br />
'''5/20'''<br />
*Infected ER2738 cells with M13-Litmus28i phage<br />
::-Plated only on Ampicillin(100ug/mL) (should have also plated on kanamycin)<br />
::-Infected for 4-5 hours-> should have only infected for 30 minutes maximum. This extra time gives the cells that were infected with M13-M13K07 the time to produced M13-M13K07 phage and reinfect<br />
<br />
'''5/21'''<br />
*Results from M13-Litmus28i infection of ER2738<br />
::-Solid lawn of growth for diluted and non-diluted<br />
::-Also sickly looking growth<br />
*Set up overnights<br />
::-ER2738 cells containing Litmus28i for freeze down<br />
::-BW23115 with F’ episome to make chemically competent cells <br />
::-ER2738 to redo infection<br />
<br />
'''5/22'''<br />
*Tested absorbance of phage produced through M13 amplification on 5/19<br />
::-Low absorbance of 0.018 at 269nm but no detection at wavelength 320nm<br />
::-Decided to redo M13 amplification<br />
*Made chemically competent BW23115 with f-episome<br />
*Made freeze down of ER2738 containing Litmus28i<br />
*Set up overnight of ER2738 containing Litmus28i to redo M13 amplification tomorrow<br />
<br />
'''5/23'''<br />
*Protocol switch to make phage using phagemid<br />
::-“M13 Amplification” protocol should only be used to make more M13-M13K07, not to make M13 phage containing a different phagemid<br />
::-Switched to new protocol (“Use of M13K07 Helper Phage for isolation of single stranded phagemid DNA” by NEB. Made modifications (see our protocols) to isolate phage rather than single-stranded DNA)<br />
::-Making phage….<br />
{| class = "wikitable"<br />
|-<br />
! Helper phage<br />
! Phagemid<br />
! Cells<br />
! Notes<br />
<br />
|-<br />
| M13K07<br />
| None<br />
| ER2738<br />
| Make more M13-M13K07<br />
<br />
|-<br />
| M13K07<br />
| Litmus28i<br />
| ER2738<br />
| Test packaging of Litmus28i<br />
<br />
|-<br />
|}<br />
*Made fresh antibiotics<br />
::-Chloramphenicol (34 ng/mL)<br />
:::*1.44g chloramphenicol into 42mL EtOH<br />
::-Ampicillin (50 ng/mL)<br />
:::*4g ampicillin into 80mL mili-Q H2O<br />
<br />
'''5/24'''<br />
*Isolated phage using new protocol<br />
::-Resuspended pellet in 200ul TBS and 200ul 30% glycerol<br />
::-Measured absorbance with UV-vis<br />
:::*concentration (phage/mL) = 6x10^16 x (A269-A320)/ (#of base pairs in the phage genome)<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! Abs (269nm)<br />
! Abs (320nm)<br />
! Genome size<br />
! Concentration (phage/mL)<br />
<br />
|-<br />
| M13-M13K07<br />
| 0.721<br />
| 0.060<br />
| <br />
| 4.57 x10^12<br />
<br />
|-<br />
| M13-Litmus28i<br />
| 0.250<br />
| 0.028<br />
| 2823<br />
| 4.72 x10^12<br />
<br />
|-<br />
|}<br />
*Infect ER2738 cells with M13-Litmus28i<br />
::-Wanted 1:10 phage:cell ratio. Math….<br />
:::*At 1 OD (e.coli), cell/mL = 5x10^8<br />
:::*5x10^7 phage * (1mL/4.72x10^12 phage) = 0.011ul phage<br />
*Set up overnights<br />
::-ER2738 for infection with M13-Litmus28i<br />
::-BW23115 F’ for infection with M13-Litmus28i to test infectivity of conjugated strain<br />
<br />
==Week 4==<br />
'''5/25'''<br />
*Infect ER and BWF’ cells with M13-Litmus28i<br />
::*Made 5mL culture of ER and BW that was at 1 OD<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! OD<br />
! mL sample for 1OD in 5mL<br />
! mL LB to 5mL<br />
<br />
|-<br />
| ER2738<br />
| 2.5<br />
| 2 mL<br />
| 3 mL<br />
<br />
|-<br />
| BW23115<br />
| 2.0<br />
| 2.5 mL<br />
| 2.5 mL<br />
<br />
|-<br />
|}<br />
::-Based on calculations from 5/24, we needed to add 0.011 ul phage per 1 mL of cells at 1 OD. This equates to 0.055 ul of phage into 5 mL cells; therefore we made a 1:10 dilution so we could add 0.5ul. Unfortunately, the pipet would not take up 0.5ul so we added 0.8ul of M13-Litmus28i phage<br />
::-Grew the cells for 20 minutes at 37C<br />
::-Plated 300ul onto Kanamycin (50ug/mL) and 300ul onto Ampicillin (100ug/mL) for each sample<br />
:::*Incubated overnight at 37C<br />
*'''Note:''' During the production of phage, the phagemid SHOULD be packaged preferentially over the Helper Phagemid but some Helper Phagemid will still be packaged. We plated on Amp to select for cells that were infected with phage containing Phagemid. We plated on Kan to select for cells that were infected with phage containing Helper Phagemid. This allows us to compare the packaging efficiency of Helper Phagemid: Phagemid.<br />
'''5/26'''<br />
*Results from 5/25 infection with M13-Litmus28i<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Result<br />
! Significance<br />
<br />
|-<br />
| ER2738 on Amp<br />
| Lawn<br />
| Litmus28i phagemid was successfully packaged into the M13 phage and is infectable<br />
<br />
|-<br />
| ER2738 on Kan<br />
| 100-200 colonies<br />
| Some M13 helper phage is packaged into the M13 phage but at a much lower rate than Litmus28i<br />
<br />
|-<br />
| BW23115 on Amp<br />
| Lawn<br />
| BW23115 is ‘equally’ infectable by M13 as ER2738<br />
<br />
|-<br />
| BW23115 on Kan<br />
| Lawn<br />
| BW23115 contains Kan resistance in its genome so this tells us nothing<br />
<br />
|-<br />
|}<br />
:*Conclusions:<br />
::-Cells grew on Ampicillin; therefore, Litmus28i phagemid was successfully packaged into M13 phage. <br />
::-For ER2738 samples, there was significant growth on Ampicillin compared to Kanamycin; therefore, Litmus28i phagemid is packaged preferentially over M13K07 Helper Phagemid<br />
::-M13-Litmus28i retains its infectivity of cells containing the F’ episome<br />
:*Because we received lawns, we have to redo the infection and plate less cells so we can calculate the uptake ratio between the phagemid and helper phage based on the number of colonies<br />
*Started 50 mL overnight of K12 ER2738 and BW23115<br />
<br />
'''5/27'''<br />
*Redo the infection done on 5/25<br />
::-Infectable cells: ER2738 and BW23115<br />
:::*Plated non-infected samples of each (non-diluted) to check for contaminants<br />
::-Diluted M13-Litmus28i (1) phage by a factor of 10. Added 5.5ul to each sample<br />
::-Grew samples for 20 minutes at 37C, 250rpm<br />
::-Plated 100ul of onto an Ampicillin (100ug/mL) plate and onto a Kanamycin (50ug/mL) plate. Incubated overnight at 37C.<br />
:::*Dilutions = 1:10; 1:100; and 1:1000<br />
'''5/28'''<br />
*Results from 5/27<br />
::-Controls were as expected<br />
:::*No growth for ER2738 non-infected grown on Amp, ER2738 non-infected grown on Kan, or BW23115 non-infected grown on Amp<br />
:::*Growth for BW23115 non-infected grown on Kan (BW23115 has Kan R in genome)<br />
::-Many colonies were received for all dilutions (1:10, 1:100, and 1:1000) of the following<br />
:::*ER2738 infected and plated on Amp<br />
:::*BW23115 infected and plated on Amp<br />
:::*BW23115 infected and plated on Kan<br />
::-Many (100s to 1000?) colonies grew on 1:10 and 1:100 dilutions of ER2738. 50-100 colonies grew on the 1:1000 dilution of ER2738<br />
:::*Compare this to the 100-200 colonies that grew from 2/25 infection (which was 300ul non-diluted, infected cells)<br />
:::*Reasons for increased yield<br />
::::*Added too much phage?<br />
::::*volume changed between experiment (5mL to 50mL)<br />
::::*Overnight culture may not have been saturated. If still in log phase, the cells would continue to grow<br />
*Made 50mL O/N cultures of ER2738 and BW23115 so we can repeat the infection tomorrow and plate further dilutions starting at 1:1000<br />
::-Carry out infection in 5mL and 50mL to test volume effect?<br />
<br />
'''5/29'''<br />
*Measured OD of overnights<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! OD<br />
! mL to have .1OD in 50mL<br />
! mL to have 1OD in 5mL<br />
<br />
|-<br />
| K12 ER2738<br />
| 3.0<br />
| 1.7<br />
| 1.7<br />
<br />
|-<br />
| BW23115<br />
| 2.9<br />
| 1.7<br />
| 1.7<br />
<br />
|-<br />
|}<br />
<br />
<br />
*Experiment 1: Infect cells using same method as 5/25 (in a 5mL culture)<br />
::-Started with 1OD cells in 5mL<br />
::-Added about 0.7ul (inaccuracies in pipet) of 1:10 diluted M13-Litmus28i phage<br />
::-Incubated (rotating) for 20 minutes<br />
::-Made 1:1,000 and 1:10,000 dilutions<br />
::-Plated 100ul on Ampicillin (100ug/mL) plates and on Kanamycin (50ugmL) plates<br />
:::*Included non-infected samples diluted by 1:1000<br />
::::*This negative control can be used for Experiment 2 since the non-infected parent solution is the same<br />
::-Incubate overnight at 37C<br />
*Experiment 2: Infect cells using protocol from “Eliminating helper phage from phage display”<br />
::-Diluted O/Ns to OD of 0.1 in 50 mL culture<br />
::-Grew samples until of OD of ER2728 = 0.59 and the OD of BW23115 = 0.60<br />
:::*Missed OD of 0.5 mark, but the two samples are close to each other<br />
::-Chilled samples on ice for 30 minutes<br />
::-Warmed in incubator for 35 minutes (should have been 30)<br />
::-Amount of phage. Rather than use 1:1 as mentioned in protocol, we used multiplicity of 1:10 (phage:cell)<br />
:::*Added 3.3ul of 1:10 diluted M13-Litmus28i (1) phage<br />
::::*(On 5/27 we added 5.5ul of diluted phage to 50mL of cells at OD of 1. Our cells were at OD of .6; therefore, 5.5*.6 = 3.3ul)<br />
::-Incubated for 30 minutes at 37C, not shaking<br />
:::*We later change this to shaking<br />
::-Dilutions<br />
:::*1:1,000; 1:5,000; 1:10,000; 1:50,000; 1:100,000; 1:1,000,000<br />
:::*Plated ER2738 and BW23115 on Ampicillin (100ug/mL)<br />
:::*Plated ER2738 on Kanamycin (50ug/mL)<br />
*Experiment 3: Growth Test (for growth curve)<br />
::-We were concerned by the low OD of the Overnights from the last few days. Wanted to be sure that 2.0-3.0 was not still in log phase. Cultures looked saturated but the OD seemed low.<br />
{| class = "wikitable"<br />
|-<br />
! Time<br />
! Elapsed time (min)<br />
! ER2738 (no antibiotic)<br />
! ER2738 (Tetracycline (20ug/mL))<br />
! BW23115 (with F’ episome) no antibiotic<br />
<br />
|-<br />
| 10:08<br />
| 0<br />
| 0.1<br />
| 0.1<br />
| 0.1<br />
<br />
|-<br />
| 11:10<br />
| 62<br />
| 0.24<br />
| 0.21<br />
| 0.21<br />
<br />
|-<br />
| 12:00<br />
| 112<br />
| 0.49<br />
| 0.45<br />
| 0.44<br />
<br />
|-<br />
| 13:00<br />
| 172<br />
| 1.00<br />
| 0.93<br />
| 0.98<br />
<br />
|-<br />
| 14:15<br />
| 217<br />
| 1.29<br />
| 1.21<br />
| 1.31<br />
<br />
|-<br />
| 15:40<br />
| 302<br />
| 2.1<br />
| 1.8<br />
| 2.3<br />
<br />
|-<br />
| 16:47<br />
| 369<br />
| 2.5<br />
| 1.9<br />
| 3.4<br />
<br />
|-<br />
| 18:05<br />
| 447<br />
| 2.6<br />
| 2.2<br />
| 2.5<br />
<br />
|-<br />
| 19:00<br />
| 502<br />
| 2.9<br />
| 2.3<br />
| 2.5<br />
<br />
|-<br />
| 20:00<br />
| 562<br />
| 3.0<br />
| 2.6<br />
| 2.8<br />
<br />
|-<br />
|}<br />
<br />
:::*The time point at 16:47 (369 minutes elapsed) for BW23115 conjugated (without antibiotics) is most likely an error. It has been removed from the growth plot<br />
'''[[File:UCB-Phage Delivery-140529.JPG]]'''<br />
*Other<br />
::-Made Amp and Kan plates (1 sleeve of each)<br />
::-Made 50mL O/N of ER2738 and BW23115F’ in case we need further dilutions<br />
::-Made 5mL O/N of ER2738, BW23115F’, and BW23115 (without F’ episome) to make chemically competent tomorrow<br />
:::*Did not have plate of BW23115 (without F’ episome) so used freeze down. Hoping to get O/N of a picked colony from CRISPR Team tomorrow morning<br />
<br />
'''5/30'''<br />
*Made chemically competent cells of…<br />
::-ER2738<br />
::-BW23115F’ (conjugated with F’ episome)<br />
::-BW23115 (not conjugated- without F’ episome)<br />
:::*Culture started from plate<br />
::-BW23115* not conjugated (without F’ episome)<br />
:::*Culture started from freeze down<br />
*Results from infections<br />
::-Negative Controls (cells were not infected; cells were diluted 1:1000)<br />
'''[[File:UCB-Phage Delivery-140530.JPG]]'''<br />
::-Results from Experiment 1 (5/29)<br />
'''[[File:UCB-Phage Delivery-140530-02.JPG]]'''<br />
::-Results from Experiment 2 (5/29)<br />
'''[[File:UCB-Phage Delivery-140530-03.JPG]]'''<br />
*Math<br />
::-If there are 5.00E+8 cells in 1mL of culture at OD of 1, then in 1mL of culture at OD of 0.59, there are 2.95E+8 cells. In a 50mL culture at OD of 0.59, there are 1.48E+10 cells.<br />
::-We added 3.3ul (0.0033mL) phage at concentration 4.62E+11 phage/mL which amounts to 1.52E+9 total phage<br />
::-Assuming that 1 phage infects 1 bacterium, we can assume that 1.52E+9 bacterial have the potential to be infected in the 50mL culture<br />
::-We plated 100ul of culture at various dilutions. If not diluted, the number of cells that can be potentially infected in 0.1mL equals 1.52E+9/500, or 3.05E+06 cells. We then accounted for the dilutions (for 1:1000 dilution, we divided 3.05E+06 by 1000 to receive 3.05E+03)<br />
::-The following table contains the number of cells with the potential to be infected assuming a 100% infectivity rate by M13 phage and that 1 cell is infected only once.<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Dilution<br />
! Potential infected cells<br />
! Colonies on Amp<br />
! % Potential (Amp)<br />
! Colonies on Kan<br />
! % Potential(Kan)<br />
! Kan:Amp<br />
<br />
|-<br />
| 1:1000<br />
| 1.52E+06<br />
| 476<br />
| 15.62%<br />
| 15<br />
| 0.820%<br />
| 1:19.04<br />
<br />
|-<br />
| 1:5000<br />
| 3.05E+05<br />
| 131<br />
| 21.49%<br />
| 13<br />
| 2.133%<br />
| 1:10.08<br />
<br />
|-<br />
| 1:10000<br />
| 1.52E+05<br />
| 93<br />
| 30.51%<br />
| 5<br />
| 1.640%<br />
| 1:18.60<br />
<br />
|-<br />
| 1:50000<br />
| 3.05E+04<br />
| 17<br />
| 44.29%<br />
| 0<br />
| 0.000%<br />
| <br />
<br />
|-<br />
|}<br />
<br />
*Conclusions from infections<br />
::-Results between and within the three trials are inconsistent. For example, the number of colonies received in experiments 1 and 2 from 5/29 differ greatly. Due to the differences in protocol, variation was expected but not to this extent. <br />
::-Our dilutions did not yield the expected 10 fold (or 5 fold) decrease in growth that was expected.<br />
::-Plates from 5/29 could be plated better to reduce dense areas of growth and growth around the rim.<br />
::-Though the experiment contained many errors we can say that the phagemid (Litmus 28i) is preferentially packaged compared to the helper phage (M13K07) but not to the degree we expected.<br />
::-Could receive increased occurrences of cells containing M13k07 due to infection, phage production, further infection<br />
<br />
==Week 5==<br />
<br />
'''6/2'''<br />
*Tested chemically competent cells through transformation<br />
::-Are cells contaminated?<br />
::-Are cells competent?<br />
*The samples for transformation<br />
{| class = "wikitable"<br />
|-<br />
! #<br />
! Cells (Tube label)<br />
! DNA (Tube label)<br />
! Resistance before transformation<br />
! Resistance after transformation<br />
<br />
|-<br />
| 1<br />
| K12 ER2738 5/20<br />
| p110+RBS (2) 4/23<br />
| Tet<br />
| Tet, Chlor<br />
<br />
|-<br />
| 2<br />
| BW (-f) 5/30<br />
| p110+RBS (2) 4/23<br />
| Kan<br />
| Kan, Chlor<br />
<br />
|-<br />
| 3<br />
| BW f-ep comp 5/22<br />
| p110+RBS (2) 4/23<br />
| Kan, Tet<br />
| Kan, Tet, Chlor<br />
<br />
|-<br />
| 4<br />
| BW (+f) 5/30<br />
| p110+RBS (2) 4/23<br />
| Kan, Tet<br />
| Kan, Tet, Chlor<br />
<br />
|-<br />
| 5<br />
| *BW23115 5/30<br />
| p110+RBS (2) 4/23<br />
| Kan<br />
| Kan, Chlor<br />
<br />
|-<br />
| 2B<br />
| K12 ER2738<br />
| 2B [from dis. kit]<br />
| Tet<br />
| Tet, Chlor<br />
<br />
|-<br />
| 2P<br />
| BW f-ep comp 5/22<br />
| 2P [from dis. kit]<br />
| Kan, Tet<br />
| Kan, Tet, Chlor<br />
<br />
|-<br />
|}<br />
<br />
'''6/3'''<br />
*Results from 6/2 Transformation<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Growth on Chlor<br />
! Growth on Kan<br />
! Growth on Kan+Tet<br />
! Growth on Amp<br />
<br />
|-<br />
| 1 N<br />
| X<br />
| X<br />
| X<br />
| X<br />
<br />
|-<br />
| 2 N<br />
| X<br />
| +<br />
| X<br />
| X<br />
<br />
|-<br />
| 3 N<br />
| X<br />
| +<br />
| +<br />
| X<br />
<br />
|-<br />
| 4 N<br />
| X<br />
| +<br />
| +<br />
| X<br />
<br />
|-<br />
| 5 N<br />
| X<br />
| +<br />
| X<br />
| X<br />
<br />
|-<br />
| 1<br />
| +<br />
| X<br />
| X<br />
| <br />
<br />
|-<br />
| 2<br />
| +<br />
| +<br />
| X<br />
| <br />
<br />
|-<br />
| 3<br />
| +<br />
| +<br />
| +<br />
| <br />
<br />
|-<br />
| 4<br />
| +<br />
| +<br />
| +<br />
| <br />
<br />
|-<br />
| 5<br />
| +<br />
| +<br />
| Many colonies but close to samp. 4<br />
| <br />
<br />
|-<br />
| 2P<br />
| + (~100)<br />
| +<br />
| +<br />
| <br />
<br />
|-<br />
| 1- JW<br />
| +<br />
| X<br />
| X<br />
| <br />
<br />
|-<br />
| 2- JW<br />
| +<br />
| +<br />
| X<br />
| <br />
<br />
|-<br />
| 3- JW<br />
| +<br />
| +<br />
| +<br />
| <br />
<br />
|-<br />
| 4- JW<br />
| +<br />
| +<br />
| +<br />
| <br />
<br />
|-<br />
| 5-JW<br />
| +<br />
| +<br />
| X<br />
| <br />
<br />
|-<br />
| 2B- JW<br />
| + (24)<br />
| X<br />
| X<br />
| <br />
<br />
|-<br />
|}<br />
*The transformations with DNA from the well (B2 and P2) had lower efficiencies than those with DNA from a mini-prep. Most likely this is due to the differences in DNA concentration (p110+RBS (2) 4/23 was at 254.4ng/ul)<br />
*Conclusions<br />
::-None of the competent cells were contaminated<br />
::-All of the competent cells are in fact, competent<br />
*Set up O/N of DH5-alpha cells to make competent tomorrow<br />
<br />
'''6/4'''<br />
*Isolation of single-stranded phagemid DNA using M13K07<br />
::-Added ER2738 colony to 50mL LB<br />
:::*Plate was cold. Next time warm plate before pricking<br />
::::*Best to use freshly grown plate<br />
::-After 4 hours, OD was at 0.02. Waited 45 minutes and OD was at 0.08. Therefore, we infected at OD 0.08<br />
::-Had started another culture when we did not think the first was growing. In incubator for about 1 hour. OD was 0.00. We infected anyway because last time it worked.<br />
::-Let infection proceed for 60 minutes then added 70ul of Kanamycin to be a final concentration of 70ug/mL<br />
*Primers came in to biobrick M13ori (packaging signal on Litmus28i)<br />
::-Resuspended primers and diluted 1:10<br />
<br />
'''6/5'''<br />
*Isolated single-stranded M13K07 DNA<br />
::-Final concentration = 5724 ng/ul (calculated from a 1:10 dilution)<br />
::-For second sample in pair, we resuspended it in TE but did not proceed to DNA extraction teps<br />
::-For the second culture we started 6/4, we resuspended pellet in TBS and glycerol to preserve the M13 phage. Measured absorbances (before glycerol was added)<br />
: #1<br />
::269 => 1.690A<br />
::320 => 0.103A<br />
: #2<br />
::269 => 1.453<br />
::320 => 0.059<br />
*For our first biobrick, we wanted to isolate the M13 origin, a segment ~500bp that allows for packaging into the M13 phage. We tried to achieve this by biobrick assembly and by Gibson Assembly.<br />
::-To biobrick M13 ori through biobrick assembly (the old-school way)<br />
:::PCR on Litmus 28i to amplify/biobrick M13ori<br />
::::*Used primers Gem003 F & R<br />
::::*Diluted Litmus 28i DNA 1:10<br />
:::Digestion of p11+RBS (1) to digest pSB1C3 bb with EcoRI-HF and PstI-HF<br />
:::Ran samples on gel and gel extracted pieces. We recieved very low yields (out of range for nano drop)<br />
::::*M13 ori: 4.0 ng/ul<br />
::::*pSB1C3: 1.8 ng/ul<br />
:::Digested M13 ori fragment despite poor extraction yield with EcoRI-HF and PstI-HF<br />
::::*Used 1.5x as much DNA as instructed based on inaccurate concentration<br />
'''[[File:UCB-Phage Delivery-140605.JPG]]'''<br />
::-Gel extracted red rectangles<br />
::-Ligation<br />
:::*10hr @ 16C, 10min @ 65C, 4ever @ 4C<br />
*To biobrick M13 ori through Gibson Assembly (the cool-kids way)<br />
::-PCR on Litmus 28i<br />
:::*Used primers Gem002 F & R<br />
:::*Diluted Litmus 28i DNA 1:10<br />
::-PCR on pSB1C3 (p11+RBS (1))<br />
:::*Used primers Gem001 F & R<br />
:::*Diluted pSB1C3 DNA 1:3<br />
<br />
'''6/6'''<br />
*Ran gel of PCR products from (6/5). Products will be used for Gibson Assembly<br />
::-Recieved bands for pSB1C3 around 2000bp and M13ori around 500bp<br />
::-No contamination in pSB1C3 PCR negative control<br />
::-Band in M13ori negative control that is the same size as sample. Contaminated by sample?<br />
::-Gel of PCR products #1 and #2 from 6/5<br />
'''[[File:UCB-Phage Delivery-140606.JPG]]'''<br />
:1. pSB1C3 with promoter+RBS as insert. Amplified with Gem001<br />
:2. No DNA control for Gem001<br />
:3. M13ori amplified with Gem002 from Litmus28i<br />
:4. No DNA control for Gem002<br />
*Gibson Assembly<br />
{| class = "wikitable"<br />
|-<br />
! Total Amount of Frag.<br />
! .02-.5pmol<br />
! 10 ul total<br />
<br />
|-<br />
| Gibson Assembly MM (2x)<br />
| 10 ul<br />
| 10<br />
<br />
|-<br />
| Dionized H2O<br />
| 10-x<br />
| <br />
<br />
|-<br />
|}<br />
::-Diluted pSB1C3 and M13ori PCR products 1:10<br />
::-Incubated 60min @ 50C<br />
::-Also used provided pUC16 as positive control<br />
*Transformation<br />
:1. p110+RBS Positive control<br />
:2. No DNA Negative control<br />
:3. Cas9 from distribution kit so we can have more<br />
:4. Thaw and refreeze cells Test competency of comp cells after thawed<br />
:5. Not chem comp cells Negative control for the above<br />
:6. Ligation Product<br />
:7. Gibson product<br />
::*7.2. Gibson product diluted 1:4<br />
:8. Gibson positive control<br />
::*7.2. Gibson positive control diluted 1:4<br />
::-For the Gibson product and the positive control, we transformed 2ul of product and 2ul of 1:4 diluted product. NEB recommends the first if using their competent cells and the second if using cells from other companies. Our cells are from NEB but we made them competent ourselves so we tried both ways<br />
:::*Plated on Chlor at concentrations of 170, 85, and 33 ug/mL<br />
*Primers came in<br />
::-Resuspended and made 1:10 dilutions<br />
<br />
'''6/7'''<br />
*Results from 6/6 transformation<br />
:1. Positive control<br />
::*Lots of growth, ~300-400 on 1:10 dilution<br />
:2. No DNA negative control<br />
::*No growth<br />
:3. Cas9 from distribution kit<br />
::*7 potential colonies (some are close to edges through) on non-diluted<br />
:4. Thawed then refroze cells<br />
::*Looks like (1)<br />
:5. Not chemically competent cells<br />
::*No growth<br />
:6. Ligation product<br />
::*13 potential colonies (some are close to edge)<br />
:7. Gibson Assembly Product<br />
::*170 -> No colonies<br />
::*85 -> No colonies<br />
::*33 -> 3 specks<br />
:7.2. Gibson Assembly product diluted 1:10<br />
::*170 -> 1 speck<br />
::*85 -> 3 colonies<br />
::*33 -> 13 colonies<br />
:8. Gibson positive control<br />
::*No colonies<br />
:8.2. Gibson positive control diluted 1:4<br />
::*No colonies<br />
*Made 6mL O/N cultures<br />
::-4 from (3) cas9 plate<br />
:::See Constitutive CRISPR notebook for more info on these samples<br />
::-7 from (6) Ligation product<br />
::-5 from (7.2 [85]) Diluted Gibson product on 85 ug/mL Chlor<br />
::-8 from (7.2 [33]) Diluted Gibson product on 33 ug/mL Chlor<br />
<br />
==Week 6==<br />
'''6/8'''<br />
*Check colonies for correct constructs.<br />
::-Mini-prepped all 24 O/Ns<br />
:::*Yielded low concentrations for samples 12, 15, 19, and 22<br />
::-Digested all with EcoRI and PstI (10ul reactions)<br />
::-Ran results on gel<br />
:::*All 4 cas9 samples had the expected bands of 2000 and 5000bp<br />
:::*All 7 ligation products have expected bands of 2000 and ~570bp<br />
:::*3 of 5 Gibson assemblies from 85ug/mL Chlor plate had expected bands of 2000 and ~500bp<br />
:::*3 of 8 Gibson assemblies from 33ug/mL Chlor plate had expected bands of 2000 and ~500bp<br />
'''[[File:UCB-Phage Delivery-140608-01.JPG]]'''<br />
<br />
'''[[File:UCB-Phage Delivery-140608-02.JPG]]'''<br />
::1-4: Cas9 from Stanford-Brown team<br />
::5-11: pSB1C3-M13ori cloned through ligation<br />
::12-24: pSB1C3-M13ori cloned through Gibson Assembly<br />
:::*12-16: Grown with 85 ug/mL Chlor<br />
:::*17-24: Grown with 33 ug/mL Chlor<br />
*Conclusions from gel<br />
::-We have cas9 safely in cells<br />
::-Our ligation reactions successfully yielded M13ori on pSB1C3<br />
::-Combined, we had a 46% success rate for the Gibson Assembly in yielding M13ori on pSB1C3<br />
:::*The 4 samples that had the lowest concentration after being mini-prepped (12,15, 19, and 22) correlate with samples that had the correct band pattern<br />
*We selected 4 samples from each (4 total between the two Gibson reactions) type<br />
::-For non-Gibson Assembled samples<br />
:::Plated 25ul on 170ug/mL Chlor<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Our usual method<br />
! Gibson Method<br />
<br />
|-<br />
| 1. Thaw on ice<br />
| 1. Thaw on ice <br />
<br />
|-<br />
| 2. Transfer 40ul cells to tube<br />
| 2. Transfer 50ul cell to tube<br />
<br />
|-<br />
| 3. Add DNA. 1ul for mini-prep OR up to 10ul for ligation<br />
| 3. Add 2ul to NEB cells OR 2ul of 1:4 diluted to other cells<br />
<br />
|-<br />
| 4. Mix by pipet Let sit 30min on ice<br />
| 4. Mix by pipet or flicking Let sit 30 min. on ice<br />
<br />
|-<br />
| 5. Heat shock: 42C for 45s<br />
| 5. Heat shock: 42C for 30s<br />
<br />
|-<br />
| 6. Ice for 5 minutes<br />
| 6. Ice for 2 min.<br />
<br />
|-<br />
| 7. Transfer to culture tube; Add 200ul SOC<br />
| 7. Add 950ul SOC to tube<br />
<br />
|-<br />
| 8. Shake or rotate for 60-120min at 37C<br />
| 8. Shake (250rpm) or rotate for 60 min. at 37C<br />
<br />
|-<br />
| 9.<br />
| 9. Warm plates to 37C<br />
<br />
|-<br />
| 10. Plate 100ul onto selection plate<br />
| 10. Plate 100ul onto plate<br />
<br />
|-<br />
| 11. Incubate O/N @ 37C<br />
| 11. Incubate O/N @ 37C<br />
<br />
|-<br />
|}<br />
::-Added 6mL LB and Chlor at concentration of 170ug/mL to grow O/N<br />
:*For Gibson Assembled samples<br />
::-Plated 25ul onto 170, 85, and 33ug/mL Chlor<br />
::-Samples from 85ng/mL plate<br />
:::Transferred 100ul to new tube, added media, added Chlor at 170ng/mL<br />
::-Samples from 33ng/mL plate<br />
:::Tranfered 100ul to new tubes, added media, added Chlor at 85ng/mL to one and 33ng/mL to the other<br />
:*Tomorrow, may send for sequencing and make freeze downs<br />
<br />
*Transformation<br />
:1. Positive control (p110+RBS diluted 1:10)<br />
:2. Non-diluted Gibson product<br />
:3. Gibson product diluted 1:4<br />
:4. Gibson product diluted 1:10<br />
::-Transformed each sample using our usual method and using the protocol given by Gibson<br />
<br />
::-Due to not have plates ready before transformation, in step 4, the samples sat for about 50 minutes. Then in step 8, they both recovered for about 150 minutes. Though not specified in our protocol, we did warm the plates to 37C. In step 10 for our protocol, since we only added 200ul SOC and wanted to plate on 3 selection plates (see below), we only plated 50ul (except for the positive control). <br />
::-Plated on three concentrations of Chloramphenicol (33 ug/mL, 85 ug/mL, and 170 ug/mL) to determine the differences in yield due to differences in concentration.Obvious hypothesis: more colonies will grow on plates that have a lower concentration of chlor.<br />
<br />
<br />
'''6/9'''<br />
*Made chemically competent 5alpha cells with Dan and Alex from main campus<br />
::-Waiting to hear results on competency<br />
*Will eventually make phage containing CRISPR-Cas9 that targets Kanamycin resistance. M13K07 has Kanamycin resistance so we need to switch the resistance on the M13 genes.<br />
::-PCR on pwp 2.po (plasmid that Sam gave us that contains the zeoR gene adjacent to ori) to amplify zeoR and ori. Zeo is on EM7 promoter<br />
:::-Primers: Gem008 R & R<br />
:::-Anneal temp from NEBuilder: 63.7C<br />
:::-Extension time: 90s<br />
:::-Expected band size on gel: 1300bp<br />
:::-Used phusion polymerase<br />
*PCR on M13K07 DNA to amplify M13 phage genes (also removes majority of M13 ori, all of KanR, and all of p15 ori)<br />
:::-Primers: Gem007 F & R<br />
:::-Anneal temp from NEBuilder: 60.2C<br />
:::-Extension time: 4:30<br />
:::-Expected band size on gel: about 6000bp<br />
:::-Used phusion polymerase<br />
*Freeze downs<br />
::-Note: Phagemid 1C3 was the original name for ‘pSB1C3-M13ori’<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Top Label<br />
! Side label<br />
! What?<br />
<br />
|-<br />
| Phagemid 1C3 6/9<br />
| From Lig<br />
| M13 ori inserted into 1C3 (biobricked); Done through ligation; Contains extra bases as spacer between biobrick prefrix/suffix and part for primer design<br />
<br />
|-<br />
| Phagemid 1C3 6/9<br />
| From Lig<br />
| “ “<br />
<br />
|-<br />
| Phagemid 1C3 6/9<br />
| Gibson<br />
| M13 ori inserted into 1C3 (biobricked); Done through Gibson cloning<br />
<br />
|-<br />
| Phagemid 1C3 6/9<br />
| Gibson<br />
| “ “<br />
<br />
|-<br />
|}<br />
'''6/10'''<br />
*Ran gel of PCRs from 6/9<br />
<br />
'''[[File:UCB-Phage Delivery-140610.JPG]]'''<br />
:1. Amplification of M13 genes from M13K07 (~6000bp)<br />
:2. No DNA control for (1) amplification<br />
:3. Amplification of Zeo resistance gene + plasmid ori (~1300bp)<br />
:4. No DNA control for (3) amplification<br />
*Gibson Assembly of above parts (did not gel extract)<br />
::-Diluted the PCR products 1:10 then added 3ul of M13K07 genes product and 7ul of ZeoR+ori product<br />
::-Incubated at 50C for 60 min.<br />
::-Transformed Gibson Assembly product into 5alpha cells<br />
:::*Used our usual protocol<br />
:::*Add 2ul of DNA<br />
::::-In one sample, diluted DNA 1:4 and in the other, we diluted DNA 1:10<br />
*Started Phage Amplification Protocol<br />
::-ER2738 transformed with Litmus 28i<br />
:::*Grew for ~2.5hr before reaching an OD of 0.04<br />
::-ER2738 transformed with pSB1C3-M13ori (M13ori on pSB1C3)<br />
:::*Grew for ~ 3.5hr before reaching an OD of 0.01, then in the next 1.5 hours, spiked to 0.19<br />
:::*We gave up and went home, and will restart tomorrow<br />
*Analyzed transformation results from 6/8<br />
<br />
'''6/11'''<br />
*Made chemically competent 5alpha cells<br />
*Restarted Phage Amplification Protocol<br />
::-Forgot to add phagemid antibiotic at start of growth. Added phagemid antibiotic when we added phage. Incubated for 90 minutes before adding Kanamycin (to select for cells that were infected by M13K07)<br />
::-Phage is at concentration 4.57x10^12 phage/mL<br />
:::Protocol calls for final concentration of 1 x10^8 phage/mL<br />
::::*(4.57 x10^12)*V = (1 x10^8)(50mL)<br />
::::*V = 0.00109mL<br />
:::Added 1.1ul of phage<br />
*Transformations<br />
{| class="wikitable"<br />
|-<br />
!DNA<br />
!Plate Selection<br />
|-<br />
|No DNA control<br />
|(all)<br />
|-<br />
|Positive control (p110+RBS)<br />
|(C)<br />
|-<br />
|M13 genes + ZeoR ori<br />
|(Z)<br />
|-<br />
|M13 genes +ZeoR ori diluted 1:4<br />
|(z)<br />
|} <br />
<br />
'''6/12'''<br />
*Transformation Results from 6/10 [Took ~36 hours to be clearly visible]<br />
::No DNA control<br />
:::-Amp: 0 <br />
:::-Zeo (50ug/mL): 200 colonies<br />
::M13 genes + ZeoR ori (1:4 dilution) <br />
:::-Zeo (25ug/mL): 300<br />
:::-Zeo (50ug/mL): 200<br />
:::-Zeo (100ug/mL): 100<br />
::M13 genes + ZeoR ori (1:10 dilution) <br />
:::-Zeo (25ug/mL): 300<br />
:::-Zeo (50ug/mL): 150<br />
:::-Zeo (100ug/mL): 150<br />
*Transformation Results for 6/11<br />
::-Positive (p110+RBS) on Chloramphenicol: 500 colonies<br />
::-No DNA on Amp: 0 colonies Zeo (100 ug/mL): specks<br />
::-No apparent growth on any other plate<br />
:::*Realized later that we grew our samples on the wrong plates. Will repeat transformation today<br />
*Transformation #1<br />
::This morning there were no colonies on positive (p110+RBS) coltrol from 6/11 even though we observed fast growth in the past. Without waiting for colonies to appear, we started a control transformation<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Diluted<br />
! Time at 42C<br />
<br />
|-<br />
| p110+RBS<br />
| No<br />
| 45s<br />
<br />
|-<br />
| p110+RBS<br />
| 1:10<br />
| 45s<br />
<br />
|-<br />
| p110+RBS<br />
| 1:10<br />
| 30s<br />
<br />
|-<br />
| p110+RBS<br />
| 1:10<br />
| No timer. ~43s<br />
<br />
|-<br />
| No DNA control<br />
| No<br />
| 45<br />
<br />
|-<br />
|}<br />
*Because ‘No DNA control 6/10’ yielded colonies, we researched Zeocin plates<br />
::-According to Life Technologies (Invitrogen), Zeocin requires low salt medium and a pH of 7.5<br />
::-Low Salt LB Medium (1L)<br />
:::*Ingredients<br />
::::10g Tryptone<br />
::::5g NaCl<br />
::::5g Yeast Extract<br />
:::-Mix ingredients<br />
:::-Adjust pH to 7.5 using NaOH (If go over, use HCl)<br />
:::-Add agar for plates at 15g/L. Autoclave<br />
:::-Thaw Zeocin on ice. Vortex<br />
:::-Add Zeocin to final concentration of 25ug/mL<br />
*Transformation #2<br />
::-Repeat of transformation on 6/11 but this time we will plate on the correct plates<br />
::-Also remade Zeocin plates<br />
*Finished isolation of M13 Litmus phage and M13 pSB1C3-M13ori phage<br />
::*Note: Phagemid 1C3 was the original name for ‘pSB1C3-M13ori’<br />
{| class = "wikitable"<br />
|-<br />
! Phage Sample<br />
! A269<br />
! A320<br />
! Concentration (phage/mL)<br />
<br />
|-<br />
| Litmus phage (1)<br />
| 0.181<br />
| 0.034<br />
| 3.12 x10^12<br />
<br />
|-<br />
| Litmus phage (2)<br />
| 0.227<br />
| 0.047<br />
| 3.83 x10^12<br />
<br />
|-<br />
| Phagemid 1C3 phage (1)<br />
| 0.101<br />
| 0.020<br />
| 1.87 x10^12<br />
<br />
|-<br />
| Phagemid 1C3 phage (2)<br />
| 0.126<br />
| 0.021<br />
| 2.42 x10^12<br />
<br />
|-<br />
|}<br />
::::phage/mL = 6x10^16 x (A269-A320)/ (#of base pairs in the phage genome)<br />
*Set up 50mL O/N of K12 ER2738 (containing f-episome) for infection tomorrow with Litmus phage and pSB1C3-M13ori phage<br />
<br />
<br />
'''6/13'''<br />
*Transformation Results for 6/11<br />
::-Several hundred colonies on Positive Control (p110+RBS) on Chlor<br />
::-No colonies for GA positive control on Amp<br />
::-No colonies for M13ori + ZeoR mistakenly plated on Amp<br />
::-No colonies for No DNA control on Amp<br />
::-100-ish colonies for No DNA control on Zeo<br />
::-100-ish colonies for cas9+AmpR+gRNA mistakenly plated on Zeo<br />
::-These last 3 points suggest/confirm that Zeo plates are no good<br />
*Transformation Results from 6/12 control test<br />
::-Colonies grew is about equal amounts on all plates, including No DNA control<br />
:::Either plates don’t contain Chlor or competent cells are contaminated<br />
::-Streaked competent cells onto new and old Chlor plates<br />
*Infection test of ER2738: Is Litmus preferentially packaged over M13K07 Helper phage? Is pSB1C3-M13ori preferentially packaged?<br />
::-Infect ER2738 with phage produced 6/12<br />
:::*Phage should have packaged Litmus 28i phagemid or pSB1C3-M13ori<br />
:::*Cells infected with phage packaging Litmus 28i will grow on Amp<br />
:::*Cells infected with phage packaging pSB1C3-M13ori will grow on Chlor<br />
:::*Cells infected with phage packaging M13K07 will grow on Kan<br />
::-After we plate, we can count the colonies and calculate a ratio of Litmus28i: M13K07 or pSB1C3-M13ori:M13K07 packaging<br />
<br />
'''6/14'''<br />
*Results of contamination test (streaked competent cells onto new and old Chlor plates)<br />
::-Colonies grew in low amounts on both plates. most likely the cells are contaminated<br />
*Transformation results for 6/12<br />
::-Many colonies for No DNA control on Zeo<br />
:::-Some colonies are turning pinkish-red<br />
::-Many colonies for diluted and non-diluted M13 genes+ZeoR on Zeo<br />
:::-Some colonies are turning pinkish-red<br />
*Could the white colonies be the designed colonies and can we kill the red colonies with Zeocin before killing the white colonies (aka. Use high Zeocin concentrations to select for the correct construct)<br />
::-Selected 1 red colony from No DNA Zeo control plate and 1 white colony from M13genes+ZeoR sample plate<br />
::-Added 100ul H2O then divided amongst 5 culture tubes each (with 5mL of low-salt LB, pH 7.5)<br />
::-Then added Zeocin to a final concentration of: 0, 25, 50, 75, 100ug/mL.<br />
*Results of 6/13 infection<br />
::-Litmus 28i infected cells<br />
:::*on Kan: Individual colonies for 1:10, 1:100, and 1:1000. No colonies on 1:5000<br />
:::*on Amp: lawn for 1:10, 1:100, and near lawn for 1:1000. Single colonies for 1:5000<br />
::-pSB1C3-M13ori infected cells<br />
:::*on Kan: Same as Litmus 28i samples on Kan<br />
:::*on Chlor: Lawn for all dilutions. Must discredit due to recent Chlor contamination<br />
<br />
==Week 7==<br />
<br />
'''6/15'''<br />
*Results of Zeocin experiment on 6/14<br />
::-Healthy growth for both at 0ug/mL Zeo<br />
::-No growth for white colony with any Zeo<br />
::-Good growth for red colony even at 100ug/mL Zeo<br />
::-This suggests that the red colonies are naturally resistant to Zeocin. Also, our plates must not contain active Neocin. We are sure that we are adding enough. Possibly, we add it while the media is too hot or leave the plates at room temperature (decondensing) for too long and this deactivates the antibiotic? Perhaps we are not attaining the correct pH<br />
*In light of recent contamination problems on both Chlor and Zeo, we made new competent cells<br />
::-5alpha<br />
::-BW23115<br />
::-BW23115 conjugated (contains f-episome)<br />
<br />
'''6/16'''<br />
*Made new Chlor antibiotic. Made to Chlor plates<br />
*Contamination Test: Streaked Zeo and new Chlor plates each with<br />
::-Colony from Zeo contaminated plate<br />
::-Colony from Chlor contaminated plate<br />
::-Non-transformed OLD chemically competent cells<br />
::-Non-transformed NEW chemically competent cells<br />
*Redid bacterial infection<br />
::-For both samples, we used ER2738 (not from the same ‘O/N’ (also, not a real O/N))<br />
::-Infected one sample with isolated ‘Litmus28i phage’ and one with ‘pSB1C3-M13ori’<br />
::-Alterations to protocol<br />
:::*Did not started from saturated O/N. Started each with a colony, waited several hours until at OD ~1. We then added these cells to fresh 50mL LB to have an OD of 0.1.<br />
:::*Missed the 30 minute infection mark. Infected for ~45 minutes.<br />
::-Plated on Kan and Amp (Litmus28i sample) or Chlor (pSB1C3-M13ori sample) at dilutions of<br />
:::*1:100, 1:1k, 1:10k, and 1:100k<br />
<br />
'''6/17'''<br />
*Q5 PCR to replace KanR with ZeoR in M13K07<br />
::-Used recommendations<br />
::-Unfortunately, I (Jo) don’t know the difference between tightening and loosening the thermocycler lid; therefore, our M13K07 sample to amplify the M13K07 genes evaporated. But that’s ok, because we were sick of Zeocin anyway and decided mid-PCR to not waste our time with a Gibson Assembly and transformation. Instead we will be using the Old-School method of digestion and ligation because let’s face it, it’s a classic (and Gibson sucks) =)<br />
*Results from infection Test<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Litmus 28i<br />
! 1:100<br />
! 1:1k<br />
! 1:10k<br />
! 1:100k<br />
<br />
|-<br />
| Amp<br />
| ~6000?<br />
| ~2056<br />
| 377<br />
| 36<br />
<br />
|-<br />
| Kan<br />
| 130<br />
| 8<br />
| 3<br />
| 0<br />
<br />
|-<br />
|}<br />
{| class = "wikitable"<br />
|-<br />
! pSB1C3-M13ori<br />
! 1:100<br />
! 1:1k<br />
! 1:10k<br />
! 1:100k<br />
<br />
|-<br />
| Chlor<br />
| 8<br />
| 1<br />
| 0<br />
| 0<br />
<br />
|-<br />
| Kan<br />
| 97<br />
| 9<br />
| 0<br />
| 0<br />
<br />
|-<br />
|}<br />
::-We consider the Litmus 28i to have been a success. The pSB1C3-M13ori …. not so much. Looking back at the Litmus28i and the M13K07, we noticed that the M13ori is facing the opposite direction as the plasmid ori. Ours faces the same direction as the plasmid ori. Because we are working with phagemids that are single stranded, we think that by flipping the M13ori, we may be able to recover functionality. We will also look into other reasons.<br />
*Results from contamination test (6/16)<br />
::-Old Chlor plate (6/12)<br />
:::-Non-transformed chemically competent cells<br />
::::*Old: some colonies<br />
::::*New: No colonies<br />
::-New Chlor plate (6/16)<br />
:::-Colony from<br />
::::*Zeo contaminated plate: No growth<br />
::::*Chlor contaminated plate: Much growth<br />
:::-Non-transformed chemically competent cells<br />
::::*Old: some colonies<br />
::::*New: No growth<br />
::-Zeo (25ug/mL, Low NaCl, pH 7.5) (6/12)<br />
:::-Colony from<br />
::::*Zeo contaminated plate: Much growth<br />
::::*Chlor contaminated plate: No growth<br />
:::-Non-transformed chemically competent cells<br />
::::*Old: some specks<br />
::::*New: some specks<br />
<br />
'''6/18'''<br />
*Ordered primers to…<br />
::-biobrick M13ori (in other direction)<br />
::-biobrick M13K07 genes<br />
'''6/19'''<br />
*Waited for primers<br />
*Set up O/N cultures to test last infection (6/16) for colonies containing pSB1C3-M13ori<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample #<br />
! Selection<br />
! Dilution<br />
! Presumably<br />
! Notes<br />
<br />
|-<br />
| 1-8<br />
| Chlor<br />
| 1:100<br />
| pSB1C3-M13ori<br />
| <br />
<br />
|-<br />
| 9<br />
| Chlor<br />
| 1:1k<br />
| pSB1C3-M13ori<br />
| <br />
<br />
|-<br />
| 10-14<br />
| Chlor<br />
| 1:1k<br />
| pSB1C3-M13ori<br />
| These colonies were not present on plate during initial counting on 6/17<br />
<br />
|-<br />
| 15-16<br />
| Kan<br />
| 1:1k<br />
| M13K07<br />
| <br />
<br />
|-<br />
|}<br />
:::-Selection and dilution refer to the plate. Cells were then grown under selection.<br />
:::-More colonies were seen on all Chlor plates. No new colonies appeared on Kan plates<br />
*Tomorrow, we will mini-prep, digest, and run the samples on a gel to verify gene transfer.<br />
<br />
'''6/20'''<br />
*Primers are in!<br />
*Cloning of pSB1C3-M13ori(New)<br />
::-PCR to amplify M13ori (packaging signal) from Litmus 28i to be in the other direction<br />
:::-Primers (Gem011 F&R) add cut sites to make part biobrick compatible<br />
:::-Was able to gel extract<br />
::::*Band at the same size as sample….. contaminated primers?<br />
:::-Then digested with E and P<br />
::-Digested pSB1C3 plasmid with E and P to linearize backbone<br />
::::*Was able to gel extract<br />
'''[[File:UCB-Phage Delivery-140620.JPG]]'''<br />
<br />
:1. Spill over from 2<br />
:2 and 3. pSB1C3 digested with EcoRI and PstI<br />
::-Insert <100bp so cannot be seen<br />
:4. M13ori<br />
*Ligation of M13ori (packaging signal) into pSB1C3<br />
::10h at 16C<br />
::20m at 80C<br />
::Hold at 4C<br />
<br />
*PCR to amplify M13K07 genes from M13K07 DNA isolated from phage<br />
::-Primers (Gem012 F&R) add cut sites to make part biobrick compatible<br />
::-Very light bands and not band might correlate with 6000pb but not enough resolution on gel to be certain. Bands were too light so did not extract<br />
::-Set up PCR again using Q5 to run O/N<br />
:::*Received only a feint, smudgy band that was too large. We did not bother with extracting the DNA<br />
*Test of 6/19 O/Ns (14 that are presumably pSB1C3-M13ori and 2 that are presumably M13K07)<br />
::-Mini-prepped all O/Ns<br />
::-Digested all with Pst-I (common RE between pSB1C3-M13ori and M13K07)<br />
:::-Even though these are phagemids, we assumed that because they were still in the cell, the plasmids were still double stranded so could be recognized by RE. Our assumption was valid<br />
:::-Results of digestion<br />
::::*1,2,4-14: all had bands that corresponded to pSB1C3-M13ori cut once<br />
::::*3: Slight band at expected size but very feint<br />
::::*15, 16: as expected, they contain bands of ~9000bp, correlating with the M13K07 phagemid. Interestingly, sample 15 also contained a (brighter) band that corresponds with pSB1C3-M13ori. We assume that this was an incident of double infection (chance or did this occur at high frequency?)<br />
'''[[File:UCB-Phage Delivery-140620-02.JPG]]'''<br />
::Check table from 6/19 for more details<br />
:::-1-14: pSB1C3-M13ori<br />
:::-15-16: M13K07 from 1:1k diluted plate<br />
<br />
'''6/21'''<br />
*Needed to transform our pSB1C3-M13ori(New) (6/20) into cells. First, we transformed into 5alpha cells; however, we need to infect these cells later in order to make phage. Our 5alphas are not competent so we repeated the transformation, this time using ER2738 cells which contain the F’ episome, allowing us to infect with the M13 phage.<br />
::-Also transformed sample #1 from 6/20 mini-preps (pSB1C3-M13ori(Old) into ER2738 cells. Do not see any notes about this plasmid being in infectable cells during the initial experiment. Reason for experiment failure???<br />
*Ran 6/20 O/N PCR (to amplify M13K07 genes) on gel (also re-ran the previous sample from the day with more DNA)<br />
::-Still, band looks too big. Brightest band still occurs between 8000 and 9000bp. Lighter band around 7000bp-- might be ~6000pb but too light to tell<br />
*Alternative plan for amplifying M13K07 genes<br />
::-Digest sample with PstI (cuts outside of the target region)<br />
'''[[File:UCB-Phage Delivery-140621.JPG]]'''<br />
::Used sample #16 from 6/20 b/c it is double stranded so will cut<br />
:-PCR amplified the linearized digestion product<br />
:::1. PCR of M13genes<br />
:::2. PCR of M13genes + DMSO<br />
::::Gel extracted pieces boxed in red<br />
::-Used primers Gem012 F&R<br />
::-Gel extracted piece but received very low yield<br />
*Alternative to alternative plan for amplifying M13K07 genes<br />
::-Digested with PstI and AgeI<br />
'''[[File:UCB-Phage Delivery-140621-02.JPG]]'''<br />
:1. Cut with PstI and AgeI<br />
:2. Uncut<br />
:3. Cut with PstI<br />
:4. Just PCR<br />
::-Gel extracted ~6000pb band after double digestion<br />
::-PCR (Phusion)<br />
:::-Digestion (A+P) product<br />
:::-Gel extraction product<br />
:::-No DNA control<br />
<br />
==Week 8==<br />
<br />
'''6/22'''<br />
*Did receive colonies from 6/21 transformations. Selected colonies for overnight<br />
{|class="wikitable"<br />
|-<br />
!number<br />
!colonies<br />
|-<br />
|1-7<br />
|pSB1C3-M13ori(NEW) in 5alpha<br />
|-<br />
|8-14<br />
|pSB1C3-M13ori(NEW) in ER2738<br />
|-<br />
|15-18<br />
|pSB1C3-M13ori(OLD) in ER2738<br />
|-<br />
|19<br />
|p110+RBS in 5alpha as control<br />
|-<br />
|20<br />
|p110+RBS in ER2738 as control<br />
|}<br />
<br />
*Gel of 6/21 O/N PCR<br />
::Digestion (AgeI+PstI) product ? Yielded the three bands that appear with every PCR of M13K07<br />
'''[[File:UCB-Phage Delivery-140622.JPG]]'''<br />
<br />
:1. M13K07-> digested (AgeI + PstI) -> PCR<br />
:2. M13K07-> digested (AgeI+ PstI) -> extraction-> PCR<br />
:3. No DNA control for PCR<br />
::-Gel extraction product ? No bands<br />
::-No DNA control ? No bands<br />
*PCR purified the above PCR (Digestion (AgeI+PstI) product) and PCR from 6/21<br />
*Digested PCR purified sample with EcoRI+PstI and ran gel<br />
'''[[File:UCB-Phage Delivery-140622-02.JPG]]'''<br />
:1. PCR-> PCR purified<br />
:2. Sample after DpnI digest<br />
:3. Sample after digestion with EcoRI and PstI<br />
:4. Samples after digestion with DpnI then EcoRI and PstI<br />
*Ligation of above digestion (M13K07 genes) with pSB1C3<br />
<br />
<br />
'''6/23'''<br />
*Transformed pSB1C3-M13ori(New) (6/22 ligation)<br />
*Check overnights from 6/22 for the correct insert<br />
::-Mini-prep O/Ns<br />
::-Digested with EcoRI and PstI<br />
'''[[File:UCB-Phage Delivery-140622-03.JPG]]'''<br />
:::1-7: pSB1C3-M13ori(New) in 5alpha<br />
:::8-14: pSB1C3-M13ori(New) in ER2738<br />
:::15-18: pSB1C3-M13ori(Old)<br />
::-All were the expected size (though gel wiggled)<br />
::-Sent 4 samples for sequencing (iGEM primers: VF2 and VR)<br />
:::# 4: Divergent (new) phagemid 1C3 transformed into 5alpha<br />
:::# 11, 13: Divergent (new) phagemid 1C3 transformed into ER2738<br />
:::# 16: Convergent (old) phagemid 1C3 transformed into ER2738<br />
<br />
'''6/24'''<br />
*Results of 6/23 transformation (pSB1C3-M13genes) from 6/22 ligation)<br />
::-Received ~100 colonies<br />
::-Set up O/N cultures for 8 of the colonies<br />
::-Time passed…..<br />
::-Mini-prepped the 8 O/N samples mentioned above (yes, it was a long day)<br />
:::*Digested samples (E+P)<br />
*Started phage amplification protocol<br />
::-Amount of phage added = 10.9ul of 1:10 diluted phage (M13 phage (1) from 5/24 (4.575 x10^12 phage/mL)) to a final concentration of 1x10^8 phage/mL in 50mL<br />
::-Managed to get both samples to the 14-28hr incubation<br />
<br />
'''6/25'''<br />
*Checked mini-prep samples from 6/24 (pSB1C3-M13genes)<br />
::Ran the digestion overnight (6/24 to 6/25)<br />
'''[[File:UCB-Phage Delivery-140625.JPG]]'''<br />
::-All contained a band at 2000bp. Mostly empty vector or contained small, light band. Sample 1 had prominent band at ~1250bp.<br />
::-None of samples contained M13genes<br />
*M13K07 is on P15A ori (10-12 copy number) whereas the pSB1C3 ori is on pUC19 (500-700 copies). It’s possible that this overexpression is detrimental to cell<br />
::Alternative low copy plasmids found in distribution kit<br />
:::*2013 (plate 5)<br />
::::pSB6A1 (1K)<br />
::::pSB3C5 (3C)<br />
::::pSB3K3 (5E)<br />
:::*2014 (plate 4)<br />
::::pSB3C5 (4D)<br />
::::pSB6A1 (2L)<br />
::-Suspended and transformed the above plasmids into 5alphas<br />
::-Next, we will select colonies, mini-prep, digest, gel extract, ligate with M13genes<br />
*Started phage amplification protocol <br />
::-Phagemids in ER2738<br />
:::pSB1C3-M13ori(New)<br />
:::pSB1C3-M13ori(Old) <br />
::-Set up O/N of ER2738 for infection tomorrow<br />
<br />
'''6/26'''<br />
*Results of transformation of plasmids from the distribution kit<br />
::-Only received colonies pSB6A1 (2L from 2014) and pSB3C5 (4D from 2014)<br />
::-Pricked colonies for O/N<br />
*PCR of M13 genes so we can ligate it into the above backbones tomorrow<br />
::-DNA: M13K07 digested with EcoRI+PstI (6/21)<br />
::-Primers: Gem012 F & R<br />
*Finished phage amplification protocol<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Samples<br />
! A269<br />
! A320<br />
! Concentration<br />
<br />
|-<br />
| Old phagemid 1C3 (1)<br />
| 0.832<br />
| 0.492<br />
| 7.828 x10^12<br />
<br />
|-<br />
| Old phagemid 1C3 (2)<br />
| 0.324<br />
| 0.083<br />
| 5.549 x10^12<br />
<br />
|-<br />
| New phagemid 1C3 (1)<br />
| 0.391<br />
| 0.077<br />
| 7.229 x10^12<br />
<br />
|-<br />
| New phagemid 1C3 (2)<br />
| 0.402<br />
| 0.639<br />
| -5.457 x10^12<br />
<br />
|-<br />
|}<br />
::::*While resuspending “New phagemid 1C3 (2)”, the tip fell off and we lost half of sample. Evidently, we lost most of phage so we tossed sample<br />
::-Prepped and Infected ER2738 with “Old phagemid 1C3 (1)” and “New phagemid 1C3 (2)”<br />
:::Added 1.6ul of 1:10 diluted “Old phagemid 1C3 (1) to ER2738 cells<br />
:::Added 1.8ul of 1:10 diluted “New phagemid 1C3 (1) to ER2738 cells<br />
::-Plated the infected cells at dilutions<br />
:::1:1 onto Chlor+Kan plates<br />
:::1:10 onto Chlor+Kan plates<br />
:::1:100 onto Chlor and onto Kan plates<br />
:::1:1000 onto Chlor and onto Kan plates<br />
:::1:10000 onto Chlor and onto Kan plates<br />
:::1:100000 onto Chlor and onto Kan plates<br />
<br />
'''6/27'''<br />
*To ligate M13genes onto different backbones<br />
::-Mini-prepped the O/Ns from 6/26 to get backbones with low copy number<br />
:::pSB6A1<br />
:::pSB3C5 <br />
::::*did not grow as well<br />
::-PCR purified PCR (to amplify M13 genes with Gem012 F & R) from 6/26<br />
::-Digestions (50ul)<br />
:::Digest pSB6A1 with E + P<br />
:::Digest pSB3C5 with E + P<br />
:::Digest PCR purification product with E + P<br />
'''[[File:UCB-Phage Delivery-140627.JPG]]'''<br />
:1. pSB6A1<br />
:2. pSB3C5<br />
:3. M13genes<br />
::-Gel extraction of the above digestions<br />
:::For each: Added 10ul of 6x loading dye to 50ul digestions and divided the total volume between 2 wells<br />
::-Ligation<br />
:::1) pSB6A1-M13genes<br />
:::2) pSB3C5-M13genes<br />
*Results from 6/26 infection (after 22hrs in incubator)<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Anti-Sense Phagemid 1C3<br />
! 1:100<br />
! 1:1000<br />
! 1:10000<br />
! 1:100000<br />
<br />
|-<br />
| Chlor<br />
| 85<br />
| 6<br />
| 2<br />
| 0<br />
<br />
|-<br />
| Kan<br />
| 405<br />
| 13<br />
| 2<br />
| 1<br />
<br />
|-<br />
|}<br />
{| class = "wikitable"<br />
|-<br />
! Sense Phagemid1C3<br />
! 1:100<br />
! 1:1000<br />
! 1:10000<br />
! 1:100000<br />
<br />
|-<br />
| Chlor<br />
| 121<br />
| 24<br />
| 3<br />
| 0<br />
<br />
|-<br />
| Kan<br />
| 25<br />
| 1<br />
| 0<br />
| 0<br />
<br />
|-<br />
|}<br />
-We also plated the infected cells on plates containing Chlor and Kan to test for the possibility of double infection<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Phagemid 1C3<br />
! 1:1<br />
! 1:10<br />
<br />
|-<br />
| Anti-Sense<br />
| 59<br />
| 0<br />
<br />
|-<br />
| Sense<br />
| 4<br />
| 0<br />
<br />
|-<br />
|}<br />
<br />
<br />
'''6/28'''<br />
*We noticed that there were more colonies on our infection plates from 6/26 than on 6/27; therefore, we recounted colonies<br />
::-No increase of colonies on Kan plates<br />
::-Significant increase of colonies on Chlor plates<br />
::-Sense refers to the first phagemid 1C3 where the M13ori is in the sense direction compared to the plasmid ori<br />
::-Anti-Sense refers to the new phagemid 1C3 where the M13ori is in the anti-sense direction compared to the plasmid ori<br />
'''[[File:UCB-Phage Delivery-140628.JPG]]'''<br />
::Numbers on the left are after 22 hours. Numbers on the right are after 38.75 hours<br />
*Results of transformation from 6/27 (pSB6A1-M13genes and pSB3C5-M13genes)<br />
::-We have many colonies. Unfortunately, some are red, suggesting that original insert (J04450) was not successfully separated from backbone through gel extraction<br />
::-Selected colonies to grow overnight in 5mL LB<br />
<br />
==Week 9==<br />
<br />
'''6/29'''<br />
*Check O/N cultures for correct constructs (pSB6A1-M13genes and pSB3C5-M13genes)<br />
::-Mini-prep samples<br />
::-Digested with EcoRI and PstI to check insert sizes<br />
::-Gel<br />
'''[[File:UCB-Phage Delivery-140629.JPG]]'''<br />
::lanes….<br />
:::1-11: pSB6A1-M13genes<br />
:::12-18: pSB3C5-M13genes<br />
::-Epic failure<br />
::-All pSB6A1 backbones are empty<br />
::-Half of pSB3C5 backbones were empty. The others contained random inserts (1700 OR 3500). We don’t know what these inserts are. 1700 band is likely the digestion product that appears when we digest M13genes<br />
*Question: Can we use empty vectors from these mini-preps as ligation vectors?<br />
::-Digest pSB6A1 mini-prep with…. (see if both cut sites were retained during re-ligation)<br />
:::no enzyme<br />
:::EcoRI<br />
:::PstI<br />
:::EcoRI + PstI<br />
'''[[File:UCB-Phage Delivery-140629-02.JPG]]'''<br />
::lanes....<br />
:::1-2: last two samples of pSB3C5-M13genes from above gel<br />
:::3. Uncut<br />
:::4. Cut with EcoRI<br />
:::5. Cut with PstI<br />
:::6. Cut with EcoRI and PstI<br />
:-Appears that two backbones are ligated together<br />
<br />
'''6/30'''<br />
*Made chemically competent ER2738 cells that contain Litmus28i DNA<br />
*To amplify M13genes in order to retry ligation<br />
::-PCR of M13K07 DNA (diluted 1:100) to amplify the M13 genes<br />
::-PCR purify PCR product<br />
::-Run on gel: PCR purification, PCR, noDNAcontrol<br />
:::*PCR and purification showed bands at ~9k; therefore, did not get product<br />
:::*No DNA control was clean<br />
*Made freeze downs of<br />
::pSB3C5-J04450<br />
::pSB6A1-J04450<br />
::‘empty’ pSB3C5<br />
::‘empty’ pSB6A1<br />
<br />
'''7/1'''<br />
*Digestion of PCR product from 6/30 to figure out where mistake is<br />
:-Not really useful<br />
'''[[File:UCB-Phage Delivery-140701.JPG]]'''<br />
:Lanes...<br />
:1. PCR pur -> digested with AgeI<br />
:2. PCR pur -> digested with NgoMIV<br />
:3. PCR pur -> digested with PstI<br />
:4. PCR pur -> uncut<br />
:5. PCR pur -> digested with dpnI<br />
:6. Uncut plasmid DNA<br />
*Talked to Mary: She says we were adding to much DNA<br />
::-PCR again. Used 1:100 dilution of 1:100 diluted M13K07 DNA. (aka 1:10000 dilution)<br />
'''[[File:UCB-Phage Delivery-140701-02.JPG]]'''<br />
*Received a beautiful band at 6k bp<br />
:lanes<br />
:1. 1:100 dilution<br />
:2. 1:1000 dilution<br />
:3. 1:10 000 dilution<br />
:4. No DNA control<br />
<br />
'''7/2'''<br />
*To biobrick M13 genes (pSB6A1-M13genes and pSB3C5-M13genes)<br />
::-PCR purified 7/1 PCR product (Primers = Gem012)<br />
::-Digested with EcoRI-HF and PstI-HF<br />
::-Ligation to<br />
::::pSBA61 (digested and gel extracted)<br />
::::pSB3C5 (digested and gel extracted)<br />
<br />
'''7/3'''<br />
*Transformation<br />
::-pSB6A1-M13genes into 5alpha cells and ER2738 with pSB1C3-M13ori<br />
::-pSB3C5-M13genes into 5alpha cells and ER2738 with Litmus28i<br />
::-Transformed into cells containing a phagemid in order to skip some steps<br />
<br />
'''7/3-7/7 Vacation!'''<br />
<br />
==Week 10==<br />
<br />
'''7/7'''<br />
*Made O/N cultures of 7/3 transformation colonies<br />
::-Transformation results were not recorded until 7/8 (see below)<br />
<br />
'''7/8'''<br />
*Transformation results ( CC = Chemically comp cells, ER = ER2738)<br />
::-No growth on No DNA controls<br />
:::ER-Litmus28i CC on AMP-Chlor-Tet<br />
:::ER-phagemid1C3 CC on AMP-Chlor-Tet <br />
:::5alpha CC on Amp <br />
:::5alpha CC on Chlor <br />
::-Lots of red colonies on positive controls, no white colonies<br />
:::ER-Litmus28i CC + pSB3C5 on AMP-Chlor-Tet<br />
:::ER-phagemid1C3 CC + pSB6A1 on AMP-Chlor-Tet<br />
::-Lots of white colonies on sample plates, some red colonies<br />
:::ER-Lit CC + M13genes-pSB3C5 on AMP-Chlor-Tet<br />
:::ER-phagemid1C3 CC + M13genes-pSB6A1 on AMP-Chlor-Tet<br />
:::5alpha CC + M13genes-pSB3C5 on Chlor<br />
:::5alpha CC + M13genes-pSB6A1 on Amp<br />
*Overnights from 7/7 look healthy<br />
::-Renamed O/N to have numbers instead of long names<br />
{| class = "wikitable"<br />
|-<br />
! Sample #<br />
! Cells<br />
! DNA<br />
<br />
|-<br />
| 1-5<br />
| ER-Lit<br />
| M13genes-pSB3C5<br />
<br />
|-<br />
| 6-10<br />
| ER-phagemid1C3<br />
| M13genes-pSB6A1<br />
<br />
|-<br />
| 11-15<br />
| 5alpha<br />
| M13genes-pSB3C5<br />
<br />
|-<br />
| 16-20<br />
| 5alpha<br />
| M13genes-pSB6A1<br />
<br />
|-<br />
|}<br />
::-Mini-prepped DNA<br />
::-Digest the mini-preps with EcoRI-HF and PstI-HF<br />
::-Run digestions on gel to check sizes<br />
'''[[File:UCB-Phage Delivery-140708.JPG]]'''<br />
<br />
:lanes<br />
::top-left: pSB3C5-M13genes in ER2738 with Litmus28i<br />
::top-right: pSB6A1-M13genes in ER2738 with pSB1C3-1C3<br />
::bottom-left: pSB3C5-M13genes in 5alpha<br />
::bottom-right: pSB6A1-M13genes in 5alpha<br />
:None are correct<br />
*Primers Gem013 came in. Resuspend and diluted primers<br />
::-O/N of pSB3C5 to use for PCR tomorrow<br />
<br />
'''7/9'''<br />
*Wanted to make Litmus28i biobrick compatible for use as a phagemid backbone for us and other iGEM teams<br />
::-Mini-prepped O/N of pSB3C5<br />
::-PCR of pSB3C5 to amplify J04450 with Litmus28i compatible cut sites<br />
:::Primers: Gem013<br />
::::*At the 3’ end, these primers are the same as VF2 and VR so will bind the region flanking J04450. This conserves the terminators that exist between the biobrick prefix and VF2 on one side and those between the biobrick suffix and VR on the other side. Therefore, we are amplifying, VF2 priming site, terminators, J04450, terminators, and VR priming site<br />
::::*At the 5’ end, these primers contain unique restriction sites found in the Litmus28i MCS<br />
*PCR of M13 genes…. again (did 4 samples)<br />
::-Used the 1:10000 dilutions<br />
<br />
'''7/10'''<br />
*Note: The following two projects were done in parallel when possible<br />
*To make Litmus28i biobrick compatible<br />
::-Ran gel of PCR from 7/9 (J04450 amplification with Gem013)<br />
'''[[File:UCB-Phage Delivery-140710.JPG]]'''<br />
<br />
:1-4: PCRs of M13genes<br />
:5. No DNA control for M13genes PCR<br />
:6. PCR of pSB3C5 backbone<br />
:7. No DNA control for pSB3C5 backbone<br />
::-PRC purified the PCR product<br />
::-Digestion #1<br />
:::Restriction enzymes had the same buffer conditions but different activation temperatures so we had to do a 2 part digestion<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Restriction enzymes<br />
! Notes<br />
<br />
|-<br />
| J04450<br />
| Sac1<br />
| PCR purified<br />
<br />
|-<br />
| Litmus 28i<br />
| Sac1<br />
| From NEB tube<br />
<br />
|-<br />
|}<br />
:::*Incubate 1hr at 37 C<br />
:::*Heat inactivated 20 minutes at 80C<br />
::-Digestion #2<br />
:::-Added 1ul BsmI to both samples<br />
:::-Incubated 1hr at 65C<br />
:::-Heat inactivated 20 minutes at 80C<br />
::Ran gel<br />
:::-See gel below<br />
:::-Tried to extract J04450 segment but received very low yield. Since band otherwise looked clean, we decided to redo digestion then skip straight to ligation<br />
::Repeat digestion #1 for J04450<br />
::Repeat digestion #2 for J04450<br />
::Ligation (10hr at 16C, 10 min at 80C)<br />
:::3. Litmus28i + J04450<br />
*To retry ligation to biobrick backbone<br />
::-Ran gel of PCR from 7/9 (M13genes amplified with Gem012)<br />
:::See gel below<br />
::-PCR purified the PCR product<br />
::-Digestion<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Restriction enzymes<br />
! Notes<br />
<br />
|-<br />
| M13 genes<br />
| EcoRI-HF + PstI-HF<br />
| PCR purified<br />
<br />
|-<br />
| 6A1<br />
| EcoRI-HF + PstI-HF<br />
| ‘empty’ pSB6A1<br />
<br />
|-<br />
| 3C5<br />
| EcoRI-HF + PstI-HF<br />
| contains J04450<br />
<br />
|-<br />
|}<br />
:Incubate 1hr at 37C<br />
:Heat inactivated 20 minutes at 80C<br />
::-Ran gel<br />
:::*See gel below<br />
:::*Tried to extract M13genes and pSB3C5 segments but received very low yields. Since bands otherwise looked clean, we decided to redo digestion then skip straight to ligation<br />
::-Repeat digestion for M13genes and pSB3C5<br />
::-Ligation (10h at 16C, 10m at 80C)<br />
:::1. pSB3C5 + M13genes<br />
:::2. pSB6A1 + M13genes<br />
*Gel<br />
'''[[File:UCB-Phage Delivery-140710-02.JPG]]'''<br />
:1. pSB1A3<br />
:2. Limtus28i<br />
:3. M13genes<br />
:4. pSB3C5<br />
:5. J04450<br />
<br />
'''7/11'''<br />
*Transformed ligations from 7/10 into 5alpha cells<br />
::1. pSB3C5 + M13genes<br />
::2. pSB6A1 + M13genes<br />
::3. Litmus28i + J04450<br />
<br />
<br />
'''7/12'''<br />
*Results of 7/11 transformation<br />
::-No growth on no DNA control (Amp or Chlor)<br />
::-Lots of growth on Lit-J04450 -> some red-> colonies are too close together to prick individual colonies<br />
:::-It’s possible that these red colonies are satellites =(<br />
:::-Swiped some and plated on new Amp plate (restreak)<br />
::-Lawn of positive control (Litmus 28i) on Amp<br />
::-Many colonies for L1 (pSB3C5-M13genes) and L2 (pSB6A1-M13genes)<br />
:::-Due to high number of red colonies on Lit-J04450 plate, we assumed that most of these colonies contain empty vector<br />
:::-Did not make O/N<br />
*Ligated M13 genes to pSB6A1<br />
::-Used the remained of our digested M13genes.<br />
::-Both digestions from 7/10<br />
*Infection Experiment<br />
::-had 3 cell stocks (each taken from a different colony the night before)<br />
::-Tested Litmus 28i, Tandem phagemid 1C3, and double infection<br />
:::*Therefore, there were 9 flasks total.<br />
::-negative control: Streaked parent cells (non-infected) onto Chlor, Amp, and kan<br />
::-Included double infection plates for Litmus 28i and phagemid 1C3<br />
<br />
==Week 11==<br />
<br />
'''7/13'''<br />
*Transformed 7/12 ligation (pSB6A1-M13genes)<br />
*Litmus28i-J04450<br />
::-All growth from 7/12 restreak was white<br />
::-Pricked some red colonies for liquid O/N-will hopefully see red tomorrow<br />
::-Pricked a few red colonies and put into 200ul H2O (a few colonies per tube- 2 tubes total).<br />
:::*Plated 150ul onto Amp plates<br />
::-Put original plate into incubator to hopefully get bigger colonies<br />
<br />
'''7/14'''<br />
*Finished TWIV ppt<br />
*Set up 2 liquid culture of red Litmus 28i colonies (Litmus28i-J04450)-- slow growth <br />
::-Mini-prepped red Litmus 28i ‘O/N’ from earlier in day<br />
::-Digested samples—to check for insert and correct cut sites<br />
*Made O/Ns of pSB6A1-M13genes colonies from 7/13 transformation<br />
<br />
<br />
'''7/15'''<br />
*Ran gel of Litmus28i-J04450 samples<br />
'''[[File:UCB-Phage Delivery-140715.JPG]]'''<br />
:1. J04450-Litmus28ibb #1 (EcoRI+PstI)<br />
:2. J04450-Litmus28ibb #1 (uncut)<br />
:3. J04450-Litmus28ibb #2 (EcoRI+PstI)<br />
:4. J04450-Litmus28ibb #2 (EcoRI+PstI)<br />
::*Accidentally added restriction enzymes<br />
:5. Litmus28i (EcoRI+PstI)<br />
::*Only has PstI site<br />
<br />
::-Verifies that Litmus28i-J00450 had correct cut sites<br />
::-From this point on, biobricked Litmus28i is called Litmus28ibb<br />
*Check 7/14 O/Ns for pSB6A1-M13genes<br />
::-Mini-prepped liquid cultures<br />
::-Digested with EcoRI and PstI<br />
::::Received bold bands of just under 4000bp. Some lanes had a very feint band ~330bp. None were the correct size<br />
*Transformed Litmus28ibb-J04450 into ER2738 cells<br />
*Remake phage packaging<br />
::-pSB1C3-M13ori(New)<br />
::-pSB1C3-M13ori(Old)<br />
<br />
'''7/16'''<br />
*Results of 7/15 transformation of Litmus28i into 6/30 ER2738 cells<br />
::-lawn on no DNA control-- most likely contaminated cells<br />
::::Streaked chem comp 5alpha and ER onto Amp and Amp+Tet plates to determine if problem is with cells or plates<br />
::-O/N culture of ER to make new chem comp cells<br />
<br />
'''7/17'''<br />
*Finished phage isolation<br />
::Note:<br />
:::*pSB1C3-M13ori (New): M13ori and plasmid ori are convergent<br />
:::*pSB1C3-M13ori (Old): M13ori and plasmid ori are tandem<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! 269nm<br />
! 320nm<br />
! [] phage/mL<br />
<br />
|-<br />
| pSB1C3-M13ori (New)<br />
| 0.386<br />
| 0.034<br />
| 8.126 E12<br />
<br />
|-<br />
| pSB1C3-M13ori (New)<br />
| 0.419<br />
| 0.036<br />
| 8.842 E12<br />
<br />
|-<br />
| pSB1C3-M13ori (Old)<br />
| 0.283<br />
| 0.025<br />
| 5.956 E12<br />
<br />
|-<br />
| pSB1C3-M13ori (Old)<br />
| 0.326<br />
| 0.024<br />
| 6.972 E12<br />
<br />
|-<br />
|}<br />
*Infection to compare the new and the old pSB1C3-M13ori<br />
::-Prepped cells for infection<br />
::-Infected cells with pSB1C3-M13ori (New) or pSB1C3-M13ori (New)<br />
::-and plated on dilutions of 1:100, 1:1000, and 1:10000 on Chlor and kan<br />
::-Also plated 1:1000 dilution on lowered Chlor concentration (34ug/mL)<br />
*Sent Litmus28ibb-J04450 for sequencing<br />
<br />
'''7/18'''<br />
*Results from contamination test<br />
::-5alpha on Amp = no growth<br />
::-5alpha on Amp+Tet = colonies<br />
::-ER on Amp = lawn<br />
::-ER on Amp+Tet = lawn<br />
*Made new chemically competent ER2738<br />
*Transformed Litmus28ibb-J04450 into ER2738 made on 3/30 and ER2738 made on 7/18<br />
::-Had two samples of Litmus28ibb-J04450 and two cell stocks, so 4 samples total<br />
<br />
'''7/19'''<br />
*Results of 7/17 infection<br />
::-pSB1C3-M13ori(Old) (tandem-when plasmid ori and M13ori point in the same direction) packages better than pSB1C3-M13ori(New) (when ori and M13ori are convergent), implying that directionality matters.<br />
'''[[File:UCB-phage lab7-19-141012.jpg]]'''<br />
<br />
==Week 12==<br />
<br />
'''7/20'''<br />
*Freeze downs<br />
:-Litmus28i-J04450 Litmus 28i is now biobrick compatible<br />
<br />
'''7/21'''<br />
*Send samples for sequencing<br />
::-pSB1C3-M13ori(Old)<br />
::-pSB1C3-M13ori(New)<br />
*To ligate M13ori to a kanamycin backbone (pSB1K3)<br />
::-Resuspended pSB1K3-J04450 from distribution kit (6B on plate 4)<br />
:::*Contains J04450 as insert (full RFP construct)<br />
::-Transform pSB1K3 into 5alpha cells<br />
<br />
'''7/22'''<br />
*Pricked colony from pSB1K3-J04450 transformation<br />
::-Mini-prepped DNA to get a supply of DNA<br />
*To put M13ori (M13 phackaging signal) onto Kanamycin resistance o we can test packaging ratios with M13K07 on the same antibiotic<br />
::-Digested pSB1K3 with EcoRI-HF and XbaI<br />
::-Digested pSB1C3-M13ori (Old) with EcoRI-HF and SpeI-HF<br />
:::*DNA was not sufficiently cut. Too much DNA? Problem with enzyme (SpeI-HF)?<br />
'''[[File:UCB-Phage Delivery-140722.JPG]]'''<br />
:1. pSB1K3<br />
:2. M13ori<br />
<br />
'''7/23'''<br />
*Sequencing samples from 7/21 were lost in the mail. Resent samples<br />
*Yet another attempt to biobrick M13genes<br />
::1. using primers that would amplify genes and M13 ori parts from M13K07<br />
::2. using primers that would amplify genes, M13 ori parts, and plasmid ori from M13K07<br />
'''[[File:UCB-Phage Delivery-140723.JPG]]'''<br />
:1. Amplified only the M13genes and M13ori<br />
:2. No DNA control for 1<br />
:3. Amplified M13genes, M13ori, and plasmid ori<br />
:4. No DNA control for 3<br />
::-When run on a gel, samples were clean with only one band at around 7kb. No contamination in no DNA controls<br />
*Digestions<br />
::-pSB1K3 with EcoRI-HF and PstI-HF to check for correct insert<br />
::-pSB1C3-M13ori (Old and new) with EcoRI-HF and SpeI-HF to test for efficient cutting with different stock of restriction enzyme<br />
'''[[File:UCB-Phage Delivery-140723-02.JPG]]'''<br />
:1-3: pSB1K3<br />
:4. pSB1C3-M13ori (Old)<br />
:5. pSB1C3-M13ori (New)<br />
*Still had inefficient cutting. Tested M13ori next to Litmus28i to disern the problem<br />
:*Uncut<br />
:*Cut once with (E, X, S, or P)<br />
:*Cut twice with (E+S or E+P)<br />
'''[[File:UCB-Phage Delivery-140723-03.JPG]]'''<br />
:1. M13ori uncut<br />
:2. M13ori Ecori-HF<br />
:3. M13ori SpeI-HF<br />
:4. M13ori XbaI<br />
:5. M13ori PstI-HF<br />
:6. M13ori EcoRI-HF + SpeI-HF<br />
:7. M13ori EcoRI-HF + PstI-HF<br />
:8. Litmus28i uncut<br />
:9. Litmus28i EcoRI-HF<br />
:10. Litmus28i SpeI-HF<br />
:11. Litmus28i XbaI<br />
:12. Litmus28i PstI-HF<br />
:13. Litmus28i EcoRI-HF + SpeI-HF<br />
:14. Litmus28i EcoRI-HF + PstI-HF<br />
*Only partial digest with only SpeI-HF for both. Complete digestion with all others, including E+S<br />
<br />
'''7/24'''<br />
*To Biobrick M13genes using pSB6A1<br />
::-PCR purified M13 genes (did both samples at the same time)<br />
::-Digested PCR purification and pSB6A1 with EcoRI-HF and PstI-HF<br />
'''[[File:UCB-Phage Delivery-140724.JPG]]'''<br />
*Gel extracted M13genes<br />
<br />
:1. M13genes<br />
:2. pSB6A1<br />
:-Bands of pSB6A1 were too light to gel extract<br />
*To swap the kanamycin resistance marker on M13K07 with ampicillin resistance<br />
::-PCR amplified AmpR from pSB6A1<br />
*made O/N culture of pSB6A1 from freeze down to mini-prep<br />
::-Also streaked cells onto plate<br />
<br />
'''7/25'''<br />
*To change resistance marker on M13K07<br />
:1. Biobrick method<br />
::*Tried to mini-prep O/N but pellet (after liquid culture was spun) was not red even after 16+ hours. Set up O/N from plate colonies<br />
:2. Swap only resistance method<br />
::*PCR purified 7/24 PCR<br />
::*Ran product on gel-> band of correct size<br />
'''[[File:UCB-Phage Delivery-140725.JPG]]'''<br />
<br />
:1. PCR product<br />
:2. PCR product-> PCR purified<br />
:3. No DNA control<br />
::-Digested the PCR purification products of M13genes+ori and AmpR with AgeI-HF and NotI-HF<br />
::*Bands on previous gel looked clean enough for both so did not gel extract<br />
::-Overnight ligation<br />
*Set up O/Ns of pSB1K3 colonies<br />
<br />
'''7/26'''<br />
*Transform<br />
::-pSB6A1+M13genes<br />
*To biobrick M13genes<br />
::-Digestion<br />
:::*pSB6A1 with EcoRI and PstI<br />
'''[[File:UCB-Phage Delivery-140726.JPG]]'''<br />
:Gel extracted backbone (band ~4000bp)<br />
*Ligations (10hrs 16C, 10min at 80C)<br />
::-pSB6A1+M13genes<br />
<br />
==Week 13==<br />
<br />
'''7/27'''<br />
*Transformation results from 7/26<br />
::-No growth for no DNA controls ''on Amp''<br />
::-No growth for M13 genes onto pSB6A1 ''on Amp''<br />
*Set up O/Ns of 5alpha and ER2738 to make competent cell tomorrow<br />
*Made freeze down of pSB1K3<br />
*Plated ER competent cells from 5/15 and 6/16 Onto Chlor plates to check for contamination<br />
<br />
'''7/28'''<br />
*No growth of ER2738 on Chlor (either sample)<br />
::-ER is not contaminated with ChlorR<br />
*Make new 5alpha chem comp cells <br />
*Transformations<br />
::-pSB6A1-M13genes<br />
::-positive control for Amp (pSB6A1)<br />
::-No DNA control<br />
<br />
'''7/29'''<br />
*Set up O/Ns of pSB6A1-M13genes<br />
::-Plated sample on reduced Amp and regular Tet<br />
::-Later in day…<br />
::-Mini-prepped O/Ns<br />
::-Digested and ran on a gel to check for insert sizes<br />
'''[[File:UCB-Phage Delivery-140729.JPG]]'''<br />
:*All but the 4th lane with 3 bands look correct<br />
<br />
'''7/30'''<br />
*Lawn on last night’s plating-> replate<br />
*Digest pSB3C5 and pSB6A1-M13genes with E and P to move M13genes to a Chlor backbone<br />
'''[[File:UCB-Phage Delivery-140730.JPG]]'''<br />
::-Extracted bands<br />
::-Ligations<br />
:::*pSB3C5 + M13genes<br />
*Sent samples for sequencing<br />
**M13 genes on pSB6A1 VF2<br />
**M13 genes on pSB6A1 VR<br />
<br />
'''Test experiment for high school kids coming to lab (7/29-7/31)'''<br />
*General idea: Have 2 strands of DNA, one has an EcoRI site while the other contains a SNP, abolishing the cut sight. We pretend that one is pathogenic (the one that is not cut) and tells kids to figure out which one is which <br />
*PCR with Dream-Taq<br />
::-Did not have special fastdigest enzyme<br />
::-Hypothesized that green dye will interfere with enzyme effectivity<br />
*PCR with Dream-Taq (no green dye)<br />
::-Digest with EcoRI fastdigest-> did not cut<br />
*Redid experiment several times<br />
::-Should digest with XbaI<br />
::-PCR purified PCR products<br />
<br />
'''7/31'''<br />
*Spent the morning with Heritage High School<br />
*Made phage<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Phage<br />
! A269<br />
! A320<br />
! genome size<br />
! [ ]<br />
<br />
|-<br />
| Litmus28ibb-J04450 (1)<br />
| 0.823<br />
| 0.197<br />
| <br />
| 9.35 x10^12<br />
<br />
|-<br />
| Litmus28ibb-J04450 (2)<br />
| 0.940<br />
| 0.168<br />
| <br />
| 1.14 x10^13<br />
<br />
|-<br />
|}<br />
<br />
'''8/2'''<br />
*Digestion<br />
::-pSB6A1-M13genes (E+P)<br />
::-pSB3C5 (E+P)<br />
<br />
==Week 14==<br />
<br />
'''8/3'''<br />
*Another attempt to clone M13genes onto pSB3C5<br />
::-Run 8/2 digestions on gel<br />
'''[[File:UCB-Phage Delivery-140803.JPG]]'''<br />
:1. M13genes (E+P)<br />
:2. pSB3C5 (E+P)<br />
::-Gel extracted <br />
:::*M13genes away from pSB6A1 backbone<br />
:::*pSB3C5 backbone away from J04450<br />
::-Ligations<br />
:::*pSB3C5 + M13genes<br />
:::*pSB3C5 + no insert<br />
::-Transformed ligations into ER2738<br />
<br />
'''8/5'''<br />
*Transformation of pSB3C5+M13genes failed<br />
*Made phage<br />
::-Fd-CAT DNA packaged with Fd-CAT<br />
::-phagemid 1C3 packaged with M13K07<br />
::-amilCP on pSB1C3 packaged with M13K07<br />
::-Litmus28ibb-J04450 packaged with M13K07<br />
<br />
'''8/6'''<br />
*Isolated phage<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Phage<br />
! [ ]<br />
<br />
|-<br />
| Fd CAT<br />
| 2.72 x10^12<br />
<br />
|-<br />
| Litmus28ibb-J04450<br />
| 8.37 x10^12<br />
<br />
|-<br />
| pSB1C3-amilCP<br />
| 1.42 x10^13<br />
<br />
|-<br />
| pSB1C3-M13ori<br />
| 4.32 x10^12<br />
<br />
|-<br />
|}<br />
<br />
'''8/6'''<br />
*Started making phage with Fd CAT the should contain Litmus28ibb-RFP<br />
*Infection<br />
::-Infected ER2738 cells with Litmus28ibb-RFP, pSB1C3-M13ori, or pSB1C3-amilCP<br />
::-Plated Litmus samples on 100ug/mL Amp and 50ug/mL Kan<br />
::-Plated other two samples on 34ug/mL Chlor and 25ug/mL Kan<br />
<br />
'''8/7'''<br />
*Isolated phage containing Litmus28ibb-RFP using helper phage<br />
::-M13K07 (as control)<br />
::-Fd-CAT (no phage pellet was observed-worried that there was no phage)<br />
*Went to CSU to have them test M13ori part compared to amilCP. Grew the samples in 5mL O/Ns then diluted to 0.5OD and grew 30 minutes rather than starting from a fresh colony<br />
<br />
'''8/8'''<br />
*At CSU<br />
::-Finished phage protocol (Test packaging of M13ori part)<br />
::-Only used 20mL infection samples<br />
::-grew to 0.55 OD<br />
::-When making phage (after 14 hour incubation), there was very little growth<br />
::-Plated non-diluted and diluted 1:1000 of M13ori and amilCP sample<br />
*Infected cells using phage isolated 8/7. Plated at dilutions of 1:100 and 1:100k<br />
*Want to test progeny Fd-CAT phage (made 8/7) for infectability<br />
::-Start phage isolation prodocol using Fd-CAT phage from 8/7 do deliver the helper phagemid<br />
:::*Phagemid:<br />
::::*None: will make Fd-CAT phage packaing Fd-CAT phagemid<br />
::::*Litmus28ibb-J04450: make Fd-CAT phage packaging Litmus28ibb-J04450 phagemid<br />
*Remake phage (Fd-CAT) using fresh stock from Mike<br />
<br />
<br />
'''8/9'''<br />
*Results of 8/8 infection<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Helper Phagemid<br />
! phagemid<br />
! Selection<br />
! 1:1000 dilution<br />
! 1:00k dilution<br />
<br />
|-<br />
| M13K07<br />
| Litmus28ibb-RFP<br />
| Kan+Tet<br />
| 400-500 (some red)<br />
| 61 white; 20 red<br />
<br />
|-<br />
| same<br />
| same<br />
| Amp+Tet<br />
| lawn<br />
| 2<br />
<br />
|-<br />
| Fd-CAT<br />
| Litmus28ibb-RFP<br />
| Chlor+Tet<br />
| 0<br />
| 0<br />
<br />
|-<br />
| same<br />
| same<br />
| Amp+Tet<br />
| 10<br />
| 707<br />
<br />
|-<br />
|}<br />
*NOTE: The same sample was plated on two different plates. For example, the sample using M13K07 as the helper phage was plated on Kan+Tet and Amp+Tet<br />
*Mini-prep<br />
::-pSC3C5-J04450 -- very little growth, low DNA yield<br />
::-Fd-CAT infected cells<br />
:::*Digest and run on a gel to verify presence of a band- there was a very feint band<br />
*Finished isolating phage…. messed up and used 0.8MgCl2, 0.2M NaCl instead of PEG during precipitation step. Also used the wrong phage<br />
::-Infected ER2738 cells anyway…. no growth by 8/12<br />
<br />
==Week 15==<br />
<br />
'''8/10'''<br />
*Are the progeny phage from 8/6 (original progeny from first Fd-Tet application) viable/ able to reproduce?<br />
::-Used these progeny to make phage that should amplify Fd-CAT phage containing either the Fd-CAT or Litmus28ibb-J04450 phagemid<br />
<br />
'''8/11'''<br />
*Finished isolating phage<br />
{| class = "wikitable"<br />
|-<br />
! Helper Phagemid<br />
! Phagemid<br />
! A269<br />
! A320<br />
! genome size<br />
! [ ]<br />
<br />
|-<br />
| Fd-CAT<br />
| Litmus28ibb (1)<br />
| 0.029<br />
| 0.013<br />
| 4080<br />
| 2.35 x10^11<br />
<br />
|-<br />
| Fd-CAT<br />
| Ltimus28ibb (2)<br />
| 0.033<br />
| 0.015<br />
| 4080<br />
| 2.65 x10^11<br />
<br />
|-<br />
| Fd-CAT<br />
| Fd-CAT (1)<br />
| 0.326<br />
| 0.082<br />
| 7775<br />
| 1.88 x10^12<br />
<br />
|-<br />
| Fd-CAT<br />
| Fd-CAT (2)<br />
| 0.320<br />
| 0.079<br />
| 7775<br />
| 1.86 x10^12<br />
<br />
|-<br />
| M13g6A1<br />
| M13g6A1 (1)<br />
| 0.322<br />
| 0.148<br />
| 10,029<br />
| 1.04 x10^12<br />
<br />
|-<br />
| M13g6A1<br />
| M13g6A1 (2)<br />
| 0.503<br />
| 0.130<br />
| 10,029<br />
| 2.23 x10^12<br />
<br />
|-<br />
|}<br />
<br />
'''8/13'''<br />
*Infected ER2738 with Fd-CAT packaging Litmus28ibb-J04450<br />
<br />
'''8/16'''<br />
*Results from Infection on 8/11<br />
{| class = "wikitable"<br />
|-<br />
! Helper Phagemid<br />
! Phagemid<br />
! 1:1<br />
! 1:10<br />
! 1:100<br />
! 1:1000<br />
<br />
|-<br />
| M13g6A1<br />
| M13g6A1<br />
| almost lawn<br />
| 728<br />
| 287<br />
| 55<br />
<br />
|-<br />
| Fd-CAT<br />
| Fd-CAT<br />
| lawn<br />
| almost lawn<br />
| 1260<br />
| 640<br />
<br />
|-<br />
| Fd-CAT<br />
| Litmus28ibb-J04450<br />
| lawn<br />
| almost lawn<br />
| 2004<br />
| 304<br />
<br />
|-<br />
|}</div>Leighlahttp://2014.igem.org/Team:CU-Boulder/Notebook/CC9Phage_TeamTeam:CU-Boulder/Notebook/CC9Phage Team2014-10-16T04:22:08Z<p>Leighla: </p>
<hr />
<div>{{Template:UCB-Main}}<br />
{{UCB-NavBar}}<br />
__FORCETOC__<br />
<br><br />
<br><br />
<br><br />
<br />
'''Note:'''<br />
'''Litmus28ibb is a biobricked version of the Litmus28i (from NEB) vector which contains the M13ori (packaging signal) which allows for uptake into M13 or Fd phage. While making this part biobrick compatible, we added J04450 as the insert.'''<br />
'''pCas9 (BBa_K1218011) contains and tracrRNA and Cas9 gene under native promoters, and a minimal CRISPR arrays. The guide RNA (referred here as gRNA) is inserted into the CRISPR array.'''<br />
<br><br />
<br><br />
<br><br />
<br />
==Week 1==<br />
<br />
'''7/21'''<br />
*To ligate pCas9 to Litmus28ibb<br />
::-Digestion<br />
:::-Litmus28ibb-J04450 with E+P<br />
:::-pCas9 with E+P<br />
'''[[File:UCB-Cas9Phage-140721.JPG]]'''<br />
:1. pCas9<br />
:2. Litmus28ibb<br />
::-Overnight ligations (10hrs at 37C, 10min at 80C)<br />
<br />
'''7/22'''<br />
*Transformed ligation from 7/21 (Litmus28ibb + pCas9) into ER2738 cells<br />
::-Plated on Amp + Tet<br />
::-Later in day, pricked colonies from morning transformation (Litmus28ibb and pCas9 in ER)<br />
<br />
'''7/23'''<br />
*Miniprepped O/N from 7/22 of Litmus28ibb-pCas9<br />
::-Digested<br />
:::*None were the correct size. Smaller bands looked like pSB1C3. Plated cells onto Chlor and there was significant growth. pSB1C3 from the pCas9?<br />
<br />
'''7/24'''<br />
*To put pCas9 onto Litmus28i<br />
::-Digested<br />
:::-Litmus28ibb with EcoRI+PstI<br />
:::-pSB1C3-pCas9 with EcoRI+SpeI<br />
:::-PSB3C5-J04450 with XbaI + PstI<br />
'''[[File:UCB-Cas9Phage-140724.JPG]]'''<br />
:1. Litmus28ibb<br />
:2. pCas9<br />
:3. J04450<br />
::-Overnight ligation (10hr @ 16C)<br />
<br />
'''7/25'''<br />
*Transformed ligation from 7/24 (Litmus28ibb+pCas9) into 5alpha cells<br />
<br />
'''7/26'''<br />
*No growth from 7/25 transformation<br />
*Tranform<br />
::-Litmus28ibb + pCas9 + J04450<br />
*Retry making Litmus28i with pCas9<br />
::-Ligation (10hrs at 16C, 10min at 80C)<br />
:::*Litmus28ibb + pCas9 + J04450<br />
<br />
==Week 2==<br />
<br />
'''7/27'''<br />
*Transformation results<br />
::-No growth for no DNA controls (on Chlor)<br />
::-4 colonies for Litmus28bb-JO4450-Cas9 (on Amp-> none are red)<br />
*Picked all 4 colonies for O/Ns<br />
<br />
'''7/28'''<br />
*3 new red colonies on Litmus28bb-Cas9-J04450 plate<br />
::-Set up cultures<br />
*O/Ns grew but did not bother<br />
*Transformations<br />
::-Litmus28bb-J04450-Cas9<br />
::-No DNA control<br />
<br />
'''7/29'''<br />
*Litmus28bb-Cas9-J04450 O/Ns<br />
::-Incubator shut off during night. Samplers were at RT by morning<br />
::-Add 1mL to fresh 4mL LB and Amp<br />
::-Mini-prep DNA<br />
::-Digest with EcoRI and PstI to check insert for correct size<br />
'''[[File:UCB-Cas9Phage-140729.JPG]]'''<br />
::-Lane 2 contains the expected 3000bp band for the Litmus28ibb backbone and the ~7000bp band for the pCas9-J04450 insert. Unfortunately, this sample also contains a 4000bp band.<br />
*Plated the above sample<br />
<br />
'''7/30'''<br />
*Digested Litmus28ibb-Cas9-RFP with EcoRI-HF<br />
'''[[File:UCB-Cas9Phage-140730.JPG]]'''<br />
::-Extracted large band ~9000<br />
::-Re-ligated piece<br />
*Sent samples for sequencing<br />
::-Litmus28bb with Cas9 and RFP (LCR) gRNA sequence<br />
<br />
'''8/1'''<br />
*Mini-prepped O/Ns of<br />
::-Litmus28ibb-Cas9-J04450 (LCR)<br />
:::*Digested with EcoRI-HF and PstI-HF => all samples had band ~3700<br />
*Digestions of Litmus and Cas9 to retry ligation<br />
'''[[File:UCB-Cas9Phage-140801.JPG]] '''<br />
:1. Litmus28ibb (E+P)<br />
:2. pCas9(E+P)<br />
:3. Litmus28ibb (E+P)<br />
:4. pCas9(E+S)<br />
:5. pSB3C5 (X+P)<br />
::-Gel extractions<br />
::-Ligations<br />
:::*Litmus28ibb (E+P) to pCas9 (E+P)<br />
:::*Litmus28ibb(E+P) to pCas9(E+S) and J04450(E+P)<br />
<br />
'''8/2'''<br />
*Transformation into BW cells (5/30)<br />
::-Hundreds of colonies for LCR Miniprep<br />
*Digestion to get a better idea of what this DNA is<br />
<br />
'''8/3'''<br />
*Isolate the LCR from the extra band in the gel<br />
::-Run 8/2 digestions on gel<br />
'''[[File:UCB-Cas9Phage-140803.JPG]]'''<br />
:1. LCR (PstI)<br />
:2. LCR (EcoRI)<br />
::-Gel extracted <br />
:::-LCR digested with PstI-HF (top band<br />
::-Re-ligate extracted LCR with no insert (should re-ligate to itself)<br />
::-Transformed ligation into ER2738<br />
*Made O/Ns<br />
::-colonies from 8/2 transformation<br />
::-restreak of LCR from freeze down<br />
<br />
==Week 3==<br />
<br />
'''8/4'''<br />
*Check overnights from 8/3 (LCR = Litmus28ibb+pCas9+RFP) for correct inserts<br />
::-Mini-prep samples<br />
::-Digest with EcoRI and PstI<br />
:::*All had sizes ~3700bp<br />
:::*No correct inserts<br />
*Attempt to ligate Litmus28ibb and pCas9(gRNA3)<br />
::-Digestions<br />
:::*Litmus28ibb-RFP E+P extracted Lit back bone<br />
:::*pCas9 gRNA3 E+P extracted pCas9 insert<br />
'''[[File:UCB-Cas9Phage-140804.JPG]]'''<br />
:1. Litmus28ibb<br />
:2. pCas9(gRNA3)<br />
:3. pCas9(unmod.)<br />
::-Ligations (10 hrs at 16C, 10 min at 80C)<br />
:::*Litmus28ibb + pCas9(gRNA3)<br />
<br />
'''8/5'''<br />
*Transformation into ER2738 (7/18), 5ul of ligation products<br />
::1. Litmus28ibb-Cas9gRNA3<br />
<br />
'''8/6'''<br />
*Another attempt at ligating pCas9 and Litmus28ibb<br />
::-Digest unmodified pCas9 with EcoRI-HF and PstI-HF<br />
::-Digest pCas9 containing pRNA1 with EcoRI-HF and PstI-HF<br />
'''[[File:UCB-Cas9Phage-140806.JPG]]'''<br />
:1. pCas9(unmod.)<br />
:2. pCas9(gRNA1)<br />
::-Gel extract pieces<br />
::-Ligate separately to Litmus28ibb<br />
*Results of 8/5 transformation<br />
::-6 colonies for Litmus28ibb+pCas9(gRNA3) on Tet+reduced Amp<br />
:::*Picked colonies for overnights<br />
::-No growth for no DNA control<br />
*Transformation of ligations from earlier today<br />
::-Litmus28ibb + pSB1C3-pCas9(unmod.)<br />
::-Litmus28ibb + pSB1C3-pCas9(gRNA1)<br />
*Mini-prep Litmus28ibb+pCas9(gRNA3) O/N samples from earlier in day<br />
::-Digested with EcoRI-HF and PstI-HF<br />
:::*All samples had bands of 3000 and 2000bp. None were correct<br />
<br />
'''8/8'''<br />
*M13ori into SB Cas9<br />
::-Digestions<br />
:::-unmodified pCas9 with EcoRI-HF and XbaI<br />
:::-M13ori with EcoRI-HF and SpeI-HF<br />
:::-pCas9(gRNA1) with EcoRI-HF and XbaI<br />
:::-pCas9(gRNA3) with EcoRI-HF and XbaI<br />
<br />
'''8/9'''<br />
*To clone M13ori onto pSB1C3-pCas9<br />
::-Run 8/8 digestion on a gel<br />
'''[[File:UCB-Cas9Phage-140809.JPG]]'''<br />
:1. M13ori<br />
:2. pCas9(gRNA3)<br />
:3. pCas9(gRNA1)<br />
:-Gel extracted<br />
::-Ligation Ligated M13ori to….<br />
:::-pSB1C3-pCas9(gRNA1)<br />
:::-pSB1C3-pCas9(gRNA3)<br />
:::-pSB1C3-pCas9(unmod.)<br />
::-Transform and plate onto low Chlor 934 ug/mL)<br />
<br />
==Week 4==<br />
<br />
'''8/10'''<br />
*Digested ligation products from 8/6 and 8/4. Saw many large bands that may have been undigested<br />
*O/Ns of 8/9 transformation<br />
::-1 colony for M13ori-pCas9(gRNA3) (only 1 colony grew)<br />
::-11 colonies for M13ori-pCas9(gRNA1)<br />
::-No colonies grew for M13ori-pCas9(unmodified)<br />
<br />
'''8/11'''<br />
*Test 8/10 O/Ns for correct inserts<br />
::-Digest 8/10 O/Ns with EcoRI-HF and PstI-HF<br />
'''[[File:UCB-Cas9Phage-140811.JPG]]'''<br />
:1. M13ori-pCas9(gRNA3)<br />
:2-12. M13ori-pCas9(gRNA1)<br />
:*Lanes 1, 8, and 9 are about the right size. Sent these samples for sequencing<br />
*To ligate M13ori to pSB1C3-pCas9(unmod.)<br />
::-Digest<br />
:::*pSB1C3-pCas9(unmod.) (E+P)<br />
:::*M13 digested 8/8<br />
::-Ligation: See L5 below<br />
*To ligate pCas9 and M13ori to pSB1A3<br />
::-Digest<br />
:::*pSB1A3 (E+P)<br />
:::*M13ori (E+S)<br />
:::*Unmodified pCas9 (X+P)<br />
:::*pCas9(gRNA1)-M13ori (E+P)<br />
:::*pCas9(gRNA3)-M13ori (E+P)<br />
::-Ligation: See L2-4 below<br />
*Another try to put pCas9 onto Litmus28ibb<br />
::-Digested<br />
:::*Unmodified Litmus28i (E+P)<br />
:::*pCas9(unmod.) (E+P)<br />
:::*pCas9(gRNA1)-M13ori (E+P)<br />
:::*pCas9(gRNA3)-M13ori (E+P)<br />
::-Ligation: See L1 and L6 below<br />
*Gel for all<br />
'''[[File:UCB-Cas9Phage-140811-02.JPG]]'''<br />
<br />
:1. Unmodified Litmus28i<br />
:2. pSB1C3-pCas9(unmodified)<br />
:3. pSB1A3<br />
:4. pSB1C3-M13ori<br />
'''[[File:UCB-Cas9Phage-140811-03.JPG]]'''<br />
:5. pSB1C3-pCas9(unmod.)<br />
:6. pSB1C3-M13ori-pCas9(gRNA1)<br />
:7. pSB1C3-M13ori-pCas9(gRNA3)<br />
<br />
*Ligations for both projects<br />
<br />
{| class = "wikitable"<br />
|-<br />
! #<br />
! Backbone<br />
! Insert 1<br />
! Insert 2<br />
<br />
|-<br />
| 1<br />
| Unmod. Litmus28i (E+P)<br />
| pCas9(unmod.) (E+P)<br />
| N/A<br />
<br />
|-<br />
| 2<br />
| pSB1A3 (E+P)<br />
| pCas9(unmod.) (X+P)<br />
| M13ori (E+S)<br />
<br />
|-<br />
| 3<br />
| pSB1A3 (E+P)<br />
| pCas9(gRNA1)-M13ori (E+P)<br />
| N/A<br />
<br />
|-<br />
| 4<br />
| pSB1A3 (E+P)<br />
| pCas9(gRNA3)-M13ori (E+P)<br />
| N/A<br />
<br />
|-<br />
| 5<br />
| pSB1C3-pCas9(unmod.) (E+X)<br />
| M13ori (E+S)<br />
| N/A<br />
<br />
|-<br />
| 6<br />
| Litmus28ibb (E+P)<br />
| pCas9(gRNA3) (E+P)<br />
| N/A<br />
<br />
|-<br />
| 7<br />
| Litmus28ibb (E+P)<br />
| pCas9(gRNA1) (E+P)<br />
| N/A<br />
<br />
|-<br />
| 8<br />
| Litmus28ibb (E+P)<br />
| pCas9(unmod.) (E+P)<br />
| N/A<br />
<br />
|-<br />
|}<br />
<br />
'''8/12'''<br />
*Transform ligation from 8/11 into ER2738 cells<br />
*Make phage using M13g6A1 as Helper Phage<br />
::-used pCas9(gRNA1)-M13ori or pCas9(gRNA3)-M13ori as phagemid<br />
*Freeze downs<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Top<br />
! Side<br />
<br />
|-<br />
| M. gRNA3 Cas9 (8/12)<br />
| M13ori Cas9 gRNA3 pSB1C3<br />
<br />
|-<br />
| M. gRNA1 Cas9 (8/12)<br />
| M13ori(8) Cas9 gRNA1 pSB1C3<br />
<br />
|-<br />
| M. gRNA1 Cas9 (8/12)<br />
| M13ori (9) Cas9 gRNA1 pSB1C3<br />
<br />
|-<br />
|}<br />
<br />
'''8/13'''<br />
*Finish phage isolation<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Helper phage<br />
! Phage<br />
! A269<br />
! A320<br />
! genome size<br />
! [ ]<br />
<br />
|-<br />
| Fd-CAT<br />
| M. gRNA1<br />
| 0.353<br />
| 0.025<br />
| 7687<br />
| 2.56 x10^12<br />
<br />
|-<br />
| Fd-CAT<br />
| M. gRNA3<br />
| 0.554<br />
| 0.037<br />
| 7687<br />
| 4.40 x10^12<br />
<br />
|-<br />
|}<br />
<br />
*Infection<br />
::-Infecting ER2738 and BWF’<br />
::-Phage<br />
:::*pSB1C3-pCas9(gRNA1)<br />
:::*pSB1C3-pCas9(gRNA3)<br />
*Transformation Results from 8/12<br />
::-Growth for the following… (made O/Ns)<br />
:::*pSB1A3-M13ori-pCas9(gRNA3)<br />
:::*pSB1C3-M13ori-pCas9(unmod)-M13ori<br />
:::*Litmus28ibb-pCas9(gRNA3)<br />
:::*Litmus28ibb-pCas9(gRNA1)<br />
:::*Litmus28ibb-pCas9(unmod.)<br />
::-Hundreds of colonies for pCas9(unmod.) on Unmod. Litmus28i. Didn’t trust result so didn’t pick coloneis. EcoRI and PstI sites touch so most likely, Litmus28i was only cut once<br />
<br />
'''8/15'''<br />
*Check O/Ns from 8/14 for correct inserts<br />
::-Mini-prepped samples<br />
::-Digested with E and P<br />
::-Results<br />
:::*No bands of the right size for<br />
:::-Litmus28ibb-pCas9(gRNA3)<br />
:::-Litmus28ibb-pCas9(gRNA1)<br />
:::-Litmus28ibb-pCas9(unmod.)<br />
:::-Expected band sizes for… (see below)<br />
*pCas9(gRNA3)-M13ori on pSB1A3<br />
*pCas9(unmod)-M13ori on pSB1C3<br />
'''[[File:UCB-Cas9Phage-140815.JPG]]'''<br />
<br />
*Transform into BWF’ to test for functionality of Cas9 when associated with m13ori, independent of infectivity<br />
::-M13ori-pCas9(gRNA1)<br />
::-M13ori-pCas9(gRNA3)<br />
<br />
'''8/16'''<br />
*PCR on LCR (Litmus28ibb with pCas9 and RFP to maybe get a good product to religate<br />
::-Didn’t work<br />
Results of 8/13 infection of BWF’ with M13ori-gRNA(1 or 3) to test pCas9 killing<br />
No growth on DNA controls on Chlor(34ug/mL) or Kan(50ug/mL)+Chlor(34ug/mL)<br />
ON Kan(50ug/mL) + Chlor (170ug/mL) in BWF’<br />
gRNA1 22 colonies<br />
gRNA3 2 colonies<br />
On Kan(25ug/mL)+Chlor(34ug/mL) in BWF’<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! 1:100<br />
! 1:1000<br />
<br />
|-<br />
| gRNA1<br />
| 617<br />
| 84<br />
<br />
|-<br />
| gRNA3<br />
| 469<br />
| 42<br />
<br />
|-<br />
|}<br />
On Chlor (34ug/mL) in BWF’<br />
<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! 1:100<br />
! 1:1000<br />
<br />
|-<br />
| gRNA1<br />
| 508<br />
| 67<br />
<br />
|-<br />
| gRNA3<br />
| 447<br />
| 41<br />
<br />
|-<br />
|}<br />
On Chlor (34ug/mL) in ER<br />
<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! 1:100<br />
! 1:1000<br />
<br />
|-<br />
| gRNA1<br />
| 702<br />
| 93<br />
<br />
|-<br />
| gRNA3<br />
| 518<br />
| 58<br />
<br />
|-<br />
|}<br />
<br />
Results of 8/15 transformation of BWF’ with M13ori—gRNA(1 or 3) to test killing<br />
Chlor (34ug/mL) + Kan (50ug/mL) <br />
<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! 1:1<br />
! 1:10<br />
<br />
|-<br />
| gRNA1<br />
| Lawn<br />
| 1904<br />
<br />
|-<br />
| gNRA3<br />
| Slightly thinner lanw<br />
| 1616<br />
<br />
|-<br />
|}<br />
<br />
Chlor (34 ug/mL)<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! Trial 1<br />
! Trial 2<br />
<br />
|-<br />
| gRNA1<br />
| 1788<br />
| 1548<br />
<br />
|-<br />
| gRNA3<br />
| 1564<br />
| 1416<br />
<br />
|-<br />
|}<br />
Samples were diluted 1:10<br />
8/17<br />
Make phage<br />
pSB1C3-M13ori-pCas9(unmod)<br />
pSB1A3-M13ori-pCas9(gRNA3)<br />
Ran uncut LCR on a gel and gel extracted the three bands<br />
<br />
8/18<br />
While making phage…. We threw away the samples….<br />
Remake phage. Same as 8/17<br />
Transform LCR gel extractions into 5alpha cells<br />
<br />
8/19<br />
Sent samples for sequencing<br />
pSB1C3-M13ori-pCas9(unmodified)<br />
pSB1A3-M13ori-pCas9(gRNA3)<br />
<br />
Finish making phage<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Name<br />
! A269<br />
! A320<br />
! Genome Size<br />
! [ ]<br />
<br />
|-<br />
| pSB1A3-M13ori-pCas9(gRNA3)<br />
| 0.697<br />
| 0.162<br />
| <br />
| Note: We later found that this sample did not actually contain the M13ori<br />
<br />
|-<br />
| pSB1C3-M13ori-pCas9(unmod)<br />
| 0.764<br />
| 0.146<br />
| 7687<br />
| 3.044 x10^13<br />
<br />
|-<br />
|}<br />
Infect cells (ER and BWF’)<br />
Grew a single 75mL sample. When OD ~0.5, divided samples into 3<br />
Added 20mL of sample to 125mL flask (3 for each strain)<br />
Added phage to a final concentration of 1 x10^8 phage/mL<br />
Incubated at 250rpm, 37C, for 30 minutes<br />
Plated at dilutions (1:10, 1:100, and 1:1k)<br />
On Chlor(170ug/mL), Kan(50ug/mL)+Chlor(85ug/mL), Kan(50ug/mL)+Chlor(170ug/mL)<br />
<br />
<br />
8/21<br />
Finished phage amplification protocol from 8/20 but did not receive any phage. <br />
Infect BWF’ cells with the phage having pSB1C3-M13ori-pCas9 containing the scramble, gRNA1, and gRNA3.<br />
<br />
8/22<br />
Present project to department<br />
<br />
8/25<br />
Results of 8/21 infection experiment<br />
Results on Kan and Chlor<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! Scramble<br />
! gRNA1<br />
! gRNA3<br />
<br />
|-<br />
| 1:1<br />
| 149<br />
| 143<br />
| 13<br />
<br />
|-<br />
| 1:10<br />
| 14<br />
| 16<br />
| 1<br />
<br />
|-<br />
|}<br />
<br />
Results on Chlor<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! Scramble<br />
! gRNA1<br />
! gRNA3<br />
<br />
|-<br />
| 1:1<br />
| 147<br />
| 117<br />
| 14<br />
<br />
|-<br />
| 1:10<br />
| 14<br />
| NA<br />
| 3<br />
<br />
|-<br />
|}<br />
<br />
8/28<br />
Retry ligation of pCas9 to litmus28ibb<br />
Digest Litmus28ibb-J04450, pCas9, and amilCP with EcoRI and PstI. Gel extract pieces<br />
Ligate pCas9 into Litmus28ibb and amilCP into Limus28ibb<br />
<br />
8/29<br />
Transform 8/28 ligation products into 5alpha cells<br />
<br />
8/29<br />
Transform pSB1C3-pCas9(unmodified) into BW and BWF’ to test targeting of f’ episome. Due to poor infection results we wanted to test the guide RNAs for targeting of the F’ episome. We have searched the vast webs for the sequence of the F’ episome but have yet to (and never will) find it.<br />
<br />
8/30<br />
Transformation results of pCas9 into BW and BWF’. Results were unexpected, we expected more growth for BW sample or at least equal growth. Differences in competence or concentration could explain results in initial count<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Sample<br />
! Initial count<br />
! After another 24 hours<br />
<br />
|-<br />
| No DNA controls (for BW and BWF’)<br />
| No growth<br />
| No growth<br />
<br />
|-<br />
| BW cells<br />
| 9 colonies<br />
| 222<br />
<br />
|-<br />
| BWF’ cells<br />
| ~100 colonies<br />
| 158<br />
<br />
|-<br />
|}<br />
<br />
Transformation results for ligation of pCas9 to Litmus28ibb<br />
<br />
{| class = "wikitable"<br />
|-<br />
! <br />
! Amp (50ug/mL)<br />
! Amp (100ug/mL)<br />
! Notes<br />
<br />
|-<br />
| No DNA control<br />
| 3<br />
| 10<br />
| All bright white<br />
<br />
|-<br />
| Litmus28ibb-pCas9<br />
| ~50 (many are red)<br />
| 25-30<br />
| High proportion are red. Some are shadowy white. A few are bright white<br />
<br />
|-<br />
| Litmus28ibb-amilCP<br />
| >300<br />
| >300<br />
| Mostly shadowy, some red, some bright white<br />
<br />
|-<br />
|}<br />
<br />
Made overnight cultures of shadowy and bright coloneis from Litmus28ibb-pCas9 samples (from both plates) and of shadowy colonies from Litmus28ibb-amilCP. Since growth on no DAN control was bright white, we assumed that bright colonies on sample plates were more likely to be contamination, but didn’t really know. Since there were fewer colonies on no DNA control plates, we figures we could randomly select colonies from the sample plates that were not a result of contamination.<br />
<br />
8/31<br />
Mini-prep overnight cultures from 8/30 and digest samples with EcoRI and PstI. When run on a gel, none of the Litmus28ibb-pCas9 samples yielded bands of the correct size. All Litmus28ibb-amilCP samples did yield the expected band sizes. <br />
<br />
9/2<br />
Sent some of mini-prep samples from 8/31 for sequencing<br />
<br />
9/3<br />
Digestion of pCas9 with EcoRI and speI and the unmodified NEB version of Litmus28i with EcoRI and XbaI. Gel extracted pieces and ligated together (No, this part would not have the BioBrick prefix/suffix)<br />
<br />
9/4<br />
Transform 9/3 Litmus28i-pCas9 ligation into 5alpha cells. Selected on low ampicillin (50ug/mL) and high ampicillin (100ug/mL)<br />
<br />
9/5<br />
Did receive colonies from 9/4 transformation. Selected 24 colonies for colony PCR with a primer pair that would span the junction of pCas9 and Litmus28i. (forward primer bound to CRISPR region of pCas9 while reverse primer bound in the middle of the Litmus28i backbone)<br />
Colony PCR failed to reveal any promising samples. Set up overnight cultures in a hopeless effort to yield different results.<br />
<br />
9/7<br />
Mini-prepped overnight cultures from 9/5. Digested each with EcoRI and PstI and ran reults on gel. Results were consistent with colony PCR: failure. A few samples had very feint bands that were approximately the correct size<br />
<br />
9/8<br />
for the samples on 9/7 with light bands of approximately the correct size, we sent samples for sequencing (another hopeless attempt to get different results)<br />
<br />
9/9<br />
Sequencing results showed that Limtus28i religated<br />
<br />
9/17<br />
New method to clone pCas9 onto Litmus28ibb vector: PCR->Digestion->ligation method<br />
PCR amplified pCas9 construct from pSB1C3-pCas9. PCR amplified Litmus28ibb backbone from Litmus28ibb-J04550<br />
PCR purified both samples. Digested both samples with EcoRI and PstI and gel extracted the pieces<br />
Ligated pCas9 to Litmus28ibb vector<br />
<br />
Sent pSB1C3-M13ori-pCas9 sample for sequencing to sequence the entire plasmid<br />
<br />
9/18<br />
Transform 9/17 ligation (Litmus28ibb-pCas9) into 10beta cells<br />
<br />
9/19<br />
There was growth on no DNA control. Some colonies for sample.<br />
<br />
9/20 <br />
Make chemically competent ER and 5alpha cells using DMSO from bottle instead of fancy tubes. We think this is the reason for later transformation failures<br />
<br />
Digested pCas9 and Litmus28ibb PCR purifications from 9/17 with EcoRI, PstI, and DpnI. PCR purified digestion samples then ligated pieces together.<br />
<br />
9/21<br />
New idea: Cloning pCas9 onto Litmus28i has not worked up until this point. What if we constructed a vector that looked similar to litmus28i. pSB1A3 has the same origin of replication and almost the same ampicillin resistance marker (with a few SNPs). Need to add M13ori (which was taken from Litmus28i) and pCas9. M13ori would not be in the correct position on plasmid but this is as close as we can get with the remaining time<br />
Digestions: M13ori-pCas9(unmod), pCas9(unmod), pSB1A3 with EcoRI and PstI. Gel showed incomplete digestion so did not gel extract<br />
<br />
Transformation into 5alpha cells: no DNA control, 9/20 ligation (Litmus28ibb-pCas9), pSB1C3-amilCP, and pSB1C3-M13ori-pCas9<br />
<br />
9/22<br />
Re-digested M13ori-pCas9(unmod) and pSB1A3-J04450 with EcoRI and PstI. pCas9(unmod) was PCR purified (product looked clean on gel so did not bother to gel extract). <br />
Ligated M13ori-pCas9(unmod) to pSB1A3 and pCas9(unmod) to pSB1A3<br />
Sent pSB1A3 to M13ori-pCas9 for sequencing to cover region not covered by previous sequencing<br />
<br />
9/23<br />
Sequencing results came back<br />
Litmus28ibb-J04450 contains two nucleotides that are flipped. They are located within the ampicillin resistance gene and do change two amino acids; however, sample grows on ampicillin so these are not loss of function mutation. <br />
pSB1C3-M13ori-pCas9 was perfect<br />
the above two samples are ready to submit to the registry<br />
<br />
Transform ligations from 9/22 (pSB1A3- M13ori-pCas9(unmod) and pCas9(unmod)) into 5 alpha cells<br />
Colony PCR 18 samples from 9/20 Limtus28ibb-pCas9(unmod) plate.<br />
<br />
9/24<br />
Run gel of 9/23 colony PCR to check for desired Litmus28ibb-pCas9(unmod). There was a band of approximately the correct size in each samples (including the no DNA control). Set up overnight cultures of first 5 samples for further testing<br />
Results of 9/23 transformation<br />
No growth for no DNA control on chlor (170ug/mL), Amp (50ug/mL) or Amp (100ug/mL)<br />
7 colonies for pSB1C3-M13genes on chlor (170ug/mL)<br />
27 colonies for pSB1A3-pCas9 on Amp (100ug/mL) and 41 colonies on Amp (50ug/mL)<br />
0 colonies for pSB1A3-M13ori-pCas9 on Amp(50 and 100ug/mL)<br />
Set up overnight cultures of some of the samples.<br />
<br />
<br />
9/25<br />
Mini-prep overnights from 9/24<br />
Digest samples to check for insert. When run on a gel, 3 samples had large bands of ~5000bp and one had an additional band at ~6000. We made overnights of these 3 colonies<br />
<br />
9/26<br />
Digested mini-prepped DNA from the three samples that looked promising from 9/25. Each sample was digested with only EcoRI, only PstI, or EcoRI and PstI. When run on a gel, it can be seen that each samples, when cut once, yielded one band 8000bp while when cut twice, yielded 2 bands of ~2000 and 5000 as expected.<br />
Sent parts to iGEM registry: pSB1C3-M13ori-pCas9 and Litmus28ibb-J04450<br />
To add the M13ori to pSB1A3-Cas9<br />
Digested pSB1A3-pCas9 with EcoRI and XbaL<br />
Digested pSB1C3-M13ori with EcoRI and SpeI<br />
Gel extracted bands of the correct sizes and combined these into a ligation reaction.<br />
<br />
9/27<br />
Transform pSB1A3-M13ori-pCas9 into Amp(50ug/mL) and Amp(100ug/mL)<br />
Later in the day…. Set up overnight cultures of colonies<br />
<br />
<br />
9/28<br />
Mini-prepped overnights made 9/27<br />
Digested overnight samples with EcoRI and PstI<br />
Did receive pSB1A3-M13ori-pCas9 samples with bands of the correct size (as compared to pSB1A3-pCas9)<br />
Set up overnight cultures of the samples that looked promising<br />
Transformed pSB1A3-M13ori-pCas9(unmod) and pSB1A3-M13ori-pCas9(gRNA3) into ER2738 cells<br />
<br />
9/29<br />
Did receive colonies from 9/28 transformation. Set up overnight cultures for both.<br />
Also set up overnight cultures of ER2738 and BWF’ for upcoming infection.<br />
Start phage amplification protocol using pSB1A3-M13ori-pCas9(unmod) and pSB1A3-M13ori-pCas9(gRNA3) as the phagemid. Used Fd-Cat as the helper phagemid<br />
<br />
9/30<br />
Finished phage amplification/isolation protocol for Fd phage packaging pSB1A3-M13ori-pCas9(unmod) and pSB1A3-M13ori-pCas9(gRNA3)<br />
<br />
10/1<br />
Measured concentration of phage isolated on 9/30. There were no phage for the gRNA3 sample. The pSB1A3-M13ori-pCas9(unmod) sample contained some phage but had a high level of contamination/impurity.<br />
Send samples pSB1A3-M13ori-pCas9(unmod) and pSB1A3-M13ori-pCas9(gRNA3) samples for sequencing to verify the presence of the M13ori<br />
<br />
10/2<br />
Sequencing results came in<br />
pSB1A3-M13ori-pCas9(unmod) looks good<br />
pSB1A3-M13ori-pCas9(gRNA3) doesn’t have the M13ori<br />
Plan<br />
1. SDM on pSB-M13ori-pCas9(unmod) to remove BsaI cut site in pSB1A3 backbone<br />
2. Change spacer from unmodified to the gRNA3<br />
3. Redo the infection experiment<br />
SDM<br />
PCR to amplify pSB1A3-M13ori-pCas9(unmod)<br />
Treat with kinase, Ligase, and DpnI<br />
Transform into NEB 5alpha cells<br />
<br />
<br />
10/3<br />
Set up O/N cultures of 10/2 transformation<br />
<br />
10/4<br />
Mini-prep overnights. Samples are called pSB1A3(delta BasI)-M13ori-pCas9(unmod) to indicate each part of the construct and the removed BsaI site in the pSB1A3 backbone.<br />
Changing the gRNA spacer in pCas9<br />
Vector digest: digested pSB1A3(delta BasI)-M13ori-pCas9(unmod) with BsaI to remove unmodified spacer. Saw only one band on the gel (cut around spacer) which shows that the additional BsaI site in the pSB1A3 backbone was removed. Gel extracted the digested vector<br />
Ligation: Used the annealed oligos for gRNA3 (our targeting sample) from the last time we changed the spacer. Ligated these oligos to the digested pSB1A3(delta BasI)--M13ori-pCas9<br />
<br />
10/5<br />
Transform pSB1A3(delta BasI)-M13ori-pCas9(unmod) and pSB1A3(delta BasI)-M13ori-pCas9(gRNA3) in ER2738 cells<br />
<br />
10/6<br />
Transformation results from 10/5<br />
No growth for no DNA control<br />
pSB1A3(delta BasI)-M13ori-pCas9(unmod) = many colonies<br />
pSB1A3(delta BasI)-M13ori-pCas9(gRNA3) = no colonies<br />
<br />
Re-anneal primers then relegate<br />
Phosphorylation of oligos for gRNA3. Incubate for 1 hour at 37C then 20 minutes at 65C<br />
Add 2.5ul 1M NaCl<br />
Anneal in thermocycler. 5 minutes at 95C then a slow cool-down for ~2 hours<br />
Dilute product 1:10<br />
Ligation of newly annealed primers to digest and gel extracted pSB1A3(delta BasI)-M13ori-pCas9(unmod) from 10/5<br />
<br />
10/7<br />
Transform 10/6 ligation into ER2738 cells<br />
<br />
10/8<br />
No growth for 10/7 transformation<br />
Transform ligation from 10/6 into 5alpha cells<br />
Last try to make pSB1A3-M13ori-pCas9(gRNA3)<br />
Know from 10/1 sequencing that we have pSB1A3-pCas9(gRNA3). Therefore, only need to add M13ori upstream of pCas9 part<br />
Digest pSB1A3-pCas9(gRNA3) with EcoRI and XbaI and pSB1C3-M13ori with ExoRI and SpeI<br />
Gel extract digested pieces<br />
Ligate gel extracted pieces together<br />
<br />
10/9<br />
No growth for 10/8 transformation<br />
Transform 10/8 ligation into 5alpha cells<br />
<br />
10/10<br />
Transform <br />
pSB1C3-M13ori-pCas9(unmod) from 10/9 into ER2738 cells<br />
pSB1A3 (delta BsaI)-M13ori-pCas9(gRNA3) from 10/6 into 5 alpha cells<br />
pSB1A3-M13ori-pCas9(gRNA3) from 10/8 into 5 alpha cells<br />
<br />
10/11<br />
Transformation results of 10/10<br />
1 colony for pSB1C3-M13ori-pCas9(unmod) in ER2738 (does not seem trustworthy)<br />
No colonies for no DNA control<br />
No colonies for the 2 other transformations<br />
Transform pSB1C3-M13ori-pCas9(unmod) into ER2738 cells again<br />
Start phage amplification protocol to make the following phage<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Phagemid<br />
! Helper Phagemid<br />
<br />
|-<br />
| pSB1C3-M13ori-pCas9(unmod)<br />
| M13g6A1<br />
<br />
|-<br />
| pSB1C3-M13ori-pCas9(unmod)<br />
| M13K07<br />
<br />
|-<br />
| pSB1C3-M13ori-pCas9(gRNA3)<br />
| M13g6A1<br />
<br />
|-<br />
| pSB1C3-M13ori-pCas9(gRNA3)<br />
| M13K07<br />
<br />
|-<br />
|}<br />
<br />
pSB1C3-M13ori-pCas9(unmod) grew unexpectedly fast so we missed the OD mark. Growth rate compared to pSB1C3-M13ori-pCas9(gRNA3) sample suggests contamination. Did not proceed with making these phage for today.<br />
<br />
10/12<br />
No growth for 10/11 transformation – conclude that ER2738 cells are not competent (these are the cells made 9/20 with DMSO from a room temp bottle instead of the alliquotes from NEB.<br />
Remake phage<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Phagemid<br />
! Helper Phagemid<br />
<br />
|-<br />
| pSB1C3-M13ori-pCas9(unmod)<br />
| M13g6A1<br />
<br />
|-<br />
| pSB1C3-M13ori-pCas9(unmod)<br />
| M13K07<br />
<br />
|-<br />
|}<br />
10/13<br />
Finish phage isolation protocol and measure<br />
<br />
{| class = "wikitable"<br />
|-<br />
! Phagemid<br />
! Helper Phagemid<br />
! Dilution<br />
! A269<br />
! A320<br />
! Genome size<br />
! Diluted [ ]<br />
! Actual [ ]<br />
<br />
|-<br />
| pSB1C3-M13ori-pCas9(gRNA3)<br />
| M13g6A1<br />
| 1:40<br />
| 0.090<br />
| 0.015<br />
| 7687<br />
| 5.85 E11<br />
| 2.34 E13<br />
<br />
|-<br />
| pSB1C3-M13ori-pCas9(gRNA3)<br />
| M13K07<br />
| 1:40<br />
| 0.085<br />
| 0.013<br />
| 7687<br />
| 5.62 E11<br />
| 2.25 E13<br />
<br />
|-<br />
| pSB1C3-M13ori-pCas9(unmod)<br />
| M13g6A1<br />
| 1:40<br />
| 0.104<br />
| 0.022<br />
| 7687<br />
| 6.40 E11<br />
| 2.56 E13<br />
<br />
|-<br />
| pSB1C3-M13ori-pCas9(unmod)<br />
| M13K07<br />
| 1:20<br />
| 0.091<br />
| 0.016<br />
| 7687<br />
| 5.85 E11<br />
| 1.17 E13<br />
<br />
|-<br />
|}<br />
<br />
Infection with phage of BW23115F’ cells<br />
Grew a single stock of cells then dived into 4 samples each with 50mL cells. Added the above phage<br />
Incubated for 30 minutes at 37C, shaking<br />
Plated onto Chlor<br />
<br />
10/14<br />
Infection results<br />
No growth on Chlor (170ul/mL)<br />
Very little to no growth on pCas9(unmod) samples. We think this is b/c the colony picked on 10/11 did not actually contain pSB1C3-M13ori-pCas9(unmod) so nothing was packaged into phage<br />
~100 colonies for pCas9(gRNA3) samples.<br />
The samples made with M13g6A1 were also plated on ampicillin. These plates show considerable growth. There is more growth on pCas9(unmod) than pCas9(gRNA3) sample plate. We believe this further supports our hypothesis that our pCas9(unmod) sample did not contain pSB1C3-M13ori-pCas9(unmod) so packaged M13g6A1 in the absence of a better phagemid. Also, for the pCas9(gRNA3) sample, there was considerably more growth on the ampicillin plate than on the chlor plate. This could be because pSB1C3-M13ori-pCas9(gRNA3) successfully killed the BW23115F’ cells as desired or the phagemid was not effectively packaged<br />
<br />
Transformed pSB1C3-M13ori-pCas9(unmod) into chemically competent ER2738 cells that we know work from July.<br />
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