http://2014.igem.org/wiki/index.php?title=Special:Contributions/Kristanguyen&feed=atom&limit=50&target=Kristanguyen&year=&month=2014.igem.org - User contributions [en]2024-03-28T13:52:12ZFrom 2014.igem.orgMediaWiki 1.16.5http://2014.igem.org/Team:Washington/Our_TeamTeam:Washington/Our Team2014-10-18T01:58:39Z<p>Kristanguyen: </p>
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<h1> UW iGEM 2014 Team </h1><br />
<br />
<h2> Undergraduate Team Members </h2><br />
<br />
<table><br />
<tr><br />
<td>[[File:Edward.jpg|200px|thumb|left|]]</td><br />
<td>Edward Chang, Biochemistry<br><br />
Poster</td><br />
</tr><br />
<tr><br />
<td>[[File:Andrew2.jpg|200px|thumb|left|]]</td><br />
<td>Andrew Chau, Chemistry<br><br />
Flow Cytometry and Team Page</td><br />
</tr><br />
<tr><br />
<td>[[File:JoshC.jpeg|200px|thumb|left|]]</td><br />
<td>Joshua Cho, Molecular, Cellular, & Developmental Biology<br><br />
Fluorescence Activated Cell Sorting, Flow Cytometry, and Presentation</td><br />
</tr><br />
<tr><br />
<td>[[File:ChrisC.jpg|200px|thumb|left|]]</td><br />
<td>Chris Choe, Molecular, Cellular, & Developmental Biology<br><br />
Expression and Stability Analysis</td><br />
</tr><br />
<tr><br />
<td>[[File:William.jpg|200px|thumb|left|]]</td><br />
<td>William Harvey, Biochemistry<br><br />
Fluorescence Activated Cell Sorting, Outreach, and Presentation</td><br />
</tr><br />
<tr><br />
<td>[[File:AlexK.jpg|200px|thumb|left|]]</td><br />
<td>Alex Kang, Biochemistry<br><br />
Expression and Stability Analysis and Outreach</td><br />
</tr><br />
<tr><br />
<td>[[File:Julia.jpg|200px|thumb|left|]]</td><br />
<td>Julia Lim, Microbiology<br><br />
BioBricks, Flow Cytometry, Outreach, and Team Page</td><br />
</tr><br />
<tr><br />
<td>[[File:Harman.jpg|200px|thumb|left|]]</td><br />
<td>Harman Malhi, Molecular, Cellular, & Developmental Biology<br><br />
Expression and Stability Analysis</td><br />
</tr><br />
<tr><br />
<td>[[File:Colton.jpg|200px|thumb|left|]]</td><br />
<td>Colton McDavid, Microbiology<br><br />
Expression and Stability Analysis, Poster, and Team Page</td><br />
</tr><br />
<tr><br />
<td>[[File:Krista.jpg|200px|thumb|left|]]</td><br />
<td>Krista Nguyen, Biochemistry<br><br />
Flow Cytometry, Outreach, Poster, and Team Page</td><br />
</tr><br />
<tr><br />
<td>[[File:Nicolov photo.JPG|200px|thumb|left|]]</td><br />
<td>Anastasia Nicolov, Bioengineering and Violin Performance<br><br />
Expression and Stability Analysis</td><br />
</tr><br />
<tr><br />
<td>[[File:Ahmed.jpg|200px|thumb|left|]]</td><br />
<td>Ahmed Qureshi, Biochemistry<br><br />
BioBricks, Outreach, and Poster</td><br />
</tr><br />
<tr><br />
<td>[[File:Rettie.jpg|200px|thumb|left|]]</td><br />
<td>Stephen Rettie, Microbiology<br><br />
BioBricks, Fluorescence Activated Cell Sorting, Outreach, and Presentation</td><br />
</tr><br />
</table><br />
<br />
<br> All experiments and data collection were preformed by the students with only instructional and experimental design help from the advisors. <br />
<br />
<br />
<h2> Advisors </h2><br />
<table><br />
<tr><br />
<td>[[File:Bolten.png|200px|thumb|left|]]</td><br />
<td>Nick Bolten, Electrical Engineering</td><br />
</tr><br />
<tr><br />
<td>[[File:Cassie.JPG|200px|thumb|left|]]</td><br />
<td>Cassie Bryan, Biochemistry</td><br />
</tr><br />
<tr><br />
<td>[[File:Arjun.jpg|200px|thumb|left|]]</td><br />
<td>Arjun Khakhar, Bioengineering</td><br />
</tr><br />
<tr><br />
<td>[[File:Bob.png|200px|thumb|left|]]</td><br />
<td>Bob Lamm, Bioengineering</td><br />
</tr><br />
<tr><br />
<td>[[File:Erik.jpeg|200px|thumb|left|]]</td><br />
<td>Erik Murphy, Molecular, Cellular, & Developmental Biology</td><br />
</tr><br />
<tr><br />
<td>[[File:Rashmi.jpg|200px|thumb|left|]]</td><br />
<td>Rashmi Ravichandran, Biochemistry</td><br />
</tr><br />
<tr><br />
<td>[[File:DavidY.jpg|200px|thumb|left|]]</td><br />
<td>David Younger, Bioengineering</td><br />
</tr><br />
</table><br />
<br />
<h2> Faculty </h2><br />
<table><br />
<tr><br />
<td>[[File:Baker.png|200px|thumb|left|]]</td><br />
<td>David Baker, Biochemistry</td><br />
</tr><br />
<tr><br />
<td>[[File:Klavins.jpg|200px|thumb|left|]]</td><br />
<td>Eric Klavins, Electrical Engineering</td><br />
</tr><br />
</table></div>Kristanguyenhttp://2014.igem.org/Team:Washington/Our_TeamTeam:Washington/Our Team2014-10-18T01:56:24Z<p>Kristanguyen: </p>
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<br />
<h1> UW iGEM 2014 Team </h1><br />
<br />
<h2> Undergraduate Team Members </h2><br />
<br />
<table><br />
<tr><br />
<td>[[File:Edward.jpg|200px|thumb|left|]]</td><br />
<td>Edward Chang, Biochemistry<br><br />
Poster</td><br />
</tr><br />
<tr><br />
<td>[[File:Andrew2.jpg|200px|thumb|left|]]</td><br />
<td>Andrew Chau, Chemistry<br><br />
Flow Cytometry and Team Page</td><br />
</tr><br />
<tr><br />
<td>[[File:JoshC.jpeg|200px|thumb|left|]]</td><br />
<td>Joshua Cho, Molecular, Cellular, & Developmental Biology<br><br />
Fluorescence Activated Cell Sorting, Flow Cytometry, and Presentation</td><br />
</tr><br />
<tr><br />
<td>[[File:ChrisC.jpg|200px|thumb|left|]]</td><br />
<td>Chris Choe, Molecular, Cellular, & Developmental Biology<br><br />
Expression and Stability Analysis</td><br />
</tr><br />
<tr><br />
<td>[[File:William.jpg|200px|thumb|left|]]</td><br />
<td>William Harvey, Biochemistry<br><br />
Fluorescence Activated Cell Sorting and Presentation</td><br />
</tr><br />
<tr><br />
<td>[[File:AlexK.jpg|200px|thumb|left|]]</td><br />
<td>Alex Kang, Biochemistry<br><br />
Expression and Stability Analysis</td><br />
</tr><br />
<tr><br />
<td>[[File:Julia.jpg|200px|thumb|left|]]</td><br />
<td>Julia Lim, Microbiology<br><br />
BioBricks, Flow Cytometry, Outreach, and Team Page</td><br />
</tr><br />
<tr><br />
<td>[[File:Harman.jpg|200px|thumb|left|]]</td><br />
<td>Harman Malhi, Molecular, Cellular, & Developmental Biology<br><br />
Expression and Stability Analysis</td><br />
</tr><br />
<tr><br />
<td>[[File:Colton.jpg|200px|thumb|left|]]</td><br />
<td>Colton McDavid, Microbiology<br><br />
Expression and Stability Analysis, Poster, and Team Page</td><br />
</tr><br />
<tr><br />
<td>[[File:Krista.jpg|200px|thumb|left|]]</td><br />
<td>Krista Nguyen, Biochemistry<br><br />
Flow Cytometry, Outreach, Poster, and Team Page</td><br />
</tr><br />
<tr><br />
<td>[[File:Nicolov photo.JPG|200px|thumb|left|]]</td><br />
<td>Anastasia Nicolov, Bioengineering and Violin Performance<br><br />
Expression and Stability Analysis</td><br />
</tr><br />
<tr><br />
<td>[[File:Ahmed.jpg|200px|thumb|left|]]</td><br />
<td>Ahmed Qureshi, Biochemistry<br><br />
BioBricks and Poster</td><br />
</tr><br />
<tr><br />
<td>[[File:Rettie.jpg|200px|thumb|left|]]</td><br />
<td>Stephen Rettie, Microbiology<br><br />
BioBricks, Fluorescence Activated Cell Sorting, and Presentation</td><br />
</tr><br />
</table><br />
<br />
<br> All experiments and data collection were preformed by the students with only instructional and experimental design help from the advisors. <br />
<br />
<br />
<h2> Advisors </h2><br />
<table><br />
<tr><br />
<td>[[File:Bolten.png|200px|thumb|left|]]</td><br />
<td>Nick Bolten, Electrical Engineering</td><br />
</tr><br />
<tr><br />
<td>[[File:Cassie.JPG|200px|thumb|left|]]</td><br />
<td>Cassie Bryan, Biochemistry</td><br />
</tr><br />
<tr><br />
<td>[[File:Arjun.jpg|200px|thumb|left|]]</td><br />
<td>Arjun Khakhar, Bioengineering</td><br />
</tr><br />
<tr><br />
<td>[[File:Bob.png|200px|thumb|left|]]</td><br />
<td>Bob Lamm, Bioengineering</td><br />
</tr><br />
<tr><br />
<td>[[File:Erik.jpeg|200px|thumb|left|]]</td><br />
<td>Erik Murphy, Molecular, Cellular, & Developmental Biology</td><br />
</tr><br />
<tr><br />
<td>[[File:Rashmi.jpg|200px|thumb|left|]]</td><br />
<td>Rashmi Ravichandran, Biochemistry</td><br />
</tr><br />
<tr><br />
<td>[[File:DavidY.jpg|200px|thumb|left|]]</td><br />
<td>David Younger, Bioengineering</td><br />
</tr><br />
</table><br />
<br />
<h2> Faculty </h2><br />
<table><br />
<tr><br />
<td>[[File:Baker.png|200px|thumb|left|]]</td><br />
<td>David Baker, Biochemistry</td><br />
</tr><br />
<tr><br />
<td>[[File:Klavins.jpg|200px|thumb|left|]]</td><br />
<td>Eric Klavins, Electrical Engineering</td><br />
</tr><br />
</table></div>Kristanguyenhttp://2014.igem.org/Team:Washington/SafetyTeam:Washington/Safety2014-10-18T01:52:23Z<p>Kristanguyen: </p>
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<p><strong>1. Do the biological materials used in your lab work pose any of the following risks? Please describe.</strong></p><br />
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<p><strong>1. Risks to the safety and health of team members or others working in the lab?</strong></p><br />
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<blockquote><br />
<p>Using <i> E.coli </i> poses a potential risk to the health and safety to our team members working in the lab if it is handled improperly or consumed. May cause irritation to skin, eyes, and respiratory tract; may affect kidneys.</p><br />
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<p><strong>2. Risks to the safety and health of the general public, if released by design or by accident?</strong></p><br />
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<p>The risks to the safety and health of the general public are the same as those for individuals directly working with these biological materials.</p><br />
</blockquote><br />
<p><strong>3. Risks to the environment, if released by design or by accident?</strong></p><br />
<blockquote><br />
<p>The risks to the are the same as those for individuals directly working with these biological materials.</p><br />
</blockquote><br />
<p><strong>4. Risks to security through malicious misuse by individuals, groups, or countries?</strong></p><br />
</blockquote><br />
<ol><br />
<blockquote><br />
<p>There is slight concern for the misuse of our systems, since any gene could be substituted in place of the existing outputs.</p><br />
</blockquote><br />
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<blockquote><br />
<p><strong>2. What safety training have you received (or plan to receive in the future)? Provide a brief description, and a link to your institution&rsquo;s safety training requirements, if available.</strong></p><br />
<blockquote><br />
<p>All student members of our team have been trained by our postdoc and graduate student advisers about proper lab techniques, lab etiquette, and biohazard waste disposal. All team members have completed the University of Washington's online training in biosafety, fume hood use, and managing lab chemicals. Advisers have been trained by their respective labs in accordance with the University of Washington Environmental Health and Safety committee's regulations. www.ehs.washington.edu/ and have had regular university EH&S inspections to make sure the lab is up to university lab standards</p><br />
</blockquote><br />
<p><strong>3. Under what biosafety provisions will / do you work?</strong></p><br />
<blockquote><br />
<p><strong>a. Please provide a link to your institution biosafety guidelines.</strong></p><br />
<blockquote><br />
<p>- www.ehs.washington.edu -</p><br />
</blockquote><br />
<p><strong>b. Does your institution have an Institutional Biosafety Committee, or an equivalent group? If yes, have you discussed your project with them? </strong></p><br />
<blockquote><br />
<p> The University of Washington Environmental Health and Safety (UW EHS) committee determines biosafety regulations and guidelines for all labs associated with our campus. We have not discussed this specific iGEM project with members of the EHS committee; however, we are working closely with our sponsor labs and have been trained according to the guidelines which they follow.</p><br />
</blockquote><br />
<p><strong>c. Does your country have national biosafety regulations or guidelines? If so, please provide a link to these regulations or guidelines if possible?</strong></p><br />
<blockquote><br />
<p>The United States of America has national biosafety regulations and guidelines determined by the Centers for Disease Control and Prevention (CDC). Specifics about their guidelines can be found at www.cdc.gov/biosafety/</p><br />
</blockquote><br />
<p><strong>d. Does your country have national biosafety regulations or guidelines? If so, please provide a link to these regulations or guidelines if possible?</strong></p><br />
<blockquote><br />
<p>The United States of America has national biosafety regulations and guidelines determined by the Centers for Disease Control and Prevention (CDC). Specifics about their guidelines can be found at www.cdc.gov/biosafety/</p><br />
</blockquote><br />
<p><strong>e. According to the WHO Biosafety Manual, what is the BioSafety Level rating of your lab? (Check the summary table on page 3, and the fuller description that starts on page 9.)</strong> If your lab does not fit neatly into category 1, 2, 3, or 4, please describe its safety features [see 2013.igem.org/Safety for help]. </p><br />
<blockquote><br />
<p>The BioSafety level of our lab is category 2. The lab room used is equipped to deal with category 2 hazards, for example, it contains a fume hood. However, for this project, only category 1 cells were used; namely, non-pathogenic <i> E. coli </i> and yeast. </p><br />
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</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/OutreachTeam:Washington/Outreach2014-10-18T01:50:10Z<p>Kristanguyen: </p>
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<img> <img src="https://static.igem.org/mediawiki/parts/7/7c/UWDiscDays2014_1.png" width="389" height="258" alt="discovery days table" </img> <img> <img src="https://static.igem.org/mediawiki/parts/b/b6/UWDiscDays2014_2.png" width="170" height="258" alt discovery days overhead </img> <img> <img> <img src="https://static.igem.org/mediawiki/parts/c/cc/Uwplate.jpg" width="258" height="258" alt="UW plate" </img> <br><br />
<p>UW Engineering Discovery Days is an annual event sponsored by the University of Washington College of Engineering and acts as an opportunity for K-12 students to explore the extensive world of engineering through the means of hands-on experience, demonstrations, presentations, as well as questions and answers. Over the two-day span of this event the campus is overflowing with young students eager to learn from undergraduates engaged across the field of engineering. The UW iGEM team took part by educating students on the basics of synthetic biology and encouraging them to be inventive in the activity we designed for them. This project was similarly presented at the Family Science Night of the Bennett Elementary School Young Scientist Week by members of the iGEM team.<br><br />
<br><br />
<br />
We decided to show how synthetic biology can also be applied to art and design by making art with <i> E. coli </i>. Prior to the event, some iGEM students painted pictures onto agar plates using <i> E. coli </i> expressing different colors: green, red, purple, and blue. These plates were parafilmed and displayed at our table for students to take a closer look at.<br><br />
<br><br />
A short slideshow was made to cover the very basics of how <i> E. coli </i> bacteria are capable of appearing different colors. We encouraged students to first recall what they understood about DNA and proteins, then we filled in the gaps on how proteins are coded by nucleotide sequences in DNA, the genetic material in cells. It was explained that proteins are the foundation of living organisms and are essential to how they look, respond to the environment, and other aspects of how they live. Once this was established, we gave them the example of proteins which possessed the function of expression of color; green proteins deriving from jellyfish while blue proteins were taken from sea anemones.<br><br />
<br><br />
<br />
Genes coding for a protein can be taken from one organism and inserted into the DNA of another organism to express said protein in the new organism. In our case we inserted the coding for color expression from organisms like jellyfish and sea anemone into different strains of <i> E. coli </i> to observe several colors through the proteins that were produced.<br><br />
<br><br />
Students were then provided a sheet of paper with a circle outlined on it to represent an agar plate and invited to come up with ideas of images that could be made from color fluorescing proteins expressed by <i> E. coli </i>. Piles of paper were collected and some of these drawings were chosen to be made by our iGEM students back in the lab. Pictures were taken and e-mailed out to the students so they could see their illustrations in the form of <i> E. coli </i> bacteria.<br />
<br />
</p><br />
<img> <img src="https://static.igem.org/mediawiki/parts/5/56/Tubeplate.jpg" width="250" height="250" alt="test tube plate" </img> <img> <img src="https://static.igem.org/mediawiki/parts/b/ba/Igemplate.jpg" width="250" height="250" alt="igem plate" </img> <img> <img> <img src="https://static.igem.org/mediawiki/parts/5/57/Butterflyplate.jpg" width="250" height="250" alt="butterfly plate" </img><br />
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</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/OutreachTeam:Washington/Outreach2014-10-18T01:49:07Z<p>Kristanguyen: </p>
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<img> <img src="https://static.igem.org/mediawiki/parts/7/7c/UWDiscDays2014_1.png" width="389" height="258" alt="discovery days table" </img> <img> <img src="https://static.igem.org/mediawiki/parts/b/b6/UWDiscDays2014_2.png" width="170" height="258" alt discovery days overhead </img> <img> <img> <img src="https://static.igem.org/mediawiki/parts/c/cc/Uwplate.jpg" width="258" height="258" alt="UW plate" </img> <br><br />
<p>UW Engineering Discovery Days is an annual event sponsored by the University of Washington College of Engineering and acts as an opportunity for K-12 students to explore the extensive world of engineering through the means of hands-on experience, demonstrations, presentations, as well as questions and answers. Over the two-day span of this event the campus is overflowing with young students eager to learn from undergraduates engaged across the field of engineering. The UW iGEM team took part by educating students on the basics of synthetic biology and encouraging them to be inventive in the activity we designed for them. This project was similarly presented at the Family Science Night of the Bennett Elementary School Young Scientist Week by members of the iGEM team.<br><br />
<br><br />
<br />
We decided to show how synthetic biology can also be applied to art and design by making art with E. coli. Prior to the event, some iGEM students painted pictures onto agar plates using E. coli expressing different colors: green, red, purple, and blue. These plates were parafilmed and displayed at our table for students to take a closer look at.<br><br />
<br><br />
A short slideshow was made to cover the very basics of how <i> E. coli </i> bacteria are capable of appearing different colors. We encouraged students to first recall what they understood about DNA and proteins, then we filled in the gaps on how proteins are coded by nucleotide sequences in DNA, the genetic material in cells. It was explained that proteins are the foundation of living organisms and are essential to how they look, respond to the environment, and other aspects of how they live. Once this was established, we gave them the example of proteins which possessed the function of expression of color; green proteins deriving from jellyfish while blue proteins were taken from sea anemones.<br><br />
<br><br />
<br />
Genes coding for a protein can be taken from one organism and inserted into the DNA of another organism to express said protein in the new organism. In our case we inserted the coding for color expression from organisms like jellyfish and sea anemone into different strains of <i> E. coli </i> to observe several colors through the proteins that were produced.<br><br />
<br><br />
Students were then provided a sheet of paper with a circle outlined on it to represent an agar plate and invited to come up with ideas of images that could be made from color fluorescing proteins expressed by <i> E. coli </i>. Piles of paper were collected and some of these drawings were chosen to be made by our iGEM students back in the lab. Pictures were taken and e-mailed out to the students so they could see their illustrations in the form of <i> E. coli </i> bacteria.<br />
<br />
</p><br />
<img> <img src="https://static.igem.org/mediawiki/parts/5/56/Tubeplate.jpg" width="250" height="250" alt="test tube plate" </img> <img> <img src="https://static.igem.org/mediawiki/parts/b/ba/Igemplate.jpg" width="250" height="250" alt="igem plate" </img> <img> <img> <img src="https://static.igem.org/mediawiki/parts/5/57/Butterflyplate.jpg" width="250" height="250" alt="butterfly plate" </img><br />
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</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/BioBricksTeam:Washington/BioBricks2014-10-18T01:46:15Z<p>Kristanguyen: </p>
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<h1> Submitted Parts </h1><br />
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<center><img src="https://static.igem.org/mediawiki/2014/a/a2/Bio-brick_image.jpg" alt="Submitted Biobricks" style="width:700px;height:263px"><br />
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<br><br />
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<sup><b>Fig 1.</b> Our submitted BioBricks consist of three parts: the Degron, the Gal4-VP16 Transcriptional activator, and Gal4-Degron-VP16 complex.</sup></center><br />
<br />
<h3> <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1408000" target="_blank">Degron (BBa_K1408000)</a> </h3><br />
<br />
<p> <br />
<br />
The Degron is an unstable protein domain that, when fused to a protein, acts as a source of instability. The presence of the Degron leads to degradation of the protein by the cell via ubiquitination. This is a new part we are submitting to the registry. </p><br />
<br />
<h3> <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1408001" target="_blank"> Gal4-VP16 Transcriptional Activator (BBa_K1408001) </a> </h3><br />
<br />
<p> <br />
<br />
This part consists of our Gal4-VP16 fusion protein, which is a transcriptional activator. Gal4 protein binds to a Gal1 promoter, while VP16 recruits transcription machinery, promoting transcription to anything under the Gal1 promoter. The two must be co-localized to work as a transcriptional activator. <br> <br />
<br />
<br><br />
<br />
This transcrptional activator has been previously submitted to the registry (BBa_K1179014 submitted by the iGEM 2013 MIT team). However, the part was incomplete compared to ours due to the presence of an illegal restriction site that cut the VP16 gene, rendering it useless. Also, this part had no user data related to it. In order to make the complete part available, together with experimental data related to the part, we decided to "re-submit" this part to the registry. <br />
<br />
</p><br />
<br />
<h3> <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1408002" target="_blank"> Gal4-Degron-VP16 (BBa_K1408002) </a> </h3><br />
<br />
<p> <br />
<br />
This part is a combination of our previous two parts submitted to the registry. By utilizing this system, the relative stabilities of proteins can be compared. A sequence for a protein inserted between the Degron and VP16 will produce a fusion protein which acts as a transcriptional activator for anything under a Gal1 promoter. If the protein inserted is unstable it will be degraded by the cell and the Gal4-VP16 transcriptional activator will no longer function. If a quantifiable marker protein such as Green Fluorescent Protein (GFP) is under Gal1 then the level of GFP output of the cell is related to the stability of the inserted protein. <br />
<br />
</p><br />
<br />
</body><br />
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<html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/Future_PlansTeam:Washington/Future Plans2014-10-18T01:41:50Z<p>Kristanguyen: </p>
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<h1> Future Plans </h1><br />
<br />
<h3> Examination of more proteins </h3><br />
<br />
<p><br />
Like any new up and coming technique, the Degron system will require further testing with a larger variety of well studied mutant variants of a single protein, as well as a larger number of well studied proteins in general, before the system can truly be accepted. This is especially important because the proteins we have used to test the Degron system are mostly alpha-helical. In order to make sure that our system is generalizable to all protein topologies, we must test to see if we can obtain similar results with proteins of other topologies. <br><br />
<br />
<br><br />
<br />
Our current plans for the future are to test a protein 33RM2 and its less stable variant, 33CL1, both of which bind to PD-1 (a negative t-cell regulator that prevents the recognition of tumorous cells by the immune system). Since the stability of both of these proteins are known and have been verified using other techniques, such as thermal melts, they are very suitable candidates for testing our Degron system.<br />
<br />
<br><br />
<br />
</p><br />
<br />
<br />
<h3> Further evolving more stable variants of existing proteins </h3><br />
<br />
<p><br />
Throughout this past summer, our team has been evolving more stable variants of BINDI through error-prone PCR. Going forwards, we will continue this process and will continually analyze the mutants with flow cytometry and select cells that exhibit higher fluorescence with FACS.<br />
<br />
</p><br />
<br />
<br />
<h3> Recommendations for Degron system use </h3><br />
<br />
<p><br />
<br />
We offer a couple recommendations for those who would like to utilize our novel method of protein stabilization:<br />
<br />
<br><br />
<br />
1. Use only Deg0, Deg1, and Deg2 for your stability testing. We offer this recommendation because Deg3 and Deg4 are just a parallel version of Deg2 and Deg1, respectively. That is, making all five constructs would be redundant as, theoretically, Deg1 and Deg4 (or Deg2 and Deg3) should have a similar stability, and hence GFP production. In order to minimize extra work, we recommend you utilize only Deg0, Deg1, and Deg2.<br />
<br />
<br><br />
<br />
2. Utilize <i>E. coli</i> as your test organism, rather than <i>S. cervisiae </i>. Generally, <i> E. coli </i> is easier to work with as it has a faster growth and is very commonly used in laboratories.<br />
<br />
</p><br />
<br />
<br />
<h3> Future Applications </h3><br />
<br />
<p><br />
<br />
Protein engineering is highly important in many areas. Our system allows a generalizable and high throughput method of engineering proteins, and is also less time consuming than current stabilization methods.<br />
<br />
Engineered proteins selected through this method could be produced in bacteria and aid in the development of thermostable, de novo protein therapeutics.<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
</body><br />
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</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/Our_ProjectTeam:Washington/Our Project2014-10-18T01:40:06Z<p>Kristanguyen: </p>
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<h1> Our System </h1><br />
<br />
<h2>Natural Cell Processes</h2><br />
<p><br />
<br />
We sought to make use of a cell's natural methods of degrading misfolded proteins. If a protein is misfolded in a cell it is targeted by the E3 ligase which attaches a ubiquitin to it. This marks the protein for degradation by the proteasome. If a protein is unstable it is likely at very low levels within the cell as it is being degraded by this system.<br />
</p><br />
<br />
<h2>Gal4-VP16</h2><br />
<br />
<p><br />
Our system relies on GFP being produced at different levels depending on the stability of a protein of interest. To do this our system puts the protein of interest in between Gal4 and VP16.<br> </p><br />
<br />
<center> <img src="https://static.igem.org/mediawiki/2014/d/d2/Frontcover.jpg" alt="Gal4-VP16 Construct"><br />
<br />
<br><br />
<br />
<sup> <b> Fig 1. </b> Our project utilizes the Gal4-VP16 transcriptional activator to test protein stability in terms of GFP output. </sup> </center><br />
<br />
<br> <br><br />
<br />
<p>Together Gal4 and VP16 up-regulate the expression of a gene under certain promoters, in our case we used Gal1. Gal4 binds to the DNA and then VP16 recruits RNA polymerase to begin transcription of the gene under Gal1. Independently Gal4 simply binds to Gal1, and VP16 is not localized to the DNA. So if our gene located in between the two, degradation of the gene leads to no transcription of anything under Gal1. <br />
</p><br />
<br />
<br />
<br />
<h2>Using a Degron to Exaggerate Differences in Stability</h2><br />
<br />
<p><br />
<br />
We sought to make a versatile system that could be used for proteins of various native stabilities. If a protein is stable enough to avoid ubiquitination but not stable enough for its engineered purpose our system would not be useful. To deal with this we used a degron. <br><br />
<br><br />
A degron is an inherently unstable protein domain. By inserting this into the fusion protein produced by our plasmid we expect a protein which is just stable enough to avoid degredation will become unstable and be degraded. We expect a very stable protein to be able to overcome this source of instability and this will allow us to the measure differences in stability of more stable protein variants where without the degron they would give very similar measurements in our system.<br />
</p><br />
<h2> Components of Our Plasmid </h2><br />
<p><br />
<br />
The fusion protein produced by expression of our plasmid is made up of the Gal4-VP16 transactivating complex with a protein of interest in between. Positioning of the degron is determined by the native stability of the protein of interest. <br />
<br />
</p><br />
<center> <img src="https://static.igem.org/mediawiki/2014/1/1d/UWPlasmid.png" alt="Degron Constructs" style="width:500px;height:406px"><br />
<br />
<br><br />
<br />
<sup> <b> Fig 2. </b> Potential Degron insert sites for our system. </sup> </center><br />
<br />
<br> <br><br />
<br />
<br />
<br />
<p><br />
<br />
There are 5 possible degron positions: <br><br />
-Deg0: This construct contains only the Gal4-VP16 transcriptional activator complex with the protein of interest in between the two (shortened as Gal4-Protein-VP16). <br><br />
-Deg1: This construct contains the Degron in front of our Gal4-Protein-VP16 complex. <br><br />
-Deg2: This construct contains the Degron in between Gal4 and the protein in our Gal4-Protein-VP15 complex. <br><br />
-Deg3: This construct contains the Degron in between the protein and VP16 in our Gal4-Protein-VP15 complex. <br><br />
-Deg4: This construct contains the Degron at the end of our Gal4-Protein-VP16 complex. <br><br />
<br />
</p><br />
<br />
<h3> Relative Stability Analyzed via Flow Cytometry </h3><br />
<br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/d/d9/Degron_construct.jpg" alt="Degron Constructs" style="width:750px;height:379px"><br />
<br />
<br><br />
<br />
<sup> <b> Fig 3. </b> Expected GFP output based on our Degron constructs </sup> </center> <br />
<br />
<br><br />
<br />
<p><br />
<br />
Flow cytometry is a high throughput method of analyzing cells for various optical outputs, namely fluorescence. A flow cytometer is an analytical instrument in which cells that have been suspended in a solution are passed through a narrow channel in which fluorescence of individual cells can be measured. <br><br />
<br><br />
By utilizing Flow Cytometry, we can measure the amount of GFP output within cells from each degron construct. Based on where the Degron is inserted, we expected a different level of fluorescence. As such, we expected to see the highest GFP production in our Deg0 construct, as it only contains the Gal4-Protein-VP16 complex with no Degron inserted, therefore we expect it to be the most stable. We expected that Deg2 and Deg3 would have a lower GFP production than Deg0 but higher than Deg1 and Deg4. This rationale was based on the fact that the Deg1 and Deg4 have the Degron exposed, making it more likely to be degraded by ubiquitination than in Deg2 and Deg3 which has the Degron buried inside the Gal4-Protein-VP16 complex.<br><br />
<br />
</p><br />
<br />
<br />
<br />
<h2> Test Protein </h2><br />
<br />
<p align = left> <br />
<br />
The test protein that must be chosen in testing a novel and new system must be a protein that has been well studied and rigorously examined through other existing and well accepted protein stability testing methods. <br />
Therefore,our team decided to use the protein known as BINDI. <br />
BINDI and two of its less stable variants, BbpD04 and BbpD04.3 were studied and examined in "A Computationally Designed Inhibitor of an Epstein-Barr Viral Bcl-2 Protein Induces Apoptosis in Infected Cells" by Procko et al<sup>1</sup>. <br />
We would like to acknowledge and thank Dr. Procko for giving us his genes to work with. <br />
<br />
</p> <br />
<br />
<br />
<h2> PyE1 a strain of <i> Saccharomyces cerevisiae </i> </h2><br />
<p><br />
<br />
We use a strain of <i> Saccharomyces cerevisiae </i> deveoloped in Stan Fields' lab at the University of Washington called PyE1. <br />
Its genome has been engineered to contain a gene from Green Fluorescent Protein (GFP) under a Gal1 promoter. <br />
When the Gal4 DNA-binding domain and the VP16 transcription activation domain are colocalized to the Gal1 promoter, expression of GFP is induced. <br />
Therefore, using our test plasmids in PyE1 generates GFP relative to the level of Gal4/VP16 peptide in the cell. <br />
The more stable the degron protein construct is, the more likely it is that more GFP will be expressed.<br />
This relationship between stability and GFP forms the basis from which we will measure the relative protein stability of our degron constructs as well as the protein of interest degron construct.<br />
<br />
</p><br />
<br />
<p><br />
<sup>1</sup>Procko, E, et al. "A Computationally Designed Inhibitor of an Epstein-Barr Viral Bcl-2 Protein Induces Apoptosis in Infected Cells" Cell 157 (2014): 1644-56.<br />
</p><br />
<br />
</body><br />
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</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/MethodsTeam:Washington/Methods2014-10-18T01:39:25Z<p>Kristanguyen: </p>
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<br />
<h1> Method </h1><br />
<br />
<p align =left><br />
<br />
The essential process of our system involves cloning and manufacturing of a plasmid in <i> E. coli </i>. Once, the plasmids have been constructed and verified in <i> E. coli </i> they are transformed into <i>S. cerevisiae</i>. The plasmid constructs are then expressed. Following several days of growth the yeast cultures are passed through a Flow Cytometer and the fluorescence of each cell is measured. Higher fluorescence is associated with higher expression of the protein of interest which in-turn is indicative of higher protein stability. <br />
<br />
</p><br />
<br />
<br />
<h2> Cloning in <i> Escherichia coli </i> </h2><br />
<br />
<p align = left> <br />
<br />
There are five possible degron constructs corresponding to five different positions the degron can take in our construct. Four of the five vectors for our protein of interest DNA code contains contain a degron (Deg1-4) as well as an EcoRI and Nhel111 restriction enzyme cutsite between Gal4 and VP16. Furthermore, the vector also contains a region that encodes Ampicillin resistance as well as autotrophic region that encodes for uracil synthesis in yeast strains. The DNA that encodes our protein of interest, i.e. the insert, is amplified to include both restriction endonuclease cutsites through a polymerase chain reaction. In a subsequent step the amplified fragment is then digested and then ligated with the appropriate vector and transformed into chemically competent<i> E.coli </i> either XL-1 Blue or XL10-Gold strains. Similarly, Deg0(no degron) vector has EcoRI along with a Hind111 cutsite. Using the different cutsites our DNA fragment is prepared using PCR and ligated into the Deg0 vector and then transformed. Once the transformation is complete, the cells are plated onto LB-agar plates supplimented with ampicillin in order to ensure that all <i> E.coli </i> colonies contain our recombinant plasmid. After being grown for a day, several colonies are swiped and added to an overnight culture. The overnight culture is grown overnight and their recombinant plasmid is harvested and sequenced (Sanger sequencing is used through Genewiz Inc.) the next morning. If the plasmids are correct, we then proceed to create a glycerol stock of the cell culture as well as a miniprep stock of the plasmid in order to conduct further experimentation in yeast. <br><br />
<br />
</p> <br />
<br />
<br />
<h2> Preparation and Passaging of <i> Saccharomyces cerevisiae </i> </h2><br />
<p align = left> <br />
<br />
Once, plasmids of the five possible degron constructs have been cloned with our three test proteins and verified, they are subsequently transformed in PyE1 a strain of <i> S. cerevisiae </i> with the ability to produce green fluorescent proteins. Following the transformation, the cells are plated onto plates with on a Selective Dropout (C-Uracil) media and incubated at 30<sup>o</sup>C for 2 days. The purpose of the dropout media is to ensure that only cells that contain our plasmid survive as the recombinant plasmid is encoded with a region that allows cells to produce uracil, an essential amino acid. Without the recombinant plasmid, the cell would be fatally deprived of uracil which has been "knocked out" of the plating media. After two days, three colonies are swiped from the plate and added to an overnight culture of 2-3mL Selective Dropout Media C-Uracil and 2% Glucose then incubated for another two days at 30<sup>o</sup>C. After another two days of incubation, a 20-50uL aliquot of each culture is "passaged" into another 3mL culture prepared in the same manner as before and incubated for the same duration and temperature as the previous culture. The passaging is done several times after each passage after the second passage, a glycerol stock is prepare from the culture and Flow Cytometry is run on the culture. <br><br />
<br />
</p><br />
<br />
<p align = left> <br />
<br />
The purpose of passaging is to gradually remove excess copies of the plasmid constructs. Excess copies, exceeding one per cell will lead to multiple fold increase in the expression of the degron protein construct. As a result of this, GFP expression will also be increased thus reducing the viability and accuracy of the Flow Cytometry measurements conducted on each cell culture. This is problematic as GFP output will become related to the number of plasmids as well as the stability of the various degron constructs which will likely invalidate any results.<br />
<br />
</p> <br />
<br />
<h2> Construct Stability Analysis with Flow Cytometry </h2><br />
<br />
<p> <br />
<br />
After PyE1 cells have been passaged several times and each cell has roughly one copy of our degron construct containing plasmid, the cells are now ready to be run through a flow cytometer. On the morning of the day that the instrument is to be used, the optical density of cultures is read and an aliquot is taken from each culture and diluted to similar optical densities. This is to ensure that all cultures are in the same growth phase and will express our protein of interest degron construct equally so there will be no discrepancies in expression levels that could skew our fluorescence measurements. Once diluted, the cultures are incubated for roughly six hours (3-4 cell doublings). After the short incubation, approximately 200 microliters of each culture is pelleted, and resuspened in PBSF buffer and run through the flow cytometer. <br><br />
<br><br />
The instrument reads forward and side scatter allowing us to see cell debris or contaminants which can then be gated (excluded) from the actual fluorescent readings. Each construct containing culture is analyzed and both mean fluorescence of a culture as well as fluorescent culture’s population data is collected. Both of which can be used to analyze the relative stabilities of each construct and protein of interest.<br />
<br />
</p> <br />
<br />
<br />
<br />
<h2> Mutagenesis through Error Prone Polymerase Chain Reactions (E-PCRs) </h2><br />
<br />
<p><br />
<br />
In order to validate our system as being capable of selecting more stable protein variants, we have to produce mutations in our protein of interest. Those mutations could potentially be beneficial and could carry a stabilizing affect on the protein of interest. Error-prone PCR utilizes DNA-polymerase's error-prone nature and further increases the likelyhood of mutations by manipulating the conditions in which, DNA-polymerase operates in, thereby causing the polymerase to create errors in DNA sequencing which in turn will create changes in the protein construct. Once, the DNA coding of our protein has been changed we can express the mutations and analyze those stabilizing or de-stabilizing affects they have on our protein. The possible mutations can then be analyzed using the degron system and more stable mutations can be easily seen then sequenced.<br />
<br />
</p><br />
<br />
<br />
<h2> Selecting Stable Variants through Fluorescence Activated Cell Sorting (FACS) </h2><br />
<br />
<p><br />
<br />
Once the DNA coding for a protein of interest has been mutated reassembled into our plasmid and transformed into PyE1, we can then analyze the fluorescent output and select cells<br />
that exhibit higher fluorescence. The nature of our system allows us to conclude that higher fluorescence output corresponds to higher stability<br />
of the protein of interest. Cells with higher fluorescence are then selected, cultured and the plasmid within them is sequenced.<br />
Fluorescence Activated Cell Sorting (FACS) makes our system very high throughput and allows us to analyze a large number of cells and possible mutations. <br />
<br />
<br />
</p> <br />
<br />
</body><br />
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</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/Our_ProjectTeam:Washington/Our Project2014-10-18T01:32:48Z<p>Kristanguyen: </p>
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<div>{{Template:Team:UW/CSS}}<br />
<br />
<html><br />
<br />
<body><br />
<br />
<h1> Our System </h1><br />
<br />
<h2>Natural Cell Processes</h2><br />
<p><br />
<br />
We sought to make use of a cell's natural methods of degrading misfolded proteins. If a protein is misfolded in a cell it is targeted by the E3 ligase which attaches a ubiquitin to it. This marks the protein for degradation by the proteasome. If a protein is unstable it is likely at very low levels within the cell as it is being degraded by this system.<br />
</p><br />
<br />
<h2>Gal4-VP16</h2><br />
<br />
<p><br />
Our system relies on GFP being produced at different levels depending on the stability of a protein of interest. To do this our system puts the protein of interest in between Gal4 and VP16.<br> </p><br />
<br />
<center> <img src="https://static.igem.org/mediawiki/2014/d/d2/Frontcover.jpg" alt="Gal4-VP16 Construct"><br />
<br />
<br><br />
<br />
<sup> <b> Fig 1. </b> Our project utilizes the Gal4-VP16 transcriptional activator to test protein stability in terms of GFP output. </sup> </center><br />
<br />
<br> <br><br />
<br />
<p>Together Gal4 and VP16 up-regulate the expression of a gene under certain promoters, in our case we used Gal1. Gal4 binds to the DNA and then VP16 recruits RNA polymerase to begin transcription of the gene under Gal1. Independently Gal4 simply binds to Gal1, and VP16 is not localized to the DNA. So if our gene located in between the two, degradation of the gene leads to no transcription of anything under Gal1. <br />
</p><br />
<br />
<br />
<br />
<h2>Using a Degron to Exaggerate Differences in Stability</h2><br />
<br />
<p><br />
<br />
We sought to make a versatile system that could be used for proteins of various native stabilities. If a protein is stable enough to avoid ubiquitination but not stable enough for its engineered purpose our system would not be useful. To deal with this we used a degron. <br><br />
<br><br />
A degron is an inherently unstable protein domain. By inserting this into the fusion protein produced by our plasmid we expect a protein which is just stable enough to avoid degredation will become unstable and be degraded. We expect a very stable protein to be able to overcome this source of instability and this will allow us to the measure differences in stability of more stable protein variants where without the degron they would give very similar measurements in our system.<br />
</p><br />
<h2> Components of Our Plasmid </h2><br />
<p><br />
<br />
The fusion protein produced by expression of our plasmid is made up of the Gal4-VP16 transactivating complex with a protein of interest in between. Positioning of the degron is determined by the native stability of the protein of interest. <br />
<br />
</p><br />
<center> <img src="https://static.igem.org/mediawiki/2014/1/1d/UWPlasmid.png" alt="Degron Constructs" style="width:500px;height:406px"><br />
<br />
<br><br />
<br />
<sup> <b> Fig 2. </b> Potential Degron insert sites for our system. </sup> </center><br />
<br />
<br> <br><br />
<br />
<br />
<br />
<p><br />
<br />
There are 5 possible degron positions: <br><br />
-Deg0: This construct contains only the Gal4-VP16 transcriptional activator complex with the protein of interest in between the two (shortened as Gal4-Protein-VP16). <br><br />
-Deg1: This construct contains the Degron in front of our Gal4-Protein-VP16 complex. <br><br />
-Deg2: This construct contains the Degron in between Gal4 and the protein in our Gal4-Protein-VP15 complex. <br><br />
-Deg3: This construct contains the Degron in between the protein and VP16 in our Gal4-Protein-VP15 complex. <br><br />
-Deg4: This construct contains the Degron at the end of our Gal4-Protein-VP16 complex. <br><br />
<br />
</p><br />
<br />
<h3> Relative Stability Analyzed via Flow Cytometry </h3><br />
<br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/d/d9/Degron_construct.jpg" alt="Degron Constructs" style="width:750px;height:379px"><br />
<br />
<br><br />
<br />
<sup> <b> Fig 3. </b> Expected GFP output based on our Degron constructs </sup> </center> <br />
<br />
<br><br />
<br />
<p><br />
<br />
Flow cytometry is a high throughput method of analyzing cells for various optical outputs, namely fluorescence. A flow cytometer is an analytical instrument in which cells that have been suspended in a solution are passed through a narrow channel in which fluorescence of individual cells can be measured. <br><br />
<br><br />
By utilizing Flow Cytometry, we can measure the amount of GFP output within cells from each degron construct. Based on where the Degron is inserted, we expected a different level of fluorescence. As such, we expected to see the highest GFP production in our Deg0 construct, as it only contains the Gal4-Protein-VP16 complex with no Degron inserted, therefore we expect it to be the most stable. We expected that Deg2 and Deg3 would have a lower GFP production than Deg0 but higher than Deg1 and Deg4. This rationale was based on the fact that the Deg1 and Deg4 have the Degron exposed, making it more likely to be degraded by ubiquitination than in Deg2 and Deg3 which has the Degron buried inside the Gal4-Protein-VP16 complex.<br><br />
<br />
</p><br />
<br />
<br />
<br />
<h2> Test Protein </h2><br />
<br />
<p align = left> <br />
<br />
The test protein that must be chosen in testing a novel and new system must be a protein that has been well studied and rigorously examined through other existing and well accepted protein stability testing methods. <br />
Therefore,our team decided to use the protein known as BINDI. <br />
BINDI and two of its less stable variants, BbpD04 and BbpD04.3 were studied and examined in "A Computationally Designed Inhibitor of an Epstein-Barr Viral Bcl-2 Protein Induces Apoptosis in Infected Cells" by Procko et al<sup>1</sup>. <br />
We would like to acknowledge and thank Dr. Procko for giving us his genes to work with. <br />
<br />
</p> <br />
<br />
<br />
<h2> PYE1 a strain of <i> Saccharomyces cerevisiae </i> </h2><br />
<p><br />
<br />
We use a strain of <i> Saccharomyces cerevisiae </i> deveoloped in Stan Fields' lab at the University of Washington called PyE1. <br />
Its genome has been engineered to contain a gene from Green Fluorescent Protein (GFP) under a Gal1 promoter. <br />
When the Gal4 DNA-binding domain and the VP16 transcription activation domain are colocalized to the Gal1 promoter, expression of GFP is induced. <br />
Therefore, using our test plasmids in PYE1 generates GFP relative to the level of Gal4/VP16 peptide in the cell. <br />
The more stable the degron protein construct is, the more likely it is that more GFP will be expressed.<br />
This relationship between stability and GFP forms the basis from which we will measure the relative protein stability of our degron constructs as well as the protein of interest degron construct.<br />
<br />
</p><br />
<br />
<p><br />
<sup>1</sup>Procko, E, et al. "A Computationally Designed Inhibitor of an Epstein-Barr Viral Bcl-2 Protein Induces Apoptosis in Infected Cells" Cell 157 (2014): 1644-56.<br />
</p><br />
<br />
</body><br />
<br />
</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/Our_ProjectTeam:Washington/Our Project2014-10-18T01:32:09Z<p>Kristanguyen: </p>
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<br />
<html><br />
<br />
<body><br />
<br />
<h1> Our System </h1><br />
<br />
<h2>Natural Cell Processes</h2><br />
<p><br />
<br />
We sought to make use of a cell's natural methods of degrading misfolded proteins. If a protein is misfolded in a cell it is targeted by the E3 ligase which attaches a ubiquitin to it. This marks the protein for degradation by the proteasome. If a protein is unstable it is likely at very low levels within the cell as it is being degraded by this system.<br />
</p><br />
<br />
<h2>Gal4-VP16</h2><br />
<br />
<p><br />
Our system relies on GFP being produced at different levels depending on the stability of a protein of interest. To do this our system puts the protein of interest in between Gal4 and VP16.<br> </p><br />
<br />
<center> <img src="https://static.igem.org/mediawiki/2014/d/d2/Frontcover.jpg" alt="Gal4-VP16 Construct"><br />
<br />
<br><br />
<br />
<sup> <b> Fig 1. </b> Our project utilizes the Gal4-VP16 transcriptional activator to test protein stability in terms of GFP output. </sup> </center><br />
<br />
<br> <br><br />
<br />
<p>Together Gal4 and VP16 up-regulate the expression of a gene under certain promoters, in our case we used Gal1. Gal4 binds to the DNA and then VP16 recruits RNA polymerase to begin transcription of the gene under Gal1. Independently Gal4 simply binds to Gal1, and VP16 is not localized to the DNA. So if our gene located in between the two, degradation of the gene leads to no transcription of anything under Gal1. <br />
</p><br />
<br />
<br />
<br />
<h2>Using a Degron to Exaggerate Differences in Stability</h2><br />
<br />
<p><br />
<br />
We sought to make a versatile system that could be used for proteins of various native stabilities. If a protein is stable enough to avoid ubiquitination but not stable enough for its engineered purpose our system would not be useful. To deal with this we used a degron. <br><br />
<br><br />
A degron is an inherently unstable protein domain. By inserting this into the fusion protein produced by our plasmid we expect a protein which is just stable enough to avoid degredation will become unstable and be degraded. We expect a very stable protein to be able to overcome this source of instability and this will allow us to the measure differences in stability of more stable protein variants where without the degron they would give very similar measurements in our system.<br />
</p><br />
<h2> Components of Our Plasmid </h2><br />
<p><br />
<br />
The fusion protein produced by expression of our plasmid is made up of the Gal4-VP16 transactivating complex with a protein of interest inbetween. Positioning of the degron is determined by the native stability of the protein of interest. <br />
<br />
</p><br />
<center> <img src="https://static.igem.org/mediawiki/2014/1/1d/UWPlasmid.png" alt="Degron Constructs" style="width:500px;height:406px"><br />
<br />
<br><br />
<br />
<sup> <b> Fig 2. </b> Potential Degron insert sites for our system. </sup> </center><br />
<br />
<br> <br><br />
<br />
<br />
<br />
<p><br />
<br />
There are 5 possible degron positions: <br><br />
-Deg0: This construct contains only the Gal4-VP16 transcriptional activator complex with the protein of interest in between the two (shortened as Gal4-Protein-VP16). <br><br />
-Deg1: This construct contains the Degron in front of our Gal4-Protein-VP16 complex. <br><br />
-Deg2: This construct contains the Degron in between Gal4 and the protein in our Gal4-Protein-VP15 complex. <br><br />
-Deg3: This construct contains the Degron in between the protein and VP16 in our Gal4-Protein-VP15 complex. <br><br />
-Deg4: This construct contains the Degron at the end of our Gal4-Protein-VP16 complex. <br><br />
<br />
</p><br />
<br />
<h3> Relative Stability Analyzed via Flow Cytometry </h3><br />
<br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/d/d9/Degron_construct.jpg" alt="Degron Constructs" style="width:750px;height:379px"><br />
<br />
<br><br />
<br />
<sup> <b> Fig 3. </b> Expected GFP output based on our Degron constructs </sup> </center> <br />
<br />
<br><br />
<br />
<p><br />
<br />
Flow cytometry is a high throughput method of analyzing cells for various optical outputs, namely fluorescence. A flow cytometer is an analytical instrument in which cells that have been suspended in a solution are passed through a narrow channel in which fluorescence of individual cells can be measured. <br><br />
<br><br />
By utilizing Flow Cytometry, we can measure the amount of GFP output within cells from each degron construct. Based on where the Degron is inserted, we expected a different level of fluorescence. As such, we expected to see the highest GFP production in our Deg0 construct, as it only contains the Gal4-Protein-VP16 complex with no Degron inserted, therefore we expect it to be the most stable. We expected that Deg2 and Deg3 would have a lower GFP production than Deg0 but higher than Deg1 and Deg4. This rationale was based on the fact that the Deg1 and Deg4 have the Degron exposed, making it more likely to be degraded by ubiquitination than in Deg2 and Deg3 which has the Degron buried inside the Gal4-Protein-VP16 complex.<br><br />
<br />
</p><br />
<br />
<br />
<br />
<h2> Test Protein </h2><br />
<br />
<p align = left> <br />
<br />
The test protein that must be chosen in testing a novel and new system must be a protein that has been well studied and rigorously examined through other existing and well accepted protein stability testing methods. <br />
Therefore,our team decided to use the protein known as BINDI. <br />
BINDI and two of its less stable variants, BbpD04 and BbpD04.3 were studied and examined in "A Computationally Designed Inhibitor of an Epstein-Barr Viral Bcl-2 Protein Induces Apoptosis in Infected Cells" by Procko et al<sup>1</sup>. <br />
We would like to acknowledge and thank Dr. Procko for giving us his genes to work with. <br />
<br />
</p> <br />
<br />
<br />
<h2> PYE1 a strain of <i> Saccharomyces cerevisiae </i> </h2><br />
<p><br />
<br />
We use a strain of <i> Saccharomyces cerevisiae </i> deveoloped in Stan Fields' lab at the University of Washington called PyE1. <br />
Its genome has been engineered to contain a gene from Green Fluorescent Protein (GFP) under a Gal1 promoter. <br />
When the Gal4 DNA-binding domain and the VP16 transcription activation domain are colocalized to the Gal1 promoter, expression of GFP is induced. <br />
Therefore, using our test plasmids in PYE1 generates GFP relative to the level of Gal4/VP16 peptide in the cell. <br />
The more stable the degron protein construct is, the more likely it is that more GFP will be expressed.<br />
This relationship between stability and GFP forms the basis from which we will measure the relative protein stability of our degron constructs as well as the protein of interest degron construct.<br />
<br />
</p><br />
<br />
<p><br />
<sup>1</sup>Procko, E, et al. "A Computationally Designed Inhibitor of an Epstein-Barr Viral Bcl-2 Protein Induces Apoptosis in Infected Cells" Cell 157 (2014): 1644-56.<br />
</p><br />
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</html></div>Kristanguyenhttp://2014.igem.org/Team:WashingtonTeam:Washington2014-10-18T01:26:47Z<p>Kristanguyen: </p>
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<br />
<p><br />
Stabilizing proteins is an incredibly important and time consuming task in the field of protein engineering. Current methods require using intimate knowledge of the protein to hypothesize point mutations that could possibly improve stability. Extensive in vitro testing follows involving cloning the new construct into your model organism, expressing the construct, purifying your protein of interest, and producing a melting curve to verify if indeed your mutation did improve stability. In addition to being time intensive, this method is unreliably successful. <br />
<br />
<br><br><br />
<br />
Our team has developed a generalizable, high throughput method to select for the increased expression and stability of engineered proteins, making them more amenable to large-scale production in <i> Escherichia coli </i> and other downstream applications. Our method involves the insertion of proteins into a Gal4-VP16 transactivator that binds a promoter directly upstream of a GFP gene. The protein of interest is inserted into the middle of this complex using polypeptide linkers allowing for the subsequent selection of mutants associated with higher GFP output. We hypothesize that more stable proteins and their complexes will not be degraded by the cell’s natural machinery which will allow the Gal4-VP16 construct to produce higher levels of GFP. Less stable proteins will be degraded through natural mechanisms and will not produce GFP. The difference between these two populations can be evaluated using Flow Cytometry or Fluorescence Activated Cell Sorting. <br><br><br />
<br />
</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/2/24/UWIgemsystem.png" alt="Overview of Project" style="width:800px;height:308px"><br />
<br />
<br><br />
<br />
<br />
<br />
<sup><b> Fig. 1. </b> This novel method of protein stabilization is potentially generalizable, as it utilizes cells' natural pathways for maintaining protein folding, and high throughput when coupled with Fluorescence-Activated Cell Sorting (FACS). </sup></center><br />
<br />
<br> <br><br />
<br />
<p><br />
<br />
Our method utilizes degrons to produce the desired range of GFP based off of the proteins relative stability while applying a destabilizing influence to the protein complex. The degron illuminates the differences between proteins of varying stabilities. This allows the system to operate with a higher clarity between stable and unstable protein variants. While the degron exaggerates differences between stabilities, it also can be used as a variable tool that can be adjusted to fit your protein of interest. By placing the degron in different positions along the protein complex, you can impose different destabilizing effects on the construct. <br><br><br />
<br />
The end goal of our project is to create a system that can be used in today’s protein engineering laboratories. By using Fluorescence Activated Cell Sorting of variants in a random mutagenesis library of our construct, we can sort out the highest GFP output variants which will correlate to the most stable variants. Through successive sorts the population will converge on a variant that improves stability of the protein complex. This revolutionary method is a generalizable alternative to current, labor intensive approaches for the selection of stable protein variants. Engineered proteins selected through this method could be produced in bacteria and aid in the development of thermostable, de novo protein therapeutics. </p><br />
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</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2014-10-18T01:10:11Z<p>Kristanguyen: </p>
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<h1> Protocols </h1><br />
<br />
<br />
<html> <a name="Media, Buffers and Solutions"></a> </html><br />
<br />
<br />
<h2> Media, Plates, and Solutions </h2><br />
<br />
<br />
<h3> Competent Cell Media Buffer (CCMB) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container:<br />
</p><br />
<p><br />
<br />
<UL><br />
<br />
<Li> 100 g glycerol (liquid) <br><br />
<br />
<LI> 10 mL x 1 M potassium acetate <br><br />
<br />
<LI> 11.8 g CaCl2*H2O <br><br />
<br />
<LI> 4 g MnCl2 <br><br />
<br />
<LI> 2 g MgCl2 <br><br />
<br />
<LI> 1 L of dH2O<br />
<br />
</UL><br />
</p><br />
<p><br />
Sterile filter or autoclave in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Super Optimal Broth (SOB) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br />
<br />
</p><br />
<br />
<p><br />
<br />
<UL><br />
<br />
<LI> 20 g tryptone <br><br />
<br />
<LI> 5 g yeast extract <br><br />
<br />
<LI> 10 mL x 1 M NaCl <br><br />
<br />
<LI> 2.5 mL x 1 M KCl <br><br />
<br />
<LI> 1 L of dH2O<br />
<br />
</UL><br />
<br />
</p><br />
<br />
<p><br />
<br />
Sterile filter or autoclave in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Phosphate Buffered Saline (PBS) Solution </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following in a 2 L container or 1 L beaker: <br />
<br />
</p><br />
<br />
<p><br />
<br />
<UL><br />
<br />
<LI>8 g NaCl <br><br />
<br />
<LI>1.44 g Na2HPO4 <br><br />
<br />
<LI>0.8 g KCl <br><br />
<br />
<LI>0.24 g KH2PO4 <br><br />
<br />
<LI>1 L of dH2O <br />
<br />
</UL><br />
<br />
</p><br />
<br />
<p><br />
<br />
Buffer to pH 7.4 <br><br />
<br />
Sterile filter or autoclave in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3>PBSF (PBS for Flow)</h3><br />
<br />
<br />
<p><br />
<br />
Mix the following in a 1 L beaker:<br />
<br />
</p><br />
<br />
<p><br />
<br />
<UL><br />
<br />
<LI> 25 mL 20X PBS, pH 7.4<br><br />
<br />
<LI> 475 mL H2O<br><br />
<br />
<LI> 2.5 g BSA (0.5%)*<br />
<br />
</UL><br />
<br />
</p><br />
<br />
<p><br />
<br />
Sterile filter in a 1 L bottle and store at 4 °C <br />
<br />
</p><br />
<br />
<br />
<h3> Yeast Extract Peptone Dextrose (YPD) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following into 950 mL of dH2O in a 1 L bottle: <br />
<br />
</p><br />
<br />
<p><br />
<br />
<UL><br />
<br />
<LI> 20 g peptone <br><br />
<br />
<LI> 10 g yeast extract<br />
<br />
</UL><br />
<br />
</p><br />
<p><br />
<br />
Autoclave <br><br />
<br />
Add 50 mL 40% glucose <br><br />
<br />
Sterile filter into a 1 L bottle <br><br />
<br />
<br><br />
<br />
Note: For long-term liquid media storage, do not add 40% glucose. Instead add the glucose directly into cell cultures. <br><br />
<br />
Note: For YPD-plates add 24 g agar to the peptone and yeast extract before autoclaving. <br> <br />
<br />
</p><br />
<br />
<br />
<h3> Selective Dropout Media, C-Uracil and C-Histidine (C-Ura and C-His) </h3><br />
<br />
<br />
<p><br />
<br />
Synthesized by the Yeast Resource Center at the University of Washington's <br />
<br />
Department of Genome Sciences and Department of Biochemistry.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<h3> Guanidinium Hydrogen Chloride </h3><br />
<br />
<br />
<p><br />
<br />
For maximum effectiveness, final concentration should be approximately 8.5 M in PBS<br><br />
<br />
Add the following to a 500mL beaker and mix:<br />
<br />
</p><br />
<br />
<p><br />
<br />
<UL><br />
<br />
<LI> 203 g guanidinium hydrogen chloride <br><br />
<br />
<LI> 250 mL PBS solution* <br><br />
<br />
<LI> Add dilute HCl to pH 7.4<br />
<br />
</UL> <br />
<br />
</p><br />
<br />
<p><br />
<br />
*It is not necessary to filter or autoclave. <br><br />
<br />
*Alternatively add slightly less than 250 mL of PBS in order to buffer the solution to the appropriate volume, then add more dH2O as necessary. <br />
<br />
</p><br />
<br />
<br />
<html><br />
<br />
<br><a href="#top">Back To Top</a> <br><br />
</html><br />
<br />
<html><a name="Basic Cloning"></a><br />
<br />
</html><br />
<br />
<h2> Basic Cloning </h2><br />
<br />
<br />
<br />
<h3> Polymerase Chain Reaction </h3><br />
<br />
<br />
<p><br />
<br />
All PCRs were done using a standard 50 μL reaction volume with GoTaq® Green Master Mix 2X purchased from PROMEGA Corporation. <br><br />
<br />
Mix the following in a 0.2 mL microcentrifuge tube on ice: <br><br />
<br />
25 μL GoTaq® Green Master Mix 2X <br><br />
<br />
1-5 μL of 10 μM forward primer <br><br />
<br />
1-5 μL of 10 μM reverse primer <br><br />
<br />
<250 ng of DNA template <br><br />
<br />
QS 50 μl nuclease-free H2O <br><br />
<br />
Conduct the reaction in a thermocycler, adjusting anneal temperature and extension times accordingly. See your polymerase supplier protocol for more details on thermocycling.<br />
<br />
</p><br />
<br />
<h3> Error-prone Polymerase Chain Reaction </h3><br />
<br />
<br />
<p> <br />
<br />
<br />
<br />
Prepare 50 μL reaction: <br><br />
<br />
5 μL 10X Mutazyme II Rxn Buffer <br><br />
<br />
1 μL 40 mM dNTP mix (200 μM each final) <br><br />
<br />
1 μL 20 μM forward primer <br><br />
<br />
1 μL 20 μM reverse primer <br><br />
<br />
1 μL Mutazyme II DNA polymerase (2.5 U/μL) <br><br />
<br />
0.01 ng template <br><br />
<br />
QS 50 μL diH2O <br><br />
<br />
<br><br />
<br />
Program thermocycler as follows: <br><br />
<br />
95 °C, 2 min <br><br />
<br />
95 °C, 30 sec <br><br />
<br />
XX °C*, 30 sec <br><br />
<br />
72 °C, X min** <br><br />
<br />
32 cycles <br><br />
<br />
72 °C, 10 min <br><br />
<br />
4 °C, hold<br />
<br />
<br><br />
<br />
*Adjust annealing temperature according to Tm of primer. <br><br />
<br />
**Adjust extension time according to the length of amplified DNA. <br><br />
<br />
<br><br />
<br />
Note: Use 0.01 ng of template (calculate by insert and not by total plasmid). <br><br />
<br />
Calculate amount of template to use as follows: <br><br />
<br />
(bp for amplified region) / (bp in total plasmid) = % amplified region <br><br />
<br />
(conc. of total plasmid) x (% amplified region as a decimal) = conc. of amplified region <br><br />
<br />
Note: Never pipette less than 0.5 μL. <br><br />
<br />
(0.01 ng of template) / (conc. of amplified region) = vol of template to add to PCR <br><br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Restriction Endonuclease Reaction (Digestion) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
All restriction enzyme reactions were done using a 50 μl reaction volume. Restriction enzymes and buffers were purchased from New England Biolabs® Inc. <br><br />
<br />
Mix the following in a 0.2 mL PCR tube: <br><br />
<br />
<br />
1 μg of DNA <br><br />
<br />
5 μL of the appropriate 10X New England Biolab® Buffer <br><br />
<br />
1 μL of each restriction enzyme (add last) <br><br />
<br />
QS 50μL nuclease-free H2O <br><br />
<br />
Incubate the reaction for 1 hr <br><br />
<br />
Heat inactive the reaction at the appropriate temperature <br><br />
<br><br />
<br />
Note: Thaw the restriction enzyme(s) on ice to improve shelf life. <br><br />
<br />
<br />
</p> <br />
<br />
<br />
<h3> Ligation </h3><br />
<br />
<br />
<p><br />
<br />
T4 DNA Ligase and Buffer were purchased from New England Biolabs® Inc. <br><br />
<br />
1. Prepare the following in a 0.2 mL microcentrifuge tube: <br><br />
<br />
50.0 ng vector DNA* <br><br />
<br />
37.5 ng vector DNA* <br><br />
<br />
2 μL 10X T4 DNA Ligase Buffer <br><br />
<br />
1 μL T4 DNA Ligase <br><br />
<br />
QS 20 μL diH2O <br><br />
<br />
2. Incubate the reaction at room temperature for 10-30 minutes or at 16 °C overnight. <br><br />
<br />
3. Heat inactivate at 65 °C for 10 minutes. <br><br />
<br />
4. Chill on ice before starting a transformation reaction. <br><br />
<br />
<br><br />
<br />
*The exact amount of DNA is dependent on the number of base pairs. In order to conduct a proper reaction consult the New England Biolab Ligation Calculator at:<br />
<br />
http://nebiocalculator.neb.com/#!/<br />
<br />
</p><br />
<br />
<html><br />
<br />
<br><a href="#top">Back To Top</a> <br><br />
</html><br />
<br />
<html><a name="Escherichia coli Protocols"></a><br />
<br />
</html><br />
<br />
<br />
<h2> <i> Escherichia coli </i> Protocols (XL1-Blue and XL10-Gold) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<p> <br />
Competent cells take two days to culture and aliquot. <br><br />
Day 1: <br><br />
1. Streak an aliquot of competent cells onto two LB-plates without antibiotics.* <br><br />
2. Incubate at 37 °C overnight. <br><br />
Day 2: <br><br />
1. In two 250 mL baffle flasks add 50 mL of SOB media. <br><br />
2. Scrape as many single colonies into either flask. <br><br />
3. Incubate and shake at 37 °C and 250 rpm for 2-3 hours. <br><br />
4. Check the optical density of the cells at 600 nm after 2 hours. <br><br />
5. Stop incubation when cultures reach approximately 0.5 optical density. <br><br />
6. Add the contents of the flask into separate 50 mL flat bottomed centrifuge tubes. <br><br />
7. Spin down the cells at 2500 rpm at 4 °C for 15 minutes. <br><br />
8. Decant the supernatant. <br><br />
9. Resuspend the cells in 16 mL of CCMB by pipetting or gently vortexing. <br><br />
10. Incubate the cells on ice for 20 minutes. <br><br />
11. Spin down the cells at 2500 rpm at 4 °C for 10 minutes. <br> <br />
12. Decant the supernatant. <br><br />
13. Resuspend the cells in 4 mL of CCMB. <br><br />
14. Quickly aliquot the cells into 1.7 mL cryogenic vials or 1.5 mL centrifuge tubes.** <br><br />
15. Store the competent cell aliquots at -80 °C. <br><br />
<br><br />
*Streak in such a way that there should be individual colony growth and no clumps after the incubation. <br><br />
**We did this in a -20 °C cold room and using an automated repeater pipette. The volume of each aliquot depends on the number of transformations you intend to do at a time. <br><br />
<br><br />
Note: After removing the cells from incubation keep them on ice or as cold as possible. <br><br />
</p><br />
<br />
<br />
<h3> Chemically Competent Cell Transformations </h3><br />
<br />
<br />
<p> <br />
1. Thaw competent <i> E. coli </i> cells on ice (XL1-Blue or XL10-Gold).* <br><br />
2. Add 50 μL of competent cells to sterile 14 mL culture tube. <br><br />
3. Add 1 μL (~100-200 ng)* of the mini-prep to each culture tube. <br><br />
4. Equilibrate the cells on ice for 10 minutes. <br><br />
5. Heat shock the cells at 42 °C for 30-45 seconds.** <br><br />
6. Immediately place the cells back on ice for 3 minutes. <br><br />
7. Add 250 μL LB media without antibiotics and shake at 250 rpm and 37 °C for 30 minutes. <br><br />
8. Spread 10 μL and 290 μL on an appropriate LB-antibiotic plate. <br><br />
9. Invert the plate and incubate at 37 °C overnight. <br><br />
<br><br />
*The exact amount of DNA to add depends on your cell's transformation efficiency. However, it is acceptable to add a larger amount to increase the number of transformed cells. <br><br />
**Do not heat shock for an extended duration as this may damage and/or kill your cells.<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnights </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14 mL round-bottom tube, add 3 mL of LB and 3 μL of 1000X antibiotic(s). <br><br />
2. Pick one isolated colony, do not collect satellites or colony clumps, with a pipette tip. <br><br />
3. Swirl the colony tip in the tube, there should be no visible cell clumps. <br><br />
4. Incubate and shake the tube at 37 °C at 250 rpm for 12-16 hours and no longer than 20 hours. <br><br />
<br />
</p><br />
<br />
<br />
<h3> DNA Extraction and Mini-Preps </h3><br />
<br />
<br />
<p><br />
<br />
All DNA Mini-Preps were prepared using EPOCH Mini-Prep Kits and following the supplied protocols.<br />
<br />
</p><br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
1. Take 1-2 mL from an overnight culture and transfer into a 1.5 mL centrifuge tube. <br><br />
2. Spin down the culture at 3000 rpm for 3 minutes. <br><br />
3. Decant the supernatant. <br><br />
4. Resuspend the cells in 500 μL of 40% glycerol and 500 μL of LB (no antibiotics) or water. <br><br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
6. Store the glycerol stock at -80 °C. <br><br />
<br />
<br />
</p><br />
<br />
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</html><br />
<br />
<html><a name="Saccharomyces cerevisiae"></a><br />
<br />
</html><br />
<br />
<h2> <i> Saccharomyces cerevisiae </i> (PyE1 Yeast) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<br />
<p><br />
<br />
This process take four days in lab with a one day wait for incubation. <br><br />
<br />
<br />
Day 1: <br><br />
1. Streak yeast cells onto a YPD plate.* <br><br />
2. Invert the plate and incubate at 30 °C for 2 days. <br><br />
<br />
Day 3: <br><br />
1. Add 50 mL of YPD liquid media into a 250 mL baffle flask. <br><br />
2. Swipe as many individual colonies as you can see into the YPD media.** <br><br />
3. Incubate and shake the culture at 30 °C at 250 rpm overnight approximately 24 hours. <br><br />
<br />
Day 4: <br><br />
1. Take an optical density measurement. <br><br />
2. In three 250 mL baffle flask add the portions of the overnight liquid culture. <br><br />
3. Dilute each culture to approximately 0.4 optical density with YPD. <br><br />
4. Incubate and shake the cultures at 30 °C at 250 rpm until the optical density reaches 1.2-1.6. <br><br />
5. Collect each culture into separate 50 mL flat-bottomed centrifuge tubes. <br><br />
6. Spin down the cells at 4000 x g for 5 minutes at 4 °C. <br><br />
7. Decant the supernatant. <br><br />
8. Resuspend the cells in 100 mL total for all three culture of dH2O. <br><br />
9. Combine the suspensions into two 50 mL flat-bottomed centrifuge tubes. <br><br />
10. Spin down the cells as above. <br><br />
11. Decant the supernatant. <br><br />
12. Resuspend each in 3 mL of 100 mM lithium acetate. <br><br />
13. Transfer both cultures into a single 15 mL conical centrifuge tube. <br><br />
14. Spin down the cells at 3000 rpm for 5 minutes. <br><br />
15. Resuspend the cells in 0.75 mL of 100 mM lithium acetate, total volume is roughly 2 mL. <br><br />
16. Qualitatively bring up the volume to 3.5 mL by adding 40% glycerol. <br><br />
17. Aliquot the cells into 1.5 mL centrifuge tubes or 1.7 mL cryogenic vials.*** <br><br />
<br><br />
*Streak in such a way that there are individual colonies visible on the plate without clumps or satellite colonies. <br><br />
**Collect only individual visible colonies. Do not collect clumps or satellite colonies. <br><br />
***The volume of aliquots depends on the number to transformations you intend to do at a time. <br><br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Chemically Competent Transformations </h3><br />
<br />
<br />
<p><br />
<br />
This protocol assumes a 50 μL aliquot of yeast competent cells were made. Furthermore, this protocol prepares enough cells for six yeast transformations. <br><br />
<br><br />
1. Add the following to 50 μL of yeast competent cells: <br><br />
240 μL of polyethylene glycol - 3350 (PEG-3350) <br><br />
36 μL of 1 M lithium acetate <br><br />
32 μL of milliQ H2O <br><br />
<br />
2. Mix the mixture by gently pipetting or vortexing. <br><br />
3. Aliquot 59 μL of the mixture into a 0.2 mL microcentrifuge tube. <br><br />
4. Add 1 μL (~100-200 ng) of DNA.* <br><br />
5. Mix the mixture by gentle pipetting or vortexing. <br><br />
6. Incubate the mixture at 30 °C for 30 minutes. <br><br />
7. Heat shock the mixture at 42 °C for 20 minutes. <br><br />
8. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
9. Decant the supernatant. <br><br />
10. Resuspend the cell pellets in 200 μL of dH2O. <br><br />
11. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
12. Resuspend the cell pellets in 200 μL of dH2O. <br><br />
13. Plate 50-150 μL of the mixture onto an appropriate selective dropout media plate. <br><br />
14. Invert and incubate at 30 °C for 2 days. <br><br />
<br><br />
*The exact amount of DNA depends on the transformation efficiency of your competent cells. <br />
<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnight Culturing </h3><br />
<br />
<br />
<p><br />
<br />
1. In a 14 mL round-bottomed culture tube add 1.8 mL selective dropout media and 0.2 mL 20% glucose. <br><br />
2. Swipe 3 isolated yeast colonies and add them to the culture tube media. <br><br />
3. Incubate and shake at 37 °C at 250 rpm for 2 days. <br><br />
<br><br />
Note: You can also make 3 mL cultures (2.7 mL S.D. media and 0.3 mL 20% glucose) or larger cultures, just make sure to dilute the glucose from 20% to 2%.<br />
<br />
</p><br />
<br />
<br />
<h3> Culture Passaging </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Take 20-50μL from a previous overnight or passage culture and add it to the culture media.<br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br><br />
<br />
Note: You can also do 3 mL cultures (2.7 mL S.D. media and 0.3 mL 20% glucose) or larger cultures, just make sure to dilute the glucose from 20% to 2%. <br><br />
Note: The exact amount of culture that you take from a previous culture is irrelevant as long as at least 1 living cell is passaged.<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
1. Take 1-2 mL from an overnight culture and transfer into a 1.5 mL centrifuge tube. <br><br />
2. Spin down the culture at 3000 rpm for 3 minutes. <br><br />
3. Decant the supernatant. <br><br />
4. Resuspend the cells in 500 μL of 40% glycerol and 500 μL of selective dropout media or water. <br><br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
6. Store the glycerol stock at -80 °C. <br><br />
<br />
<br />
</p><br />
<br />
<html><br />
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</html><br />
<br />
<html><a name="Flow Cytometry"></a><br />
<br />
</html><br />
<br />
<h2> Flow Cytometry </h2><br />
<br />
<br />
<h3> Dilutions </h3><br />
<br />
<p><br />
<br />
1. From an overnight culture measure the optical density at 660 nm by making 1:10 dilutions. <br><br />
2. Take enough culture to make a 1 mL aliquot with an OD of 0.4. <br><br />
3. Spin down the aliquot in a 1.5 mL centrifuge tube at 3000 rpm for 3 minutes. <br><br />
4. Decant the supernatant. <br><br />
5. Resuspend the cell pellet in 800 μL of the appropriate selective dropout media and 200 μL of 20% glucose. <br><br />
6. Transfer the new culture to a 14 mL culture tube. <br><br />
7. Incubate and shake at 30 °C and 250 rpm for at least 6 hours (1.2-1.6 optical density).<br />
<br />
</p><br />
<br />
<br />
<h3> Preparations for Analysis using C6 Accuri Flow Cytometer </h3><br />
<br />
<br />
<p><br />
<br />
1. From the dilution previously made, measure the optical density, roughly 1.2-1.6. <br><br />
2. Make an aliquot of 500 μL of the dilution culture in a 1.5 mL centrifuge tube. <br><br />
3. Spin down the aliquot at 3000 rpm for 3 minutes. <br><br />
4. Decant the supernatant. <br><br />
5. Resuspend the cell pellet in 500 μL of PBSF. <br><br />
6. Spin down the resuspension at 3000 rpm for 3 minutes. <br><br />
7. Decant the supernatant. <br><br />
8. Resuspend the cell pellet in another 500 μL of PBSF.* <br><br />
9. Prepare the C6 Accuri Flow Cytometer by running a backflush cycle and a diH2O cycle. <br><br />
10. Load the sample onto the sip. <br><br />
11. Run the sample with 100,000 cell count. <br><br />
12. Repeat for all samples and make sure to change data cells otherwise the old data will be erased. <br><br />
13. Once finished, run a cleaning cycle with Accuri approved cleaning solution, then run a diH2O cycle. <br><br />
<br> <br />
*For special cases do not resuspend all samples, instead resuspend immediately before running the sample through the flow cytometer.<br />
<br />
<br />
</p><br />
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<br />
<html><a name="Fluorescence Activated Cell Sorting"></a><br />
<br />
</html><br />
<br />
<h2> Fluorescence Activated Cell Sorting </h2><br />
<br />
<br />
<h3>Dilution of Cells:</h3><br />
<br />
<p> <br />
1. Take OD600 of the cultures.<br><br />
2. Calculate volume to spin down for OD of 0.4 in appropriate volume (typically 1 mL) - C1V1=C2V2<br>.<br />
3. Spin down appropriate volume in eppendorf tubes for 3 min at 3000 rpm.<br><br />
4. Aspirate off supernatant.<br><br />
5. Resuspend pellet in 1 mL C-Ura + 2% glucose (or other appropriate media).<br><br />
6. Shake in 14 mL culture tube at 30 °C for 6 hrs. <br><br />
</p><br />
<br />
<h3>Sample Prep:</h3><br />
<br />
<p><br />
1. Transfer 500 μL of samples and negative control to eppendorf tubes.<br><br />
2. Spin down cells (3000 rpm, 3 min). <br><br />
3. Aspirate off supernatant. Resuspend in PBSF. <br><br />
4. Spin down cells (3000 rpm, 3 min).<br><br />
5. Aspirate off supernatant. Resuspend in PBSF.<br><br />
</p><br />
<br />
<h3>Using the Fluorescence-Activated Cell Sorter</h3><br />
<p><br />
<br />
1. Load the “iGEM Template” file in the FACS Software.<br><br />
2. Make sure the stream is stable.<br><br />
3. Run all controls and record 100,000 events for analysis. <br><br />
4. While running controls, set FSC/SSC and FSC-H/FSC-W gates.<br><br />
5. Run library and record 100,000 events for analysis.<br><br />
6. Set gate for top 1.00% of GFP fluorescence.<br><br />
7. Sort cells falling in all three gates. Sort ten-fold over library size.<br><br />
8. Run bleach and diH2O through FACS to avoid cross-contamination.<br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Protein Expression"></a><br />
<br />
</html><br />
<br />
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</html><br />
<br />
<h2> Protein Expression </h2><br />
<br />
<br />
<h3> Overnight Cultures </h3><br />
<br />
<p><br />
<br />
1. Add 25 mL TB and 25 μL 1000X Kan to a 250 mL baffled flask. <br><br />
2. Stab a glycerol stock with a P1000 pipette and swirl in the flask of media. <br><br />
3. Put flask in 37 °C shaker at 250 rpm for 16-20 hrs.<br> <br />
<br />
<br />
</p><br />
<br />
<h3> Protein Expression </h3><br />
<br />
<p> <br />
<br />
1. Add 500 μL 1000X Kanamycin and 1 mL MgSO4 to 500 mL TB in 2 L baffled flask. <br><br />
2. Transfer 10 mL overnight culture to TB. <br><br />
3. Shake at 37 °C and 250 rpm until OD600 is between 0.5 and 0.8. <br><br />
4. Allow flask to rest at room temp for 30 min. <br><br />
5. Add 125 μL 1 M IPTG. <br><br />
6. Shake flask at 18 °C for ~16-20 hrs.<br />
<br />
</p><br />
<br />
<h3> Protein Extraction and Purification </h3><br />
<br />
<p><br />
1. Transfer cell culture to centrifuge tube. <br><br />
2. Centrifuge culture at 4000 x g for 10 min. <br><br />
3. Discard supernatant. <br><br />
4. Resuspend pellet in 25 mL wash buffer and add 250 μL of 100X PMSF, 250 μL of 100 mg/mL lysozyme, and 250 μL of 10 mg/mL DNAse. <br><br />
5. Sonicate sample with 0.25 inch probe for 5 min at 70% amplitude with 20 sec on and off pulses. <br><br />
6. Take 50 μL total sample. <br><br />
7. Transfer lysate to SS-34 centrifuge tube. <br><br />
8. Centrifuge for 30 min at 18000 x g. <br><br />
9. Take 50 μL soluble sample.<br />
<br />
<br />
</p><br />
<br />
<h3> Nickel Nitrotriacetic Acid Chromatography (Nickel-NTA Chromatography) </h3><br />
<br />
<p><br />
<br />
1. Add 5 mL 50%(v/v) nickel resin in ethanol to a 25 mL gravity flow column and allow to settle to 2.5 mL(CV). <br><br />
2. Rinse with 10CV dH2O. <br><br />
3. Equilibrate with 10CV lysis buffer. <br><br />
4. Load sample onto column. <br><br />
5. Wash column with 15CV lysis buffer. <br><br />
6. Perform 2 additional wash steps with 15CV. <br><br />
7. Elute sample in 10CV elution buffer and collect eluate. <br><br />
8. Take 50 μL pure sample. <br />
<br />
</p><br />
<br />
<h3> Size Exclusion Chromatography (SEC) </h3><br />
<p><br />
1. Concentrate sample to as high as possible without inducing protein aggregation.<br><br />
2. Pre-equilibrate Superdex 75 column with 48 mL PBS. <br><br />
3. Inject 500 μL sample onto column. <br><br />
4. Run 36 mL PBS through column at 0.5 mL/min, collecting 1 mL fractions. <br><br />
5. Verify presence of protein in fractions by measuring concentration on Nanodrop and running SDS-PAGE (15 kDa protein should elute at ~13 mL).<br><br />
6. Pool fractions containing protein.<br />
<br />
</p><br />
<br />
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<br />
<html><a name="Stability Analysis"></a><br />
<br />
</html><br />
<br />
<h2> Stability Analysis </h2><br />
<br />
<br />
<h3> Circular Dichroism: Wavelength Scan </h3><br />
<p><br />
1. Load 1mm cuvette with 400 μL protein solution onto CD. <br><br />
2. Take wavelength scan: <br><br />
260 nm-190 nm <br><br />
sample every 1 nm <br><br />
averaging time 3 sec <br><br />
1 scan <br><br />
step scan <br><br />
25 °C <br><br />
3. Record wavelength which gives strongest signal (222 nm).<br />
</p><br />
<br />
<h3> Circular Dichroism: Guanidine Melt </h3><br />
<p><br />
1. Load 1 cm cuvette containing 1.996 mL of 0.05 mg/mL protein solution and stirrer onto CD. <br><br />
2. Prepare 8 mL of 0.05 mg/mL protein in concentrated guanidine solution. <br><br />
3. Set up Automixer with guanidine solution on one syringe and waste tube on other syringe. <br><br />
4. Titrate up to 6 M guanidine, taking a CD measurement at 222 nm every 0.15 M interval. <br><br />
5. Also measure the fluorescence at 280 nm to ensure the total protein concentration is not changing.<br />
<br />
</p><br />
<br />
<br />
<br />
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</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2014-10-18T01:06:01Z<p>Kristanguyen: </p>
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<h1> Protocols </h1><br />
<br />
<br />
<html> <a name="Media, Buffers and Solutions"></a> </html><br />
<br />
<br />
<h2> Media, Plates, and Solutions </h2><br />
<br />
<br />
<h3> Competent Cell Media Buffer (CCMB) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container:<br />
</p><br />
<p><br />
<br />
<UL><br />
<br />
<Li> 100 g glycerol (liquid) <br><br />
<br />
<LI> 10 mL x 1 M potassium acetate <br><br />
<br />
<LI> 11.8 g CaCl2*H2O <br><br />
<br />
<LI> 4 g MnCl2 <br><br />
<br />
<LI> 2 g MgCl2 <br><br />
<br />
<LI> 1 L of dH2O<br />
<br />
</UL><br />
</p><br />
<p><br />
Sterile filter or autoclave in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Super Optimal Broth (SOB) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br />
<br />
</p><br />
<br />
<p><br />
<br />
<UL><br />
<br />
<LI> 20 g tryptone <br><br />
<br />
<LI> 5 g yeast extract <br><br />
<br />
<LI> 10 mL x 1 M NaCl <br><br />
<br />
<LI> 2.5 mL x 1 M KCl <br><br />
<br />
<LI> 1 L of dH2O<br />
<br />
</UL><br />
<br />
</p><br />
<br />
<p><br />
<br />
Sterile filter or autoclave in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Phosphate Buffered Saline (PBS) Solution </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following in a 2 L container or 1 L beaker: <br />
<br />
</p><br />
<br />
<p><br />
<br />
<UL><br />
<br />
<LI>8 g NaCl <br><br />
<br />
<LI>1.44 g Na2HPO4 <br><br />
<br />
<LI>0.8 g KCl <br><br />
<br />
<LI>0.24 g KH2PO4 <br><br />
<br />
<LI>1 L of dH2O <br />
<br />
</UL><br />
<br />
</p><br />
<br />
<p><br />
<br />
Buffer to pH 7.4 <br><br />
<br />
Sterile filter or autoclave in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3>PBSF (PBS for Flow)</h3><br />
<br />
<br />
<p><br />
<br />
Mix the following in a 1 L beaker:<br />
<br />
</p><br />
<br />
<p><br />
<br />
<UL><br />
<br />
<LI> 25 mL 20X PBS, pH 7.4<br><br />
<br />
<LI> 475 mL H2O<br><br />
<br />
<LI> 2.5 g BSA (0.5%)*<br />
<br />
</UL><br />
<br />
</p><br />
<br />
<p><br />
<br />
Sterile filter in a 1 L bottle and store at 4 °C <br />
<br />
</p><br />
<br />
<br />
<h3> Yeast Extract Peptone Dextrose (YPD) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following into 950 mL of dH2O in a 1 L bottle: <br />
<br />
</p><br />
<br />
<p><br />
<br />
<UL><br />
<br />
<LI> 20 g peptone <br><br />
<br />
<LI> 10 g yeast extract<br />
<br />
</UL><br />
<br />
</p><br />
<p><br />
<br />
Autoclave <br><br />
<br />
Add 50 mL 40% glucose <br><br />
<br />
Sterile filter into a 1 L bottle <br><br />
<br />
<br><br />
<br />
Note: For long-term liquid media storage, do not add 40% glucose. Instead add the glucose directly into cell cultures. <br><br />
<br />
Note: For YPD-plates add 24 g agar to the peptone and yeast extract before autoclaving. <br> <br />
<br />
</p><br />
<br />
<br />
<h3> Selective Dropout Media, C-Uracil and C-Histidine (C-Ura and C-His) </h3><br />
<br />
<br />
<p><br />
<br />
Synthesized by the Yeast Resource Center at the University of Washington's <br />
<br />
Department of Genome Sciences and Department of Biochemistry.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<h3> Guanidinium Hydrogen Chloride </h3><br />
<br />
<br />
<p><br />
<br />
For maximum effectiveness, final concentration should be approximately 8.5 M in PBS<br><br />
<br />
Add the following to a 500mL beaker and mix:<br />
<br />
</p><br />
<br />
<p><br />
<br />
<UL><br />
<br />
<LI> 203 g guanidinium hydrogen chloride <br><br />
<br />
<LI> 250 mL PBS solution* <br><br />
<br />
<LI> Add dilute HCl to pH 7.4<br />
<br />
</UL> <br />
<br />
</p><br />
<br />
<p><br />
<br />
*It is not necessary to filter or autoclave. <br><br />
<br />
*Alternatively add slightly less than 250 mL of PBS in order to buffer the solution to the appropriate volume, then add more dH2O as necessary. <br />
<br />
</p><br />
<br />
<br />
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<br />
<html><a name="Basic Cloning"></a><br />
<br />
</html><br />
<br />
<h2> Basic Cloning </h2><br />
<br />
<br />
<br />
<h3> Polymerase Chain Reaction </h3><br />
<br />
<br />
<p><br />
<br />
All PCRs were done using a standard 50 μL reaction volume with GoTaq® Green Master Mix 2X purchased from PROMEGA Corporation. <br><br />
<br />
Mix the following in a 0.2 mL microcentrifuge tube on ice: <br><br />
<br />
25 μL GoTaq® Green Master Mix 2X <br><br />
<br />
1-5 μL of 10 μM forward primer <br><br />
<br />
1-5 μL of 10 μM reverse primer <br><br />
<br />
<250 ng of DNA template <br><br />
<br />
QS 50 μl nuclease-free H2O <br><br />
<br />
Conduct the reaction in a thermocycler, adjusting anneal temperature and extension times accordingly. See your polymerase supplier protocol for more details on thermocycling.<br />
<br />
</p><br />
<br />
<h3> Error-prone Polymerase Chain Reaction </h3><br />
<br />
<br />
<p> <br />
<br />
<br />
<br />
Prepare 50 μL reaction: <br><br />
<br />
5 μL 10X Mutazyme II Rxn Buffer <br><br />
<br />
1 μL 40 mM dNTP mix (200 μM each final) <br><br />
<br />
1 μL 20 μM forward primer <br><br />
<br />
1 μL 20 μM reverse primer <br><br />
<br />
1 μL Mutazyme II DNA polymerase (2.5 U/μL) <br><br />
<br />
0.01 ng template <br><br />
<br />
QS 50 μL diH2O <br><br />
<br />
<br><br />
<br />
Program thermocycler as follows: <br><br />
<br />
95 °C, 2 min <br><br />
<br />
95 °C, 30 sec <br><br />
<br />
XX °C*, 30 sec <br><br />
<br />
72 °C, X min** <br><br />
<br />
32 cycles <br><br />
<br />
72 °C, 10 min <br><br />
<br />
4 °C, hold<br />
<br />
<br><br />
<br />
*Adjust annealing temperature according to Tm of primer. <br><br />
<br />
**Adjust extension time according to the length of amplified DNA. <br><br />
<br />
<br><br />
<br />
Note: Use 0.01 ng of template (calculate by insert and not by total plasmid). <br><br />
<br />
Calculate amount of template to use as follows: <br><br />
<br />
(bp for amplified region) / (bp in total plasmid) = % amplified region <br><br />
<br />
(conc. of total plasmid) x (% amplified region as a decimal) = conc. of amplified region <br><br />
<br />
Note: Never pipette less than 0.5 μL. <br><br />
<br />
(0.01 ng of template) / (conc. of amplified region) = vol of template to add to PCR <br><br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Restriction Endonuclease Reaction (Digestion) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
All restriction enzyme reactions were done using a 50 μl reaction volume. Restriction enzymes and buffers were purchased from New England Biolabs® Inc. <br><br />
<br />
Mix the following in a 0.2 mL PCR tube: <br><br />
<br />
<br />
1 μg of DNA <br><br />
<br />
5 μL of the appropriate 10X New England Biolab® Buffer <br><br />
<br />
1 μL of each restriction enzyme (add last) <br><br />
<br />
QS 50μL nuclease-free H2O <br><br />
<br />
Incubate the reaction for 1 hr <br><br />
<br />
Heat inactive the reaction at the appropriate temperature <br><br />
<br><br />
<br />
Note: Thaw the restriction enzyme(s) on ice to improve shelf life <br><br />
<br />
<br />
</p> <br />
<br />
<br />
<h3> Ligation </h3><br />
<br />
<br />
<p><br />
<br />
T4 DNA Ligase and Buffer were purchased from New England Biolabs® Inc. <br><br />
<br />
1. Prepare the following in a 0.2 mL microcentrifuge tube: <br><br />
<br />
50.0 ng vector DNA* <br><br />
<br />
37.5 ng vector DNA* <br><br />
<br />
2 μL 10X T4 DNA Ligase Buffer <br><br />
<br />
1 μL T4 DNA Ligase <br><br />
<br />
QS 20 μL diH2O <br><br />
<br />
2. Incubate the reaction at room temperature for 10-30 minutes or at 16 °C overnight. <br><br />
<br />
3. Heat inactivate at 65 °C for 10 minutes. <br><br />
<br />
4. Chill on ice before starting a transformation reaction. <br><br />
<br />
<br><br />
<br />
*The exact amount of DNA is dependent on the number of base pairs. In order to conduct a proper reaction consult the New England Biolab Ligation Calculator at:<br />
<br />
http://nebiocalculator.neb.com/#!/<br />
<br />
</p><br />
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<html><a name="Escherichia coli Protocols"></a><br />
<br />
</html><br />
<br />
<br />
<h2> <i> Escherichia coli </i> Protocols (XL1-Blue and XL10-Gold) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<p> <br />
Competent cells take two days to culture and aliquot. <br><br />
Day 1: <br><br />
1. Streak an aliquot of competent cells onto two LB-plates without antibiotics.* <br><br />
2. Incubate at 37 °C overnight. <br><br />
Day 2: <br><br />
1. In two 250 mL baffle flasks add 50 mL of SOB media. <br><br />
2. Scrape as many single colonies into either flask. <br><br />
3. Incubate and shake at 37 °C and 250 rpm for 2-3 hours. <br><br />
4. Check the optical density of the cells at 600 nm after 2 hours. <br><br />
5. Stop incubation when cultures reach approximately 0.5 optical density. <br><br />
6. Add the contents of the flask into separate 50 mL flat bottomed centrifuge tubes. <br><br />
7. Spin down the cells at 2500 rpm at 4 °C for 15 minutes. <br><br />
8. Decant the supernatant. <br><br />
9. Resuspend the cells in 16 mL of CCMB by pipetting or gently vortexing. <br><br />
10. Incubate the cells on ice for 20 minutes. <br><br />
11. Spin down the cells at 2500 rpm at 4 °C for 10 minutes. <br> <br />
12. Decant the supernatant. <br><br />
13. Resuspend the cells in 4 mL of CCMB. <br><br />
14. Quickly aliquot the cells into 1.7 mL cryogenic vials or 1.5 mL centrifuge tubes.** <br><br />
15. Store the competent cell aliquots at -80 °C. <br><br />
<br><br />
*Streak in such a way that there should be individual colony growth and no clumps after the incubation. <br><br />
**We did this in a -20 °C cold room and using an automated repeater pipette. The volume of each aliquot depends on the number of transformations you intend to do at a time. <br><br />
<br><br />
Note: After removing the cells from incubation keep them on ice or as cold as possible. <br><br />
</p><br />
<br />
<br />
<h3> Chemically Competent Cell Transformations </h3><br />
<br />
<br />
<p> <br />
1. Thaw competent <i> E. coli </i> cells on ice (XL1-Blue or XL10-Gold).* <br><br />
2. Add 50 μL of competent cells to sterile 14 mL culture tube. <br><br />
3. Add 1 μL (~100-200 ng)* of the mini-prep to each culture tube. <br><br />
4. Equilibrate the cells on ice for 10 minutes. <br><br />
5. Heat shock the cells at 42 °C for 30-45 seconds.** <br><br />
6. Immediately place the cells back on ice for 3 minutes. <br><br />
7. Add 250 μL LB media without antibiotics and shake at 250 rpm and 37 °C for 30 minutes. <br><br />
8. Spread 10 μL and 290 μL on an appropriate LB-antibiotic plate. <br><br />
9. Invert the plate and incubate at 37 °C overnight. <br><br />
<br><br />
*The exact amount of DNA to add depends on your cell's transformation efficiency. However, it is acceptable to add a larger amount to increase the number of transformed cells. <br><br />
**Do not heat shock for an extended duration as this may damage and/or kill your cells.<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnights </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14 mL round-bottom tube, add 3 mL of LB and 3 μL of 1000X antibiotic(s). <br><br />
2. Pick one isolated colony, do not collect satellites or colony clumps, with a pipette tip. <br><br />
3. Swirl the colony tip in the tube, there should be no visible cell clumps. <br><br />
4. Incubate and shake the tube at 37 °C at 250 rpm for 12-16 hours and no longer than 20 hours. <br><br />
<br />
</p><br />
<br />
<br />
<h3> DNA Extraction and Mini-Preps </h3><br />
<br />
<br />
<p><br />
<br />
All DNA Mini-Preps were prepared using EPOCH Mini-Prep Kits and following the supplied protocols.<br />
<br />
</p><br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
1. Take 1-2 mL from an overnight culture and transfer into a 1.5 mL centrifuge tube. <br><br />
2. Spin down the culture at 3000 rpm for 3 minutes. <br><br />
3. Decant the supernatant. <br><br />
4. Resuspend the cells in 500 μL of 40% glycerol and 500 μL of LB (no antibiotics) or water. <br><br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
6. Store the glycerol stock at -80 °C. <br><br />
<br />
<br />
</p><br />
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<br />
<html><a name="Saccharomyces cerevisiae"></a><br />
<br />
</html><br />
<br />
<h2> <i> Saccharomyces cerevisiae </i> (PyE1 Yeast) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<br />
<p><br />
<br />
This process take four days in lab with a one day wait for incubation. <br><br />
<br />
<br />
Day 1: <br><br />
1. Streak yeast cells onto a YPD plate.* <br><br />
2. Invert the plate and incubate at 30 °C for 2 days. <br><br />
<br />
Day 3: <br><br />
1. Add 50 mL of YPD liquid media into a 250 mL baffle flask. <br><br />
2. Swipe as many individual colonies as you can see into the YPD media.** <br><br />
3. Incubate and shake the culture at 30 °C at 250 rpm overnight approximately 24 hours. <br><br />
<br />
Day 4: <br><br />
1. Take an optical density measurement. <br><br />
2. In three 250 mL baffle flask add the portions of the overnight liquid culture. <br><br />
3. Dilute each culture to approximately 0.4 optical density with YPD. <br><br />
4. Incubate and shake the cultures at 30 °C at 250 rpm until the optical density reaches 1.2-1.6. <br><br />
5. Collect each culture into separate 50 mL flat-bottomed centrifuge tubes. <br><br />
6. Spin down the cells at 4000 x g for 5 minutes at 4 °C. <br><br />
7. Decant the supernatant. <br><br />
8. Resuspend the cells in 100 mL total for all three culture of dH2O. <br><br />
9. Combine the suspensions into two 50 mL flat-bottomed centrifuge tubes. <br><br />
10. Spin down the cells as above. <br><br />
11. Decant the supernatant. <br><br />
12. Resuspend each in 3 mL of 100 mM lithium acetate. <br><br />
13. Transfer both cultures into a single 15 mL conical centrifuge tube. <br><br />
14. Spin down the cells at 3000 rpm for 5 minutes. <br><br />
15. Resuspend the cells in 0.75 mL of 100 mM lithium acetate, total volume is roughly 2 mL. <br><br />
16. Qualitatively bring up the volume to 3.5 mL by adding 40% glycerol. <br><br />
17. Aliquot the cells into 1.5 mL centrifuge tubes or 1.7 mL cryogenic vials.*** <br><br />
<br><br />
*Streak in such a way that there are individual colonies visible on the plate without clumps or satellite colonies. <br><br />
**Collect only individual visible colonies. Do not collect clumps or satellite colonies. <br><br />
***The volume of aliquots depends on the number to transformations you intend to do at a time. <br><br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Chemically Competent Transformations </h3><br />
<br />
<br />
<p><br />
<br />
This protocol assumes a 50 μL aliquot of yeast competent cells were made. Furthermore, this protocol prepares enough cells for six yeast transformations. <br><br />
<br><br />
1. Add the following to 50 μL of yeast competent cells: <br><br />
240 μL of polyethylene glycol - 3350 (PEG-3350) <br><br />
36 μL of 1 M lithium acetate <br><br />
32 μL of milliQ H2O <br><br />
<br />
2. Mix the mixture by gently pipetting or vortexing. <br><br />
3. Aliquot 59 μL of the mixture into a 0.2 mL microcentrifuge tube. <br><br />
4. Add 1 μL (~100-200 ng) of DNA.* <br><br />
5. Mix the mixture by gentle pipetting or vortexing. <br><br />
6. Incubate the mixture at 30 °C for 30 minutes. <br><br />
7. Heat shock the mixture at 42 °C for 20 minutes. <br><br />
8. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
9. Decant the supernatant. <br><br />
10. Resuspend the cell pellets in 200 μL of dH2O. <br><br />
11. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
12. Resuspend the cell pellets in 200 μL of dH2O. <br><br />
13. Plate 50-150 μL of the mixture onto an appropriate selective dropout media plate. <br><br />
14. Invert and incubate at 30 °C for 2 days. <br><br />
<br><br />
*The exact amount of DNA depends on the transformation efficiency of your competent cells. <br />
<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnight Culturing </h3><br />
<br />
<br />
<p><br />
<br />
1. In a 14 mL round-bottomed culture tube add 1.8 mL selective dropout media and 0.2 mL 20% glucose. <br><br />
2. Swipe 3 isolated yeast colonies and add them to the culture tube media. <br><br />
3. Incubate and shake at 37 °C at 250 rpm for 2 days. <br><br />
<br><br />
Note: You can also make 3 mL cultures (2.7 mL S.D. media and 0.3 mL 20% glucose) or larger cultures, just make sure to dilute the glucose from 20% to 2%.<br />
<br />
</p><br />
<br />
<br />
<h3> Culture Passaging </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Take 20-50μL from a previous overnight or passage culture and add it to the culture media.<br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br><br />
<br />
Note: You can also do 3 mL cultures (2.7 mL S.D. media and 0.3 mL 20% glucose) or larger cultures, just make sure to dilute the glucose from 20% to 2%. <br><br />
Note: The exact amount of culture that you take from a previous culture is irrelevant as long as at least 1 living cell is passaged.<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
1. Take 1-2 mL from an overnight culture and transfer into a 1.5 mL centrifuge tube. <br><br />
2. Spin down the culture at 3000 rpm for 3 minutes. <br><br />
3. Decant the supernatant. <br><br />
4. Resuspend the cells in 500 μL of 40% glycerol and 500 μL of selective dropout media or water. <br><br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
6. Store the glycerol stock at -80 °C. <br><br />
<br />
<br />
</p><br />
<br />
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<html><a name="Flow Cytometry"></a><br />
<br />
</html><br />
<br />
<h2> Flow Cytometry </h2><br />
<br />
<br />
<h3> Dilutions </h3><br />
<br />
<p><br />
<br />
1. From an overnight culture measure the optical density at 660 nm by making 1:10 dilutions. <br><br />
2. Take enough culture to make a 1 mL aliquot with an OD of 0.4. <br><br />
3. Spin down the aliquot in a 1.5 mL centrifuge tube at 3000 rpm for 3 minutes. <br><br />
4. Decant the supernatant. <br><br />
5. Resuspend the cell pellet in 800 μL of the appropriate selective dropout media and 200 μL of 20% glucose. <br><br />
6. Transfer the new culture to a 14 mL culture tube. <br><br />
7. Incubate and shake at 30 °C and 250 rpm for at least 6 hours (1.2-1.6 optical density).<br />
<br />
</p><br />
<br />
<br />
<h3> Preparations for Analysis using C6 Accuri Flow Cytometer </h3><br />
<br />
<br />
<p><br />
<br />
1. From the dilution previously made, measure the optical density, roughly 1.2-1.6. <br><br />
2. Make an aliquot of 500 μL of the dilution culture in a 1.5 mL centrifuge tube. <br><br />
3. Spin down the aliquot at 3000 rpm for 3 minutes. <br><br />
4. Decant the supernatant. <br><br />
5. Resuspend the cell pellet in 500 μL of PBSF. <br><br />
6. Spin down the resuspension at 3000 rpm for 3 minutes. <br><br />
7. Decant the supernatant. <br><br />
8. Resuspend the cell pellet in another 500 μL of PBSF.* <br><br />
9. Prepare the C6 Accuri Flow Cytometer by running a backflush cycle and a diH2O cycle. <br><br />
10. Load the sample onto the sip. <br><br />
11. Run the sample with 100,000 cell count. <br><br />
12. Repeat for all samples and make sure to change data cells otherwise the old data will be erased. <br><br />
13. Once finished, run a cleaning cycle with Accuri approved cleaning solution, then run a diH2O cycle. <br><br />
<br> <br />
*For special cases do not resuspend all samples, instead resuspend immediately before running the sample through the flow cytometer.<br />
<br />
<br />
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<html><a name="Fluorescence Activated Cell Sorting"></a><br />
<br />
</html><br />
<br />
<h2> Fluorescence Activated Cell Sorting </h2><br />
<br />
<br />
<h3>Dilution of Cells:</h3><br />
<br />
<p> <br />
1. Take OD600 of the cultures.<br><br />
2. Calculate volume to spin down for OD of 0.4 in appropriate volume (typically 1 mL) - C1V1=C2V2<br>.<br />
3. Spin down appropriate volume in eppendorf tubes for 3 min at 3000 rpm.<br><br />
4. Aspirate off supernatant.<br><br />
5. Resuspend pellet in 1 mL C-Ura + 2% glucose (or other appropriate media).<br><br />
6. Shake in 14 mL culture tube at 30 °C for 6 hrs. <br><br />
</p><br />
<br />
<h3>Sample Prep:</h3><br />
<br />
<p><br />
1. Transfer 500 μL of samples and negative control to eppendorf tubes.<br><br />
2. Spin down cells (3000 rpm, 3 min). <br><br />
3. Aspirate off supernatant. Resuspend in PBSF. <br><br />
4. Spin down cells (3000 rpm, 3 min).<br><br />
5. Aspirate off supernatant. Resuspend in PBSF.<br><br />
</p><br />
<br />
<h3>Using the Fluorescence-Activated Cell Sorter</h3><br />
<p><br />
<br />
1. Load the “iGEM Template” file in the FACS Software.<br><br />
2. Make sure the stream is stable.<br><br />
3. Run all controls and record 100,000 events for analysis. <br><br />
4. While running controls, set FSC/SSC and FSC-H/FSC-W gates.<br><br />
5. Run library and record 100,000 events for analysis.<br><br />
6. Set gate for top 1.00% of GFP fluorescence.<br><br />
7. Sort cells falling in all three gates. Sort ten-fold over library size.<br><br />
8. Run bleach and diH2O through FACS to avoid cross-contamination.<br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Protein Expression"></a><br />
<br />
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<br />
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<br />
<h2> Protein Expression </h2><br />
<br />
<br />
<h3> Overnight Cultures </h3><br />
<br />
<p><br />
<br />
1. Add 25 mL TB and 25 μL 1000X Kan to a 250 mL baffled flask. <br><br />
2. Stab a glycerol stock with a P1000 pipette and swirl in the flask of media. <br><br />
3. Put flask in 37 °C shaker at 250 rpm for 16-20 hrs.<br> <br />
<br />
<br />
</p><br />
<br />
<h3> Protein Expression </h3><br />
<br />
<p> <br />
<br />
1. Add 500 μL 1000X Kanamycin and 1 mL MgSO4 to 500 mL TB in 2 L baffled flask. <br><br />
2. Transfer 10 mL overnight culture to TB. <br><br />
3. Shake at 37 °C and 250 rpm until OD600 is between 0.5 and 0.8. <br><br />
4. Allow flask to rest at room temp for 30 min. <br><br />
5. Add 125 μL 1 M IPTG. <br><br />
6. Shake flask at 18 °C for ~16-20 hrs.<br />
<br />
</p><br />
<br />
<h3> Protein Extraction and Purification </h3><br />
<br />
<p><br />
1. Transfer cell culture to centrifuge tube. <br><br />
2. Centrifuge culture at 4000 x g for 10 min. <br><br />
3. Discard supernatant. <br><br />
4. Resuspend pellet in 25 mL wash buffer and add 250 μL of 100X PMSF, 250 μL of 100 mg/mL lysozyme, and 250 μL of 10 mg/mL DNAse. <br><br />
5. Sonicate sample with 0.25 inch probe for 5 min at 70% amplitude with 20 sec on and off pulses. <br><br />
6. Take 50 μL total sample. <br><br />
7. Transfer lysate to SS-34 centrifuge tube. <br><br />
8. Centrifuge for 30 min at 18000 x g. <br><br />
9. Take 50 μL soluble sample.<br />
<br />
<br />
</p><br />
<br />
<h3> Nickel Nitrotriacetic Acid Chromatography (Nickel-NTA Chromatography) </h3><br />
<br />
<p><br />
<br />
1. Add 5 mL 50%(v/v) nickel resin in ethanol to a 25 mL gravity flow column and allow to settle to 2.5 mL(CV). <br><br />
2. Rinse with 10CV dH2O. <br><br />
3. Equilibrate with 10CV lysis buffer. <br><br />
4. Load sample onto column. <br><br />
5. Wash column with 15CV lysis buffer. <br><br />
6. Perform 2 additional wash steps with 15CV. <br><br />
7. Elute sample in 10CV elution buffer and collect eluate. <br><br />
8. Take 50 μL pure sample. <br />
<br />
</p><br />
<br />
<h3> Size Exclusion Chromatography (SEC) </h3><br />
<p><br />
1. Concentrate sample to as high as possible without inducing protein aggregation.<br><br />
2. Pre-equilibrate Superdex 75 column with 48 mL PBS. <br><br />
3. Inject 500 μL sample onto column. <br><br />
4. Run 36 mL PBS through column at 0.5 mL/min, collecting 1 mL fractions. <br><br />
5. Verify presence of protein in fractions by measuring concentration on Nanodrop and running SDS-PAGE (15 kDa protein should elute at ~13 mL).<br><br />
6. Pool fractions containing protein.<br />
<br />
</p><br />
<br />
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<html><a name="Stability Analysis"></a><br />
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<br />
<h2> Stability Analysis </h2><br />
<br />
<br />
<h3> Circular Dichroism: Wavelength Scan </h3><br />
<p><br />
1. Load 1mm cuvette with 400 μL protein solution onto CD. <br><br />
2. Take wavelength scan: <br><br />
260 nm-190 nm <br><br />
sample every 1 nm <br><br />
averaging time 3 sec <br><br />
1 scan <br><br />
step scan <br><br />
25 °C <br><br />
3. Record wavelength which gives strongest signal (222 nm).<br />
</p><br />
<br />
<h3> Circular Dichroism: Guanidine Melt </h3><br />
<p><br />
1. Load 1 cm cuvette containing 1.996 mL of 0.05 mg/mL protein solution and stirrer onto CD. <br><br />
2. Prepare 8 mL of 0.05 mg/mL protein in concentrated guanidine solution. <br><br />
3. Set up Automixer with guanidine solution on one syringe and waste tube on other syringe. <br><br />
4. Titrate up to 6 M guanidine, taking a CD measurement at 222 nm every 0.15 M interval. <br><br />
5. Also measure the fluorescence at 280 nm to ensure the total protein concentration is not changing.<br />
<br />
</p><br />
<br />
<br />
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</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2014-10-17T23:00:18Z<p>Kristanguyen: </p>
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<br />
<h1> Protocols </h1><br />
<br />
<br />
<html> <a name="Media, Buffers and Solutions"></a> </html><br />
<br />
<br />
<h2> Media, Plates, and Solutions </h2><br />
<br />
<br />
<h3> Competent Cell Media Buffer (CCMB) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container:<br />
</p><br />
<p><br />
<br />
<UL><br />
<br />
<Li> 100 g glycerol (liquid) <br><br />
<br />
<LI> 10 mL x 1 M potassium acetate <br><br />
<br />
<LI> 11.8 g CaCl2*H2O <br><br />
<br />
<LI> 4 g MnCl2 <br><br />
<br />
<LI> 2 g MgCl2 <br><br />
<br />
<LI> 1 L of dH2O<br />
<br />
</UL><br />
</p><br />
<p><br />
Sterile filter or autoclave in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Super Optimal Broth (SOB) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br />
<br />
</p><br />
<br />
<p><br />
<br />
<UL><br />
<br />
<LI> 20 g tryptone <br><br />
<br />
<LI> 5 g yeast extract <br><br />
<br />
<LI> 10 mL x 1 M NaCl <br><br />
<br />
<LI> 2.5 mL x 1 M KCl <br><br />
<br />
<LI> 1 L of dH2O<br />
<br />
</UL><br />
<br />
</p><br />
<br />
<p><br />
<br />
Sterile filter or autoclave in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Phosphate Buffered Saline (PBS) Solution </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following in a 2 L container or 1 L beaker: <br />
<br />
</p><br />
<br />
<p><br />
<br />
<UL><br />
<br />
<LI>8 g NaCl <br><br />
<br />
<LI>1.44 g Na2HPO4 <br><br />
<br />
<LI>0.8 g KCl <br><br />
<br />
<LI>0.24 g KH2PO4 <br><br />
<br />
<LI>1 L of dH2O <br />
<br />
</UL><br />
<br />
</p><br />
<br />
<p><br />
<br />
Buffer to pH 7.4 <br><br />
<br />
Sterile filter or autoclave in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3>PBSF (PBS for Flow)</h3><br />
<br />
<br />
<p><br />
<br />
Mix the following in a 1 L beaker:<br />
<br />
</p><br />
<br />
<p><br />
<br />
<UL><br />
<br />
<LI> 25 mL 20X PBS, pH 7.4<br><br />
<br />
<LI> 475 mL H2O<br><br />
<br />
<LI> 2.5 g BSA (0.5%)*<br />
<br />
</UL><br />
<br />
</p><br />
<br />
<p><br />
<br />
Sterile filter in a 1L bottle and store at 4 °C <br />
<br />
</p><br />
<br />
<br />
<h3> Yeast Extract Peptone Dextrose (YPD) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following into 950 mL of dH2O in a 1 L bottle: <br />
<br />
</p><br />
<br />
<p><br />
<br />
<UL><br />
<br />
<LI> 20 g peptone <br><br />
<br />
<LI> 10 g yeast extract<br />
<br />
</UL><br />
<br />
</p><br />
<p><br />
<br />
Autoclave <br><br />
<br />
Add 50 mL 40% glucose <br><br />
<br />
Sterile filter into a 1 L bottle <br><br />
<br />
<br><br />
<br />
Note: For long-term liquid media storage, do not add 40% glucose. Instead add the glucose directly into cell cultures. <br><br />
<br />
Note: For YPD-plates add 24 g agar to the peptone and yeast extract before autoclaving. <br> <br />
<br />
</p><br />
<br />
<br />
<h3> Selective Dropout Media, C-Uracil and C-Histidine (C-Ura and C-His) </h3><br />
<br />
<br />
<p><br />
<br />
Synthesized by the Yeast Resource Center at the University of Washington's <br />
<br />
Department of Genome Sciences and Department of Biochemistry.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<h3> Guanidinium Hydrogen Chloride </h3><br />
<br />
<br />
<p><br />
<br />
For maximum effectiveness, final concentration should be approximately 8.5 M in PBS<br><br />
<br />
Add the following to a 500mL beaker and mix:<br />
<br />
</p><br />
<br />
<p><br />
<br />
<UL><br />
<br />
<LI> 203 g guanidinium hydrogen chloride <br><br />
<br />
<LI> 250 mL PBS solution* <br><br />
<br />
<LI> Add dilute HCl to pH 7.4<br />
<br />
</UL> <br />
<br />
</p><br />
<br />
<p><br />
<br />
*It is not necessary to filter or autoclave. <br><br />
<br />
*Alternatively add slightly less than 250 mL of PBS in order to buffer the solution to the appropriate volume, then add more dH2O as necessary. <br />
<br />
</p><br />
<br />
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<html><a name="Basic Cloning"></a><br />
<br />
</html><br />
<br />
<h2> Basic Cloning </h2><br />
<br />
<br />
<br />
<h3> Polymerase Chain Reaction </h3><br />
<br />
<br />
<p><br />
<br />
All PCRs were done using a standard 50 μL reaction volume with GoTaq® Green Master Mix 2X purchased from PROMEGA Corporation. <br><br />
<br />
Mix the following in a 0.2 mL microcentrifuge tube on ice: <br><br />
<br />
25 μL GoTaq® Green Master Mix 2X <br><br />
<br />
1-5 μL of 10 μM forward primer <br><br />
<br />
1-5 μL of 10 μM reverse primer <br><br />
<br />
<250 ng of DNA template <br><br />
<br />
QS 50 μl nuclease-free H2O <br><br />
<br />
Conduct the reaction in a thermocycler, adjusting anneal temperature and extension times accordingly. See your polymerase supplier protocol for more details on thermocycling.<br />
<br />
</p><br />
<br />
<h3> Error-prone Polymerase Chain Reaction </h3><br />
<br />
<br />
<p> <br />
<br />
<br />
<br />
Prepare 50 μL reaction: <br><br />
<br />
5 μL 10X Mutazyme II Rxn Buffer <br><br />
<br />
1 μL 40 mM dNTP mix (200 μM each final) <br><br />
<br />
1 μL 20 μM forward primer <br><br />
<br />
1 μL 20 μM reverse primer <br><br />
<br />
1 μL Mutazyme II DNA polymerase (2.5 U/μL) <br><br />
<br />
0.01 ng template <br><br />
<br />
QS 50 μL diH2O <br><br />
<br />
<br><br />
<br />
Program thermocycler as follows: <br><br />
<br />
95 °C, 2 min <br><br />
<br />
95 °C, 30 sec <br><br />
<br />
XX °C*, 30 sec <br><br />
<br />
72 °C, X min** <br><br />
<br />
32 cycles <br><br />
<br />
72 °C, 10 min <br><br />
<br />
4 °C, hold<br />
<br />
<br><br />
<br />
*Adjust annealing temperature according to Tm of primer. <br><br />
<br />
**Adjust extension time according to the length of amplified DNA. <br><br />
<br />
<br><br />
<br />
Note: Use 0.01 ng of template (calculate by insert and not by total plasmid). <br><br />
<br />
Calculate amount of template to use as follows: <br><br />
<br />
(bp for amplified region) / (bp in total plasmid) = % amplified region <br><br />
<br />
(conc. of total plasmid) x (% amplified region as a decimal) = conc. of amplified region <br><br />
<br />
Note: Never pipette less than 0.5 μL. <br><br />
<br />
(0.01 ng of template) / (conc. of amplified region) = vol of template to add to PCR <br><br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Restriction Endonuclease Reaction (Digestion) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
All restriction enzyme reactions were done using a 50 μl reaction volume. Restriction enzymes and buffers were purchased from New England Biolabs® Inc. <br><br />
<br />
Mix the following in a 0.2 mL PCR tube: <br><br />
<br />
<br />
1 μg of DNA <br><br />
<br />
5 μL of the appropriate 10X New England Biolab® Buffer <br><br />
<br />
1 μL of each restriction enzyme (add last) <br><br />
<br />
QS 50μL nuclease-free H2O <br><br />
<br />
Incubate the reaction for 1 hr <br><br />
<br />
Heat inactive the reaction at the appropriate temperature <br><br />
<br><br />
<br />
Note: Thaw the restriction enzyme(s) on ice to improve shelf life <br><br />
<br />
<br />
</p> <br />
<br />
<br />
<h3> Ligation </h3><br />
<br />
<br />
<p><br />
<br />
T4 DNA Ligase and Buffer were purchased from New England Biolabs® Inc. <br><br />
<br />
1. Prepare the following in a 0.2 mL microcentrifuge tube: <br><br />
<br />
50.0 ng vector DNA* <br><br />
<br />
37.5 ng vector DNA* <br><br />
<br />
2 μL 10X T4 DNA Ligase Buffer <br><br />
<br />
1 μL T4 DNA Ligase <br><br />
<br />
QS 20 μL diH2O <br><br />
<br />
2. Incubate the reaction at room temperature for 10-30 minutes or at 16 °C overnight. <br><br />
<br />
3. Heat inactivate at 65 °C for 10 minutes. <br><br />
<br />
4. Chill on ice before starting a transformation reaction. <br><br />
<br />
<br><br />
<br />
*The exact amount of DNA is dependent on the number of base pairs. In order to conduct a proper reaction consult the New England Biolab Ligation Calculator at:<br />
<br />
http://nebiocalculator.neb.com/#!/<br />
<br />
</p><br />
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<html><a name="Escherichia coli Protocols"></a><br />
<br />
</html><br />
<br />
<br />
<h2> <i> Escherichia coli </i> Protocols (XL1-Blue and XL10-Gold) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<p> <br />
Competent cells take two days to culture and aliquot. <br><br />
Day 1: <br><br />
1. Streak an aliquot of competent cells onto two LB-plates without antibiotics.* <br><br />
2. Incubate at 37 °C overnight. <br><br />
Day 2: <br><br />
1. In two 250 mL baffle flasks add 50 mL of SOB media. <br><br />
2. Scrape as many single colonies into either flask. <br><br />
3. Incubate and shake at 37 °C and 250 rpm for 2-3 hours. <br><br />
4. Check the optical density of the cells at 600 nm after 2 hours. <br><br />
5. Stop incubation when cultures reach approximately 0.5 optical density. <br><br />
6. Add the contents of the flask into separate 50 mL flat bottomed centrifuge tubes. <br><br />
7. Spin down the cells at 2500 rpm at 4 °C for 15 minutes. <br><br />
8. Decant the supernatant. <br><br />
9. Resuspend the cells in 16 mL of CCMB by pipetting or gently vortexing. <br><br />
10. Incubate the cells on ice for 20 minutes. <br><br />
11. Spin down the cells at 2500 rpm at 4 °C for 10 minutes. <br> <br />
12. Decant the supernatant. <br><br />
13. Resuspend the cells in 4 mL of CCMB. <br><br />
14. Quickly aliquot the cells into 1.7 mL cryogenic vials or 1.5 mL centrifuge tubes.** <br><br />
15. Store the competent cell aliquots at -80 °C. <br><br />
<br><br />
*Streak in such a way that there should be individual colony growth and no clumps after the incubation. <br><br />
**We did this in a -20 °C cold room and using an automated repeater pipette. The volume of each aliquot depends on the number of transformations you intend to do at a time. <br><br />
<br><br />
Note: After removing the cells from incubation keep them on ice or as cold as possible. <br><br />
</p><br />
<br />
<br />
<h3> Chemically Competent Cell Transformations </h3><br />
<br />
<br />
<p> <br />
1. Thaw competent <i> E. coli </i> cells on ice (XL1-Blue or XL10-Gold).* <br><br />
2. Add 50 μL of competent cells to sterile 14 mL culture tube. <br><br />
3. Add 1 μL (~100-200 ng)* of the mini-prep to each culture tube. <br><br />
4. Equilibrate the cells on ice for 10 minutes. <br><br />
5. Heat shock the cells at 42 °C for 30-45 seconds.** <br><br />
6. Immediately place the cells back on ice for 3 minutes. <br><br />
7. Add 250 μL LB media without antibiotics and shake at 250 rpm and 37 °C for 30 minutes. <br><br />
8. Spread 10 μL and 290 μL on an appropriate LB-antibiotic plate. <br><br />
9. Invert the plate and incubate at 37 °C overnight. <br><br />
<br><br />
*The exact amount of DNA to add depends on your cell's transformation efficiency. However, it is acceptable to add a larger amount to increase the number of transformed cells. <br><br />
**Do not heat shock for an extended duration as this may damage and/or kill your cells.<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnights </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14 mL round-bottom tube, add 3 mL of LB and 3 μL of 1000X antibiotic(s). <br><br />
2. Pick one isolated colony, do not collect satellites or colony clumps, with a pipette tip. <br><br />
3. Swirl the colony tip in the tube, there should be no visible cell clumps. <br><br />
4. Incubate and shake the tube at 37 °C at 250 rpm for 12-16 hours and no longer than 20 hours. <br><br />
<br />
</p><br />
<br />
<br />
<h3> DNA Extraction and Mini-Preps </h3><br />
<br />
<br />
<p><br />
<br />
All DNA Mini-Preps were prepared using EPOCH Mini-Prep Kits and following the supplied protocols.<br />
<br />
</p><br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
1. Take 1-2 mL from an overnight culture and transfer into a 1.5 mL centrifuge tube. <br><br />
2. Spin down the culture at 3000 rpm for 3 minutes. <br><br />
3. Decant the supernatant. <br><br />
4. Resuspend the cells in 500 μL of 40% glycerol and 500 μL of LB (no antibiotics) or water. <br><br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
6. Store the glycerol stock at -80 °C. <br><br />
<br />
<br />
</p><br />
<br />
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<html><a name="Saccharomyces cerevisiae"></a><br />
<br />
</html><br />
<br />
<h2> <i> Saccharomyces cerevisiae </i> (PyE1 Yeast) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<br />
<p><br />
<br />
This process take four days in lab with a one day wait for incubation. <br><br />
<br />
<br />
Day 1: <br><br />
1. Streak yeast cells onto a YPD plate.* <br><br />
2. Invert the plate and incubate at 30 °C for 2 days. <br><br />
<br />
Day 3: <br><br />
1. Add 50 mL of YPD liquid media into a 250 mL baffle flask. <br><br />
2. Swipe as many individual colonies as you can see into the YPD media.** <br><br />
3. Incubate and shake the culture at 30 °C at 250 rpm overnight approximately 24 hours. <br><br />
<br />
Day 4: <br><br />
1. Take an optical density measurement. <br><br />
2. In three 250 mL baffle flask add the portions of the overnight liquid culture. <br><br />
3. Dilute each culture to approximately 0.4 optical density with YPD. <br><br />
4. Incubate and shake the cultures at 30 °C at 250 rpm until the optical density reaches 1.2-1.6. <br><br />
5. Collect each culture into separate 50 mL flat-bottomed centrifuge tubes. <br><br />
6. Spin down the cells at 4000 x g for 5 minutes at 4 °C. <br><br />
7. Decant the supernatant. <br><br />
8. Resuspend the cells in 100 mL total for all three culture of dH2O. <br><br />
9. Combine the suspensions into two 50 mL flat-bottomed centrifuge tubes. <br><br />
10. Spin down the cells as above. <br><br />
11. Decant the supernatant. <br><br />
12. Resuspend each in 3 mL of 100 mM lithium acetate. <br><br />
13. Transfer both cultures into a single 15 mL conical centrifuge tube. <br><br />
14. Spin down the cells at 3000 rpm for 5 minutes. <br><br />
15. Resuspend the cells in 0.75 mL of 100 mM lithium acetate, total volume is roughly 2 mL. <br><br />
16. Qualitatively bring up the volume to 3.5 mL by adding 40% glycerol. <br><br />
17. Aliquot the cells into 1.5 mL centrifuge tubes or 1.7 mL cryogenic vials.*** <br><br />
<br><br />
*Streak in such a way that there are individual colonies visible on the plate without clumps or satellite colonies. <br><br />
**Collect only individual visible colonies. Do not collect clumps or satellite colonies. <br><br />
***The volume of aliquots depends on the number to transformations you intend to do at a time. <br><br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Chemically Competent Transformations </h3><br />
<br />
<br />
<p><br />
<br />
This protocol assumes a 50 μL aliquot of yeast competent cells were made. Furthermore, this protocol prepares enough cells for six yeast transformations. <br><br />
<br><br />
1. Add the following to 50 μL of yeast competent cells: <br><br />
240 μL of polyethylene glycol - 3350 (PEG-3350) <br><br />
36 μL of 1 M lithium acetate <br><br />
32 μL of milliQ H2O <br><br />
<br />
2. Mix the mixture by gently pipetting or vortexing. <br><br />
3. Aliquot 59 μL of the mixture into a 0.2 mL microcentrifuge tube. <br><br />
4. Add 1 μL (~100-200 ng) of DNA.* <br><br />
5. Mix the mixture by gentle pipetting or vortexing. <br><br />
6. Incubate the mixture at 30 °C for 30 minutes. <br><br />
7. Heat shock the mixture at 42 °C for 20 minutes. <br><br />
8. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
9. Decant the supernatant. <br><br />
10. Resuspend the cell pellets in 200 μL of dH2O. <br><br />
11. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
12. Resuspend the cell pellets in 200 μL of dH2O. <br><br />
13. Plate 50-150 μL of the mixture onto an appropriate selective dropout media plate. <br><br />
14. Invert and incubate at 30 °C for 2 days. <br><br />
<br><br />
*The exact amount of DNA depends on the transformation efficiency of your competent cells. <br />
<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnight Culturing </h3><br />
<br />
<br />
<p><br />
<br />
1. In a 14 mL round-bottomed culture tube add 1.8 mL selective dropout media and 0.2 mL 20% glucose. <br><br />
2. Swipe 3 isolated yeast colonies and add them to the culture tube media. <br><br />
3. Incubate and shake at 37 °C at 250 rpm for 2 days. <br><br />
<br><br />
Note: You can also make 3 mL cultures (2.7 mL S.D. media and 0.3 mL 20% glucose) or larger cultures, just make sure to dilute the glucose from 20% to 2%.<br />
<br />
</p><br />
<br />
<br />
<h3> Culture Passaging </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Take 20-50μL from a previous overnight or passage culture and add it to the culture media.<br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br><br />
<br />
Note: You can also do 3 mL cultures (2.7 mL S.D. media and 0.3 mL 20% glucose) or larger cultures, just make sure to dilute the glucose from 20% to 2%. <br><br />
Note: The exact amount of culture that you take from a previous culture is irrelevant as long as at least 1 living cell is passaged.<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
1. Take 1-2 mL from an overnight culture and transfer into a 1.5 mL centrifuge tube. <br><br />
2. Spin down the culture at 3000 rpm for 3 minutes. <br><br />
3. Decant the supernatant. <br><br />
4. Resuspend the cells in 500 μL of 40% glycerol and 500 μL of selective dropout media or water. <br><br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
6. Store the glycerol stock at -80 °C. <br><br />
<br />
<br />
</p><br />
<br />
<html><br />
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<br><a href="#top">Back To Top</a> <br><br />
</html><br />
<br />
<html><a name="Flow Cytometry"></a><br />
<br />
</html><br />
<br />
<h2> Flow Cytometry </h2><br />
<br />
<br />
<h3> Dilutions </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. From an overnight culture measure the optical density at 660 nm by making 1:10 dilutions. <br><br />
2. Take enough culture to make a 1 mL aliquot with an OD of 0.4. <br><br />
3. Spin down the aliquot in a 1.5 mL centrifuge tube at 3000 rpm for 3 minutes. <br><br />
4. Decant the supernatant. <br><br />
5. Resuspend the cell pellet in 800 μL of the appropriate selective dropout media and 200 μL of 20% glucose. <br><br />
6. Transfer the new culture to a 14 mL culture tube. <br><br />
7. Incubate and shake at 30 °C and 250 rpm for at least 6 hours (1.2-1.6 optical density).<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Preparations for Analysis using C6 Accuri Flow Cytometer </h3><br />
<br />
<br />
<p><br />
<br />
1. From the dilution previously made, measure the optical density, roughly 1.2-1.6. <br><br />
2. Make an aliquot of 500 μL of the dilution culture in a 1.5 mL centrifuge tube. <br><br />
3. Spin down the aliquot at 3000 rpm for 3 minutes. <br><br />
4. Decant the supernatant. <br><br />
5. Resuspend the cell pellet in 500 μL of PBSF. <br><br />
6. Spin down the resuspension at 3000 rpm for 3 minutes. <br><br />
7. Decant the supernatant. <br><br />
8. Resuspend the cell pellet in another 500 μL of PBSF.* <br><br />
9. Prepare the C6 Accuri Flow Cytometer by running a backflush cycle and a diH2O cycle. <br><br />
10. Load the sample onto the sip. <br><br />
11. Run the sample with 100,000 cell count. <br><br />
12. Repeat for all samples and make sure to change data cells otherwise the old data will be erased. <br><br />
13. Once finished, run a cleaning cycle with Accuri approved cleaning solution, then run a diH2O cycle. <br><br />
<br> <br />
*For special cases do not resuspend all samples, instead resuspend immediately before running the sample through the flow cytometer.<br />
<br />
<br />
</p><br />
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<br />
<html><a name="Fluorescence Activated Cell Sorting"></a><br />
<br />
</html><br />
<br />
<h2> Fluorescence Activated Cell Sorting </h2><br />
<br />
<br />
<h3> Final Preparations </h3><br />
<br />
<br />
<br />
<p> <br />
<br />
Sample Prep:<br />
<br />
<br />
Spin down samples and negative control (5000 RPM, 1 min), keeping in mind the library size. Aspirate off supernatant. Resuspend in PBSF. Spin down cells. Aspirate off supernatant. Resuspend in PBSF. <br><br />
<br />
<br><br />
<br />
1. Open the “iGEM Template” file in the FACS Software and change name to current date and sort cycle<br><br />
<br />
2. Make sure the stream is stable (look for green light in bottom right corner). If not, run the Sort Calibration order. <br><br />
<br />
3. Run Negative Control from the PyE1 cells. <br><br />
<br />
a. Load cells onto carrier and into the machine. Press play button on screen. <br><br />
<br />
b. Set gate around lower left quadrant of cells to ensure single cell analysis using Forward Scatter Area and Side Scatter Area as your axes. Make sure oval gate covers around 80% of cell population. <br><br />
<br />
c. Set second gate on the first gated population by double-clicking on the gated population and using Forward Scatter Height and Forward Scatter Width as your axes. You will notice two distinct populations. Try to focus on the single cell portion of the plot. <br><br />
<br />
NOTE: If you see a large portion of the second gated population existing near the upper right edge of the first gate, you may need to enlarge the first gate to fit more of the population. <br><br />
<br />
d. Press record. Record 100,000 events and stop run. Move to Next Tube. <br><br />
<br />
4. Run first control (Gene clone) <br><br />
<br />
a. Follow step 3 to run the first control <br><br />
<br />
5. Run first library sample <br><br />
<br />
a. Follow steps 3b and 3c to set first two gates correctly. <br><br />
<br />
b. Set final gate for sort which includes top 1% of GFP producers from second gated population. <br><br />
<br />
c. Use final gate to set up the sort. <br><br />
<br />
d. Select sort conditions at the bottom of the screen. <br><br />
<br />
e. Insert and load collection tube. <br><br />
<br />
f. Record 100,000 events, and sort 10x the library size <br><br />
<br />
6. Run Bleach and diH2O through FACS to avoid cross-contamination. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Protein Expression"></a><br />
<br />
</html><br />
<br />
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</html><br />
<br />
<h2> Protein Expression </h2><br />
<br />
<br />
<h3> Overnight Cultures </h3><br />
<br />
<p><br />
<br />
1. Add 25 mL TB and 25 μL 1000X Kan to a 250 mL baffled flask. <br><br />
2. Stab a glycerol stock with a P1000 pipette and swirl in the flask of media. <br><br />
3. Put flask in 37 °C shaker at 250 rpm for 16-20 hrs.<br> <br />
<br />
<br />
</p><br />
<br />
<h3> Protein Expression </h3><br />
<br />
<p> <br />
<br />
1. Add 500 μL 1000X Kanamycin and 1 mL MgSO4 to 500 mL TB in 2 L baffled flask. <br><br />
2. Transfer 10 mL overnight culture to TB. <br><br />
3. Shake at 37 °C and 250 rpm until OD600 is between 0.5 and 0.8. <br><br />
4. Allow flask to rest at room temp for 30 min. <br><br />
5. Add 125 μL 1 M IPTG. <br><br />
6. Shake flask at 18 °C for ~16-20 hrs.<br />
<br />
</p><br />
<br />
<h3> Protein Extraction and Purification </h3><br />
<br />
<p><br />
1. Transfer cell culture to centrifuge tube. <br><br />
2. Centrifuge culture at 4000 x g for 10 min. <br><br />
3. Discard supernatant. <br><br />
4. Resuspend pellet in 25 mL wash buffer and add 250 μL of 100X PMSF, 250 μL of 100 mg/mL lysozyme, and 250 μL of 10 mg/mL DNAse. <br><br />
5. Sonicate sample with 0.25 inch probe for 5 min at 70% amplitude with 20 sec on and off pulses. <br><br />
6. Take 50 μL total sample. <br><br />
7. Transfer lysate to SS-34 centrifuge tube. <br><br />
8. Centrifuge for 30 min at 18000 x g. <br><br />
9. Take 50 μL soluble sample.<br />
<br />
<br />
</p><br />
<br />
<h3> Nickel Nitrotriacetic Acid Chromatography (Nickel-NTA Chromatography) </h3><br />
<br />
<p><br />
<br />
1. Add 5 mL 50%(v/v) nickel resin in ethanol to a 25 mL gravity flow column and allow to settle to 2.5 mL(CV). <br><br />
2. Rinse with 10CV dH2O. <br><br />
3. Equilibrate with 10CV lysis buffer. <br><br />
4. Load sample onto column. <br><br />
5. Wash column with 15CV lysis buffer. <br><br />
6. Perform 2 additional wash steps with 15CV. <br><br />
7. Elute sample in 10CV elution buffer and collect eluate. <br><br />
8. Take 50 μL pure sample. <br />
<br />
</p><br />
<br />
<h3> Size Exclusion Chromatography (S.E.C.) </h3><br />
<p><br />
1. Concentrate sample to as high as possible without inducing protein aggregation.<br><br />
2. Pre-equilibrate Superdex 75 column with 48 mL PBS. <br><br />
3. Inject 500 μL sample onto column. <br><br />
4. Run 36 mL PBS through column at 0.5 mL/min, collecting 1 mL fractions. <br><br />
5. Verify presence of protein in fractions by measuring concentration on Nanodrop and running SDS-PAGE (15 kDa protein should elute at ~13 mL).<br><br />
6. Pool fractions containing protein.<br />
<br />
</p><br />
<br />
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<br />
<html><a name="Stability Analysis"></a><br />
<br />
</html><br />
<br />
<h2> Stability Analysis </h2><br />
<br />
<br />
<h3> Circular Dichroism: Wavelength Scan </h3><br />
<p><br />
1. Load 1mm cuvette with 400 μL protein solution onto CD. <br><br />
2. Take wavelength scan: <br><br />
260 nm-190 nm <br><br />
sample every 1 nm <br><br />
averaging time 3 sec <br><br />
1 scan <br><br />
step scan <br><br />
25 °C <br><br />
3. Record wavelength which gives strongest signal (222 nm).<br />
</p><br />
<br />
<h3> Circular Dichroism: Guanidine Melt </h3><br />
<p><br />
1. Load 1 cm cuvette containing 1.996 mL of 0.05 mg/mL protein solution and stirrer onto CD. <br><br />
2. Prepare 8 mL of 0.05 mg/mL protein in concentrated guanidine solution. <br><br />
3. Set up Automixer with guanidine solution on one syringe and waste tube on other syringe. <br><br />
4. Titrate up to 6 M guanidine, taking a CD measurement at 222 nm every 0.15 M interval. <br><br />
5. Also measure the fluorescence at 280 nm to ensure the total protein concentration is not changing.<br />
<br />
</p><br />
<br />
<br />
<br />
<html><br />
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</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2014-10-17T22:50:41Z<p>Kristanguyen: </p>
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<br />
<h1> Protocols </h1><br />
<br />
<br />
<html> <a name="Media, Buffers and Solutions"></a> </html><br />
<br />
<br />
<h2> Media, Plates, and Solutions </h2><br />
<br />
<br />
<h3> Competent Cell Media Buffer (CCMB) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container:<br />
</p><br />
<p><br />
<br />
<UL><br />
<br />
<Li> 100 g glycerol (liquid) <br><br />
<br />
<LI> 10 mL x 1 M potassium acetate <br><br />
<br />
<LI> 11.8 g CaCl2*H2O <br><br />
<br />
<LI> 4 g MnCl2 <br><br />
<br />
<LI> 2 g MgCl2 <br><br />
<br />
<LI> 1 L of dH2O<br />
<br />
</UL><br />
</p><br />
<p><br />
Sterile filter or autoclave in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Super Optimal Broth (SOB) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br />
<br />
</p><br />
<br />
<p><br />
<br />
<UL><br />
<br />
<LI> 20 g tryptone <br><br />
<br />
<LI> 5 g yeast extract <br><br />
<br />
<LI> 10 mL x 1 M NaCl <br><br />
<br />
<LI> 2.5 mL x 1 M KCl <br><br />
<br />
<LI> 1 L of dH2O<br />
<br />
</UL><br />
<br />
</p><br />
<br />
<p><br />
<br />
Sterile filter or autoclave in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Phosphate Buffered Saline (PBS) Solution </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following in a 2 L container or 1 L beaker: <br />
<br />
</p><br />
<br />
<p><br />
<br />
<UL><br />
<br />
<LI>8 g NaCl <br><br />
<br />
<LI>1.44 g Na2HPO4 <br><br />
<br />
<LI>0.8 g KCl <br><br />
<br />
<LI>0.24 g KH2PO4 <br><br />
<br />
<LI>1 L of dH2O <br />
<br />
</UL><br />
<br />
</p><br />
<br />
<p><br />
<br />
Buffer to pH 7.4 <br><br />
<br />
Sterile filter or autoclave in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3>PBSF (PBS for Flow)</h3><br />
<br />
<br />
<p><br />
<br />
Mix the following in a 1 L beaker:<br />
<br />
</p><br />
<br />
<p><br />
<br />
<UL><br />
<br />
<LI> 25 mL 20X PBS, pH 7.4<br><br />
<br />
<LI> 475 mL H2O<br><br />
<br />
<LI> 2.5 g BSA (0.5%)*<br />
<br />
</UL><br />
<br />
</p><br />
<br />
<p><br />
<br />
Sterile filter in a 1L bottle and store at 4 °C <br />
<br />
</p><br />
<br />
<br />
<h3> Yeast Extract Peptone Dextrose (YPD) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following into 950 mL of dH2O in a 1 L bottle: <br />
<br />
</p><br />
<br />
<p><br />
<br />
<UL><br />
<br />
<LI> 20 g peptone <br><br />
<br />
<LI> 10 g yeast extract<br />
<br />
</UL><br />
<br />
</p><br />
<p><br />
<br />
Autoclave <br><br />
<br />
Add 50 mL 40% glucose <br><br />
<br />
Sterile filter into a 1 L bottle <br><br />
<br />
<br><br />
<br />
Note: For long-term liquid media storage, do not add 40% glucose. Instead add the glucose directly into cell cultures. <br><br />
<br />
Note: For YPD-plates add 24 g agar to the peptone and yeast extract before autoclaving. <br> <br />
<br />
</p><br />
<br />
<br />
<h3> Selective Dropout Media, C-Uracil and C-Histidine (C-Ura and C-His) </h3><br />
<br />
<br />
<p><br />
<br />
Synthesized by the Yeast Resource Center at the University of Washington's <br />
<br />
Department of Genome Sciences and Department of Biochemistry.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<h3> Guanidinium Hydrogen Chloride </h3><br />
<br />
<br />
<p><br />
<br />
For maximum effectiveness, final concentration should be approximately 8.5 M in PBS<br><br />
<br />
Add the following to a 500mL beaker and mix:<br />
<br />
</p><br />
<br />
<p><br />
<br />
<UL><br />
<br />
<LI> 203 g guanidinium hydrogen chloride <br><br />
<br />
<LI> 250 mL PBS solution* <br><br />
<br />
<LI> Add dilute HCl to pH 7.4<br />
<br />
</UL> <br />
<br />
</p><br />
<br />
<p><br />
<br />
*It is not necessary to filter or autoclave. <br><br />
<br />
*Alternatively add slightly less than 250 mL of PBS in order to buffer the solution to the appropriate volume, then add more dH2O as necessary. <br />
<br />
</p><br />
<br />
<br />
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<br />
<html><a name="Basic Cloning"></a><br />
<br />
</html><br />
<br />
<h2> Basic Cloning </h2><br />
<br />
<br />
<br />
<h3> Polymerase Chain Reaction </h3><br />
<br />
<br />
<p><br />
<br />
All PCRs were done using a standard 50 μL reaction volume with GoTaq® Green Master Mix 2X purchased from PROMEGA Corporation. <br><br />
<br />
Mix the following in a 0.2 mL microcentrifuge tube on ice: <br><br />
<br />
25 μL GoTaq® Green Master Mix 2X <br><br />
<br />
1-5 μL of 10 μM forward primer <br><br />
<br />
1-5 μL of 10 μM reverse primer <br><br />
<br />
<250 ng of DNA template <br><br />
<br />
QS 50 μl nuclease-free H2O <br><br />
<br />
Conduct the reaction in a thermocycler, adjusting anneal temperature and extension times accordingly. See your polymerase supplier protocol for more details on thermocycling.<br />
<br />
</p><br />
<br />
<h3> Error-prone Polymerase Chain Reaction </h3><br />
<br />
<br />
<p> <br />
<br />
<br />
<br />
Prepare 50 μL reaction: <br><br />
<br />
5 μL 10X Mutazyme II Rxn Buffer <br><br />
<br />
1 μL 40 mM dNTP mix (200 μM each final) <br><br />
<br />
1 μL 20 μM forward primer <br><br />
<br />
1 μL 20 μM reverse primer <br><br />
<br />
1 μL Mutazyme II DNA polymerase (2.5 U/μL) <br><br />
<br />
0.01 ng template <br><br />
<br />
QS 50 μL diH2O <br><br />
<br />
<br><br />
<br />
Program thermocycler as follows: <br><br />
<br />
95 °C, 2 min <br><br />
<br />
95 °C, 30 sec <br><br />
<br />
XX °C*, 30 sec <br><br />
<br />
72 °C, X min** <br><br />
<br />
32 cycles <br><br />
<br />
72 °C, 10 min <br><br />
<br />
4 °C, hold<br />
<br />
<br><br />
<br />
*Adjust annealing temperature according to Tm of primer. <br><br />
<br />
**Adjust extension time according to the length of amplified DNA. <br><br />
<br />
<br><br />
<br />
Note: Use 0.01 ng of template (calculate by insert and not by total plasmid). <br><br />
<br />
Calculate amount of template to use as follows: <br><br />
<br />
(bp for amplified region) / (bp in total plasmid) = % amplified region <br><br />
<br />
(conc. of total plasmid) x (% amplified region as a decimal) = conc. of amplified region <br><br />
<br />
Note: Never pipette less than 0.5 μL. <br><br />
<br />
(0.01 ng of template) / (conc. of amplified region) = vol of template to add to PCR <br><br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Restriction Endonuclease Reaction (Digestion) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
All restriction enzyme reactions were done using a 50 μl reaction volume. Restriction enzymes and buffers were purchased from New England Biolabs® Inc. <br><br />
<br />
Mix the following in a 0.2 mL PCR tube: <br><br />
<br />
<br />
1 μg of DNA <br><br />
<br />
5 μL of the appropriate 10X New England Biolab® Buffer <br><br />
<br />
1 μL of each restriction enzyme (add last) <br><br />
<br />
QS 50μL nuclease-free H2O <br><br />
<br />
Incubate the reaction for 1 hr <br><br />
<br />
Heat inactive the reaction at the appropriate temperature <br><br />
<br><br />
<br />
Note: Thaw the restriction enzyme(s) on ice to improve shelf life <br><br />
<br />
<br />
</p> <br />
<br />
<br />
<h3> Ligation </h3><br />
<br />
<br />
<p><br />
<br />
T4 DNA Ligase and Buffer were purchased from New England Biolabs® Inc. <br><br />
<br />
1. Prepare the following in a 0.2 mL microcentrifuge tube: <br><br />
<br />
50.0 ng vector DNA* <br><br />
<br />
37.5 ng vector DNA* <br><br />
<br />
2 μL 10X T4 DNA Ligase Buffer <br><br />
<br />
1 μL T4 DNA Ligase <br><br />
<br />
QS 20 μL diH2O <br><br />
<br />
2. Incubate the reaction at room temperature for 10-30 minutes or at 16 °C overnight. <br><br />
<br />
3. Heat inactivate at 65 °C for 10 minutes. <br><br />
<br />
4. Chill on ice before starting a transformation reaction. <br><br />
<br />
<br><br />
<br />
*The exact amount of DNA is dependent on the number of base pairs. In order to conduct a proper reaction consult the New England Biolab Ligation Calculator at:<br />
<br />
http://nebiocalculator.neb.com/#!/<br />
<br />
</p><br />
<br />
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<html><a name="Escherichia coli Protocols"></a><br />
<br />
</html><br />
<br />
<br />
<h2> <i> Escherichia coli </i> Protocols (XL1-Blue and XL10-Gold) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<p> <br />
Competent cells take two days to culture and aliquot. <br><br />
Day 1: <br><br />
1. Streak an aliquot of competent cells onto two LB-plates without antibiotics.* <br><br />
2. Incubate at 37 °C overnight. <br><br />
Day 2: <br><br />
1. In two 250 mL baffle flasks add 50 mL of SOB media. <br><br />
2. Scrape as many single colonies into either flask. <br><br />
3. Incubate and shake at 37 °C and 250 rpm for 2-3 hours. <br><br />
4. Check the optical density of the cells at 600 nm after 2 hours. <br><br />
5. Stop incubation when cultures reach approximately 0.5 optical density. <br><br />
6. Add the contents of the flask into separate 50 mL flat bottomed centrifuge tubes. <br><br />
7. Spin down the cells at 2500 rpm at 4 °C for 15 minutes. <br><br />
8. Decant the supernatant. <br><br />
9. Resuspend the cells in 16 mL of CCMB by pipetting or gently vortexing. <br><br />
10. Incubate the cells on ice for 20 minutes. <br><br />
11. Spin down the cells at 2500 rpm at 4 °C for 10 minutes. <br> <br />
12. Decant the supernatant. <br><br />
13. Resuspend the cells in 4 mL of CCMB. <br><br />
14. Quickly aliquot the cells into 1.7 mL cryogenic vials or 1.5 mL centrifuge tubes.** <br><br />
15. Store the competent cell aliquots at -80 °C. <br><br />
<br><br />
*Streak in such a way that there should be individual colony growth and no clumps after the incubation. <br><br />
**We did this in a -20 °C cold room and using an automated repeater pipette. The volume of each aliquot depends on the number of transformations you intend to do at a time. <br><br />
<br><br />
Note: After removing the cells from incubation keep them on ice or as cold as possible. <br><br />
</p><br />
<br />
<br />
<h3> Chemically Competent Cell Transformations </h3><br />
<br />
<br />
<p> <br />
1. Thaw competent <i> E. coli </i> cells on ice (XL1-Blue or XL10-Gold).* <br><br />
2. Add 50 μL of competent cells to sterile 14 mL culture tube. <br><br />
3. Add 1 μL (~100-200 ng)* of the mini-prep to each culture tube. <br><br />
4. Equilibrate the cells on ice for 10 minutes. <br><br />
5. Heat shock the cells at 42 °C for 30-45 seconds.** <br><br />
6. Immediately place the cells back on ice for 3 minutes. <br><br />
7. Add 250 μL LB media without antibiotics and shake at 250 rpm and 37 °C for 30 minutes. <br><br />
8. Spread 10 μL and 290 μL on an appropriate LB-antibiotic plate. <br><br />
9. Invert the plate and incubate at 37 °C overnight. <br><br />
<br><br />
*The exact amount of DNA to add depends on your cell's transformation efficiency. However, it is acceptable to add a larger amount to increase the number of transformed cells. <br><br />
**Do not heat shock for an extended duration as this may damage and/or kill your cells.<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnights </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14 mL round-bottom tube, add 3 mL of LB and 3 μL of 1000X antibiotic(s). <br><br />
2. Pick one isolated colony, do not collect satellites or colony clumps, with a pipette tip. <br><br />
3. Swirl the colony tip in the tube, there should be no visible cell clumps. <br><br />
4. Incubate and shake the tube at 37 °C at 250 rpm for 12-16 hours and no longer than 20 hours. <br><br />
<br />
</p><br />
<br />
<br />
<h3> DNA Extraction and Mini-Preps </h3><br />
<br />
<br />
<p><br />
<br />
All DNA Mini-Preps were prepared using EPOCH Mini-Prep Kits and following the supplied protocols.<br />
<br />
</p><br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
1. Take 1-2 mL from an overnight culture and transfer into a 1.5 mL centrifuge tube. <br><br />
2. Spin down the culture at 3000 rpm for 3 minutes. <br><br />
3. Decant the supernatant. <br><br />
4. Resuspend the cells in 500 μL of 40% glycerol and 500 μL of LB (no antibiotics) or water. <br><br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
6. Store the glycerol stock at -80 °C. <br><br />
<br />
<br />
</p><br />
<br />
<html><br />
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</html><br />
<br />
<html><a name="Saccharomyces cerevisiae"></a><br />
<br />
</html><br />
<br />
<h2> <i> Saccharomyces cerevisiae </i> (PyE1 Yeast) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<br />
<p><br />
<br />
This process take four days in lab with a one day wait for incubation. <br><br />
<br />
<br />
Day 1: <br><br />
1. Streak yeast cells onto a YPD plate.* <br><br />
2. Invert the plate and incubate at 30 °C for 2 days. <br><br />
<br />
Day 3: <br><br />
1. Add 50 mL of YPD liquid media into a 250 mL baffle flask. <br><br />
2. Swipe as many individual colonies as you can see into the YPD media.** <br><br />
3. Incubate and shake the culture at 30 °C at 250 rpm overnight approximately 24 hours. <br><br />
<br />
Day 4: <br><br />
1. Take an optical density measurement. <br><br />
2. In three 250 mL baffle flask add the portions of the overnight liquid culture. <br><br />
3. Dilute each culture to approximately 0.4 optical density with YPD. <br><br />
4. Incubate and shake the cultures at 30 °C at 250 rpm until the optical density reaches 1.2-1.6. <br><br />
5. Collect each culture into separate 50 mL flat-bottomed centrifuge tubes. <br><br />
6. Spin down the cells at 4000 x g for 5 minutes at 4 °C. <br><br />
7. Decant the supernatant. <br><br />
8. Resuspend the cells in 100 mL total for all three culture of dH2O. <br><br />
9. Combine the suspensions into two 50 mL flat-bottomed centrifuge tubes. <br><br />
10. Spin down the cells as above. <br><br />
11. Decant the supernatant. <br><br />
12. Resuspend each in 3 mL of 100 mM lithium acetate. <br><br />
13. Transfer both cultures into a single 15 mL conical centrifuge tube. <br><br />
14. Spin down the cells at 3000 rpm for 5 minutes. <br><br />
15. Resuspend the cells in 0.75 mL of 100 mM lithium acetate, total volume is roughly 2 mL. <br><br />
16. Qualitatively bring up the volume to 3.5 mL by adding 40% glycerol. <br><br />
17. Aliquot the cells into 1.5 mL centrifuge tubes or 1.7 mL cryogenic vials.*** <br><br />
<br><br />
*Streak in such a way that there are individual colonies visible on the plate without clumps or satellite colonies. <br><br />
**Collect only individual visible colonies. Do not collect clumps or satellite colonies. <br><br />
***The volume of aliquots depends on the number to transformations you intend to do at a time. <br><br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Chemically Competent Transformations </h3><br />
<br />
<br />
<p><br />
<br />
This protocol assumes a 50 μL aliquot of yeast competent cells were made. Furthermore, this protocol prepares enough cells for six yeast transformations. <br><br />
<br><br />
1. Add the following to 50 μL of yeast competent cells: <br><br />
240 μL of polyethylene glycol - 3350 (PEG-3350) <br><br />
36 μL of 1 M lithium acetate <br><br />
32 μL of milliQ H2O <br><br />
<br />
2. Mix the mixture by gently pipetting or vortexing. <br><br />
3. Aliquot 59 μL of the mixture into a 0.2 mL microcentrifuge tube. <br><br />
4. Add 1 μL (~100-200 ng) of DNA.* <br><br />
5. Mix the mixture by gentle pipetting or vortexing. <br><br />
6. Incubate the mixture at 30 °C for 30 minutes. <br><br />
7. Heat shock the mixture at 42 °C for 20 minutes. <br><br />
8. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
9. Decant the supernatant. <br><br />
10. Resuspend the cell pellets in 200 μL of dH2O. <br><br />
11. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
12. Resuspend the cell pellets in 200 μL of dH2O. <br><br />
13. Plate 50-150 μL of the mixture onto an appropriate selective dropout media plate. <br><br />
14. Invert and incubate at 30 °C for 2 days. <br><br />
<br><br />
*The exact amount of DNA depends on the transformation efficiency of your competent cells. <br />
<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnight Culturing </h3><br />
<br />
<br />
<p><br />
<br />
1. In a 14 mL round-bottomed culture tube add 1.8 mL selective dropout media and 0.2 mL 20% glucose. <br><br />
2. Swipe 3 isolated yeast colonies and add them to the culture tube media. <br><br />
3. Incubate and shake at 37 °C at 250 rpm for 2 days. <br><br />
<br><br />
Note: You can also make 3 mL cultures (2.7 mL S.D. media and 0.3 mL 20% glucose) or larger cultures, just make sure to dilute the glucose from 20% to 2%.<br />
<br />
</p><br />
<br />
<br />
<h3> Culture Passaging </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Take 20-50μL from a previous overnight or passage culture and add it to the culture media.<br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br><br />
<br />
Note: You can also do 3 mL cultures (2.7 mL S.D. media and 0.3 mL 20% glucose) or larger cultures, just make sure to dilute the glucose from 20% to 2%. <br><br />
Note: The exact amount of culture that you take from a previous culture is irrelevant as long as at least 1 living cell is passaged.<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
1. Take 1-2 mL from an overnight culture and transfer into a 1.5 mL centrifuge tube. <br><br />
2. Spin down the culture at 3000 rpm for 3 minutes. <br><br />
3. Decant the supernatant. <br><br />
4. Resuspend the cells in 500 μL of 40% glycerol and 500 μL of selective dropout media or water. <br><br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
6. Store the glycerol stock at -80 °C. <br><br />
<br />
<br />
</p><br />
<br />
<html><br />
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</html><br />
<br />
<html><a name="Flow Cytometry"></a><br />
<br />
</html><br />
<br />
<h2> Flow Cytometry </h2><br />
<br />
<br />
<h3> Dilutions </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. From an overnight culture measure the optical density at 660nm by making 1:10 dilutions. <br><br />
<br />
2. Take enough culture to make a 1mL aliquot with OD 0.4. <br><br />
<br />
3. Spin down the aliquot in a 1.5mL centrifuge tube at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 800μL of the appropriate selective dropout media and 200μL of 20% glucose. <br><br />
<br />
6. Transfer the new culture to a 14mL culture tube. <br><br />
<br />
7. Incubate and shake at 30C and 250rpm for at least 6 hours (1.2-1.6 optical density).<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Preparations for Analysis using C6 Accuri Flow Cytometer </h3><br />
<br />
<br />
<p><br />
<br />
<br />
1. From the dilution previously made, measure the optical density, roughly 1.2-1.6. <br><br />
<br />
2. Make an aliquot of 500μL of the dilution culture in a 1.5mL centrifuge tube. <br><br />
<br />
3. Spin down the aliquot at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 500μL of PBS(F). <br><br />
<br />
6. Spin down the resuspension at 3000rpm for 3 minutes. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cell pellet in another 500uL of PBS(F).* <br><br />
<br />
9. Prepare the C6 Accuri Flow Cytometer by running a backflush cycle and a dH2O cycle. <br><br />
<br />
10. Load the sample onto the sip. <br><br />
<br />
11. Run the sample with 100,000 cell count. <br><br />
<br />
12. Repeat for all samples and make sure to change data cells otherwise the old data is erased. <br><br />
<br />
13. Once finished, run a cleaning cycle with Accuri approved cleaning solution, then run a dH2O cycle. <br><br />
<br />
<br />
<br />
*For special cases do not resuspend all samples, instead resuspend immediately before running the sample through the flow cytometer.<br />
<br />
<br />
</p><br />
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</html><br />
<br />
<html><a name="Fluorescence Activated Cell Sorting"></a><br />
<br />
</html><br />
<br />
<h2> Fluorescence Activated Cell Sorting </h2><br />
<br />
<br />
<h3> Final Preparations </h3><br />
<br />
<br />
<br />
<p> <br />
<br />
Sample Prep:<br />
<br />
<br />
Spin down samples and negative control (5000 RPM, 1 min), keeping in mind the library size. Aspirate off supernatant. Resuspend in PBSF. Spin down cells. Aspirate off supernatant. Resuspend in PBSF. <br><br />
<br />
<br><br />
<br />
1. Open the “iGEM Template” file in the FACS Software and change name to current date and sort cycle<br><br />
<br />
2. Make sure the stream is stable (look for green light in bottom right corner). If not, run the Sort Calibration order. <br><br />
<br />
3. Run Negative Control from the PyE1 cells. <br><br />
<br />
a. Load cells onto carrier and into the machine. Press play button on screen. <br><br />
<br />
b. Set gate around lower left quadrant of cells to ensure single cell analysis using Forward Scatter Area and Side Scatter Area as your axes. Make sure oval gate covers around 80% of cell population. <br><br />
<br />
c. Set second gate on the first gated population by double-clicking on the gated population and using Forward Scatter Height and Forward Scatter Width as your axes. You will notice two distinct populations. Try to focus on the single cell portion of the plot. <br><br />
<br />
NOTE: If you see a large portion of the second gated population existing near the upper right edge of the first gate, you may need to enlarge the first gate to fit more of the population. <br><br />
<br />
d. Press record. Record 100,000 events and stop run. Move to Next Tube. <br><br />
<br />
4. Run first control (Gene clone) <br><br />
<br />
a. Follow step 3 to run the first control <br><br />
<br />
5. Run first library sample <br><br />
<br />
a. Follow steps 3b and 3c to set first two gates correctly. <br><br />
<br />
b. Set final gate for sort which includes top 1% of GFP producers from second gated population. <br><br />
<br />
c. Use final gate to set up the sort. <br><br />
<br />
d. Select sort conditions at the bottom of the screen. <br><br />
<br />
e. Insert and load collection tube. <br><br />
<br />
f. Record 100,000 events, and sort 10x the library size <br><br />
<br />
6. Run Bleach and diH2O through FACS to avoid cross-contamination. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Protein Expression"></a><br />
<br />
</html><br />
<br />
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</html><br />
<br />
<h2> Protein Expression </h2><br />
<br />
<br />
<h3> Overnight Cultures </h3><br />
<br />
<p><br />
<br />
1. Add 25mL TB and 25uL Kan to a 250mL baffled flask <br><br />
<br />
2. Stab a glycerol stock with a p1000 pipette and swirl in the flask of media <br><br />
<br />
3. Put flask in 37C shaker overnight <br />
<br />
</p><br />
<br />
<h3> Protein Expression </h3><br />
<br />
<p> <br />
<br />
1. Add 500μL 1000x Kanamycin to 500mL TB in 2L baffled flask <br><br />
<br />
2. Transfer 10mL overnight culture to TB <br><br />
<br />
3. Shake at 37C (DO WE NEED THE RPM?) <br><br />
<br />
4. Remove flask from shaker when optical density is between 0.5 and 0.8 <br><br />
<br />
5. Allow flask to rest at room temp for 30 min <br><br />
<br />
6. Add 125μL 1M IPTG <br><br />
<br />
7. Shake flask at 18C overnight<br />
<br />
</p><br />
<br />
<h3> Protein Extraction and Purification </h3><br />
<br />
<p><br />
<br />
1. Transfer cell culture to centrifuge flask <br><br />
<br />
2. Centrifuge culture at 4000g for 10 min <br><br />
<br />
3. Discard supernatant <br><br />
<br />
4. Resuspend pellet in 25mL lysis buffer <br><br />
<br />
5. Add 250μL of 100x PMSF, 250μL of 100mg/mL lysozyme, and 250μL of 10mg/mL DNAse <br><br />
<br />
6. Sonicate sample with 0.25inch probe for 5 min at 70% amplitude with 20 sec on and off pulses <br><br />
<br />
7. Take 50μL total sample <br><br />
<br />
8. Transfer lysate to SS-34 centrifuge tube <br><br />
<br />
9. Centrifuge for 30 min at 18000g <br><br />
<br />
10. Take 50μL soluble sample<br />
<br />
</p><br />
<br />
<h3> Nickel Nitrotriacetic Acid Chromatography (Nickel-NTA Chromatography) </h3><br />
<br />
<p><br />
<br />
1. Add 5mL 50%(v/v) nickel resin in ethanol to 25mL column and allow to settle to ~2.5mL(CV) <br><br />
<br />
2. Rinse with 10CV H20 <br><br />
<br />
3. Equilibrate with 10CV lysis buffer <br><br />
<br />
4. Load sample onto column <br><br />
<br />
5. Wash column with 15CV lysis buffer <br><br />
<br />
6. Perform 2 additional wash steps with 15CV <br><br />
<br />
7. Elute sample in 10CV elution buffer and collect eluate <br><br />
<br />
8. Take 50μL pure sample <br />
<br />
</p><br />
<br />
<h3> Size Exclusion Chromatography (S.E.C.) </h3><br />
<p><br />
1. Concentrate sample to as high as possible without inducing protein aggregation<br><br />
2. Pre-equilibrate Superdex 75 column with 48 ml PBS <br><br />
3. Inject 500 ul sample onto column <br><br />
4. Run 36 ml PBS through column at 0.5 ml/min, collecting 1 ml fractions <br><br />
5. Verify presence of protein in fractions by measuring concentration on Nanodrop and running SDS-PAGE (15 kDa protein should elute at ~13 ml)<br><br />
6. Pool fractions containing protein <br />
</p><br />
<br />
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<br />
<html><a name="Stability Analysis"></a><br />
<br />
</html><br />
<br />
<h2> Stability Analysis </h2><br />
<br />
<br />
<h3> Circular Dichroism: Wavelength Scan </h3><br />
<p><br />
1. Load 1mm cuvette with 400uL protein solution onto CD <br><br />
2. Take wavelength scan <br><br />
260nm-190nm <br><br />
sample every 1nm <br><br />
averaging time 3sec <br><br />
1 scan <br><br />
step scan <br><br />
25C <br><br />
3. Record wavelength which gives strongest signal (222nm)<br />
</p><br />
<br />
<h3> Circular Dichroism: Guanidine Melt </h3><br />
<p><br />
1. Load 1cm cuvette containing 1.996mL of 0.05mg/mL protein solution and stirrer onto CD <br><br />
2. Prepare 8mL of 0.05mg/mL protein in concentrated guanidine solution <br><br />
3. Set up Automixer with guanidine solution on one syringe and waste tube on other syringe <br><br />
4. Titrate up to 6 M guanidine, taking a CD measurement at 222 nm every 0.15 M interval <br><br />
5. Also measure the fluorescence at 280 nm to ensure the total protein concentration is not changing<br />
</p><br />
<br />
<br />
<br />
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</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2014-10-17T22:49:18Z<p>Kristanguyen: </p>
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<h1> Protocols </h1><br />
<br />
<br />
<html> <a name="Media, Buffers and Solutions"></a> </html><br />
<br />
<br />
<h2> Media, Plates, and Solutions </h2><br />
<br />
<br />
<h3> Competent Cell Media Buffer (CCMB) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container:<br />
</p><br />
<p><br />
<br />
<UL><br />
<br />
<Li> 100 g glycerol (liquid) <br><br />
<br />
<LI> 10 mL x 1 M potassium acetate <br><br />
<br />
<LI> 11.8 g CaCl2*H2O <br><br />
<br />
<LI> 4 g MnCl2 <br><br />
<br />
<LI> 2 g MgCl2 <br><br />
<br />
<LI> 1 L of dH2O<br />
<br />
</UL><br />
</p><br />
<p><br />
Sterile filter or autoclave in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Super Optimal Broth (SOB) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br />
<br />
</p><br />
<br />
<p><br />
<br />
<UL><br />
<br />
<LI> 20 g tryptone <br><br />
<br />
<LI> 5 g yeast extract <br><br />
<br />
<LI> 10 mL x 1 M NaCl <br><br />
<br />
<LI> 2.5 mL x 1 M KCl <br><br />
<br />
<LI> 1 L of dH2O<br />
<br />
</UL><br />
<br />
</p><br />
<br />
<p><br />
<br />
Sterile filter or autoclave in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Phosphate Buffered Saline (PBS) Solution </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following in a 2 L container or 1 L beaker: <br />
<br />
</p><br />
<br />
<p><br />
<br />
<UL><br />
<br />
<LI>8 g NaCl <br><br />
<br />
<LI>1.44 g Na2HPO4 <br><br />
<br />
<LI>0.8 g KCl <br><br />
<br />
<LI>0.24 g KH2PO4 <br><br />
<br />
<LI>1 L of dH2O <br />
<br />
</UL><br />
<br />
</p><br />
<br />
<p><br />
<br />
Buffer to pH 7.4 <br><br />
<br />
Sterile filter or autoclave in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3>PBSF (PBS for Flow)</h3><br />
<br />
<br />
<p><br />
<br />
Mix the following in a 1 L beaker:<br />
<br />
</p><br />
<br />
<p><br />
<br />
<UL><br />
<br />
<LI> 25 mL 20X PBS, pH 7.4<br><br />
<br />
<LI> 475 mL H2O<br><br />
<br />
<LI> 2.5 g BSA (0.5%)*<br />
<br />
</UL><br />
<br />
</p><br />
<br />
<p><br />
<br />
Sterile filter in a 1L bottle and store at 4 °C <br />
<br />
</p><br />
<br />
<br />
<h3> Yeast Extract Peptone Dextrose (YPD) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following into 950 mL of dH2O in a 1 L bottle: <br />
<br />
</p><br />
<br />
<p><br />
<br />
<UL><br />
<br />
<LI> 20 g peptone <br><br />
<br />
<LI> 10 g yeast extract<br />
<br />
</UL><br />
<br />
</p><br />
<p><br />
<br />
Autoclave <br><br />
<br />
Add 50 mL 40% glucose <br><br />
<br />
Sterile filter into a 1 L bottle <br><br />
<br />
<br><br />
<br />
Note: For long-term liquid media storage, do not add 40% glucose. Instead add the glucose directly into cell cultures. <br><br />
<br />
Note: For YPD-plates add 24 g agar to the peptone and yeast extract before autoclaving. <br> <br />
<br />
</p><br />
<br />
<br />
<h3> Selective Dropout Media, C-Uracil and C-Histidine (C-Ura and C-His) </h3><br />
<br />
<br />
<p><br />
<br />
Synthesized by the Yeast Resource Center at the University of Washington's <br />
<br />
Department of Genome Sciences and Department of Biochemistry.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<h3> Guanidinium Hydrogen Chloride </h3><br />
<br />
<br />
<p><br />
<br />
For maximum effectiveness, final concentration should be approximately 8.5 M in PBS<br><br />
<br />
Add the following to a 500mL beaker and mix:<br />
<br />
</p><br />
<br />
<p><br />
<br />
<UL><br />
<br />
<LI> 203 g guanidinium hydrogen chloride <br><br />
<br />
<LI> 250 mL PBS solution* <br><br />
<br />
<LI> Add dilute HCl to pH 7.4<br />
<br />
</UL> <br />
<br />
</p><br />
<br />
<p><br />
<br />
*It is not necessary to filter or autoclave. <br><br />
<br />
*Alternatively add slightly less than 250 mL of PBS in order to buffer the solution to the appropriate volume, then add more dH2O as necessary. <br />
<br />
</p><br />
<br />
<br />
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<br />
<html><a name="Basic Cloning"></a><br />
<br />
</html><br />
<br />
<h2> Basic Cloning </h2><br />
<br />
<br />
<br />
<h3> Polymerase Chain Reaction </h3><br />
<br />
<br />
<p><br />
<br />
All PCRs were done using a standard 50 μL reaction volume with GoTaq® Green Master Mix 2X purchased from PROMEGA Corporation. <br><br />
<br />
Mix the following in a 0.2 mL microcentrifuge tube on ice: <br><br />
<br />
25 μL GoTaq® Green Master Mix 2X <br><br />
<br />
1-5 μL of 10 μM forward primer <br><br />
<br />
1-5 μL of 10 μM reverse primer <br><br />
<br />
<250 ng of DNA template <br><br />
<br />
QS 50 μl nuclease-free H2O <br><br />
<br />
Conduct the reaction in a thermocycler, adjusting anneal temperature and extension times accordingly. See your polymerase supplier protocol for more details on thermocycling.<br />
<br />
</p><br />
<br />
<h3> Error-prone Polymerase Chain Reaction </h3><br />
<br />
<br />
<p> <br />
<br />
<br />
<br />
Prepare 50 μL reaction: <br><br />
<br />
5 μL 10X Mutazyme II Rxn Buffer <br><br />
<br />
1 μL 40 mM dNTP mix (200 μM each final) <br><br />
<br />
1 μL 20 μM forward primer <br><br />
<br />
1 μL 20 μM reverse primer <br><br />
<br />
1 μL Mutazyme II DNA polymerase (2.5 U/μL) <br><br />
<br />
0.01 ng template <br><br />
<br />
QS 50 μL diH2O <br><br />
<br />
<br><br />
<br />
Program thermocycler as follows: <br><br />
<br />
95 °C, 2 min <br><br />
<br />
95 °C, 30 sec <br><br />
<br />
XX °C*, 30 sec <br><br />
<br />
72 °C, X min** <br><br />
<br />
32 cycles <br><br />
<br />
72 °C, 10 min <br><br />
<br />
4 °C, hold<br />
<br />
<br><br />
<br />
*Adjust annealing temperature according to Tm of primer. <br><br />
<br />
**Adjust extension time according to the length of amplified DNA. <br><br />
<br />
<br><br />
<br />
Note: Use 0.01 ng of template (calculate by insert and not by total plasmid). <br><br />
<br />
Calculate amount of template to use as follows: <br><br />
<br />
(bp for amplified region) / (bp in total plasmid) = % amplified region <br><br />
<br />
(conc. of total plasmid) x (% amplified region as a decimal) = conc. of amplified region <br><br />
<br />
<br><br />
<br />
Note: Never pipette less than 0.5 μL. <br><br />
<br />
(0.01 ng of template) / (conc. of amplified region) = vol of template to add to PCR <br><br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Restriction Endonuclease Reaction (Digestion) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
All restriction enzyme reactions were done using a 50 μl reaction volume. Restriction enzymes and buffers were purchased from New England Biolabs® Inc. <br><br />
<br />
Mix the following in a 0.2 mL PCR tube: <br><br />
<br />
<br />
1 μg of DNA <br><br />
<br />
5 μL of the appropriate 10X New England Biolab® Buffer <br><br />
<br />
1 μL of each restriction enzyme (add last) <br><br />
<br />
QS 50μL nuclease-free H2O <br><br />
<br />
Incubate the reaction for 1 hr <br><br />
<br />
Heat inactive the reaction at the appropriate temperature <br><br />
<br><br />
<br />
Note: Thaw the restriction enzyme(s) on ice to improve shelf life <br><br />
<br />
<br />
</p> <br />
<br />
<br />
<h3> Ligation </h3><br />
<br />
<br />
<p><br />
<br />
T4 DNA Ligase and Buffer were purchased from New England Biolabs® Inc. <br><br />
<br />
1. Prepare the following in a 0.2 mL microcentrifuge tube: <br><br />
<br />
50.0 ng vector DNA* <br><br />
<br />
37.5 ng vector DNA* <br><br />
<br />
2 μL 10X T4 DNA Ligase Buffer <br><br />
<br />
1 μL T4 DNA Ligase <br><br />
<br />
QS 20 μL diH2O <br><br />
<br />
2. Incubate the reaction at room temperature for 10-30 minutes or at 16 °C overnight. <br><br />
<br />
3. Heat inactivate at 65 °C for 10 minutes. <br><br />
<br />
4. Chill on ice before starting a transformation reaction. <br><br />
<br />
<br><br />
<br />
*The exact amount of DNA is dependent on the number of base pairs. In order to conduct a proper reaction consult the New England Biolab Ligation Calculator at:<br />
<br />
http://nebiocalculator.neb.com/#!/<br />
<br />
</p><br />
<br />
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<br />
<html><a name="Escherichia coli Protocols"></a><br />
<br />
</html><br />
<br />
<br />
<h2> <i> Escherichia coli </i> Protocols (XL1-Blue and XL10-Gold) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<p> <br />
Competent cells take two days to culture and aliquot. <br><br />
Day 1: <br><br />
1. Streak an aliquot of competent cells onto two LB-plates without antibiotics.* <br><br />
2. Incubate at 37 °C overnight. <br><br />
Day 2: <br><br />
1. In two 250 mL baffle flasks add 50 mL of SOB media. <br><br />
2. Scrape as many single colonies into either flask. <br><br />
3. Incubate and shake at 37 °C and 250 rpm for 2-3 hours. <br><br />
4. Check the optical density of the cells at 600 nm after 2 hours. <br><br />
5. Stop incubation when cultures reach approximately 0.5 optical density. <br><br />
6. Add the contents of the flask into separate 50 mL flat bottomed centrifuge tubes. <br><br />
7. Spin down the cells at 2500 rpm at 4 °C for 15 minutes. <br><br />
8. Decant the supernatant. <br><br />
9. Resuspend the cells in 16 mL of CCMB by pipetting or gently vortexing. <br><br />
10. Incubate the cells on ice for 20 minutes. <br><br />
11. Spin down the cells at 2500 rpm at 4 °C for 10 minutes. <br> <br />
12. Decant the supernatant. <br><br />
13. Resuspend the cells in 4 mL of CCMB. <br><br />
14. Quickly aliquot the cells into 1.7 mL cryogenic vials or 1.5 mL centrifuge tubes.** <br><br />
15. Store the competent cell aliquots at -80 °C. <br><br />
<br><br />
*Streak in such a way that there should be individual colony growth and no clumps after the incubation. <br><br />
**We did this in a -20 °C cold room and using an automated repeater pipette. The volume of each aliquot depends on the number of transformations you intend to do at a time. <br><br />
<br><br />
Note: After removing the cells from incubation keep them on ice or as cold as possible. <br><br />
</p><br />
<br />
<br />
<h3> Chemically Competent Cell Transformations </h3><br />
<br />
<br />
<p> <br />
1. Thaw competent <i> E. coli </i> cells on ice (XL1-Blue or XL10-Gold).* <br><br />
2. Add 50 μL of competent cells to sterile 14 mL culture tube. <br><br />
3. Add 1 μL (~100-200 ng)* of the mini-prep to each culture tube. <br><br />
4. Equilibrate the cells on ice for 10 minutes. <br><br />
5. Heat shock the cells at 42 °C for 30-45 seconds.** <br><br />
6. Immediately place the cells back on ice for 3 minutes. <br><br />
7. Add 250 μL LB media without antibiotics and shake at 250 rpm and 37 °C for 30 minutes. <br><br />
8. Spread 10 μL and 290 μL on an appropriate LB-antibiotic plate. <br><br />
9. Invert the plate and incubate at 37 °C overnight. <br><br />
<br><br />
*The exact amount of DNA to add depends on your cell's transformation efficiency. However, it is acceptable to add a larger amount to increase the number of transformed cells. <br><br />
**Do not heat shock for an extended duration as this may damage and/or kill your cells.<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnights </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14 mL round-bottom tube, add 3 mL of LB and 3 μL of 1000X antibiotic(s). <br><br />
2. Pick one isolated colony, do not collect satellites or colony clumps, with a pipette tip. <br><br />
3. Swirl the colony tip in the tube, there should be no visible cell clumps. <br><br />
4. Incubate and shake the tube at 37 °C at 250 rpm for 12-16 hours and no longer than 20 hours. <br><br />
<br />
</p><br />
<br />
<br />
<h3> DNA Extraction and Mini-Preps </h3><br />
<br />
<br />
<p><br />
<br />
All DNA Mini-Preps were prepared using EPOCH Mini-Prep Kits and following the supplied protocols.<br />
<br />
</p><br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
1. Take 1-2 mL from an overnight culture and transfer into a 1.5 mL centrifuge tube. <br><br />
2. Spin down the culture at 3000 rpm for 3 minutes. <br><br />
3. Decant the supernatant. <br><br />
4. Resuspend the cells in 500 μL of 40% glycerol and 500 μL of LB (no antibiotics) or water. <br><br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
6. Store the glycerol stock at -80 °C. <br><br />
<br />
<br />
</p><br />
<br />
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<br />
<html><a name="Saccharomyces cerevisiae"></a><br />
<br />
</html><br />
<br />
<h2> <i> Saccharomyces cerevisiae </i> (PyE1 Yeast) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<br />
<p><br />
<br />
This process take four days in lab with a one day wait for incubation. <br><br />
<br />
<br />
Day 1: <br><br />
1. Streak yeast cells onto a YPD plate.* <br><br />
2. Invert the plate and incubate at 30 °C for 2 days. <br><br />
<br />
Day 3: <br><br />
1. Add 50 mL of YPD liquid media into a 250 mL baffle flask. <br><br />
2. Swipe as many individual colonies as you can see into the YPD media.** <br><br />
3. Incubate and shake the culture at 30 °C at 250 rpm overnight approximately 24 hours. <br><br />
<br />
Day 4: <br><br />
1. Take an optical density measurement. <br><br />
2. In three 250 mL baffle flask add the portions of the overnight liquid culture. <br><br />
3. Dilute each culture to approximately 0.4 optical density with YPD. <br><br />
4. Incubate and shake the cultures at 30 °C at 250 rpm until the optical density reaches 1.2-1.6. <br><br />
5. Collect each culture into separate 50 mL flat-bottomed centrifuge tubes. <br><br />
6. Spin down the cells at 4000 x g for 5 minutes at 4 °C. <br><br />
7. Decant the supernatant. <br><br />
8. Resuspend the cells in 100 mL total for all three culture of dH2O. <br><br />
9. Combine the suspensions into two 50 mL flat-bottomed centrifuge tubes. <br><br />
10. Spin down the cells as above. <br><br />
11. Decant the supernatant. <br><br />
12. Resuspend each in 3 mL of 100 mM lithium acetate. <br><br />
13. Transfer both cultures into a single 15 mL conical centrifuge tube. <br><br />
14. Spin down the cells at 3000 rpm for 5 minutes. <br><br />
15. Resuspend the cells in 0.75 mL of 100 mM lithium acetate, total volume is roughly 2 mL. <br><br />
16. Qualitatively bring up the volume to 3.5 mL by adding 40% glycerol. <br><br />
17. Aliquot the cells into 1.5 mL centrifuge tubes or 1.7 mL cryogenic vials.*** <br><br />
<br><br />
*Streak in such a way that there are individual colonies visible on the plate without clumps or satellite colonies. <br><br />
**Collect only individual visible colonies. Do not collect clumps or satellite colonies. <br><br />
***The volume of aliquots depends on the number to transformations you intend to do at a time. <br><br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Chemically Competent Transformations </h3><br />
<br />
<br />
<p><br />
<br />
This protocol assumes a 50 μL aliquot of yeast competent cells were made. Furthermore, this protocol prepares enough cells for six yeast transformations. <br><br />
<br><br />
1. Add the following to 50 μL of yeast competent cells: <br><br />
240 μL of polyethylene glycol - 3350 (PEG-3350) <br><br />
36 μL of 1 M lithium acetate <br><br />
32 μL of milliQ H2O <br><br />
<br />
2. Mix the mixture by gently pipetting or vortexing. <br><br />
3. Aliquot 59 μL of the mixture into a 0.2 mL microcentrifuge tube. <br><br />
4. Add 1 μL (~100-200 ng) of DNA.* <br><br />
5. Mix the mixture by gentle pipetting or vortexing. <br><br />
6. Incubate the mixture at 30 °C for 30 minutes. <br><br />
7. Heat shock the mixture at 42 °C for 20 minutes. <br><br />
8. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
9. Decant the supernatant. <br><br />
10. Resuspend the cell pellets in 200 μL of dH2O. <br><br />
11. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
12. Resuspend the cell pellets in 200 μL of dH2O. <br><br />
13. Plate 50-150 μL of the mixture onto an appropriate selective dropout media plate. <br><br />
14. Invert and incubate at 30 °C for 2 days. <br><br />
<br><br />
*The exact amount of DNA depends on the transformation efficiency of your competent cells. <br />
<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnight Culturing </h3><br />
<br />
<br />
<p><br />
<br />
1. In a 14 mL round-bottomed culture tube add 1.8 mL selective dropout media and 0.2 mL 20% glucose. <br><br />
2. Swipe 3 isolated yeast colonies and add them to the culture tube media. <br><br />
3. Incubate and shake at 37 °C at 250 rpm for 2 days. <br><br />
<br><br />
Note: You can also make 3 mL cultures (2.7 mL S.D. media and 0.3 mL 20% glucose) or larger cultures, just make sure to dilute the glucose from 20% to 2%.<br />
<br />
</p><br />
<br />
<br />
<h3> Culture Passaging </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Take 20-50μL from a previous overnight or passage culture and add it to the culture media.<br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br><br />
<br />
Note: You can also do 3 mL cultures (2.7 mL S.D. media and 0.3 mL 20% glucose) or larger cultures, just make sure to dilute the glucose from 20% to 2%. <br><br />
Note: The exact amount of culture that you take from a previous culture is irrelevant as long as at least 1 living cell is passaged.<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
1. Take 1-2 mL from an overnight culture and transfer into a 1.5 mL centrifuge tube. <br><br />
2. Spin down the culture at 3000 rpm for 3 minutes. <br><br />
3. Decant the supernatant. <br><br />
4. Resuspend the cells in 500 μL of 40% glycerol and 500 μL of selective dropout media or water. <br><br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
6. Store the glycerol stock at -80 °C. <br><br />
<br />
<br />
</p><br />
<br />
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<br />
<html><a name="Flow Cytometry"></a><br />
<br />
</html><br />
<br />
<h2> Flow Cytometry </h2><br />
<br />
<br />
<h3> Dilutions </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. From an overnight culture measure the optical density at 660nm by making 1:10 dilutions. <br><br />
<br />
2. Take enough culture to make a 1mL aliquot with OD 0.4. <br><br />
<br />
3. Spin down the aliquot in a 1.5mL centrifuge tube at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 800μL of the appropriate selective dropout media and 200μL of 20% glucose. <br><br />
<br />
6. Transfer the new culture to a 14mL culture tube. <br><br />
<br />
7. Incubate and shake at 30C and 250rpm for at least 6 hours (1.2-1.6 optical density).<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Preparations for Analysis using C6 Accuri Flow Cytometer </h3><br />
<br />
<br />
<p><br />
<br />
<br />
1. From the dilution previously made, measure the optical density, roughly 1.2-1.6. <br><br />
<br />
2. Make an aliquot of 500μL of the dilution culture in a 1.5mL centrifuge tube. <br><br />
<br />
3. Spin down the aliquot at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 500μL of PBS(F). <br><br />
<br />
6. Spin down the resuspension at 3000rpm for 3 minutes. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cell pellet in another 500uL of PBS(F).* <br><br />
<br />
9. Prepare the C6 Accuri Flow Cytometer by running a backflush cycle and a dH2O cycle. <br><br />
<br />
10. Load the sample onto the sip. <br><br />
<br />
11. Run the sample with 100,000 cell count. <br><br />
<br />
12. Repeat for all samples and make sure to change data cells otherwise the old data is erased. <br><br />
<br />
13. Once finished, run a cleaning cycle with Accuri approved cleaning solution, then run a dH2O cycle. <br><br />
<br />
<br />
<br />
*For special cases do not resuspend all samples, instead resuspend immediately before running the sample through the flow cytometer.<br />
<br />
<br />
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<br />
<html><a name="Fluorescence Activated Cell Sorting"></a><br />
<br />
</html><br />
<br />
<h2> Fluorescence Activated Cell Sorting </h2><br />
<br />
<br />
<h3> Final Preparations </h3><br />
<br />
<br />
<br />
<p> <br />
<br />
Sample Prep:<br />
<br />
<br />
Spin down samples and negative control (5000 RPM, 1 min), keeping in mind the library size. Aspirate off supernatant. Resuspend in PBSF. Spin down cells. Aspirate off supernatant. Resuspend in PBSF. <br><br />
<br />
<br><br />
<br />
1. Open the “iGEM Template” file in the FACS Software and change name to current date and sort cycle<br><br />
<br />
2. Make sure the stream is stable (look for green light in bottom right corner). If not, run the Sort Calibration order. <br><br />
<br />
3. Run Negative Control from the PyE1 cells. <br><br />
<br />
a. Load cells onto carrier and into the machine. Press play button on screen. <br><br />
<br />
b. Set gate around lower left quadrant of cells to ensure single cell analysis using Forward Scatter Area and Side Scatter Area as your axes. Make sure oval gate covers around 80% of cell population. <br><br />
<br />
c. Set second gate on the first gated population by double-clicking on the gated population and using Forward Scatter Height and Forward Scatter Width as your axes. You will notice two distinct populations. Try to focus on the single cell portion of the plot. <br><br />
<br />
NOTE: If you see a large portion of the second gated population existing near the upper right edge of the first gate, you may need to enlarge the first gate to fit more of the population. <br><br />
<br />
d. Press record. Record 100,000 events and stop run. Move to Next Tube. <br><br />
<br />
4. Run first control (Gene clone) <br><br />
<br />
a. Follow step 3 to run the first control <br><br />
<br />
5. Run first library sample <br><br />
<br />
a. Follow steps 3b and 3c to set first two gates correctly. <br><br />
<br />
b. Set final gate for sort which includes top 1% of GFP producers from second gated population. <br><br />
<br />
c. Use final gate to set up the sort. <br><br />
<br />
d. Select sort conditions at the bottom of the screen. <br><br />
<br />
e. Insert and load collection tube. <br><br />
<br />
f. Record 100,000 events, and sort 10x the library size <br><br />
<br />
6. Run Bleach and diH2O through FACS to avoid cross-contamination. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Protein Expression"></a><br />
<br />
</html><br />
<br />
<html><br />
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</html><br />
<br />
<h2> Protein Expression </h2><br />
<br />
<br />
<h3> Overnight Cultures </h3><br />
<br />
<p><br />
<br />
1. Add 25mL TB and 25uL Kan to a 250mL baffled flask <br><br />
<br />
2. Stab a glycerol stock with a p1000 pipette and swirl in the flask of media <br><br />
<br />
3. Put flask in 37C shaker overnight <br />
<br />
</p><br />
<br />
<h3> Protein Expression </h3><br />
<br />
<p> <br />
<br />
1. Add 500μL 1000x Kanamycin to 500mL TB in 2L baffled flask <br><br />
<br />
2. Transfer 10mL overnight culture to TB <br><br />
<br />
3. Shake at 37C (DO WE NEED THE RPM?) <br><br />
<br />
4. Remove flask from shaker when optical density is between 0.5 and 0.8 <br><br />
<br />
5. Allow flask to rest at room temp for 30 min <br><br />
<br />
6. Add 125μL 1M IPTG <br><br />
<br />
7. Shake flask at 18C overnight<br />
<br />
</p><br />
<br />
<h3> Protein Extraction and Purification </h3><br />
<br />
<p><br />
<br />
1. Transfer cell culture to centrifuge flask <br><br />
<br />
2. Centrifuge culture at 4000g for 10 min <br><br />
<br />
3. Discard supernatant <br><br />
<br />
4. Resuspend pellet in 25mL lysis buffer <br><br />
<br />
5. Add 250μL of 100x PMSF, 250μL of 100mg/mL lysozyme, and 250μL of 10mg/mL DNAse <br><br />
<br />
6. Sonicate sample with 0.25inch probe for 5 min at 70% amplitude with 20 sec on and off pulses <br><br />
<br />
7. Take 50μL total sample <br><br />
<br />
8. Transfer lysate to SS-34 centrifuge tube <br><br />
<br />
9. Centrifuge for 30 min at 18000g <br><br />
<br />
10. Take 50μL soluble sample<br />
<br />
</p><br />
<br />
<h3> Nickel Nitrotriacetic Acid Chromatography (Nickel-NTA Chromatography) </h3><br />
<br />
<p><br />
<br />
1. Add 5mL 50%(v/v) nickel resin in ethanol to 25mL column and allow to settle to ~2.5mL(CV) <br><br />
<br />
2. Rinse with 10CV H20 <br><br />
<br />
3. Equilibrate with 10CV lysis buffer <br><br />
<br />
4. Load sample onto column <br><br />
<br />
5. Wash column with 15CV lysis buffer <br><br />
<br />
6. Perform 2 additional wash steps with 15CV <br><br />
<br />
7. Elute sample in 10CV elution buffer and collect eluate <br><br />
<br />
8. Take 50μL pure sample <br />
<br />
</p><br />
<br />
<h3> Size Exclusion Chromatography (S.E.C.) </h3><br />
<p><br />
1. Concentrate sample to as high as possible without inducing protein aggregation<br><br />
2. Pre-equilibrate Superdex 75 column with 48 ml PBS <br><br />
3. Inject 500 ul sample onto column <br><br />
4. Run 36 ml PBS through column at 0.5 ml/min, collecting 1 ml fractions <br><br />
5. Verify presence of protein in fractions by measuring concentration on Nanodrop and running SDS-PAGE (15 kDa protein should elute at ~13 ml)<br><br />
6. Pool fractions containing protein <br />
</p><br />
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<html><a name="Stability Analysis"></a><br />
<br />
</html><br />
<br />
<h2> Stability Analysis </h2><br />
<br />
<br />
<h3> Circular Dichroism: Wavelength Scan </h3><br />
<p><br />
1. Load 1mm cuvette with 400uL protein solution onto CD <br><br />
2. Take wavelength scan <br><br />
260nm-190nm <br><br />
sample every 1nm <br><br />
averaging time 3sec <br><br />
1 scan <br><br />
step scan <br><br />
25C <br><br />
3. Record wavelength which gives strongest signal (222nm)<br />
</p><br />
<br />
<h3> Circular Dichroism: Guanidine Melt </h3><br />
<p><br />
1. Load 1cm cuvette containing 1.996mL of 0.05mg/mL protein solution and stirrer onto CD <br><br />
2. Prepare 8mL of 0.05mg/mL protein in concentrated guanidine solution <br><br />
3. Set up Automixer with guanidine solution on one syringe and waste tube on other syringe <br><br />
4. Titrate up to 6 M guanidine, taking a CD measurement at 222 nm every 0.15 M interval <br><br />
5. Also measure the fluorescence at 280 nm to ensure the total protein concentration is not changing<br />
</p><br />
<br />
<br />
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</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2014-10-17T22:48:14Z<p>Kristanguyen: </p>
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<br />
<h1> Protocols </h1><br />
<br />
<br />
<html> <a name="Media, Buffers and Solutions"></a> </html><br />
<br />
<br />
<h2> Media, Plates, and Solutions </h2><br />
<br />
<br />
<h3> Competent Cell Media Buffer (CCMB) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container:<br />
</p><br />
<p><br />
<br />
<UL><br />
<br />
<Li> 100 g glycerol (liquid) <br><br />
<br />
<LI> 10 mL x 1 M potassium acetate <br><br />
<br />
<LI> 11.8 g CaCl2*H2O <br><br />
<br />
<LI> 4 g MnCl2 <br><br />
<br />
<LI> 2 g MgCl2 <br><br />
<br />
<LI> 1 L of dH2O<br />
<br />
</UL><br />
</p><br />
<p><br />
Sterile filter or autoclave in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Super Optimal Broth (SOB) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br />
<br />
</p><br />
<br />
<p><br />
<br />
<UL><br />
<br />
<LI> 20 g tryptone <br><br />
<br />
<LI> 5 g yeast extract <br><br />
<br />
<LI> 10 mL x 1 M NaCl <br><br />
<br />
<LI> 2.5 mL x 1 M KCl <br><br />
<br />
<LI> 1 L of dH2O<br />
<br />
</UL><br />
<br />
</p><br />
<br />
<p><br />
<br />
Sterile filter or autoclave in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Phosphate Buffered Saline (PBS) Solution </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following in a 2 L container or 1 L beaker: <br />
<br />
</p><br />
<br />
<p><br />
<br />
<UL><br />
<br />
<LI>8 g NaCl <br><br />
<br />
<LI>1.44 g Na2HPO4 <br><br />
<br />
<LI>0.8 g KCl <br><br />
<br />
<LI>0.24 g KH2PO4 <br><br />
<br />
<LI>1 L of dH2O <br />
<br />
</UL><br />
<br />
</p><br />
<br />
<p><br />
<br />
Buffer to pH 7.4 <br><br />
<br />
Sterile filter or autoclave in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3>PBSF (PBS for Flow)</h3><br />
<br />
<br />
<p><br />
<br />
Mix the following in a 1 L beaker:<br />
<br />
</p><br />
<br />
<p><br />
<br />
<UL><br />
<br />
<LI> 25 mL 20X PBS, pH 7.4<br><br />
<br />
<LI> 475 mL H2O<br><br />
<br />
<LI> 2.5 g BSA (0.5%)*<br />
<br />
</UL><br />
<br />
</p><br />
<br />
<p><br />
<br />
Sterile filter in a 1L bottle and store at 4 °C <br />
<br />
</p><br />
<br />
<br />
<h3> Yeast Extract Peptone Dextrose (YPD) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following into 950 mL of dH2O in a 1 L bottle: <br />
<br />
</p><br />
<br />
<p><br />
<br />
<UL><br />
<br />
<LI> 20 g peptone <br><br />
<br />
<LI> 10 g yeast extract<br />
<br />
</UL><br />
<br />
</p><br />
<p><br />
<br />
Autoclave <br><br />
<br />
Add 50 mL 40% glucose <br><br />
<br />
Sterile filter into a 1 L bottle <br><br />
<br />
<br><br />
<br />
Note: For long-term liquid media storage, do not add 40% glucose. Instead add the glucose directly into cell cultures. <br><br />
<br />
Note: For YPD-plates add 24 g agar to the peptone and yeast extract before autoclaving. <br> <br />
<br />
</p><br />
<br />
<br />
<h3> Selective Dropout Media, C-Uracil and C-Histidine (C-Ura and C-His) </h3><br />
<br />
<br />
<p><br />
<br />
Synthesized by the Yeast Resource Center at the University of Washington's <br />
<br />
Department of Genome Sciences and Department of Biochemistry.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<h3> Guanidinium Hydrogen Chloride </h3><br />
<br />
<br />
<p><br />
<br />
For maximum effectiveness, final concentration should be approximately 8.5 M in PBS<br><br />
<br />
Add the following to a 500mL beaker and mix:<br />
<br />
</p><br />
<br />
<p><br />
<br />
<UL><br />
<br />
<LI> 203 g guanidinium hydrogen chloride <br><br />
<br />
<LI> 250 mL PBS solution* <br><br />
<br />
<LI> Add dilute HCl to pH 7.4<br />
<br />
</UL> <br />
<br />
</p><br />
<br />
<p><br />
<br />
*It is not necessary to filter or autoclave. <br><br />
<br />
*Alternatively add slightly less than 250 mL of PBS in order to buffer the solution to the appropriate volume, then add more dH2O as necessary. <br />
<br />
</p><br />
<br />
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<html><a name="Basic Cloning"></a><br />
<br />
</html><br />
<br />
<h2> Basic Cloning </h2><br />
<br />
<br />
<br />
<h3> Polymerase Chain Reaction </h3><br />
<br />
<br />
<p><br />
<br />
All PCRs were done using a standard 50 μL reaction volume with GoTaq® Green Master Mix 2X purchased from PROMEGA Corporation. <br><br />
<br />
Mix the following in a 0.2 mL microcentrifuge tube on ice: <br><br />
<br />
25 μL GoTaq® Green Master Mix 2X <br><br />
<br />
1-5 μL of 10 μM forward primer <br><br />
<br />
1-5 μL of 10 μM reverse primer <br><br />
<br />
<250 ng of DNA template <br><br />
<br />
QS 50 μl nuclease-free H2O <br><br />
<br />
Conduct the reaction in a thermocycler, adjusting anneal temperature and extension times accordingly. See your polymerase supplier protocol for more details on thermocycling.<br />
<br />
</p><br />
<br />
<h3> Error-prone Polymerase Chain Reaction </h3><br />
<br />
<br />
<p> <br />
<br />
<br />
<br />
Prepare 50 μL reaction: <br><br />
<br />
5 μL 10X Mutazyme II Rxn Buffer <br><br />
<br />
1 μL 40 mM dNTP mix (200 μM each final) <br><br />
<br />
1 μL 20 μM forward primer <br><br />
<br />
1 μL 20 μM reverse primer <br><br />
<br />
1 μL Mutazyme II DNA polymerase (2.5 U/μL) <br><br />
<br />
0.01 ng template <br><br />
<br />
QS 50 μL diH2O <br><br />
<br />
<br><br />
<br />
Program thermocycler as follows: <br><br />
<br />
95 °C, 2 min <br><br />
<br />
95 °C, 30 sec <br><br />
<br />
XX °C*, 30 sec <br><br />
<br />
72 °C, X min** <br><br />
<br />
32 cycles <br><br />
<br />
72 °C, 10 min <br><br />
<br />
4 °C, hold<br />
<br />
<br><br />
<br />
*Adjust annealing temperature according to Tm of primer. <br><br />
<br />
**Adjust extension time according to the length of amplified DNA. <br><br />
<br />
<br><br />
<br />
Note: Use 0.01 ng of template (calculate by insert and not by total plasmid). <br><br />
<br />
Calculate amount of template to use as follows: <br><br />
<br />
(bp for amplified region) / (bp in total plasmid) = % amplified region <br><br />
<br />
(conc. of total plasmid) x (% amplified region as a decimal) = conc. of amplified region <br><br />
<br />
<br><br />
<br />
Note: Never pipette less than 0.5 μL. <br><br />
<br />
(0.01 ng of template) / (conc. of amplified region) = vol of template to add to PCR <br><br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Restriction Endonuclease Reaction (Digestion) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
All restriction enzyme reactions were done using a 50 μl reaction volume. Restriction enzymes and buffers were purchased from New England Biolabs® Inc. <br><br />
<br />
Mix the following in a 0.2 mL PCR tube: <br><br />
<br />
<br />
1 μg of DNA <br><br />
<br />
5 μL of the appropriate 10X New England Biolab® Buffer <br><br />
<br />
1 μL of each restriction enzyme (add last) <br><br />
<br />
QS 50μL nuclease-free H2O <br><br />
<br />
Incubate the reaction for 1 hr <br><br />
<br />
Heat inactive the reaction at the appropriate temperature <br><br />
<br><br />
<br />
Note: Thaw the restriction enzyme(s) on ice to improve shelf life <br><br />
<br />
<br />
</p> <br />
<br />
<br />
<h3> Ligation </h3><br />
<br />
<br />
<p><br />
<br />
T4 DNA Ligase and Buffer were purchased from New England Biolabs® Inc. <br><br />
<br />
1. Prepare the following in a 0.2 mL microcentrifuge tube: <br><br />
<br />
50.0 ng vector DNA* <br><br />
<br />
37.5 ng vector DNA* <br><br />
<br />
2 μL 10X T4 DNA Ligase Buffer <br><br />
<br />
1 μL T4 DNA Ligase <br><br />
<br />
QS 20 μL diH2O <br><br />
<br />
2. Incubate the reaction at room temperature for 10-30 minutes or at 16 °C overnight. <br><br />
<br />
3. Heat inactivate at 65 °C for 10 minutes. <br><br />
<br />
4. Chill on ice before starting a transformation reaction. <br><br />
<br />
<br><br />
<br />
*The exact amount of DNA is dependent on the number of base pairs. In order to conduct a proper reaction consult the New England Biolab Ligation Calculator at:<br />
<br />
http://nebiocalculator.neb.com/#!/<br />
<br />
</p><br />
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<html><a name="Escherichia coli Protocols"></a><br />
<br />
</html><br />
<br />
<br />
<h2> <i> Escherichia coli </i> Protocols (XL1-Blue and XL10-Gold) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<p> <br />
Competent cells take two days to culture and aliquot. <br><br />
Day 1: <br><br />
1. Streak an aliquot of competent cells onto two LB-plates without antibiotics.* <br><br />
2. Incubate at 37 °C overnight. <br><br />
Day 2: <br><br />
1. In two 250 mL baffle flasks add 50 mL of SOB media. <br><br />
2. Scrape as many single colonies into either flask. <br><br />
3. Incubate and shake at 37 °C and 250 rpm for 2-3 hours. <br><br />
4. Check the optical density of the cells at 600 nm after 2 hours. <br><br />
5. Stop incubation when cultures reach approximately 0.5 optical density. <br><br />
6. Add the contents of the flask into separate 50 mL flat bottomed centrifuge tubes. <br><br />
7. Spin down the cells at 2500 rpm at 4 °C for 15 minutes. <br><br />
8. Decant the supernatant. <br><br />
9. Resuspend the cells in 16 mL of CCMB by pipetting or gently vortexing. <br><br />
10. Incubate the cells on ice for 20 minutes. <br><br />
11. Spin down the cells at 2500 rpm at 4 °C for 10 minutes. <br> <br />
12. Decant the supernatant. <br><br />
13. Resuspend the cells in 4 mL of CCMB. <br><br />
14. Quickly aliquot the cells into 1.7 mL cryogenic vials or 1.5 mL centrifuge tubes.** <br><br />
15. Store the competent cell aliquots at -80 °C. <br><br />
<br><br />
*Streak in such a way that there should be individual colony growth and no clumps after the incubation. <br><br />
**We did this in a -20 °C cold room and using an automated repeater pipette. The volume of each aliquot depends on the number of transformations you intend to do at a time. <br><br />
<br><br />
Note: After removing the cells from incubation keep them on ice or as cold as possible. <br><br />
</p><br />
<br />
<br />
<h3> Chemically Competent Cell Transformations </h3><br />
<br />
<br />
<p> <br />
1. Thaw competent <i> E. coli </i> cells on ice (XL1-Blue or XL10-Gold).* <br><br />
2. Add 50 μL of competent cells to sterile 14 mL culture tube. <br><br />
3. Add 1 μL (~100-200 ng)* of the mini-prep to each culture tube. <br><br />
4. Equilibrate the cells on ice for 10 minutes. <br><br />
5. Heat shock the cells at 42 °C for 30-45 seconds.** <br><br />
6. Immediately place the cells back on ice for 3 minutes. <br><br />
7. Add 250 μL LB media without antibiotics and shake at 250 rpm and 37 °C for 30 minutes. <br><br />
8. Spread 10 μL and 290 μL on an appropriate LB-antibiotic plate. <br><br />
9. Invert the plate and incubate at 37 °C overnight. <br><br />
<br><br />
*The exact amount of DNA to add depends on your cell's transformation efficiency. However, it is acceptable to add a larger amount to increase the number of transformed cells. <br><br />
**Do not heat shock for an extended duration as this may damage and/or kill your cells.<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnights </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14 mL round-bottom tube, add 3 mL of LB and 3 μL of 1000X antibiotic(s). <br><br />
2. Pick one isolated colony, do not collect satellites or colony clumps, with a pipette tip. <br><br />
3. Swirl the colony tip in the tube, there should be no visible cell clumps. <br><br />
4. Incubate and shake the tube at 37 °C at 250 rpm for 12-16 hours and no longer than 20 hours. <br><br />
<br />
</p><br />
<br />
<br />
<h3> DNA Extraction and Mini-Preps </h3><br />
<br />
<br />
<p><br />
<br />
All DNA Mini-Preps were prepared using EPOCH Mini-Prep Kits and following the supplied protocols.<br />
<br />
</p><br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
1. Take 1-2 mL from an overnight culture and transfer into a 1.5 mL centrifuge tube. <br><br />
2. Spin down the culture at 3000 rpm for 3 minutes. <br><br />
3. Decant the supernatant. <br><br />
4. Resuspend the cells in 500 μL of 40% glycerol and 500 μL of LB (no antibiotics) or water. <br><br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
6. Store the glycerol stock at -80 °C. <br><br />
<br />
<br />
</p><br />
<br />
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<html><a name="Saccharomyces cerevisiae"></a><br />
<br />
</html><br />
<br />
<h2> <i> Saccharomyces cerevisiae </i> (PyE1 Yeast) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<br />
<p><br />
<br />
This process take four days in lab with a one day wait for incubation. <br><br />
<br />
<br />
Day 1: <br><br />
1. Streak yeast cells onto a YPD plate.* <br><br />
2. Invert the plate and incubate at 30 °C for 2 days. <br><br />
<br />
Day 3: <br><br />
1. Add 50 mL of YPD liquid media into a 250 mL baffle flask. <br><br />
2. Swipe as many individual colonies as you can see into the YPD media.** <br><br />
3. Incubate and shake the culture at 30 °C at 250 rpm overnight approximately 24 hours. <br><br />
<br />
Day 4: <br><br />
1. Take an optical density measurement. <br><br />
2. In three 250 mL baffle flask add the portions of the overnight liquid culture. <br><br />
3. Dilute each culture to approximately 0.4 optical density with YPD. <br><br />
4. Incubate and shake the cultures at 30 °C at 250 rpm until the optical density reaches 1.2-1.6. <br><br />
5. Collect each culture into separate 50 mL flat-bottomed centrifuge tubes. <br><br />
6. Spin down the cells at 4000 x g for 5 minutes at 4 °C. <br><br />
7. Decant the supernatant. <br><br />
8. Resuspend the cells in 100 mL total for all three culture of dH2O. <br><br />
9. Combine the suspensions into two 50 mL flat-bottomed centrifuge tubes. <br><br />
10. Spin down the cells as above. <br><br />
11. Decant the supernatant. <br><br />
12. Resuspend each in 3 mL of 100 mM lithium acetate. <br><br />
13. Transfer both cultures into a single 15 mL conical centrifuge tube. <br><br />
14. Spin down the cells at 3000 rpm for 5 minutes. <br><br />
15. Resuspend the cells in 0.75 mL of 100 mM lithium acetate, total volume is roughly 2 mL. <br><br />
16. Qualitatively bring up the volume to 3.5 mL by adding 40% glycerol. <br><br />
17. Aliquot the cells into 1.5 mL centrifuge tubes or 1.7 mL cryogenic vials.*** <br><br />
<br />
*Streak in such a way that there are individual colonies visible on the plate without clumps or satellite colonies. <br><br />
**Collect only individual visible colonies. Do not collect clumps or satellite colonies. <br><br />
***The volume of aliquots depends on the number to transformations you intend to do at a time. <br><br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Chemically Competent Transformations </h3><br />
<br />
<br />
<p><br />
<br />
This protocol assumes a 50 μL aliquot of yeast competent cells were made. Furthermore, this protocol prepares enough cells for six yeast transformations. <br><br />
<br><br />
1. Add the following to 50 μL of yeast competent cells: <br><br />
240 μL of polyethylene glycol - 3350 (PEG-3350) <br><br />
36 μL of 1 M lithium acetate <br><br />
32 μL of milliQ H2O <br><br />
<br />
2. Mix the mixture by gently pipetting or vortexing. <br><br />
3. Aliquot 59 μL of the mixture into a 0.2 mL microcentrifuge tube. <br><br />
4. Add 1 μL (~100-200 ng) of DNA.* <br><br />
5. Mix the mixture by gentle pipetting or vortexing. <br><br />
6. Incubate the mixture at 30 °C for 30 minutes. <br><br />
7. Heat shock the mixture at 42 °C for 20 minutes. <br><br />
8. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
9. Decant the supernatant. <br><br />
10. Resuspend the cell pellets in 200 μL of dH2O. <br><br />
11. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
12. Resuspend the cell pellets in 200 μL of dH2O. <br><br />
13. Plate 50-150 μL of the mixture onto an appropriate selective dropout media plate. <br><br />
14. Invert and incubate at 30 °C for 2 days. <br><br />
*The exact amount of DNA depends on the transformation efficiency of your competent cells. <br />
<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnight Culturing </h3><br />
<br />
<br />
<p><br />
<br />
1. In a 14 mL round-bottomed culture tube add 1.8 mL selective dropout media and 0.2 mL 20% glucose. <br><br />
2. Swipe 3 isolated yeast colonies and add them to the culture tube media. <br><br />
3. Incubate and shake at 37 °C at 250 rpm for 2 days. <br><br />
<br><br />
Note: You can also make 3 mL cultures (2.7 mL S.D. media and 0.3 mL 20% glucose) or larger cultures, just make sure to dilute the glucose from 20% to 2%.<br />
<br />
</p><br />
<br />
<br />
<h3> Culture Passaging </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Take 20-50μL from a previous overnight or passage culture and add it to the culture media.<br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3 mL cultures (2.7 mL S.D. media and 0.3 mL 20% glucose) or larger cultures, just make sure to dilute the glucose from 20% to 2%. <br><br />
Note: The exact amount of culture that you take from a previous culture is irrelevant as long as at least 1 living cell is passaged.<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
1. Take 1-2 mL from an overnight culture and transfer into a 1.5 mL centrifuge tube. <br><br />
2. Spin down the culture at 3000 rpm for 3 minutes. <br><br />
3. Decant the supernatant. <br><br />
4. Resuspend the cells in 500 μL of 40% glycerol and 500 μL of selective dropout media or water. <br><br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
6. Store the glycerol stock at -80 °C. <br><br />
<br />
<br />
</p><br />
<br />
<html><br />
<br />
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</html><br />
<br />
<html><a name="Flow Cytometry"></a><br />
<br />
</html><br />
<br />
<h2> Flow Cytometry </h2><br />
<br />
<br />
<h3> Dilutions </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. From an overnight culture measure the optical density at 660nm by making 1:10 dilutions. <br><br />
<br />
2. Take enough culture to make a 1mL aliquot with OD 0.4. <br><br />
<br />
3. Spin down the aliquot in a 1.5mL centrifuge tube at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 800μL of the appropriate selective dropout media and 200μL of 20% glucose. <br><br />
<br />
6. Transfer the new culture to a 14mL culture tube. <br><br />
<br />
7. Incubate and shake at 30C and 250rpm for at least 6 hours (1.2-1.6 optical density).<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Preparations for Analysis using C6 Accuri Flow Cytometer </h3><br />
<br />
<br />
<p><br />
<br />
<br />
1. From the dilution previously made, measure the optical density, roughly 1.2-1.6. <br><br />
<br />
2. Make an aliquot of 500μL of the dilution culture in a 1.5mL centrifuge tube. <br><br />
<br />
3. Spin down the aliquot at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 500μL of PBS(F). <br><br />
<br />
6. Spin down the resuspension at 3000rpm for 3 minutes. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cell pellet in another 500uL of PBS(F).* <br><br />
<br />
9. Prepare the C6 Accuri Flow Cytometer by running a backflush cycle and a dH2O cycle. <br><br />
<br />
10. Load the sample onto the sip. <br><br />
<br />
11. Run the sample with 100,000 cell count. <br><br />
<br />
12. Repeat for all samples and make sure to change data cells otherwise the old data is erased. <br><br />
<br />
13. Once finished, run a cleaning cycle with Accuri approved cleaning solution, then run a dH2O cycle. <br><br />
<br />
<br />
<br />
*For special cases do not resuspend all samples, instead resuspend immediately before running the sample through the flow cytometer.<br />
<br />
<br />
</p><br />
<html><br />
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</html><br />
<br />
<html><a name="Fluorescence Activated Cell Sorting"></a><br />
<br />
</html><br />
<br />
<h2> Fluorescence Activated Cell Sorting </h2><br />
<br />
<br />
<h3> Final Preparations </h3><br />
<br />
<br />
<br />
<p> <br />
<br />
Sample Prep:<br />
<br />
<br />
Spin down samples and negative control (5000 RPM, 1 min), keeping in mind the library size. Aspirate off supernatant. Resuspend in PBSF. Spin down cells. Aspirate off supernatant. Resuspend in PBSF. <br><br />
<br />
<br><br />
<br />
1. Open the “iGEM Template” file in the FACS Software and change name to current date and sort cycle<br><br />
<br />
2. Make sure the stream is stable (look for green light in bottom right corner). If not, run the Sort Calibration order. <br><br />
<br />
3. Run Negative Control from the PyE1 cells. <br><br />
<br />
a. Load cells onto carrier and into the machine. Press play button on screen. <br><br />
<br />
b. Set gate around lower left quadrant of cells to ensure single cell analysis using Forward Scatter Area and Side Scatter Area as your axes. Make sure oval gate covers around 80% of cell population. <br><br />
<br />
c. Set second gate on the first gated population by double-clicking on the gated population and using Forward Scatter Height and Forward Scatter Width as your axes. You will notice two distinct populations. Try to focus on the single cell portion of the plot. <br><br />
<br />
NOTE: If you see a large portion of the second gated population existing near the upper right edge of the first gate, you may need to enlarge the first gate to fit more of the population. <br><br />
<br />
d. Press record. Record 100,000 events and stop run. Move to Next Tube. <br><br />
<br />
4. Run first control (Gene clone) <br><br />
<br />
a. Follow step 3 to run the first control <br><br />
<br />
5. Run first library sample <br><br />
<br />
a. Follow steps 3b and 3c to set first two gates correctly. <br><br />
<br />
b. Set final gate for sort which includes top 1% of GFP producers from second gated population. <br><br />
<br />
c. Use final gate to set up the sort. <br><br />
<br />
d. Select sort conditions at the bottom of the screen. <br><br />
<br />
e. Insert and load collection tube. <br><br />
<br />
f. Record 100,000 events, and sort 10x the library size <br><br />
<br />
6. Run Bleach and diH2O through FACS to avoid cross-contamination. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Protein Expression"></a><br />
<br />
</html><br />
<br />
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</html><br />
<br />
<h2> Protein Expression </h2><br />
<br />
<br />
<h3> Overnight Cultures </h3><br />
<br />
<p><br />
<br />
1. Add 25mL TB and 25uL Kan to a 250mL baffled flask <br><br />
<br />
2. Stab a glycerol stock with a p1000 pipette and swirl in the flask of media <br><br />
<br />
3. Put flask in 37C shaker overnight <br />
<br />
</p><br />
<br />
<h3> Protein Expression </h3><br />
<br />
<p> <br />
<br />
1. Add 500μL 1000x Kanamycin to 500mL TB in 2L baffled flask <br><br />
<br />
2. Transfer 10mL overnight culture to TB <br><br />
<br />
3. Shake at 37C (DO WE NEED THE RPM?) <br><br />
<br />
4. Remove flask from shaker when optical density is between 0.5 and 0.8 <br><br />
<br />
5. Allow flask to rest at room temp for 30 min <br><br />
<br />
6. Add 125μL 1M IPTG <br><br />
<br />
7. Shake flask at 18C overnight<br />
<br />
</p><br />
<br />
<h3> Protein Extraction and Purification </h3><br />
<br />
<p><br />
<br />
1. Transfer cell culture to centrifuge flask <br><br />
<br />
2. Centrifuge culture at 4000g for 10 min <br><br />
<br />
3. Discard supernatant <br><br />
<br />
4. Resuspend pellet in 25mL lysis buffer <br><br />
<br />
5. Add 250μL of 100x PMSF, 250μL of 100mg/mL lysozyme, and 250μL of 10mg/mL DNAse <br><br />
<br />
6. Sonicate sample with 0.25inch probe for 5 min at 70% amplitude with 20 sec on and off pulses <br><br />
<br />
7. Take 50μL total sample <br><br />
<br />
8. Transfer lysate to SS-34 centrifuge tube <br><br />
<br />
9. Centrifuge for 30 min at 18000g <br><br />
<br />
10. Take 50μL soluble sample<br />
<br />
</p><br />
<br />
<h3> Nickel Nitrotriacetic Acid Chromatography (Nickel-NTA Chromatography) </h3><br />
<br />
<p><br />
<br />
1. Add 5mL 50%(v/v) nickel resin in ethanol to 25mL column and allow to settle to ~2.5mL(CV) <br><br />
<br />
2. Rinse with 10CV H20 <br><br />
<br />
3. Equilibrate with 10CV lysis buffer <br><br />
<br />
4. Load sample onto column <br><br />
<br />
5. Wash column with 15CV lysis buffer <br><br />
<br />
6. Perform 2 additional wash steps with 15CV <br><br />
<br />
7. Elute sample in 10CV elution buffer and collect eluate <br><br />
<br />
8. Take 50μL pure sample <br />
<br />
</p><br />
<br />
<h3> Size Exclusion Chromatography (S.E.C.) </h3><br />
<p><br />
1. Concentrate sample to as high as possible without inducing protein aggregation<br><br />
2. Pre-equilibrate Superdex 75 column with 48 ml PBS <br><br />
3. Inject 500 ul sample onto column <br><br />
4. Run 36 ml PBS through column at 0.5 ml/min, collecting 1 ml fractions <br><br />
5. Verify presence of protein in fractions by measuring concentration on Nanodrop and running SDS-PAGE (15 kDa protein should elute at ~13 ml)<br><br />
6. Pool fractions containing protein <br />
</p><br />
<br />
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<br />
<html><a name="Stability Analysis"></a><br />
<br />
</html><br />
<br />
<h2> Stability Analysis </h2><br />
<br />
<br />
<h3> Circular Dichroism: Wavelength Scan </h3><br />
<p><br />
1. Load 1mm cuvette with 400uL protein solution onto CD <br><br />
2. Take wavelength scan <br><br />
260nm-190nm <br><br />
sample every 1nm <br><br />
averaging time 3sec <br><br />
1 scan <br><br />
step scan <br><br />
25C <br><br />
3. Record wavelength which gives strongest signal (222nm)<br />
</p><br />
<br />
<h3> Circular Dichroism: Guanidine Melt </h3><br />
<p><br />
1. Load 1cm cuvette containing 1.996mL of 0.05mg/mL protein solution and stirrer onto CD <br><br />
2. Prepare 8mL of 0.05mg/mL protein in concentrated guanidine solution <br><br />
3. Set up Automixer with guanidine solution on one syringe and waste tube on other syringe <br><br />
4. Titrate up to 6 M guanidine, taking a CD measurement at 222 nm every 0.15 M interval <br><br />
5. Also measure the fluorescence at 280 nm to ensure the total protein concentration is not changing<br />
</p><br />
<br />
<br />
<br />
<html><br />
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<a href="#linktotop">Back To Top</a><br />
</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2014-10-17T22:47:08Z<p>Kristanguyen: </p>
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<br />
<h1> Protocols </h1><br />
<br />
<br />
<html> <a name="Media, Buffers and Solutions"></a> </html><br />
<br />
<br />
<h2> Media, Plates, and Solutions </h2><br />
<br />
<br />
<h3> Competent Cell Media Buffer (CCMB) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container:<br />
</p><br />
<p><br />
<br />
<UL><br />
<br />
<Li> 100 g glycerol (liquid) <br><br />
<br />
<LI> 10 mL x 1 M potassium acetate <br><br />
<br />
<LI> 11.8 g CaCl2*H2O <br><br />
<br />
<LI> 4 g MnCl2 <br><br />
<br />
<LI> 2 g MgCl2 <br><br />
<br />
<LI> 1 L of dH2O<br />
<br />
</UL><br />
</p><br />
<p><br />
Sterile filter or autoclave in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Super Optimal Broth (SOB) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br />
<br />
</p><br />
<br />
<p><br />
<br />
<UL><br />
<br />
<LI> 20 g tryptone <br><br />
<br />
<LI> 5 g yeast extract <br><br />
<br />
<LI> 10 mL x 1 M NaCl <br><br />
<br />
<LI> 2.5 mL x 1 M KCl <br><br />
<br />
<LI> 1 L of dH2O<br />
<br />
</UL><br />
<br />
</p><br />
<br />
<p><br />
<br />
Sterile filter or autoclave in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Phosphate Buffered Saline (PBS) Solution </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following in a 2 L container or 1 L beaker: <br />
<br />
</p><br />
<br />
<p><br />
<br />
<UL><br />
<br />
<LI>8 g NaCl <br><br />
<br />
<LI>1.44 g Na2HPO4 <br><br />
<br />
<LI>0.8 g KCl <br><br />
<br />
<LI>0.24 g KH2PO4 <br><br />
<br />
<LI>1 L of dH2O <br />
<br />
</UL><br />
<br />
</p><br />
<br />
<p><br />
<br />
Buffer to pH 7.4 <br><br />
<br />
Sterile filter or autoclave in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3>PBSF (PBS for Flow)</h3><br />
<br />
<br />
<p><br />
<br />
Mix the following in a 1 L beaker:<br />
<br />
</p><br />
<br />
<p><br />
<br />
<UL><br />
<br />
<LI> 25 mL 20X PBS, pH 7.4<br><br />
<br />
<LI> 475 mL H2O<br><br />
<br />
<LI> 2.5 g BSA (0.5%)*<br />
<br />
</UL><br />
<br />
</p><br />
<br />
<p><br />
<br />
Sterile filter in a 1L bottle and store at 4 °C <br />
<br />
</p><br />
<br />
<br />
<h3> Yeast Extract Peptone Dextrose (YPD) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following into 950 mL of dH2O in a 1 L bottle: <br />
<br />
</p><br />
<br />
<p><br />
<br />
<UL><br />
<br />
<LI> 20 g peptone <br><br />
<br />
<LI> 10 g yeast extract<br />
<br />
</UL><br />
<br />
</p><br />
<p><br />
<br />
Autoclave <br><br />
<br />
Add 50 mL 40% glucose <br><br />
<br />
Sterile filter into a 1 L bottle <br><br />
<br />
<br><br />
<br />
Note: For long-term liquid media storage, do not add 40% glucose. Instead add the glucose directly into cell cultures. <br><br />
<br />
Note: For YPD-plates add 24 g agar to the peptone and yeast extract before autoclaving. <br> <br />
<br />
</p><br />
<br />
<br />
<h3> Selective Dropout Media, C-Uracil and C-Histidine (C-Ura and C-His) </h3><br />
<br />
<br />
<p><br />
<br />
Synthesized by the Yeast Resource Center at the University of Washington's <br />
<br />
Department of Genome Sciences and Department of Biochemistry.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<h3> Guanidinium Hydrogen Chloride </h3><br />
<br />
<br />
<p><br />
<br />
For maximum effectiveness, final concentration should be approximately 8.5 M in PBS<br><br />
<br />
Add the following to a 500mL beaker and mix:<br />
<br />
</p><br />
<br />
<p><br />
<br />
<UL><br />
<br />
<LI> 203 g guanidinium hydrogen chloride <br><br />
<br />
<LI> 250 mL PBS solution* <br><br />
<br />
<LI> Add dilute HCl to pH 7.4<br />
<br />
</UL> <br />
<br />
</p><br />
<br />
<p><br />
<br />
*It is not necessary to filter or autoclave. <br><br />
<br />
*Alternatively add slightly less than 250 mL of PBS in order to buffer the solution to the appropriate volume, then add more dH2O as necessary. <br />
<br />
</p><br />
<br />
<br />
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</html><br />
<br />
<html><a name="Basic Cloning"></a><br />
<br />
</html><br />
<br />
<h2> Basic Cloning </h2><br />
<br />
<br />
<br />
<h3> Polymerase Chain Reaction </h3><br />
<br />
<br />
<p><br />
<br />
All PCRs were done using a standard 50 μL reaction volume with GoTaq® Green Master Mix 2X purchased from PROMEGA Corporation. <br><br />
<br />
Mix the following in a 0.2 mL microcentrifuge tube on ice: <br><br />
<br />
25 μL GoTaq® Green Master Mix 2X <br><br />
<br />
1-5 μL of 10 μM forward primer <br><br />
<br />
1-5 μL of 10 μM reverse primer <br><br />
<br />
<250 ng of DNA template <br><br />
<br />
QS 50 μl nuclease-free H2O <br><br />
<br />
Conduct the reaction in a thermocycler, adjusting anneal temperature and extension times accordingly. See your polymerase supplier protocol for more details on thermocycling.<br />
<br />
</p><br />
<br />
<h3> Error-prone Polymerase Chain Reaction </h3><br />
<br />
<br />
<p> <br />
<br />
<br />
<br />
Prepare 50 μL reaction: <br><br />
<br />
5 μL 10X Mutazyme II Rxn Buffer <br><br />
<br />
1 μL 40 mM dNTP mix (200 μM each final) <br><br />
<br />
1 μL 20 μM forward primer <br><br />
<br />
1 μL 20 μM reverse primer <br><br />
<br />
1 μL Mutazyme II DNA polymerase (2.5 U/μL) <br><br />
<br />
0.01 ng template <br><br />
<br />
QS 50 μL diH2O <br><br />
<br />
<br><br />
<br />
Program thermocycler as follows: <br><br />
<br />
95 °C, 2 min <br><br />
<br />
95 °C, 30 sec <br><br />
<br />
XX °C*, 30 sec <br><br />
<br />
72 °C, X min** <br><br />
<br />
32 cycles <br><br />
<br />
72 °C, 10 min <br><br />
<br />
4 °C, hold<br />
<br />
<br><br />
<br />
*Adjust annealing temperature according to Tm of primer. <br><br />
<br />
**Adjust extension time according to the length of amplified DNA. <br><br />
<br />
<br><br />
<br />
Note: Use 0.01 ng of template (calculate by insert and not by total plasmid). <br><br />
<br />
Calculate amount of template to use as follows: <br><br />
<br />
(bp for amplified region) / (bp in total plasmid) = % amplified region <br><br />
<br />
(conc. of total plasmid) x (% amplified region as a decimal) = conc. of amplified region <br><br />
<br />
<br><br />
<br />
Note: Never pipette less than 0.5 μL. <br><br />
<br />
(0.01 ng of template) / (conc. of amplified region) = vol of template to add to PCR <br><br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Restriction Endonuclease Reaction (Digestion) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
All restriction enzyme reactions were done using a 50 μl reaction volume. Restriction enzymes and buffers were purchased from New England Biolabs® Inc. <br><br />
<br />
Mix the following in a 0.2 mL PCR tube: <br><br />
<br />
<br />
1 μg of DNA <br><br />
<br />
5 μL of the appropriate 10X New England Biolab® Buffer <br><br />
<br />
1 μL of each restriction enzyme (add last) <br><br />
<br />
QS 50μL nuclease-free H2O <br><br />
<br />
Incubate the reaction for 1 hr <br><br />
<br />
Heat inactive the reaction at the appropriate temperature <br><br />
<br><br />
<br />
Note: Thaw the restriction enzyme(s) on ice to improve shelf life <br><br />
<br />
<br />
</p> <br />
<br />
<br />
<h3> Ligation </h3><br />
<br />
<br />
<p><br />
<br />
T4 DNA Ligase and Buffer were purchased from New England Biolabs® Inc. <br><br />
<br />
1. Prepare the following in a 0.2 mL microcentrifuge tube: <br><br />
<br />
50.0 ng vector DNA* <br><br />
<br />
37.5 ng vector DNA* <br><br />
<br />
2 μL 10X T4 DNA Ligase Buffer <br><br />
<br />
1 μL T4 DNA Ligase <br><br />
<br />
QS 20 μL diH2O <br><br />
<br />
2. Incubate the reaction at room temperature for 10-30 minutes or at 16 °C overnight. <br><br />
<br />
3. Heat inactivate at 65 °C for 10 minutes. <br><br />
<br />
4. Chill on ice before starting a transformation reaction. <br><br />
<br />
<br><br />
<br />
*The exact amount of DNA is dependent on the number of base pairs. In order to conduct a proper reaction consult the New England Biolab Ligation Calculator at:<br />
<br />
http://nebiocalculator.neb.com/#!/<br />
<br />
</p><br />
<br />
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<br />
<html><a name="Escherichia coli Protocols"></a><br />
<br />
</html><br />
<br />
<br />
<h2> <i> Escherichia coli </i> Protocols (XL1-Blue and XL10-Gold) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<p> <br />
Competent cells take two days to culture and aliquot. <br><br />
Day 1: <br><br />
1. Streak an aliquot of competent cells onto two LB-plates without antibiotics.* <br><br />
2. Incubate at 37 °C overnight. <br><br />
Day 2: <br><br />
1. In two 250 mL baffle flasks add 50 mL of SOB media. <br><br />
2. Scrape as many single colonies into either flask. <br><br />
3. Incubate and shake at 37 °C and 250 rpm for 2-3 hours. <br><br />
4. Check the optical density of the cells at 600 nm after 2 hours. <br><br />
5. Stop incubation when cultures reach approximately 0.5 optical density. <br><br />
6. Add the contents of the flask into separate 50 mL flat bottomed centrifuge tubes. <br><br />
7. Spin down the cells at 2500 rpm at 4 °C for 15 minutes. <br><br />
8. Decant the supernatant. <br><br />
9. Resuspend the cells in 16 mL of CCMB by pipetting or gently vortexing. <br><br />
10. Incubate the cells on ice for 20 minutes. <br><br />
11. Spin down the cells at 2500 rpm at 4 °C for 10 minutes. <br> <br />
12. Decant the supernatant. <br><br />
13. Resuspend the cells in 4 mL of CCMB. <br><br />
14. Quickly aliquot the cells into 1.7 mL cryogenic vials or 1.5 mL centrifuge tubes.** <br><br />
15. Store the competent cell aliquots at -80 °C. <br><br />
<br><br />
*Streak in such a way that there should be individual colony growth and no clumps after the incubation. <br><br />
**We did this in a -20 °C cold room and using an automated repeater pipette. The volume of each aliquot depends on the number of transformations you intend to do at a time. <br><br />
<br><br />
Note: After removing the cells from incubation keep them on ice or as cold as possible. <br><br />
</p><br />
<br />
<br />
<h3> Chemically Competent Cell Transformations </h3><br />
<br />
<br />
<p> <br />
1. Thaw competent <i> E. coli </i> cells on ice (XL1-Blue or XL10-Gold).* <br><br />
2. Add 50 μL of competent cells to sterile 14 mL culture tube. <br><br />
3. Add 1 μL (~100-200 ng)* of the mini-prep to each culture tube. <br><br />
4. Equilibrate the cells on ice for 10 minutes. <br><br />
5. Heat shock the cells at 42 °C for 30-45 seconds.** <br><br />
6. Immediately place the cells back on ice for 3 minutes. <br><br />
7. Add 250 μL LB media without antibiotics and shake at 250 rpm and 37 °C for 30 minutes. <br><br />
8. Spread 10 μL and 290 μL on an appropriate LB-antibiotic plate. <br><br />
9. Invert the plate and incubate at 37 °C overnight. <br><br />
<br><br />
*The exact amount of DNA to add depends on your cell's transformation efficiency. However, it is acceptable to add a larger amount to increase the number of transformed cells. <br><br />
**Do not heat shock for an extended duration as this may damage and/or kill your cells.<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnights </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14 mL round-bottom tube, add 3 mL of LB and 3 μL of 1000X antibiotic(s). <br><br />
2. Pick one isolated colony, do not collect satellites or colony clumps, with a pipette tip. <br><br />
3. Swirl the colony tip in the tube, there should be no visible cell clumps. <br><br />
4. Incubate and shake the tube at 37 °C at 250 rpm for 12-16 hours and no longer than 20 hours. <br><br />
<br />
</p><br />
<br />
<br />
<h3> DNA Extraction and Mini-Preps </h3><br />
<br />
<br />
<p><br />
<br />
All DNA Mini-Preps were prepared using EPOCH Mini-Prep Kits and following the supplied protocols.<br />
<br />
</p><br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
1. Take 1-2 mL from an overnight culture and transfer into a 1.5 mL centrifuge tube. <br><br />
2. Spin down the culture at 3000 rpm for 3 minutes. <br><br />
3. Decant the supernatant. <br><br />
4. Resuspend the cells in 500 μL of 40% glycerol and 500 μL of LB (no antibiotics) or water. <br><br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
6. Store the glycerol stock at -80 °C. <br><br />
<br />
<br />
</p><br />
<br />
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<br />
<html><a name="Saccharomyces cerevisiae"></a><br />
<br />
</html><br />
<br />
<h2> <i> Saccharomyces cerevisiae </i> (PyE1 Yeast) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<br />
<p><br />
<br />
This process take four days in lab with a one day wait for incubation. <br><br />
<br />
<br />
Day 1: <br><br />
1. Streak yeast cells onto a YPD plate.* <br><br />
2. Invert the plate and incubate at 30 °C for 2 days. <br><br />
<br />
Day 3: <br><br />
1. Add 50 mL of YPD liquid media into a 250 mL baffle flask. <br><br />
2. Swipe as many individual colonies as you can see into the YPD media.** <br><br />
3. Incubate and shake the culture at 30 °C at 250 rpm overnight approximately 24 hours. <br><br />
<br />
Day 4: <br><br />
1. Take an optical density measurement. <br><br />
2. In three 250 mL baffle flask add the portions of the overnight liquid culture. <br><br />
3. Dilute each culture to approximately 0.4 optical density with YPD. <br><br />
4. Incubate and shake the cultures at 30 °C at 250 rpm until the optical density reaches 1.2-1.6. <br><br />
5. Collect each culture into separate 50 mL flat-bottomed centrifuge tubes. <br><br />
6. Spin down the cells at 4000 x g for 5 minutes at 4 °C. <br><br />
7. Decant the supernatant. <br><br />
8. Resuspend the cells in 100 mL total for all three culture of dH2O. <br><br />
9. Combine the suspensions into two 50 mL flat-bottomed centrifuge tubes. <br><br />
10. Spin down the cells as above. <br><br />
11. Decant the supernatant. <br><br />
12. Resuspend each in 3 mL of 100 mM lithium acetate. <br><br />
13. Transfer both cultures into a single 15 mL conical centrifuge tube. <br><br />
14. Spin down the cells at 3000 rpm for 5 minutes. <br><br />
15. Resuspend the cells in 0.75 mL of 100 mM lithium acetate, total volume is roughly 2 mL. <br><br />
16. Qualitatively bring up the volume to 3.5 mL by adding 40% glycerol. <br><br />
17. Aliquot the cells into 1.5 mL centrifuge tubes or 1.7 mL cryogenic vials.*** <br><br />
<br />
*Streak in such a way that there are individual colonies visible on the plate without clumps or satellite colonies. <br><br />
**Collect only individual visible colonies. Do not collect clumps or satellite colonies. <br><br />
***The volume of aliquots depends on the number to transformations you intend to do at a time. <br><br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Chemically Competent Transformations </h3><br />
<br />
<br />
<p><br />
<br />
This protocol assumes a 50 μL aliquot of yeast competent cells were made. Furthermore, this protocol prepares enough cells for six yeast transformations. <br><br />
<br><br />
1. Add the following to 50 μL of yeast competent cells: <br><br />
240 μL of polyethylene glycol - 3350 (PEG-3350) <br><br />
36 μL of 1 M lithium acetate <br><br />
32 μL of milliQ H2O <br><br />
<br />
2. Mix the mixture by gently pipetting or vortexing. <br><br />
3. Aliquot 59 μL of the mixture into a 0.2 mL microcentrifuge tube. <br><br />
4. Add 1 μL (~100-200 ng) of DNA.* <br><br />
5. Mix the mixture by gentle pipetting or vortexing. <br><br />
6. Incubate the mixture at 30 °C for 30 minutes. <br><br />
7. Heat shock the mixture at 42 °C for 20 minutes. <br><br />
8. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
9. Decant the supernatant. <br><br />
10. Resuspend the cell pellets in 200 μL of dH2O. <br><br />
11. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
12. Resuspend the cell pellets in 200 μL of dH2O. <br><br />
13. Plate 50-150 μL of the mixture onto an appropriate selective dropout media plate. <br><br />
14. Invert and incubate at 30 °C for 2 days. <br><br />
*The exact amount of DNA depends on the transformation efficiency of your competent cells. <br />
<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnight Culturing </h3><br />
<br />
<br />
<p><br />
<br />
1. In a 14 mL round-bottomed culture tube add 1.8 mL selective dropout media and 0.2 mL 20% glucose. <br><br />
2. Swipe 3 isolated yeast colonies and add them to the culture tube media. <br><br />
3. Incubate and shake at 37 °C at 250 rpm for 2 days. <br><br />
<br><br />
Note: You can also make 3 mL cultures (2.7 mL S.D. media and 0.3 mL 20% glucose) or larger cultures, just make sure to dilute the glucose from 20% to 2%.<br />
<br />
</p><br />
<br />
<br />
<h3> Culture Passaging </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Take 20-50μL from a previous overnight or passage culture and add it to the culture media.<br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3 mL cultures (2.7 mL S.D. media and 0.3 mL 20% glucose) or larger cultures, just make sure to dilute the glucose from 20% to 2%. <br><br />
Note: The exact amount of culture that you take from a previous culture is irrelevant as long as at least 1 living cell is passaged.<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
1. Take 1-2 mL from an overnight culture and transfer into a 1.5 mL centrifuge tube. <br><br />
2. Spin down the culture at 3000 rpm for 3 minutes. <br><br />
3. Decant the supernatant. <br><br />
4. Resuspend the cells in 500 μL of 40% glycerol and 500 μL of selective dropout media or water. <br><br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
6. Store the glycerol stock at -80 °C. <br><br />
<br />
<br />
</p><br />
<br />
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<br />
<html><a name="Flow Cytometry"></a><br />
<br />
</html><br />
<br />
<h2> Flow Cytometry </h2><br />
<br />
<br />
<h3> Dilutions </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. From an overnight culture measure the optical density at 660nm by making 1:10 dilutions. <br><br />
<br />
2. Take enough culture to make a 1mL aliquot with OD 0.4. <br><br />
<br />
3. Spin down the aliquot in a 1.5mL centrifuge tube at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 800μL of the appropriate selective dropout media and 200μL of 20% glucose. <br><br />
<br />
6. Transfer the new culture to a 14mL culture tube. <br><br />
<br />
7. Incubate and shake at 30C and 250rpm for at least 6 hours (1.2-1.6 optical density).<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Preparations for Analysis using C6 Accuri Flow Cytometer </h3><br />
<br />
<br />
<p><br />
<br />
<br />
1. From the dilution previously made, measure the optical density, roughly 1.2-1.6. <br><br />
<br />
2. Make an aliquot of 500μL of the dilution culture in a 1.5mL centrifuge tube. <br><br />
<br />
3. Spin down the aliquot at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 500μL of PBS(F). <br><br />
<br />
6. Spin down the resuspension at 3000rpm for 3 minutes. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cell pellet in another 500uL of PBS(F).* <br><br />
<br />
9. Prepare the C6 Accuri Flow Cytometer by running a backflush cycle and a dH2O cycle. <br><br />
<br />
10. Load the sample onto the sip. <br><br />
<br />
11. Run the sample with 100,000 cell count. <br><br />
<br />
12. Repeat for all samples and make sure to change data cells otherwise the old data is erased. <br><br />
<br />
13. Once finished, run a cleaning cycle with Accuri approved cleaning solution, then run a dH2O cycle. <br><br />
<br />
<br />
<br />
*For special cases do not resuspend all samples, instead resuspend immediately before running the sample through the flow cytometer.<br />
<br />
<br />
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<br />
<html><a name="Fluorescence Activated Cell Sorting"></a><br />
<br />
</html><br />
<br />
<h2> Fluorescence Activated Cell Sorting </h2><br />
<br />
<br />
<h3> Final Preparations </h3><br />
<br />
<br />
<br />
<p> <br />
<br />
Sample Prep:<br />
<br />
<br />
Spin down samples and negative control (5000 RPM, 1 min), keeping in mind the library size. Aspirate off supernatant. Resuspend in PBSF. Spin down cells. Aspirate off supernatant. Resuspend in PBSF. <br><br />
<br />
<br><br />
<br />
1. Open the “iGEM Template” file in the FACS Software and change name to current date and sort cycle<br><br />
<br />
2. Make sure the stream is stable (look for green light in bottom right corner). If not, run the Sort Calibration order. <br><br />
<br />
3. Run Negative Control from the PyE1 cells. <br><br />
<br />
a. Load cells onto carrier and into the machine. Press play button on screen. <br><br />
<br />
b. Set gate around lower left quadrant of cells to ensure single cell analysis using Forward Scatter Area and Side Scatter Area as your axes. Make sure oval gate covers around 80% of cell population. <br><br />
<br />
c. Set second gate on the first gated population by double-clicking on the gated population and using Forward Scatter Height and Forward Scatter Width as your axes. You will notice two distinct populations. Try to focus on the single cell portion of the plot. <br><br />
<br />
NOTE: If you see a large portion of the second gated population existing near the upper right edge of the first gate, you may need to enlarge the first gate to fit more of the population. <br><br />
<br />
d. Press record. Record 100,000 events and stop run. Move to Next Tube. <br><br />
<br />
4. Run first control (Gene clone) <br><br />
<br />
a. Follow step 3 to run the first control <br><br />
<br />
5. Run first library sample <br><br />
<br />
a. Follow steps 3b and 3c to set first two gates correctly. <br><br />
<br />
b. Set final gate for sort which includes top 1% of GFP producers from second gated population. <br><br />
<br />
c. Use final gate to set up the sort. <br><br />
<br />
d. Select sort conditions at the bottom of the screen. <br><br />
<br />
e. Insert and load collection tube. <br><br />
<br />
f. Record 100,000 events, and sort 10x the library size <br><br />
<br />
6. Run Bleach and diH2O through FACS to avoid cross-contamination. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Protein Expression"></a><br />
<br />
</html><br />
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<br />
<h2> Protein Expression </h2><br />
<br />
<br />
<h3> Overnight Cultures </h3><br />
<br />
<p><br />
<br />
1. Add 25mL TB and 25uL Kan to a 250mL baffled flask <br><br />
<br />
2. Stab a glycerol stock with a p1000 pipette and swirl in the flask of media <br><br />
<br />
3. Put flask in 37C shaker overnight <br />
<br />
</p><br />
<br />
<h3> Protein Expression </h3><br />
<br />
<p> <br />
<br />
1. Add 500μL 1000x Kanamycin to 500mL TB in 2L baffled flask <br><br />
<br />
2. Transfer 10mL overnight culture to TB <br><br />
<br />
3. Shake at 37C (DO WE NEED THE RPM?) <br><br />
<br />
4. Remove flask from shaker when optical density is between 0.5 and 0.8 <br><br />
<br />
5. Allow flask to rest at room temp for 30 min <br><br />
<br />
6. Add 125μL 1M IPTG <br><br />
<br />
7. Shake flask at 18C overnight<br />
<br />
</p><br />
<br />
<h3> Protein Extraction and Purification </h3><br />
<br />
<p><br />
<br />
1. Transfer cell culture to centrifuge flask <br><br />
<br />
2. Centrifuge culture at 4000g for 10 min <br><br />
<br />
3. Discard supernatant <br><br />
<br />
4. Resuspend pellet in 25mL lysis buffer <br><br />
<br />
5. Add 250μL of 100x PMSF, 250μL of 100mg/mL lysozyme, and 250μL of 10mg/mL DNAse <br><br />
<br />
6. Sonicate sample with 0.25inch probe for 5 min at 70% amplitude with 20 sec on and off pulses <br><br />
<br />
7. Take 50μL total sample <br><br />
<br />
8. Transfer lysate to SS-34 centrifuge tube <br><br />
<br />
9. Centrifuge for 30 min at 18000g <br><br />
<br />
10. Take 50μL soluble sample<br />
<br />
</p><br />
<br />
<h3> Nickel Nitrotriacetic Acid Chromatography (Nickel-NTA Chromatography) </h3><br />
<br />
<p><br />
<br />
1. Add 5mL 50%(v/v) nickel resin in ethanol to 25mL column and allow to settle to ~2.5mL(CV) <br><br />
<br />
2. Rinse with 10CV H20 <br><br />
<br />
3. Equilibrate with 10CV lysis buffer <br><br />
<br />
4. Load sample onto column <br><br />
<br />
5. Wash column with 15CV lysis buffer <br><br />
<br />
6. Perform 2 additional wash steps with 15CV <br><br />
<br />
7. Elute sample in 10CV elution buffer and collect eluate <br><br />
<br />
8. Take 50μL pure sample <br />
<br />
</p><br />
<br />
<h3> Size Exclusion Chromatography (S.E.C.) </h3><br />
<p><br />
1. Concentrate sample to as high as possible without inducing protein aggregation<br><br />
2. Pre-equilibrate Superdex 75 column with 48 ml PBS <br><br />
3. Inject 500 ul sample onto column <br><br />
4. Run 36 ml PBS through column at 0.5 ml/min, collecting 1 ml fractions <br><br />
5. Verify presence of protein in fractions by measuring concentration on Nanodrop and running SDS-PAGE (15 kDa protein should elute at ~13 ml)<br><br />
6. Pool fractions containing protein <br />
</p><br />
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<html><a name="Stability Analysis"></a><br />
<br />
</html><br />
<br />
<h2> Stability Analysis </h2><br />
<br />
<br />
<h3> Circular Dichroism: Wavelength Scan </h3><br />
<p><br />
1. Load 1mm cuvette with 400uL protein solution onto CD <br><br />
2. Take wavelength scan <br><br />
260nm-190nm <br><br />
sample every 1nm <br><br />
averaging time 3sec <br><br />
1 scan <br><br />
step scan <br><br />
25C <br><br />
3. Record wavelength which gives strongest signal (222nm)<br />
</p><br />
<br />
<h3> Circular Dichroism: Guanidine Melt </h3><br />
<p><br />
1. Load 1cm cuvette containing 1.996mL of 0.05mg/mL protein solution and stirrer onto CD <br><br />
2. Prepare 8mL of 0.05mg/mL protein in concentrated guanidine solution <br><br />
3. Set up Automixer with guanidine solution on one syringe and waste tube on other syringe <br><br />
4. Titrate up to 6 M guanidine, taking a CD measurement at 222 nm every 0.15 M interval <br><br />
5. Also measure the fluorescence at 280 nm to ensure the total protein concentration is not changing<br />
</p><br />
<br />
<br />
<br />
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</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2014-10-17T21:22:27Z<p>Kristanguyen: </p>
<hr />
<div>{{Template:Team:UW/CSS}}<br />
<br />
<br />
<h1> Protocols </h1><br />
<br />
<br />
<html><a name="Media, Plates, and Sol"></a><br />
<br />
</html><br />
<br />
<br />
<h2> Media, Plates, and Solutions </h2><br />
<br />
<br />
<h3> Competent Cell Media Buffer (CCMB) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
100 g glycerol (liquid) <br><br />
<br />
10 mL x 1 M potassium acetate <br><br />
<br />
11.8 g CaCl2*H2O <br><br />
<br />
4 g MnCl2 <br><br />
<br />
2 g MgCl2 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<h3> Super Optimal Broth (SOB) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
20 g Tryptone <br><br />
<br />
5 g Yeast Extract <br><br />
<br />
10 mL x 1 M NaCl <br><br />
<br />
2.5 mL x 1 M KCl <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Phosphate Buffered Saline (PBS) Solution </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container or 1 L beaker: <br><br />
<br />
8 g NaCl <br><br />
<br />
1.44 g Na2HPO4 <br><br />
<br />
0.8 g KCl <br><br />
<br />
0.24 g KH2PO4 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Buffer to pH 7.4 <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<h3> PSBF (PBS for Flow) </h3> <br />
<br />
<p><br />
<br />
Mix the following in a 1 L beaker: <br><br />
<br />
25 mL 20X PBS, pH 7.4 <br><br />
<br />
574 mL H2O <br><br />
<br />
2.5 g BSA (0.5%)* <br><br />
<br />
Filter and store at 4 °C <br><br />
<br><br />
*Do not need to pH - should be at pH 7.4 like 20X PBS <br><br />
<br />
</p> <br />
<br />
<br />
<h3> Yeast Extract Peptone Dextrose (YPD) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following into 950 mL of dH2O in a 1 L bottle: <br><br />
<br />
20 g peptone <br><br />
<br />
10 g yeast extract <br><br />
<br />
Autoclave (20 min at 121 °C and 20 psi) <br><br />
<br />
Add 50 mL 40% glucose <br><br />
<br />
Sterile filter into a 1 L bottle <br><br />
<br />
<br><br />
<br />
Note: For long-term liquid media storage, do not add 40% glucose. Instead add the glucose directly into cell cultures. <br><br />
<br />
Note: For YPD-plates add 24 g agar to the peptone and yeast extract before autoclaving. <br />
<br />
</p><br />
<br />
<br />
<h3> Selective Dropout Media, C-Uracil and C-Histidine (C-Ura and C-His) </h3><br />
<br />
<br />
<p><br />
<br />
Synthesized by the Yeast Resource Center at the University of Washington's <br />
<br />
Department of Genome Sciences and Department of Biochemistry.<br />
<br />
</p><br />
<br />
<br />
<h3> Guanidinium Hydrogen Chloride </h3><br />
<br />
<br />
<p><br />
<br />
For maximum effectiveness, final concentration should be approximately 8.5 M in PBS.<br><br />
<br />
203 g guanidinium hydrogen chloride <br><br />
<br />
250 mL PBS solution* <br><br />
<br />
Add dilute HCl to pH 7.4 <br><br />
<br />
<br><br />
<br />
*Alternatively add slightly less than 250 mL of PBS in order to buffer the solution to the appropriate volume, then add more dH2O as necessary. <br />
<br />
</p><br />
<br />
<html><a name="Basic Cloning"></a><br />
<br />
</html><br />
<br />
<h2> Basic Cloning </h2><br />
<br />
<br />
<br />
<h3> Polymerase Chain Reaction </h3><br />
<br />
<br />
<p><br />
<br />
All PCRs were done using a standard 50 μL reaction volume with GoTaq® Green Master Mix 2X purchased from PROMEGA Corporation. <br><br />
<br />
Mix the following in a 0.2 mL microcentrifuge tube on ice: <br><br />
<br />
25 μL GoTaq® Green Master Mix 2X <br><br />
<br />
1-5 μL of 10 μM forward primer <br><br />
<br />
1-5 μL of 10 μM reverse primer <br><br />
<br />
<250 ng of DNA template <br><br />
<br />
QS 50 μl nuclease-free H2O <br><br />
<br />
Conduct the reaction in a thermocycler, adjusting anneal temperature and extension times accordingly. See your polymerase supplier protocol for more details on thermocycling.<br />
<br />
</p><br />
<br />
<h3> Error-prone Polymerase Chain Reaction </h3><br />
<br />
<br />
<p> <br />
<br />
<br />
<br />
Prepare 50 μL reaction: <br><br />
<br />
5 μL 10X Mutazyme II Rxn Buffer <br><br />
<br />
1 μL 40 mM dNTP mix (200 μM each final) <br><br />
<br />
1 μL 20 μM forward primer <br><br />
<br />
1 μL 20 μM reverse primer <br><br />
<br />
1 μL Mutazyme II DNA polymerase (2.5 U/μL) <br><br />
<br />
0.01 ng template <br><br />
<br />
QS 50 μL diH2O <br><br />
<br />
<br><br />
<br />
Program thermocycler as follows: <br><br />
<br />
95 °C, 2 min <br><br />
<br />
95 °C, 30 sec <br><br />
<br />
XX °C*, 30 sec <br><br />
<br />
72 °C, X min** <br><br />
<br />
32 cycles <br><br />
<br />
72 °C, 10 min <br><br />
<br />
4 °C, hold<br />
<br />
<br><br />
<br />
*Adjust annealing temperature according to Tm of primer. <br><br />
<br />
**Adjust extension time according to the length of amplified DNA. <br><br />
<br />
<br><br />
<br />
Note: Use 0.01 ng of template (calculate by insert and not by total plasmid). <br><br />
<br />
Calculate amount of template to use as follows: <br><br />
<br />
(bp for amplified region) / (bp in total plasmid) = % amplified region <br><br />
<br />
(conc. of total plasmid) x (% amplified region as a decimal) = conc. of amplified region <br><br />
<br />
<br><br />
<br />
Note: Never pipette less than 0.5 μL. <br><br />
<br />
(0.01 ng of template) / (conc. of amplified region) = vol of template to add to PCR <br><br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Restriction Endonuclease Reaction (Digestion) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
All restriction enzyme reactions were done using a 50 μl reaction volume. Restriction enzymes and buffers were purchased from New England Biolabs® Inc. <br><br />
<br />
Mix the following in a 0.2 mL PCR tube: <br><br />
<br />
<br />
1 μg of DNA <br><br />
<br />
5 μL of the appropriate 10X New England Biolab® Buffer <br><br />
<br />
1 μL of each restriction enzyme (add last) <br><br />
<br />
QS 50μL nuclease-free H2O <br><br />
<br />
Incubate the reaction for 1 hr <br><br />
<br />
Heat inactive the reaction at the appropriate temperature <br><br />
<br><br />
<br />
Note: Thaw the restriction enzyme(s) on ice to improve shelf life <br><br />
<br />
<br />
</p> <br />
<br />
<br />
<h3> Ligation </h3><br />
<br />
<br />
<p><br />
<br />
T4 DNA Ligase and Buffer were purchased from New England Biolabs® Inc. <br><br />
<br />
1. Prepare the following in a 0.2 mL microcentrifuge tube: <br><br />
<br />
50.0 ng vector DNA* <br><br />
<br />
37.5 ng vector DNA* <br><br />
<br />
2 μL 10X T4 DNA Ligase Buffer <br><br />
<br />
1 μL T4 DNA Ligase <br><br />
<br />
QS 20 μL diH2O <br><br />
<br />
2. Incubate the reaction at room temperature for 10-30 minutes or at 16 °C overnight. <br><br />
<br />
3. Heat inactivate at 65 °C for 10 minutes. <br><br />
<br />
4. Chill on ice before starting a transformation reaction. <br><br />
<br />
<br><br />
<br />
*The exact amount of DNA is dependent on the number of base pairs. In order to conduct a proper reaction consult the New England Biolab Ligation Calculator at:<br />
<br />
http://nebiocalculator.neb.com/#!/<br />
<br />
</p><br />
<br />
<br />
<html><a name="Escherichia coli Protocols"></a><br />
<br />
</html><br />
<br />
<br />
<h2> <i> Escherichia coli </i> Protocols (XL1-Blue and XL10-Gold) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<p> <br />
Competent cells take two days to culture and aliquot. <br><br />
Day 1: <br><br />
1. Streak an aliquot of competent cells onto two LB-plates without antibiotics.* <br><br />
2. Incubate at 37 °C overnight. <br><br />
Day 2: <br><br />
1. In two 250 mL baffle flasks add 50 mL of SOB media. <br><br />
2. Scrape as many single colonies into either flask. <br><br />
3. Incubate and shake at 37 °C and 250 rpm for 2-3 hours. <br><br />
4. Check the optical density of the cells at 600 nm after 2 hours. <br><br />
5. Stop incubation when cultures reach approximately 0.5 optical density. <br><br />
6. Add the contents of the flask into separate 50 mL flat bottomed centrifuge tubes. <br><br />
7. Spin down the cells at 2500 rpm at 4 °C for 15 minutes. <br><br />
8. Decant the supernatant. <br><br />
9. Resuspend the cells in 16 mL of CCMB by pipetting or gently vortexing. <br><br />
10. Incubate the cells on ice for 20 minutes. <br><br />
11. Spin down the cells at 2500 rpm at 4 °C for 10 minutes. <br> <br />
12. Decant the supernatant. <br><br />
13. Resuspend the cells in 4 mL of CCMB. <br><br />
14. Quickly aliquot the cells into 1.7 mL cryogenic vials or 1.5 mL centrifuge tubes.** <br><br />
15. Store the competent cell aliquots at -80 °C. <br><br />
<br><br />
*Streak in such a way that there should be individual colony growth and no clumps after the incubation. <br><br />
**We did this in a -20 °C cold room and using an automated repeater pipette. The volume of each aliquot depends on the number of transformations you intend to do at a time. <br><br />
<br><br />
Note: After removing the cells from incubation keep them on ice or as cold as possible. <br><br />
</p><br />
<br />
<br />
<h3> Chemically Competent Cell Transformations </h3><br />
<br />
<br />
<p> <br />
1. Thaw competent <i> E. coli </i> cells on ice (XL1-Blue or XL10-Gold).* <br><br />
2. Add 50 μL of competent cells to sterile 14 mL culture tube. <br><br />
3. Add 1 μL (~100-200 ng)* of the mini-prep to each culture tube. <br><br />
4. Equilibrate the cells on ice for 10 minutes. <br><br />
5. Heat shock the cells at 42 °C for 30-45 seconds.** <br><br />
6. Immediately place the cells back on ice for 3 minutes. <br><br />
7. Add 250 μL LB media without antibiotics and shake at 250 rpm and 37 °C for 30 minutes. <br><br />
8. Spread 10 μL and 290 μL on an appropriate LB-antibiotic plate. <br><br />
9. Invert the plate and incubate at 37 °C overnight. <br><br />
<br><br />
*The exact amount of DNA to add depends on your cell's transformation efficiency. However, it is acceptable to add a larger amount to increase the number of transformed cells. <br><br />
**Do not heat shock for an extended duration as this may damage and/or kill your cells.<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnights </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14 mL round-bottom tube, add 3 mL of LB and 3 μL of 1000X antibiotic(s). <br><br />
2. Pick one isolated colony, do not collect satellites or colony clumps, with a pipette tip. <br><br />
3. Swirl the colony tip in the tube, there should be no visible cell clumps. <br><br />
4. Incubate and shake the tube at 37 °C at 250 rpm for 12-16 hours and no longer than 20 hours. <br><br />
<br />
</p><br />
<br />
<br />
<h3> DNA-Extraction and Mini-Preps </h3><br />
<br />
<br />
<p><br />
<br />
All DNA Mini-Preps were prepared using EPOCH Mini-Prep Kits and following the supplied protocols.<br />
<br />
</p><br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
1. Take 1-2 mL from an overnight culture and transfer into a 1.5 mL centrifuge tube. <br><br />
2. Spin down the culture at 3000 rpm for 3 minutes. <br><br />
3. Decant the supernatant. <br><br />
4. Resuspend the cells in 500 μL of 40% glycerol and 500 μL of LB (no antibiotics) or water. <br><br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
6. Store the glycerol stock at -80 °C. <br><br />
<br />
<br />
</p><br />
<br />
<br />
<html><a name="Saccharomyces cerevisiae"></a><br />
<br />
</html><br />
<br />
<h2> <i> Saccharomyces cerevisiae </i> (PyE1 Yeast) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<br />
<p><br />
<br />
This process take 4 days in lab with a 1 day wait for incubation. <br><br />
<br />
<br />
Day 1: <br><br />
<br />
1. Streak yeast cells onto a YPD plate.* <br><br />
<br />
2. Invert the plate and incubate at 30C for 2 days. <br><br />
<br />
<br />
Day 3: <br><br />
<br />
1. Add 50mL of YPD liquid media into a 250mL baffle flask. <br><br />
<br />
2. Swipe as many individual colonies as you can see into the YPD media.** <br><br />
<br />
3. Incubate and shake the culture at 30C at 250rpm overnight approximately 24 hours. <br><br />
<br />
<br />
Day 4: <br><br />
<br />
1. Take an optical density measurement. <br><br />
<br />
2. In three 250mL baffle flask add the portions of the overnight liquid culture. <br><br />
<br />
3. Dilute each culture to approximately 0.4 optical density with YPD. <br><br />
<br />
4. Incubate and shake the cultures at 30C at 250rpm until the optical density reaches 1.2-1.6. <br><br />
<br />
5. Collect each culture into separate 50ml flat-bottomed centrifuge tubes. <br><br />
<br />
6. Spin down the cells at 4000g for 5 minutes at 4C. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cells in 100mL total for all three culture of dH2O. <br><br />
<br />
9. Combine the suspensions into two 50mL flat-bottomed centrifuge tubes. <br><br />
<br />
10. Spin down the cells as above. <br><br />
<br />
11. Decant the supernatant. <br><br />
<br />
12. Resuspend each in 3mL of 100mM Lithium Acetate. <br><br />
<br />
13. Transfer both cultures into a single 15mL conical centrifuge tube. <br><br />
<br />
14. Spin down the cells at 3000rpm for 5 minutes. <br><br />
<br />
15. Resuspend the cells in 0.75mL of 100mM Lithium Acetate, total volume is roughly 2mL. <br><br />
<br />
16. Qualitatively bring up the volume to 3.5mL by adding 40% Glycerol. <br><br />
<br />
17. Aliquot the cells into 1.5mL centrifuge tubes or 1.7mL cryogenic vials.*** <br><br />
<br />
<br />
*Streak in such a way that there are individual colonies visible on the plate without clumps or satellite colonies. <br><br />
<br />
**Collect only individual visible colonies. Do not collect clumps or satellite colonies. <br><br />
<br />
***The volume of aliquots depends on the number to transformations you intend to do at a time. <br><br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Chemically Competent Transformations </h3><br />
<br />
<br />
<p><br />
<br />
This protocol assumes a 50μL aliquot of yeast competent cells were made. <br><br />
<br />
Furthermore, this protocol prepares enough cells for 6 yeast transformations. <br><br />
<br />
<br><br />
<br />
1. Add the following to 50μL of yeast competent cells: <br><br />
<br />
240μL of Polyethylene Glycol - 3350 (PEG-3350) <br><br />
<br />
36μL of 1M Lithium Acetate <br><br />
<br />
32μL of dH2O <br><br />
<br />
<br />
<br />
2. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
3. Aliquot 59μL of the mixture into a 0.2mL microcentrifuge tube. <br><br />
<br />
4. Add 1uL (~100-200ng) of DNA. <br><br />
<br />
5. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
6. Incubate the mixture at 30C for 30 minutes. <br><br />
<br />
7. Heat shock the mixture at 42C for 20 minutes. <br><br />
<br />
8. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
9. Decant the supernatant. <br><br />
<br />
10. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
11. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
12. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
13. Plate 50-150μL of the mixture onto an appropriate Selective Dropout Media plate. <br><br />
<br />
14. Invert and incubate at 30C for 2 days. <br><br />
<br />
*The exact amount of DNA depends on the transformation efficiency of your competent cells. <br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnight Culturing </h3><br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Swipe 3 individually visible yeast colonies and add them to the culture tube media. <br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%.<br />
<br />
</p><br />
<br />
<br />
<h3> Culture Passaging </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Take 20-50μL from a previous overnight or passage culture and add it to the culture media.<br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%. <br><br />
<br />
Note: The exact amount of culture that you take from a previous culture is irrelevant as long as at least 1 living cell is passaged.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500uL of 40% glycerol and 500μL of Selective Dropout media or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Flow Cytometry"></a><br />
<br />
</html><br />
<br />
<h2> Flow Cytometry </h2><br />
<br />
<br />
<h3> Dilutions </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. From an overnight culture measure the optical density at 660nm by making 1:10 dilutions. <br><br />
<br />
2. Take enough culture to make a 1mL aliquot with OD 0.4. <br><br />
<br />
3. Spin down the aliquot in a 1.5mL centrifuge tube at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 800μL of the appropriate selective dropout media and 200μL of 20% glucose. <br><br />
<br />
6. Transfer the new culture to a 14mL culture tube. <br><br />
<br />
7. Incubate and shake at 30C and 250rpm for at least 6 hours (1.2-1.6 optical density).<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Preparations for Analysis using C6 Accuri Flow Cytometer </h3><br />
<br />
<br />
<p><br />
<br />
<br />
1. From the dilution previously made, measure the optical density, roughly 1.2-1.6. <br><br />
<br />
2. Make an aliquot of 500μL of the dilution culture in a 1.5mL centrifuge tube. <br><br />
<br />
3. Spin down the aliquot at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 500μL of PBS(F). <br><br />
<br />
6. Spin down the resuspension at 3000rpm for 3 minutes. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cell pellet in another 500uL of PBS(F).* <br><br />
<br />
9. Prepare the C6 Accuri Flow Cytometer by running a backflush cycle and a dH2O cycle. <br><br />
<br />
10. Load the sample onto the sip. <br><br />
<br />
11. Run the sample with 100,000 cell count. <br><br />
<br />
12. Repeat for all samples and make sure to change data cells otherwise the old data is erased. <br><br />
<br />
13. Once finished, run a cleaning cycle with Accuri approved cleaning solution, then run a dH2O cycle. <br><br />
<br />
<br />
<br />
*For special cases do not resuspend all samples, instead resuspend immediately before running the sample through the flow cytometer.<br />
<br />
<br />
</p><br />
<br />
<br />
<html><a name="Fluorescence Activated Cell Sorting"></a><br />
<br />
</html><br />
<br />
<h2> Fluorescence Activated Cell Sorting </h2><br />
<br />
<br />
<h3> Final Preparations </h3><br />
<br />
<br />
<br />
<p> <br />
<br />
Sample Prep:<br />
<br />
<br />
Spin down samples and negative control (5000 RPM, 1 min), keeping in mind the library size. Aspirate off supernatant. Resuspend in PBSF. Spin down cells. Aspirate off supernatant. Resuspend in PBSF. <br><br />
<br />
<br><br />
<br />
1. Open the “iGEM Template” file in the FACS Software and change name to current date and sort cycle<br><br />
<br />
2. Make sure the stream is stable (look for green light in bottom right corner). If not, run the Sort Calibration order. <br><br />
<br />
3. Run Negative Control from the PyE1 cells. <br><br />
<br />
a. Load cells onto carrier and into the machine. Press play button on screen. <br><br />
<br />
b. Set gate around lower left quadrant of cells to ensure single cell analysis using Forward Scatter Area and Side Scatter Area as your axes. Make sure oval gate covers around 80% of cell population. <br><br />
<br />
c. Set second gate on the first gated population by double-clicking on the gated population and using Forward Scatter Height and Forward Scatter Width as your axes. You will notice two distinct populations. Try to focus on the single cell portion of the plot. <br><br />
<br />
NOTE: If you see a large portion of the second gated population existing near the upper right edge of the first gate, you may need to enlarge the first gate to fit more of the population. <br><br />
<br />
d. Press record. Record 100,000 events and stop run. Move to Next Tube. <br><br />
<br />
4. Run first control (Gene clone) <br><br />
<br />
a. Follow step 3 to run the first control <br><br />
<br />
5. Run first library sample <br><br />
<br />
a. Follow steps 3b and 3c to set first two gates correctly. <br><br />
<br />
b. Set final gate for sort which includes top 1% of GFP producers from second gated population. <br><br />
<br />
c. Use final gate to set up the sort. <br><br />
<br />
d. Select sort conditions at the bottom of the screen. <br><br />
<br />
e. Insert and load collection tube. <br><br />
<br />
f. Record 100,000 events, and sort 10x the library size <br><br />
<br />
6. Run Bleach and diH2O through FACS to avoid cross-contamination. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Protein Expression"></a><br />
<br />
</html><br />
<br />
<h2> Protein Expression </h2><br />
<br />
<br />
<h3> Overnight Cultures </h3><br />
<br />
<p><br />
<br />
1. Add 25mL TB and 25uL Kan to a 250mL baffled flask <br><br />
<br />
2. Stab a glycerol stock with a p1000 pipette and swirl in the flask of media <br><br />
<br />
3. Put flask in 37C shaker overnight <br />
<br />
</p><br />
<br />
<h3> Protein Expression </h3><br />
<br />
<p> <br />
<br />
1. Add 500μL 1000x Kanamycin to 500mL TB in 2L baffled flask <br><br />
<br />
2. Transfer 10mL overnight culture to TB <br><br />
<br />
3. Shake at 37C (DO WE NEED THE RPM?) <br><br />
<br />
4. Remove flask from shaker when optical density is between 0.5 and 0.8 <br><br />
<br />
5. Allow flask to rest at room temp for 30 min <br><br />
<br />
6. Add 125μL 1M IPTG <br><br />
<br />
7. Shake flask at 18C overnight<br />
<br />
</p><br />
<br />
<h3> Protein Extraction and Purification </h3><br />
<br />
<p><br />
<br />
1. Transfer cell culture to centrifuge flask <br><br />
<br />
2. Centrifuge culture at 4000g for 10 min <br><br />
<br />
3. Discard supernatant <br><br />
<br />
4. Resuspend pellet in 25mL lysis buffer <br><br />
<br />
5. Add 250μL of 100x PMSF, 250μL of 100mg/mL lysozyme, and 250μL of 10mg/mL DNAse <br><br />
<br />
6. Sonicate sample with 0.25inch probe for 5 min at 70% amplitude with 20 sec on and off pulses <br><br />
<br />
7. Take 50μL total sample <br><br />
<br />
8. Transfer lysate to SS-34 centrifuge tube <br><br />
<br />
9. Centrifuge for 30 min at 18000g <br><br />
<br />
10. Take 50μL soluble sample<br />
<br />
</p><br />
<br />
<h3> Nickel Nitrotriacetic Acid Chromatography (Nickel-NTA Chromatography) </h3><br />
<br />
<p><br />
<br />
1. Add 5mL 50%(v/v) nickel resin in ethanol to 25mL column and allow to settle to ~2.5mL(CV) <br><br />
<br />
2. Rinse with 10CV H20 <br><br />
<br />
3. Equilibrate with 10CV lysis buffer <br><br />
<br />
4. Load sample onto column <br><br />
<br />
5. Wash column with 15CV lysis buffer <br><br />
<br />
6. Perform 2 additional wash steps with 15CV <br><br />
<br />
7. Elute sample in 10CV elution buffer and collect eluate <br><br />
<br />
8. Take 50μL pure sample <br />
<br />
</p><br />
<br />
<h3> Size Exclusion Chromatography (S.E.C.) </h3><br />
<p><br />
1. Concentrate sample to as high as possible without inducing protein aggregation<br><br />
2. Pre-equilibrate Superdex 75 column with 48 ml PBS <br><br />
3. Inject 500 ul sample onto column <br><br />
4. Run 36 ml PBS through column at 0.5 ml/min, collecting 1 ml fractions <br><br />
5. Verify presence of protein in fractions by measuring concentration on Nanodrop and running SDS-PAGE (15 kDa protein should elute at ~13 ml)<br><br />
6. Pool fractions containing protein <br />
</p><br />
<br />
<br />
<html><a name="Stability Analysis"></a><br />
<br />
</html><br />
<br />
<h2> Stability Analysis </h2><br />
<br />
<br />
<h3> Circular Dichroism: Wavelength Scan </h3><br />
<p><br />
1. Load 1mm cuvette with 400uL protein solution onto CD <br><br />
2. Take wavelength scan <br><br />
260nm-190nm <br><br />
sample every 1nm <br><br />
averaging time 3sec <br><br />
1 scan <br><br />
step scan <br><br />
25C <br><br />
3. Record wavelength which gives strongest signal (222nm)<br />
</p><br />
<br />
<h3> Circular Dichroism: Guanidine Melt </h3><br />
<p><br />
1. Load 1cm cuvette containing 1.996mL of 0.05mg/mL protein solution and stirrer onto CD <br><br />
2. Prepare 8mL of 0.05mg/mL protein in concentrated guanidine solution <br><br />
3. Set up Automixer with guanidine solution on one syringe and waste tube on other syringe <br><br />
4. Titrate up to 6 M guanidine, taking a CD measurement at 222 nm every 0.15 M interval <br><br />
5. Also measure the fluorescence at 280 nm to ensure the total protein concentration is not changing<br />
</p><br />
<br />
<br />
<br />
<html><br />
<br />
<a href="#linktotop">Back To Top</a><br />
</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2014-10-17T21:20:58Z<p>Kristanguyen: </p>
<hr />
<div>{{Template:Team:UW/CSS}}<br />
<br />
<br />
<h1> Protocols </h1><br />
<br />
<br />
<html><a name="Media, Plates, and Sol"></a><br />
<br />
</html><br />
<br />
<br />
<h2> Media, Plates, and Solutions </h2><br />
<br />
<br />
<h3> Competent Cell Media Buffer (CCMB) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
100 g glycerol (liquid) <br><br />
<br />
10 mL x 1 M potassium acetate <br><br />
<br />
11.8 g CaCl2*H2O <br><br />
<br />
4 g MnCl2 <br><br />
<br />
2 g MgCl2 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<h3> Super Optimal Broth (SOB) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
20 g Tryptone <br><br />
<br />
5 g Yeast Extract <br><br />
<br />
10 mL x 1 M NaCl <br><br />
<br />
2.5 mL x 1 M KCl <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Phosphate Buffered Saline (PBS) Solution </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container or 1 L beaker: <br><br />
<br />
8 g NaCl <br><br />
<br />
1.44 g Na2HPO4 <br><br />
<br />
0.8 g KCl <br><br />
<br />
0.24 g KH2PO4 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Buffer to pH 7.4 <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<h3> PSBF (PBS for Flow) </h3> <br />
<br />
<p><br />
<br />
Mix the following in a 1 L beaker: <br><br />
<br />
25 mL 20X PBS, pH 7.4 <br><br />
<br />
574 mL H2O <br><br />
<br />
2.5 g BSA (0.5%)* <br><br />
<br />
Filter and store at 4 °C <br><br />
<br><br />
*Do not need to pH - should be at pH 7.4 like 20X PBS <br><br />
<br />
</p> <br />
<br />
<br />
<h3> Yeast Extract Peptone Dextrose (YPD) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following into 950 mL of dH2O in a 1 L bottle: <br><br />
<br />
20 g peptone <br><br />
<br />
10 g yeast extract <br><br />
<br />
Autoclave (20 min at 121 °C and 20 psi) <br><br />
<br />
Add 50 mL 40% glucose <br><br />
<br />
Sterile filter into a 1 L bottle <br><br />
<br />
<br><br />
<br />
Note: For long-term liquid media storage, do not add 40% glucose. Instead add the glucose directly into cell cultures. <br><br />
<br />
Note: For YPD-plates add 24 g agar to the peptone and yeast extract before autoclaving. <br />
<br />
</p><br />
<br />
<br />
<h3> Selective Dropout Media, C-Uracil and C-Histidine (C-Ura and C-His) </h3><br />
<br />
<br />
<p><br />
<br />
Synthesized by the Yeast Resource Center at the University of Washington's <br />
<br />
Department of Genome Sciences and Department of Biochemistry.<br />
<br />
</p><br />
<br />
<br />
<h3> Guanidinium Hydrogen Chloride </h3><br />
<br />
<br />
<p><br />
<br />
For maximum effectiveness, final concentration should be approximately 8.5 M in PBS.<br><br />
<br />
203 g guanidinium hydrogen chloride <br><br />
<br />
250 mL PBS solution* <br><br />
<br />
Add dilute HCl to pH 7.4 <br><br />
<br />
<br><br />
<br />
*Alternatively add slightly less than 250 mL of PBS in order to buffer the solution to the appropriate volume, then add more dH2O as necessary. <br />
<br />
</p><br />
<br />
<html><a name="Basic Cloning"></a><br />
<br />
</html><br />
<br />
<h2> Basic Cloning </h2><br />
<br />
<br />
<br />
<h3> Polymerase Chain Reaction </h3><br />
<br />
<br />
<p><br />
<br />
All PCRs were done using a standard 50 μL reaction volume with GoTaq® Green Master Mix 2X purchased from PROMEGA Corporation. <br><br />
<br />
Mix the following in a 0.2 mL microcentrifuge tube on ice: <br><br />
<br />
25 μL GoTaq® Green Master Mix 2X <br><br />
<br />
1-5 μL of 10 μM forward primer <br><br />
<br />
1-5 μL of 10 μM reverse primer <br><br />
<br />
<250 ng of DNA template <br><br />
<br />
QS 50 μl nuclease-free H2O <br><br />
<br />
Conduct the reaction in a thermocycler, adjusting anneal temperature and extension times accordingly. See your polymerase supplier protocol for more details on thermocycling.<br />
<br />
</p><br />
<br />
<h3> Error-prone Polymerase Chain Reaction </h3><br />
<br />
<br />
<p> <br />
<br />
<br />
<br />
Prepare 50 μL reaction: <br><br />
<br />
5 μL 10X Mutazyme II Rxn Buffer <br><br />
<br />
1 μL 40 mM dNTP mix (200 μM each final) <br><br />
<br />
1 μL 20 μM forward primer <br><br />
<br />
1 μL 20 μM reverse primer <br><br />
<br />
1 μL Mutazyme II DNA polymerase (2.5 U/μL) <br><br />
<br />
0.01 ng template <br><br />
<br />
QS 50 μL diH2O <br><br />
<br />
<br><br />
<br />
Program thermocycler as follows: <br><br />
<br />
95 °C, 2 min <br><br />
<br />
95 °C, 30 sec <br><br />
<br />
XX °C*, 30 sec <br><br />
<br />
72 °C, X min** <br><br />
<br />
32 cycles <br><br />
<br />
72 °C, 10 min <br><br />
<br />
4 °C, hold<br />
<br />
<br><br />
<br />
*Adjust annealing temperature according to Tm of primer. <br><br />
<br />
**Adjust extension time according to the length of amplified DNA. <br><br />
<br />
<br><br />
<br />
Note: Use 0.01 ng of template (calculate by insert and not by total plasmid). <br><br />
<br />
Calculate amount of template to use as follows: <br><br />
<br />
(bp for amplified region) / (bp in total plasmid) = % amplified region <br><br />
<br />
(conc. of total plasmid) x (% amplified region as a decimal) = conc. of amplified region <br><br />
<br />
<br><br />
<br />
Note: Never pipette less than 0.5 μL. <br><br />
<br />
(0.01 ng of template) / (conc. of amplified region) = vol of template to add to PCR <br><br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Restriction Endonuclease Reaction (Digestion) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
All restriction enzyme reactions were done using a 50 μl reaction volume. Restriction enzymes and buffers were purchased from New England Biolabs® Inc. <br><br />
<br />
Mix the following in a 0.2 mL PCR tube: <br><br />
<br />
<br />
1 μg of DNA <br><br />
<br />
5 μL of the appropriate 10X New England Biolab® Buffer <br><br />
<br />
1 μL of each restriction enzyme (add last) <br><br />
<br />
QS 50μL nuclease-free H2O <br><br />
<br />
Incubate the reaction for 1 hr <br><br />
<br />
Heat inactive the reaction at the appropriate temperature <br><br />
<br><br />
<br />
Note: Thaw the restriction enzyme(s) on ice to improve shelf life <br><br />
<br />
<br />
</p> <br />
<br />
<br />
<h3> Ligation </h3><br />
<br />
<br />
<p><br />
<br />
T4 DNA Ligase and Buffer were purchased from New England Biolabs® Inc. <br><br />
<br />
1. Prepare the following in a 0.2 mL microcentrifuge tube: <br><br />
<br />
50.0 ng vector DNA* <br><br />
<br />
37.5 ng vector DNA* <br><br />
<br />
2 μL 10X T4 DNA Ligase Buffer <br><br />
<br />
1 μL T4 DNA Ligase <br><br />
<br />
QS 20 μL diH2O <br><br />
<br />
2. Incubate the reaction at room temperature for 10-30 minutes or at 16 °C overnight. <br><br />
<br />
3. Heat inactivate at 65 °C for 10 minutes. <br><br />
<br />
4. Chill on ice before starting a transformation reaction. <br><br />
<br />
<br><br />
<br />
*The exact amount of DNA is dependent on the number of base pairs. In order to conduct a proper reaction consult the New England Biolab Ligation Calculator at:<br />
<br />
http://nebiocalculator.neb.com/#!/<br />
<br />
</p><br />
<br />
<br />
<html><a name="Escherichia coli Protocols"></a><br />
<br />
</html><br />
<br />
<br />
<h2> <i> Escherichia coli </i> Protocols (XL1-Blue and XL10-Gold) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<p> <br />
Competent cells take two days to culture and aliquot. <br><br />
Day 1: <br><br />
1. Streak an aliquot of competent cells onto two LB-plates without antibiotics.* <br><br />
2. Incubate at 37 °C overnight. <br><br />
Day 2: <br><br />
1. In two 250 mL baffle flasks add 50 mL of SOB media. <br><br />
2. Scrape as many single colonies into either flask. <br><br />
3. Incubate and shake at 37 °C and 250 rpm for 2-3 hours. <br><br />
4. Check the optical density of the cells at 600 nm after 2 hours. <br><br />
5. Stop incubation when cultures reach approximately 0.5 optical density. <br><br />
6. Add the contents of the flask into separate 50 mL flat bottomed centrifuge tubes. <br><br />
7. Spin down the cells at 2500 rpm at 4 °C for 15 minutes. <br><br />
8. Decant the supernatant. <br><br />
9. Resuspend the cells in 16 mL of CCMB by pipetting or gently vortexing. <br><br />
10. Incubate the cells on ice for 20 minutes. <br><br />
11. Spin down the cells at 2500 rpm at 4 °C for 10 minutes. <br> <br />
12. Decant the supernatant. <br><br />
13. Resuspend the cells in 4 mL of CCMB. <br><br />
14. Quickly aliquot the cells into 1.7 mL cryogenic vials or 1.5 mL centrifuge tubes.** <br><br />
15. Store the competent cell aliquots at -80 °C. <br><br />
<br><br />
*Streak in such a way that there should be individual colony growth and no clumps after the incubation. <br><br />
**We did this in a -20 °C cold room and using an automated repeater pipette. The volume of each aliquot depends on the number of transformations you intend to do at a time. <br><br />
<br><br />
Note: After removing the cells from incubation keep them on ice or as cold as possible. <br><br />
</p><br />
<br />
<br />
<h3> Chemically Competent Cell Transformations </h3><br />
<br />
<br />
<p> <br />
1. Thaw competent <i> E. coli </i> cells on ice (XL1-Blue or XL10-Gold).* <br><br />
2. Add 50 μL of competent cells to sterile 14 mL culture tube. <br><br />
3. Add 1 μL (~100-200 ng)* of the mini-prep to each culture tube. <br><br />
4. Equilibrate the cells on ice for 10 minutes. <br><br />
5. Heat shock the cells at 42 °C for 30-45 seconds.** <br><br />
6. Immediately place the cells back on ice for 3 minutes. <br><br />
7. Add 250 μL LB media without antibiotics and shake at 250 rpm and 37 °C for 30 minutes. <br><br />
8. Spread 10 μL and 290 μL on an appropriate LB-antibiotic plate. <br><br />
9. Invert the plate and incubate at 37 °C overnight. <br><br />
<br><br />
*The exact amount of DNA to add depends on your cell's transformation efficiency. However, it is acceptable to add a larger amount to increase the number of transformed cells. <br><br />
**Do not heat shock for an extended duration as this may damage and/or kill your cells.<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnights </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14 mL round-bottom tube, add 3 mL of LB and 3 μL of 1000X antibiotic(s). <br><br />
2. Pick one isolated colony, do not collect satellites or colony clumps, with a pipette tip. <br><br />
3. Swirl the colony tip in the tube, there should be no visible cell clumps. <br><br />
4. Incubate and shake the tube at 37 °C at 250 rpm for 12-16 hours and no longer than 20 hours. <br><br />
<br />
</p><br />
<br />
<br />
<h3> DNA-Extraction and mini-preps </h3><br />
<br />
<br />
<p><br />
<br />
All DNA mini-preps were prepared using EPOCH mini-kits and following the supplied protocols.<br />
<br />
</p><br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500μL of 40% Glycerol and 500uL of LB (no antibiotics) or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Saccharomyces cerevisiae"></a><br />
<br />
</html><br />
<br />
<h2> <i> Saccharomyces cerevisiae </i> (PyE1 Yeast) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<br />
<p><br />
<br />
This process take 4 days in lab with a 1 day wait for incubation. <br><br />
<br />
<br />
Day 1: <br><br />
<br />
1. Streak yeast cells onto a YPD plate.* <br><br />
<br />
2. Invert the plate and incubate at 30C for 2 days. <br><br />
<br />
<br />
Day 3: <br><br />
<br />
1. Add 50mL of YPD liquid media into a 250mL baffle flask. <br><br />
<br />
2. Swipe as many individual colonies as you can see into the YPD media.** <br><br />
<br />
3. Incubate and shake the culture at 30C at 250rpm overnight approximately 24 hours. <br><br />
<br />
<br />
Day 4: <br><br />
<br />
1. Take an optical density measurement. <br><br />
<br />
2. In three 250mL baffle flask add the portions of the overnight liquid culture. <br><br />
<br />
3. Dilute each culture to approximately 0.4 optical density with YPD. <br><br />
<br />
4. Incubate and shake the cultures at 30C at 250rpm until the optical density reaches 1.2-1.6. <br><br />
<br />
5. Collect each culture into separate 50ml flat-bottomed centrifuge tubes. <br><br />
<br />
6. Spin down the cells at 4000g for 5 minutes at 4C. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cells in 100mL total for all three culture of dH2O. <br><br />
<br />
9. Combine the suspensions into two 50mL flat-bottomed centrifuge tubes. <br><br />
<br />
10. Spin down the cells as above. <br><br />
<br />
11. Decant the supernatant. <br><br />
<br />
12. Resuspend each in 3mL of 100mM Lithium Acetate. <br><br />
<br />
13. Transfer both cultures into a single 15mL conical centrifuge tube. <br><br />
<br />
14. Spin down the cells at 3000rpm for 5 minutes. <br><br />
<br />
15. Resuspend the cells in 0.75mL of 100mM Lithium Acetate, total volume is roughly 2mL. <br><br />
<br />
16. Qualitatively bring up the volume to 3.5mL by adding 40% Glycerol. <br><br />
<br />
17. Aliquot the cells into 1.5mL centrifuge tubes or 1.7mL cryogenic vials.*** <br><br />
<br />
<br />
*Streak in such a way that there are individual colonies visible on the plate without clumps or satellite colonies. <br><br />
<br />
**Collect only individual visible colonies. Do not collect clumps or satellite colonies. <br><br />
<br />
***The volume of aliquots depends on the number to transformations you intend to do at a time. <br><br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Chemically Competent Transformations </h3><br />
<br />
<br />
<p><br />
<br />
This protocol assumes a 50μL aliquot of yeast competent cells were made. <br><br />
<br />
Furthermore, this protocol prepares enough cells for 6 yeast transformations. <br><br />
<br />
<br><br />
<br />
1. Add the following to 50μL of yeast competent cells: <br><br />
<br />
240μL of Polyethylene Glycol - 3350 (PEG-3350) <br><br />
<br />
36μL of 1M Lithium Acetate <br><br />
<br />
32μL of dH2O <br><br />
<br />
<br />
<br />
2. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
3. Aliquot 59μL of the mixture into a 0.2mL microcentrifuge tube. <br><br />
<br />
4. Add 1uL (~100-200ng) of DNA. <br><br />
<br />
5. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
6. Incubate the mixture at 30C for 30 minutes. <br><br />
<br />
7. Heat shock the mixture at 42C for 20 minutes. <br><br />
<br />
8. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
9. Decant the supernatant. <br><br />
<br />
10. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
11. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
12. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
13. Plate 50-150μL of the mixture onto an appropriate Selective Dropout Media plate. <br><br />
<br />
14. Invert and incubate at 30C for 2 days. <br><br />
<br />
*The exact amount of DNA depends on the transformation efficiency of your competent cells. <br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnight Culturing </h3><br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Swipe 3 individually visible yeast colonies and add them to the culture tube media. <br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%.<br />
<br />
</p><br />
<br />
<br />
<h3> Culture Passaging </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Take 20-50μL from a previous overnight or passage culture and add it to the culture media.<br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%. <br><br />
<br />
Note: The exact amount of culture that you take from a previous culture is irrelevant as long as at least 1 living cell is passaged.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500uL of 40% glycerol and 500μL of Selective Dropout media or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Flow Cytometry"></a><br />
<br />
</html><br />
<br />
<h2> Flow Cytometry </h2><br />
<br />
<br />
<h3> Dilutions </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. From an overnight culture measure the optical density at 660nm by making 1:10 dilutions. <br><br />
<br />
2. Take enough culture to make a 1mL aliquot with OD 0.4. <br><br />
<br />
3. Spin down the aliquot in a 1.5mL centrifuge tube at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 800μL of the appropriate selective dropout media and 200μL of 20% glucose. <br><br />
<br />
6. Transfer the new culture to a 14mL culture tube. <br><br />
<br />
7. Incubate and shake at 30C and 250rpm for at least 6 hours (1.2-1.6 optical density).<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Preparations for Analysis using C6 Accuri Flow Cytometer </h3><br />
<br />
<br />
<p><br />
<br />
<br />
1. From the dilution previously made, measure the optical density, roughly 1.2-1.6. <br><br />
<br />
2. Make an aliquot of 500μL of the dilution culture in a 1.5mL centrifuge tube. <br><br />
<br />
3. Spin down the aliquot at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 500μL of PBS(F). <br><br />
<br />
6. Spin down the resuspension at 3000rpm for 3 minutes. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cell pellet in another 500uL of PBS(F).* <br><br />
<br />
9. Prepare the C6 Accuri Flow Cytometer by running a backflush cycle and a dH2O cycle. <br><br />
<br />
10. Load the sample onto the sip. <br><br />
<br />
11. Run the sample with 100,000 cell count. <br><br />
<br />
12. Repeat for all samples and make sure to change data cells otherwise the old data is erased. <br><br />
<br />
13. Once finished, run a cleaning cycle with Accuri approved cleaning solution, then run a dH2O cycle. <br><br />
<br />
<br />
<br />
*For special cases do not resuspend all samples, instead resuspend immediately before running the sample through the flow cytometer.<br />
<br />
<br />
</p><br />
<br />
<br />
<html><a name="Fluorescence Activated Cell Sorting"></a><br />
<br />
</html><br />
<br />
<h2> Fluorescence Activated Cell Sorting </h2><br />
<br />
<br />
<h3> Final Preparations </h3><br />
<br />
<br />
<br />
<p> <br />
<br />
Sample Prep:<br />
<br />
<br />
Spin down samples and negative control (5000 RPM, 1 min), keeping in mind the library size. Aspirate off supernatant. Resuspend in PBSF. Spin down cells. Aspirate off supernatant. Resuspend in PBSF. <br><br />
<br />
<br><br />
<br />
1. Open the “iGEM Template” file in the FACS Software and change name to current date and sort cycle<br><br />
<br />
2. Make sure the stream is stable (look for green light in bottom right corner). If not, run the Sort Calibration order. <br><br />
<br />
3. Run Negative Control from the PyE1 cells. <br><br />
<br />
a. Load cells onto carrier and into the machine. Press play button on screen. <br><br />
<br />
b. Set gate around lower left quadrant of cells to ensure single cell analysis using Forward Scatter Area and Side Scatter Area as your axes. Make sure oval gate covers around 80% of cell population. <br><br />
<br />
c. Set second gate on the first gated population by double-clicking on the gated population and using Forward Scatter Height and Forward Scatter Width as your axes. You will notice two distinct populations. Try to focus on the single cell portion of the plot. <br><br />
<br />
NOTE: If you see a large portion of the second gated population existing near the upper right edge of the first gate, you may need to enlarge the first gate to fit more of the population. <br><br />
<br />
d. Press record. Record 100,000 events and stop run. Move to Next Tube. <br><br />
<br />
4. Run first control (Gene clone) <br><br />
<br />
a. Follow step 3 to run the first control <br><br />
<br />
5. Run first library sample <br><br />
<br />
a. Follow steps 3b and 3c to set first two gates correctly. <br><br />
<br />
b. Set final gate for sort which includes top 1% of GFP producers from second gated population. <br><br />
<br />
c. Use final gate to set up the sort. <br><br />
<br />
d. Select sort conditions at the bottom of the screen. <br><br />
<br />
e. Insert and load collection tube. <br><br />
<br />
f. Record 100,000 events, and sort 10x the library size <br><br />
<br />
6. Run Bleach and diH2O through FACS to avoid cross-contamination. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Protein Expression"></a><br />
<br />
</html><br />
<br />
<h2> Protein Expression </h2><br />
<br />
<br />
<h3> Overnight Cultures </h3><br />
<br />
<p><br />
<br />
1. Add 25mL TB and 25uL Kan to a 250mL baffled flask <br><br />
<br />
2. Stab a glycerol stock with a p1000 pipette and swirl in the flask of media <br><br />
<br />
3. Put flask in 37C shaker overnight <br />
<br />
</p><br />
<br />
<h3> Protein Expression </h3><br />
<br />
<p> <br />
<br />
1. Add 500μL 1000x Kanamycin to 500mL TB in 2L baffled flask <br><br />
<br />
2. Transfer 10mL overnight culture to TB <br><br />
<br />
3. Shake at 37C (DO WE NEED THE RPM?) <br><br />
<br />
4. Remove flask from shaker when optical density is between 0.5 and 0.8 <br><br />
<br />
5. Allow flask to rest at room temp for 30 min <br><br />
<br />
6. Add 125μL 1M IPTG <br><br />
<br />
7. Shake flask at 18C overnight<br />
<br />
</p><br />
<br />
<h3> Protein Extraction and Purification </h3><br />
<br />
<p><br />
<br />
1. Transfer cell culture to centrifuge flask <br><br />
<br />
2. Centrifuge culture at 4000g for 10 min <br><br />
<br />
3. Discard supernatant <br><br />
<br />
4. Resuspend pellet in 25mL lysis buffer <br><br />
<br />
5. Add 250μL of 100x PMSF, 250μL of 100mg/mL lysozyme, and 250μL of 10mg/mL DNAse <br><br />
<br />
6. Sonicate sample with 0.25inch probe for 5 min at 70% amplitude with 20 sec on and off pulses <br><br />
<br />
7. Take 50μL total sample <br><br />
<br />
8. Transfer lysate to SS-34 centrifuge tube <br><br />
<br />
9. Centrifuge for 30 min at 18000g <br><br />
<br />
10. Take 50μL soluble sample<br />
<br />
</p><br />
<br />
<h3> Nickel Nitrotriacetic Acid Chromatography (Nickel-NTA Chromatography) </h3><br />
<br />
<p><br />
<br />
1. Add 5mL 50%(v/v) nickel resin in ethanol to 25mL column and allow to settle to ~2.5mL(CV) <br><br />
<br />
2. Rinse with 10CV H20 <br><br />
<br />
3. Equilibrate with 10CV lysis buffer <br><br />
<br />
4. Load sample onto column <br><br />
<br />
5. Wash column with 15CV lysis buffer <br><br />
<br />
6. Perform 2 additional wash steps with 15CV <br><br />
<br />
7. Elute sample in 10CV elution buffer and collect eluate <br><br />
<br />
8. Take 50μL pure sample <br />
<br />
</p><br />
<br />
<h3> Size Exclusion Chromatography (S.E.C.) </h3><br />
<p><br />
1. Concentrate sample to as high as possible without inducing protein aggregation<br><br />
2. Pre-equilibrate Superdex 75 column with 48 ml PBS <br><br />
3. Inject 500 ul sample onto column <br><br />
4. Run 36 ml PBS through column at 0.5 ml/min, collecting 1 ml fractions <br><br />
5. Verify presence of protein in fractions by measuring concentration on Nanodrop and running SDS-PAGE (15 kDa protein should elute at ~13 ml)<br><br />
6. Pool fractions containing protein <br />
</p><br />
<br />
<br />
<html><a name="Stability Analysis"></a><br />
<br />
</html><br />
<br />
<h2> Stability Analysis </h2><br />
<br />
<br />
<h3> Circular Dichroism: Wavelength Scan </h3><br />
<p><br />
1. Load 1mm cuvette with 400uL protein solution onto CD <br><br />
2. Take wavelength scan <br><br />
260nm-190nm <br><br />
sample every 1nm <br><br />
averaging time 3sec <br><br />
1 scan <br><br />
step scan <br><br />
25C <br><br />
3. Record wavelength which gives strongest signal (222nm)<br />
</p><br />
<br />
<h3> Circular Dichroism: Guanidine Melt </h3><br />
<p><br />
1. Load 1cm cuvette containing 1.996mL of 0.05mg/mL protein solution and stirrer onto CD <br><br />
2. Prepare 8mL of 0.05mg/mL protein in concentrated guanidine solution <br><br />
3. Set up Automixer with guanidine solution on one syringe and waste tube on other syringe <br><br />
4. Titrate up to 6 M guanidine, taking a CD measurement at 222 nm every 0.15 M interval <br><br />
5. Also measure the fluorescence at 280 nm to ensure the total protein concentration is not changing<br />
</p><br />
<br />
<br />
<br />
<html><br />
<br />
<a href="#linktotop">Back To Top</a><br />
</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2014-10-17T21:20:15Z<p>Kristanguyen: </p>
<hr />
<div>{{Template:Team:UW/CSS}}<br />
<br />
<br />
<h1> Protocols </h1><br />
<br />
<br />
<html><a name="Media, Plates, and Sol"></a><br />
<br />
</html><br />
<br />
<br />
<h2> Media, Plates, and Solutions </h2><br />
<br />
<br />
<h3> Competent Cell Media Buffer (CCMB) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
100 g glycerol (liquid) <br><br />
<br />
10 mL x 1 M potassium acetate <br><br />
<br />
11.8 g CaCl2*H2O <br><br />
<br />
4 g MnCl2 <br><br />
<br />
2 g MgCl2 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<h3> Super Optimal Broth (SOB) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
20 g Tryptone <br><br />
<br />
5 g Yeast Extract <br><br />
<br />
10 mL x 1 M NaCl <br><br />
<br />
2.5 mL x 1 M KCl <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Phosphate Buffered Saline (PBS) Solution </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container or 1 L beaker: <br><br />
<br />
8 g NaCl <br><br />
<br />
1.44 g Na2HPO4 <br><br />
<br />
0.8 g KCl <br><br />
<br />
0.24 g KH2PO4 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Buffer to pH 7.4 <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<h3> PSBF (PBS for Flow) </h3> <br />
<br />
<p><br />
<br />
Mix the following in a 1 L beaker: <br><br />
<br />
25 mL 20X PBS, pH 7.4 <br><br />
<br />
574 mL H2O <br><br />
<br />
2.5 g BSA (0.5%)* <br><br />
<br />
Filter and store at 4 °C <br><br />
<br><br />
*Do not need to pH - should be at pH 7.4 like 20X PBS <br><br />
<br />
</p> <br />
<br />
<br />
<h3> Yeast Extract Peptone Dextrose (YPD) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following into 950 mL of dH2O in a 1 L bottle: <br><br />
<br />
20 g peptone <br><br />
<br />
10 g yeast extract <br><br />
<br />
Autoclave (20 min at 121 °C and 20 psi) <br><br />
<br />
Add 50 mL 40% glucose <br><br />
<br />
Sterile filter into a 1 L bottle <br><br />
<br />
<br><br />
<br />
Note: For long-term liquid media storage, do not add 40% glucose. Instead add the glucose directly into cell cultures. <br><br />
<br />
Note: For YPD-plates add 24 g agar to the peptone and yeast extract before autoclaving. <br />
<br />
</p><br />
<br />
<br />
<h3> Selective Dropout Media, C-Uracil and C-Histidine (C-Ura and C-His) </h3><br />
<br />
<br />
<p><br />
<br />
Synthesized by the Yeast Resource Center at the University of Washington's <br />
<br />
Department of Genome Sciences and Department of Biochemistry.<br />
<br />
</p><br />
<br />
<br />
<h3> Guanidinium Hydrogen Chloride </h3><br />
<br />
<br />
<p><br />
<br />
For maximum effectiveness, final concentration should be approximately 8.5 M in PBS.<br><br />
<br />
203 g guanidinium hydrogen chloride <br><br />
<br />
250 mL PBS solution* <br><br />
<br />
Add dilute HCl to pH 7.4 <br><br />
<br />
<br><br />
<br />
*Alternatively add slightly less than 250 mL of PBS in order to buffer the solution to the appropriate volume, then add more dH2O as necessary. <br />
<br />
</p><br />
<br />
<html><a name="Basic Cloning"></a><br />
<br />
</html><br />
<br />
<h2> Basic Cloning </h2><br />
<br />
<br />
<br />
<h3> Polymerase Chain Reaction </h3><br />
<br />
<br />
<p><br />
<br />
All PCRs were done using a standard 50 μL reaction volume with GoTaq® Green Master Mix 2X purchased from PROMEGA Corporation. <br><br />
<br />
Mix the following in a 0.2 mL microcentrifuge tube on ice: <br><br />
<br />
25 μL GoTaq® Green Master Mix 2X <br><br />
<br />
1-5 μL of 10 μM forward primer <br><br />
<br />
1-5 μL of 10 μM reverse primer <br><br />
<br />
<250 ng of DNA template <br><br />
<br />
QS 50 μl nuclease-free H2O <br><br />
<br />
Conduct the reaction in a thermocycler, adjusting anneal temperature and extension times accordingly. See your polymerase supplier protocol for more details on thermocycling.<br />
<br />
</p><br />
<br />
<h3> Error-prone Polymerase Chain Reaction </h3><br />
<br />
<br />
<p> <br />
<br />
<br />
<br />
Prepare 50 μL reaction: <br><br />
<br />
5 μL 10X Mutazyme II Rxn Buffer <br><br />
<br />
1 μL 40 mM dNTP mix (200 μM each final) <br><br />
<br />
1 μL 20 μM forward primer <br><br />
<br />
1 μL 20 μM reverse primer <br><br />
<br />
1 μL Mutazyme II DNA polymerase (2.5 U/μL) <br><br />
<br />
0.01 ng template <br><br />
<br />
QS 50 μL diH2O <br><br />
<br />
<br><br />
<br />
Program thermocycler as follows: <br><br />
<br />
95 °C, 2 min <br><br />
<br />
95 °C, 30 sec <br><br />
<br />
XX °C*, 30 sec <br><br />
<br />
72 °C, X min** <br><br />
<br />
32 cycles <br><br />
<br />
72 °C, 10 min <br><br />
<br />
4 °C, hold<br />
<br />
<br><br />
<br />
*Adjust annealing temperature according to Tm of primer. <br><br />
<br />
**Adjust extension time according to the length of amplified DNA. <br><br />
<br />
<br><br />
<br />
Note: Use 0.01 ng of template (calculate by insert and not by total plasmid). <br><br />
<br />
Calculate amount of template to use as follows: <br><br />
<br />
(bp for amplified region) / (bp in total plasmid) = % amplified region <br><br />
<br />
(conc. of total plasmid) x (% amplified region as a decimal) = conc. of amplified region <br><br />
<br />
<br><br />
<br />
Note: Never pipette less than 0.5 μL. <br><br />
<br />
(0.01 ng of template) / (conc. of amplified region) = vol of template to add to PCR <br><br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Restriction Endonuclease Reaction (Digestion) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
All restriction enzyme reactions were done using a 50 μl reaction volume. Restriction enzymes and buffers were purchased from New England Biolabs® Inc. <br><br />
<br />
Mix the following in a 0.2 mL PCR tube: <br><br />
<br />
<br />
1 μg of DNA <br><br />
<br />
5 μL of the appropriate 10X New England Biolab® Buffer <br><br />
<br />
1 μL of each restriction enzyme (add last) <br><br />
<br />
QS 50μL nuclease-free H2O <br><br />
<br />
Incubate the reaction for 1 hr <br><br />
<br />
Heat inactive the reaction at the appropriate temperature <br><br />
<br><br />
<br />
Note: Thaw the restriction enzyme(s) on ice to improve shelf life <br><br />
<br />
<br />
</p> <br />
<br />
<br />
<h3> Ligation </h3><br />
<br />
<br />
<p><br />
<br />
T4 DNA Ligase and Buffer were purchased from New England Biolabs® Inc. <br><br />
<br />
1. Prepare the following in a 0.2 mL microcentrifuge tube: <br><br />
<br />
50.0 ng vector DNA* <br><br />
<br />
37.5 ng vector DNA* <br><br />
<br />
2 μL 10X T4 DNA Ligase Buffer <br><br />
<br />
1 μL T4 DNA Ligase <br><br />
<br />
QS 20 μL diH2O <br><br />
<br />
2. Incubate the reaction at room temperature for 10-30 minutes or at 16 °C overnight. <br><br />
<br />
3. Heat inactivate at 65 °C for 10 minutes. <br><br />
<br />
4. Chill on ice before starting a transformation reaction. <br><br />
<br />
<br><br />
<br />
*The exact amount of DNA is dependent on the number of base pairs. In order to conduct a proper reaction consult the New England Biolab Ligation Calculator at:<br />
<br />
http://nebiocalculator.neb.com/#!/<br />
<br />
</p><br />
<br />
<br />
<html><a name="Escherichia coli Protocols"></a><br />
<br />
</html><br />
<br />
<br />
<h2> <i> Escherichia coli </i> Protocols (XL1-Blue and XL10-Gold) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<p> <br />
Competent cells take two days to culture and aliquot. <br><br />
Day 1: <br><br />
1. Streak an aliquot of competent cells onto two LB-plates without antibiotics.* <br><br />
2. Incubate at 37 °C overnight. <br><br />
Day 2: <br><br />
1. In two 250 mL baffle flasks add 50 mL of SOB media. <br><br />
2. Scrape as many single colonies into either flask. <br><br />
3. Incubate and shake at 37 °C and 250 rpm for 2-3 hours. <br><br />
4. Check the optical density of the cells at 600 nm after 2 hours. <br><br />
5. Stop incubation when cultures reach approximately 0.5 optical density. <br><br />
6. Add the contents of the flask into separate 50 mL flat bottomed centrifuge tubes. <br><br />
7. Spin down the cells at 2500 rpm at 4 °C for 15 minutes. <br><br />
8. Decant the supernatant. <br><br />
9. Resuspend the cells in 16 mL of CCMB by pipetting or gently vortexing. <br><br />
10. Incubate the cells on ice for 20 minutes. <br><br />
11. Spin down the cells at 2500 rpm at 4 °C for 10 minutes. <br> <br />
12. Decant the supernatant. <br><br />
13. Resuspend the cells in 4 mL of CCMB. <br><br />
14. Quickly aliquot the cells into 1.7 mL cryogenic vials or 1.5 mL centrifuge tubes.** <br><br />
15. Store the competent cell aliquots at -80 °C. <br><br />
<br><br />
*Streak in such a way that there should be individual colony growth and no clumps after the incubation. <br><br />
**We did this in a -20 °C cold room and using an automated repeater pipette. The volume of each aliquot depends on the number of transformations you intend to do at a time. <br><br />
<br><br />
Note: After removing the cells from incubation keep them on ice or as cold as possible. <br><br />
</p><br />
<br />
<br />
<h3> Chemically Competent Cell Transformations </h3><br />
<br />
<br />
<p> <br />
1. Thaw competent <i> E. coli </i> cells on ice (XL1-Blue or XL10-Gold).* <br><br />
2. Add 50 μL of competent cells to sterile 14 mL culture tube. <br><br />
3. Add 1 μL (~100-200 ng)* of the mini-prep to each culture tube. <br><br />
4. Equilibrate the cells on ice for 10 minutes. <br><br />
5. Heat shock the cells at 42 °C for 30-45 seconds.** <br><br />
6. Immediately place the cells back on ice for 3 minutes. <br><br />
7. Add 250 μL LB media without antibiotics and shake at 250 rpm and 37 °C for 30 minutes. <br><br />
8. Spread 10 μL and 290 μL on an appropriate LB-antibiotic plate. <br><br />
9. Invert the plate and incubate at 37 °C overnight. <br><br />
<br><br />
*The exact amount of DNA to add depends on your cell's transformation efficiency. However, it is acceptable to add a larger amount to increase the number of transformed cells. <br><br />
**Do not heat shock for an extended duration as this may damage and/or kill your cells.<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnights </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottom tube, add 3-5mL of LB and an appropriate volume of antibiotic(s). <br><br />
<br />
2. Swipe several individual colonies, do not collect satellites or colony clumps, with a pipette tip. <br><br />
<br />
3. Swirl the colony tip in the tube, there should be no visible cell clumps. <br><br />
<br />
4. Incubate and shake the tube at 37C at 250rpm for 12-16 hours and no longer than 20 hours. <br><br />
<br />
</p><br />
<br />
<br />
<h3> DNA-Extraction and mini-preps </h3><br />
<br />
<br />
<p><br />
<br />
All DNA mini-preps were prepared using EPOCH mini-kits and following the supplied protocols.<br />
<br />
</p><br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500μL of 40% Glycerol and 500uL of LB (no antibiotics) or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Saccharomyces cerevisiae"></a><br />
<br />
</html><br />
<br />
<h2> <i> Saccharomyces cerevisiae </i> (PyE1 Yeast) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<br />
<p><br />
<br />
This process take 4 days in lab with a 1 day wait for incubation. <br><br />
<br />
<br />
Day 1: <br><br />
<br />
1. Streak yeast cells onto a YPD plate.* <br><br />
<br />
2. Invert the plate and incubate at 30C for 2 days. <br><br />
<br />
<br />
Day 3: <br><br />
<br />
1. Add 50mL of YPD liquid media into a 250mL baffle flask. <br><br />
<br />
2. Swipe as many individual colonies as you can see into the YPD media.** <br><br />
<br />
3. Incubate and shake the culture at 30C at 250rpm overnight approximately 24 hours. <br><br />
<br />
<br />
Day 4: <br><br />
<br />
1. Take an optical density measurement. <br><br />
<br />
2. In three 250mL baffle flask add the portions of the overnight liquid culture. <br><br />
<br />
3. Dilute each culture to approximately 0.4 optical density with YPD. <br><br />
<br />
4. Incubate and shake the cultures at 30C at 250rpm until the optical density reaches 1.2-1.6. <br><br />
<br />
5. Collect each culture into separate 50ml flat-bottomed centrifuge tubes. <br><br />
<br />
6. Spin down the cells at 4000g for 5 minutes at 4C. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cells in 100mL total for all three culture of dH2O. <br><br />
<br />
9. Combine the suspensions into two 50mL flat-bottomed centrifuge tubes. <br><br />
<br />
10. Spin down the cells as above. <br><br />
<br />
11. Decant the supernatant. <br><br />
<br />
12. Resuspend each in 3mL of 100mM Lithium Acetate. <br><br />
<br />
13. Transfer both cultures into a single 15mL conical centrifuge tube. <br><br />
<br />
14. Spin down the cells at 3000rpm for 5 minutes. <br><br />
<br />
15. Resuspend the cells in 0.75mL of 100mM Lithium Acetate, total volume is roughly 2mL. <br><br />
<br />
16. Qualitatively bring up the volume to 3.5mL by adding 40% Glycerol. <br><br />
<br />
17. Aliquot the cells into 1.5mL centrifuge tubes or 1.7mL cryogenic vials.*** <br><br />
<br />
<br />
*Streak in such a way that there are individual colonies visible on the plate without clumps or satellite colonies. <br><br />
<br />
**Collect only individual visible colonies. Do not collect clumps or satellite colonies. <br><br />
<br />
***The volume of aliquots depends on the number to transformations you intend to do at a time. <br><br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Chemically Competent Transformations </h3><br />
<br />
<br />
<p><br />
<br />
This protocol assumes a 50μL aliquot of yeast competent cells were made. <br><br />
<br />
Furthermore, this protocol prepares enough cells for 6 yeast transformations. <br><br />
<br />
<br><br />
<br />
1. Add the following to 50μL of yeast competent cells: <br><br />
<br />
240μL of Polyethylene Glycol - 3350 (PEG-3350) <br><br />
<br />
36μL of 1M Lithium Acetate <br><br />
<br />
32μL of dH2O <br><br />
<br />
<br />
<br />
2. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
3. Aliquot 59μL of the mixture into a 0.2mL microcentrifuge tube. <br><br />
<br />
4. Add 1uL (~100-200ng) of DNA. <br><br />
<br />
5. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
6. Incubate the mixture at 30C for 30 minutes. <br><br />
<br />
7. Heat shock the mixture at 42C for 20 minutes. <br><br />
<br />
8. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
9. Decant the supernatant. <br><br />
<br />
10. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
11. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
12. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
13. Plate 50-150μL of the mixture onto an appropriate Selective Dropout Media plate. <br><br />
<br />
14. Invert and incubate at 30C for 2 days. <br><br />
<br />
*The exact amount of DNA depends on the transformation efficiency of your competent cells. <br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnight Culturing </h3><br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Swipe 3 individually visible yeast colonies and add them to the culture tube media. <br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%.<br />
<br />
</p><br />
<br />
<br />
<h3> Culture Passaging </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Take 20-50μL from a previous overnight or passage culture and add it to the culture media.<br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%. <br><br />
<br />
Note: The exact amount of culture that you take from a previous culture is irrelevant as long as at least 1 living cell is passaged.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500uL of 40% glycerol and 500μL of Selective Dropout media or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Flow Cytometry"></a><br />
<br />
</html><br />
<br />
<h2> Flow Cytometry </h2><br />
<br />
<br />
<h3> Dilutions </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. From an overnight culture measure the optical density at 660nm by making 1:10 dilutions. <br><br />
<br />
2. Take enough culture to make a 1mL aliquot with OD 0.4. <br><br />
<br />
3. Spin down the aliquot in a 1.5mL centrifuge tube at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 800μL of the appropriate selective dropout media and 200μL of 20% glucose. <br><br />
<br />
6. Transfer the new culture to a 14mL culture tube. <br><br />
<br />
7. Incubate and shake at 30C and 250rpm for at least 6 hours (1.2-1.6 optical density).<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Preparations for Analysis using C6 Accuri Flow Cytometer </h3><br />
<br />
<br />
<p><br />
<br />
<br />
1. From the dilution previously made, measure the optical density, roughly 1.2-1.6. <br><br />
<br />
2. Make an aliquot of 500μL of the dilution culture in a 1.5mL centrifuge tube. <br><br />
<br />
3. Spin down the aliquot at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 500μL of PBS(F). <br><br />
<br />
6. Spin down the resuspension at 3000rpm for 3 minutes. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cell pellet in another 500uL of PBS(F).* <br><br />
<br />
9. Prepare the C6 Accuri Flow Cytometer by running a backflush cycle and a dH2O cycle. <br><br />
<br />
10. Load the sample onto the sip. <br><br />
<br />
11. Run the sample with 100,000 cell count. <br><br />
<br />
12. Repeat for all samples and make sure to change data cells otherwise the old data is erased. <br><br />
<br />
13. Once finished, run a cleaning cycle with Accuri approved cleaning solution, then run a dH2O cycle. <br><br />
<br />
<br />
<br />
*For special cases do not resuspend all samples, instead resuspend immediately before running the sample through the flow cytometer.<br />
<br />
<br />
</p><br />
<br />
<br />
<html><a name="Fluorescence Activated Cell Sorting"></a><br />
<br />
</html><br />
<br />
<h2> Fluorescence Activated Cell Sorting </h2><br />
<br />
<br />
<h3> Final Preparations </h3><br />
<br />
<br />
<br />
<p> <br />
<br />
Sample Prep:<br />
<br />
<br />
Spin down samples and negative control (5000 RPM, 1 min), keeping in mind the library size. Aspirate off supernatant. Resuspend in PBSF. Spin down cells. Aspirate off supernatant. Resuspend in PBSF. <br><br />
<br />
<br><br />
<br />
1. Open the “iGEM Template” file in the FACS Software and change name to current date and sort cycle<br><br />
<br />
2. Make sure the stream is stable (look for green light in bottom right corner). If not, run the Sort Calibration order. <br><br />
<br />
3. Run Negative Control from the PyE1 cells. <br><br />
<br />
a. Load cells onto carrier and into the machine. Press play button on screen. <br><br />
<br />
b. Set gate around lower left quadrant of cells to ensure single cell analysis using Forward Scatter Area and Side Scatter Area as your axes. Make sure oval gate covers around 80% of cell population. <br><br />
<br />
c. Set second gate on the first gated population by double-clicking on the gated population and using Forward Scatter Height and Forward Scatter Width as your axes. You will notice two distinct populations. Try to focus on the single cell portion of the plot. <br><br />
<br />
NOTE: If you see a large portion of the second gated population existing near the upper right edge of the first gate, you may need to enlarge the first gate to fit more of the population. <br><br />
<br />
d. Press record. Record 100,000 events and stop run. Move to Next Tube. <br><br />
<br />
4. Run first control (Gene clone) <br><br />
<br />
a. Follow step 3 to run the first control <br><br />
<br />
5. Run first library sample <br><br />
<br />
a. Follow steps 3b and 3c to set first two gates correctly. <br><br />
<br />
b. Set final gate for sort which includes top 1% of GFP producers from second gated population. <br><br />
<br />
c. Use final gate to set up the sort. <br><br />
<br />
d. Select sort conditions at the bottom of the screen. <br><br />
<br />
e. Insert and load collection tube. <br><br />
<br />
f. Record 100,000 events, and sort 10x the library size <br><br />
<br />
6. Run Bleach and diH2O through FACS to avoid cross-contamination. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Protein Expression"></a><br />
<br />
</html><br />
<br />
<h2> Protein Expression </h2><br />
<br />
<br />
<h3> Overnight Cultures </h3><br />
<br />
<p><br />
<br />
1. Add 25mL TB and 25uL Kan to a 250mL baffled flask <br><br />
<br />
2. Stab a glycerol stock with a p1000 pipette and swirl in the flask of media <br><br />
<br />
3. Put flask in 37C shaker overnight <br />
<br />
</p><br />
<br />
<h3> Protein Expression </h3><br />
<br />
<p> <br />
<br />
1. Add 500μL 1000x Kanamycin to 500mL TB in 2L baffled flask <br><br />
<br />
2. Transfer 10mL overnight culture to TB <br><br />
<br />
3. Shake at 37C (DO WE NEED THE RPM?) <br><br />
<br />
4. Remove flask from shaker when optical density is between 0.5 and 0.8 <br><br />
<br />
5. Allow flask to rest at room temp for 30 min <br><br />
<br />
6. Add 125μL 1M IPTG <br><br />
<br />
7. Shake flask at 18C overnight<br />
<br />
</p><br />
<br />
<h3> Protein Extraction and Purification </h3><br />
<br />
<p><br />
<br />
1. Transfer cell culture to centrifuge flask <br><br />
<br />
2. Centrifuge culture at 4000g for 10 min <br><br />
<br />
3. Discard supernatant <br><br />
<br />
4. Resuspend pellet in 25mL lysis buffer <br><br />
<br />
5. Add 250μL of 100x PMSF, 250μL of 100mg/mL lysozyme, and 250μL of 10mg/mL DNAse <br><br />
<br />
6. Sonicate sample with 0.25inch probe for 5 min at 70% amplitude with 20 sec on and off pulses <br><br />
<br />
7. Take 50μL total sample <br><br />
<br />
8. Transfer lysate to SS-34 centrifuge tube <br><br />
<br />
9. Centrifuge for 30 min at 18000g <br><br />
<br />
10. Take 50μL soluble sample<br />
<br />
</p><br />
<br />
<h3> Nickel Nitrotriacetic Acid Chromatography (Nickel-NTA Chromatography) </h3><br />
<br />
<p><br />
<br />
1. Add 5mL 50%(v/v) nickel resin in ethanol to 25mL column and allow to settle to ~2.5mL(CV) <br><br />
<br />
2. Rinse with 10CV H20 <br><br />
<br />
3. Equilibrate with 10CV lysis buffer <br><br />
<br />
4. Load sample onto column <br><br />
<br />
5. Wash column with 15CV lysis buffer <br><br />
<br />
6. Perform 2 additional wash steps with 15CV <br><br />
<br />
7. Elute sample in 10CV elution buffer and collect eluate <br><br />
<br />
8. Take 50μL pure sample <br />
<br />
</p><br />
<br />
<h3> Size Exclusion Chromatography (S.E.C.) </h3><br />
<p><br />
1. Concentrate sample to as high as possible without inducing protein aggregation<br><br />
2. Pre-equilibrate Superdex 75 column with 48 ml PBS <br><br />
3. Inject 500 ul sample onto column <br><br />
4. Run 36 ml PBS through column at 0.5 ml/min, collecting 1 ml fractions <br><br />
5. Verify presence of protein in fractions by measuring concentration on Nanodrop and running SDS-PAGE (15 kDa protein should elute at ~13 ml)<br><br />
6. Pool fractions containing protein <br />
</p><br />
<br />
<br />
<html><a name="Stability Analysis"></a><br />
<br />
</html><br />
<br />
<h2> Stability Analysis </h2><br />
<br />
<br />
<h3> Circular Dichroism: Wavelength Scan </h3><br />
<p><br />
1. Load 1mm cuvette with 400uL protein solution onto CD <br><br />
2. Take wavelength scan <br><br />
260nm-190nm <br><br />
sample every 1nm <br><br />
averaging time 3sec <br><br />
1 scan <br><br />
step scan <br><br />
25C <br><br />
3. Record wavelength which gives strongest signal (222nm)<br />
</p><br />
<br />
<h3> Circular Dichroism: Guanidine Melt </h3><br />
<p><br />
1. Load 1cm cuvette containing 1.996mL of 0.05mg/mL protein solution and stirrer onto CD <br><br />
2. Prepare 8mL of 0.05mg/mL protein in concentrated guanidine solution <br><br />
3. Set up Automixer with guanidine solution on one syringe and waste tube on other syringe <br><br />
4. Titrate up to 6 M guanidine, taking a CD measurement at 222 nm every 0.15 M interval <br><br />
5. Also measure the fluorescence at 280 nm to ensure the total protein concentration is not changing<br />
</p><br />
<br />
<br />
<br />
<html><br />
<br />
<a href="#linktotop">Back To Top</a><br />
</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2014-10-17T21:19:46Z<p>Kristanguyen: </p>
<hr />
<div>{{Template:Team:UW/CSS}}<br />
<br />
<br />
<h1> Protocols </h1><br />
<br />
<br />
<html><a name="Media, Plates, and Sol"></a><br />
<br />
</html><br />
<br />
<br />
<h2> Media, Plates, and Solutions </h2><br />
<br />
<br />
<h3> Competent Cell Media Buffer (CCMB) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
100 g glycerol (liquid) <br><br />
<br />
10 mL x 1 M potassium acetate <br><br />
<br />
11.8 g CaCl2*H2O <br><br />
<br />
4 g MnCl2 <br><br />
<br />
2 g MgCl2 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<h3> Super Optimal Broth (SOB) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
20 g Tryptone <br><br />
<br />
5 g Yeast Extract <br><br />
<br />
10 mL x 1 M NaCl <br><br />
<br />
2.5 mL x 1 M KCl <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Phosphate Buffered Saline (PBS) Solution </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container or 1 L beaker: <br><br />
<br />
8 g NaCl <br><br />
<br />
1.44 g Na2HPO4 <br><br />
<br />
0.8 g KCl <br><br />
<br />
0.24 g KH2PO4 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Buffer to pH 7.4 <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<h3> PSBF (PBS for Flow) </h3> <br />
<br />
<p><br />
<br />
Mix the following in a 1 L beaker: <br><br />
<br />
25 mL 20X PBS, pH 7.4 <br><br />
<br />
574 mL H2O <br><br />
<br />
2.5 g BSA (0.5%)* <br><br />
<br />
Filter and store at 4 °C <br><br />
<br><br />
*Do not need to pH - should be at pH 7.4 like 20X PBS <br><br />
<br />
</p> <br />
<br />
<br />
<h3> Yeast Extract Peptone Dextrose (YPD) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following into 950 mL of dH2O in a 1 L bottle: <br><br />
<br />
20 g peptone <br><br />
<br />
10 g yeast extract <br><br />
<br />
Autoclave (20 min at 121 °C and 20 psi) <br><br />
<br />
Add 50 mL 40% glucose <br><br />
<br />
Sterile filter into a 1 L bottle <br><br />
<br />
<br><br />
<br />
Note: For long-term liquid media storage, do not add 40% glucose. Instead add the glucose directly into cell cultures. <br><br />
<br />
Note: For YPD-plates add 24 g agar to the peptone and yeast extract before autoclaving. <br />
<br />
</p><br />
<br />
<br />
<h3> Selective Dropout Media, C-Uracil and C-Histidine (C-Ura and C-His) </h3><br />
<br />
<br />
<p><br />
<br />
Synthesized by the Yeast Resource Center at the University of Washington's <br />
<br />
Department of Genome Sciences and Department of Biochemistry.<br />
<br />
</p><br />
<br />
<br />
<h3> Guanidinium Hydrogen Chloride </h3><br />
<br />
<br />
<p><br />
<br />
For maximum effectiveness, final concentration should be approximately 8.5 M in PBS.<br><br />
<br />
203 g guanidinium hydrogen chloride <br><br />
<br />
250 mL PBS solution* <br><br />
<br />
Add dilute HCl to pH 7.4 <br><br />
<br />
<br><br />
<br />
*Alternatively add slightly less than 250 mL of PBS in order to buffer the solution to the appropriate volume, then add more dH2O as necessary. <br />
<br />
</p><br />
<br />
<html><a name="Basic Cloning"></a><br />
<br />
</html><br />
<br />
<h2> Basic Cloning </h2><br />
<br />
<br />
<br />
<h3> Polymerase Chain Reaction </h3><br />
<br />
<br />
<p><br />
<br />
All PCRs were done using a standard 50 μL reaction volume with GoTaq® Green Master Mix 2X purchased from PROMEGA Corporation. <br><br />
<br />
Mix the following in a 0.2 mL microcentrifuge tube on ice: <br><br />
<br />
25 μL GoTaq® Green Master Mix 2X <br><br />
<br />
1-5 μL of 10 μM forward primer <br><br />
<br />
1-5 μL of 10 μM reverse primer <br><br />
<br />
<250 ng of DNA template <br><br />
<br />
QS 50 μl nuclease-free H2O <br><br />
<br />
Conduct the reaction in a thermocycler, adjusting anneal temperature and extension times accordingly. See your polymerase supplier protocol for more details on thermocycling.<br />
<br />
</p><br />
<br />
<h3> Error-prone Polymerase Chain Reaction </h3><br />
<br />
<br />
<p> <br />
<br />
<br />
<br />
Prepare 50 μL reaction: <br><br />
<br />
5 μL 10X Mutazyme II Rxn Buffer <br><br />
<br />
1 μL 40 mM dNTP mix (200 μM each final) <br><br />
<br />
1 μL 20 μM forward primer <br><br />
<br />
1 μL 20 μM reverse primer <br><br />
<br />
1 μL Mutazyme II DNA polymerase (2.5 U/μL) <br><br />
<br />
0.01 ng template <br><br />
<br />
QS 50 μL diH2O <br><br />
<br />
<br><br />
<br />
Program thermocycler as follows: <br><br />
<br />
95 °C, 2 min <br><br />
<br />
95 °C, 30 sec <br><br />
<br />
XX °C*, 30 sec <br><br />
<br />
72 °C, X min** <br><br />
<br />
32 cycles <br><br />
<br />
72 °C, 10 min <br><br />
<br />
4 °C, hold<br />
<br />
<br><br />
<br />
*Adjust annealing temperature according to Tm of primer. <br><br />
<br />
**Adjust extension time according to the length of amplified DNA. <br><br />
<br />
<br><br />
<br />
Note: Use 0.01 ng of template (calculate by insert and not by total plasmid). <br><br />
<br />
Calculate amount of template to use as follows: <br><br />
<br />
(bp for amplified region) / (bp in total plasmid) = % amplified region <br><br />
<br />
(conc. of total plasmid) x (% amplified region as a decimal) = conc. of amplified region <br><br />
<br />
<br><br />
<br />
Note: Never pipette less than 0.5 μL. <br><br />
<br />
(0.01 ng of template) / (conc. of amplified region) = vol of template to add to PCR <br><br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Restriction Endonuclease Reaction (Digestion) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
All restriction enzyme reactions were done using a 50 μl reaction volume. Restriction enzymes and buffers were purchased from New England Biolabs® Inc. <br><br />
<br />
Mix the following in a 0.2 mL PCR tube: <br><br />
<br />
<br />
1 μg of DNA <br><br />
<br />
5 μL of the appropriate 10X New England Biolab® Buffer <br><br />
<br />
1 μL of each restriction enzyme (add last) <br><br />
<br />
QS 50μL nuclease-free H2O <br><br />
<br />
Incubate the reaction for 1 hr <br><br />
<br />
Heat inactive the reaction at the appropriate temperature <br><br />
<br><br />
<br />
Note: Thaw the restriction enzyme(s) on ice to improve shelf life <br><br />
<br />
<br />
</p> <br />
<br />
<br />
<h3> Ligation </h3><br />
<br />
<br />
<p><br />
<br />
T4 DNA Ligase and Buffer were purchased from New England Biolabs® Inc. <br><br />
<br />
1. Prepare the following in a 0.2 mL microcentrifuge tube: <br><br />
<br />
50.0 ng vector DNA* <br><br />
<br />
37.5 ng vector DNA* <br><br />
<br />
2 μL 10X T4 DNA Ligase Buffer <br><br />
<br />
1 μL T4 DNA Ligase <br><br />
<br />
QS 20 μL diH2O <br><br />
<br />
2. Incubate the reaction at room temperature for 10-30 minutes or at 16 °C overnight. <br><br />
<br />
3. Heat inactivate at 65 °C for 10 minutes. <br><br />
<br />
4. Chill on ice before starting a transformation reaction. <br><br />
<br />
<br><br />
<br />
*The exact amount of DNA is dependent on the number of base pairs. In order to conduct a proper reaction consult the New England Biolab Ligation Calculator at:<br />
<br />
http://nebiocalculator.neb.com/#!/<br />
<br />
</p><br />
<br />
<br />
<html><a name="Escherichia coli Protocols"></a><br />
<br />
</html><br />
<br />
<br />
<h2> <i> Escherichia coli </i> Protocols (XL1-Blue and XL10-Gold) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<p> <br />
Competent cells take two days to culture and aliquot. <br><br />
Day 1: <br><br />
1. Streak an aliquot of competent cells onto two LB-plates without antibiotics.* <br><br />
2. Incubate at 37 °C overnight. <br><br />
Day 2: <br><br />
1. In two 250 mL baffle flasks add 50 mL of SOB media. <br><br />
2. Scrape as many single colonies into either flask. <br><br />
3. Incubate and shake at 37 °C and 250 rpm for 2-3 hours. <br><br />
4. Check the optical density of the cells at 600 nm after 2 hours. <br><br />
5. Stop incubation when cultures reach approximately 0.5 optical density. <br><br />
6. Add the contents of the flask into separate 50 mL flat bottomed centrifuge tubes. <br><br />
7. Spin down the cells at 2500 rpm at 4 °C for 15 minutes. <br><br />
8. Decant the supernatant. <br><br />
9. Resuspend the cells in 16 mL of CCMB by pipetting or gently vortexing. <br><br />
10. Incubate the cells on ice for 20 minutes. <br><br />
11. Spin down the cells at 2500 rpm at 4 °C for 10 minutes. <br> <br />
12. Decant the supernatant. <br><br />
13. Resuspend the cells in 4 mL of CCMB. <br><br />
14. Quickly aliquot the cells into 1.7 mL cryogenic vials or 1.5 mL centrifuge tubes.** <br><br />
15. Store the competent cell aliquots at -80 °C. <br><br />
<br><br />
*Streak in such a way that there should be individual colony growth and no clumps after the incubation. <br><br />
**We did this in a -20 °C cold room and using an automated repeater pipette. The volume of each aliquot depends on the number of transformations you intend to do at a time. <br><br />
<br><br />
Note: After removing the cells from incubation keep them on ice or as cold as possible. <br><br />
</p><br />
<br />
<br />
<h3> Chemically Competent Cell Transformations </h3><br />
<br />
<br />
<p> <br />
1. Thaw competent <i> E. coli </i> cells on ice (XL1-Blue or XL10-Gold).* <br><br />
2. Add 50 μL of competent cells to sterile 14 mL culture tube. <br><br />
3. Add 1 μL (~100-200 ng)* of the mini-prep to each culture tube. <br><br />
4. Equilibrate the cells on ice for 10 minutes. <br><br />
5. Heat shock the cells at 42 °C for 30-45 seconds.** <br><br />
6. Immediately place the cells back on ice for 3 minutes. <br><br />
7. Add 250 μL LB media without antibiotics and shake at 250 rpm and 37 °C for 30 minutes. <br><br />
8. Spread 10 μL and 290 μL on an appropriate LB-antibiotic plate. <br><br />
9. Invert the plate and incubate at 37 °C overnight.<br />
<br><br />
*The exact amount of DNA to add depends on your cell's transformation efficiency. However, it is acceptable to add a larger amount to increase the number of transformed cells. <br><br />
**Do not heat shock for an extended duration as this may damage and/or kill your cells.<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnights </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottom tube, add 3-5mL of LB and an appropriate volume of antibiotic(s). <br><br />
<br />
2. Swipe several individual colonies, do not collect satellites or colony clumps, with a pipette tip. <br><br />
<br />
3. Swirl the colony tip in the tube, there should be no visible cell clumps. <br><br />
<br />
4. Incubate and shake the tube at 37C at 250rpm for 12-16 hours and no longer than 20 hours. <br><br />
<br />
</p><br />
<br />
<br />
<h3> DNA-Extraction and mini-preps </h3><br />
<br />
<br />
<p><br />
<br />
All DNA mini-preps were prepared using EPOCH mini-kits and following the supplied protocols.<br />
<br />
</p><br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500μL of 40% Glycerol and 500uL of LB (no antibiotics) or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Saccharomyces cerevisiae"></a><br />
<br />
</html><br />
<br />
<h2> <i> Saccharomyces cerevisiae </i> (PyE1 Yeast) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<br />
<p><br />
<br />
This process take 4 days in lab with a 1 day wait for incubation. <br><br />
<br />
<br />
Day 1: <br><br />
<br />
1. Streak yeast cells onto a YPD plate.* <br><br />
<br />
2. Invert the plate and incubate at 30C for 2 days. <br><br />
<br />
<br />
Day 3: <br><br />
<br />
1. Add 50mL of YPD liquid media into a 250mL baffle flask. <br><br />
<br />
2. Swipe as many individual colonies as you can see into the YPD media.** <br><br />
<br />
3. Incubate and shake the culture at 30C at 250rpm overnight approximately 24 hours. <br><br />
<br />
<br />
Day 4: <br><br />
<br />
1. Take an optical density measurement. <br><br />
<br />
2. In three 250mL baffle flask add the portions of the overnight liquid culture. <br><br />
<br />
3. Dilute each culture to approximately 0.4 optical density with YPD. <br><br />
<br />
4. Incubate and shake the cultures at 30C at 250rpm until the optical density reaches 1.2-1.6. <br><br />
<br />
5. Collect each culture into separate 50ml flat-bottomed centrifuge tubes. <br><br />
<br />
6. Spin down the cells at 4000g for 5 minutes at 4C. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cells in 100mL total for all three culture of dH2O. <br><br />
<br />
9. Combine the suspensions into two 50mL flat-bottomed centrifuge tubes. <br><br />
<br />
10. Spin down the cells as above. <br><br />
<br />
11. Decant the supernatant. <br><br />
<br />
12. Resuspend each in 3mL of 100mM Lithium Acetate. <br><br />
<br />
13. Transfer both cultures into a single 15mL conical centrifuge tube. <br><br />
<br />
14. Spin down the cells at 3000rpm for 5 minutes. <br><br />
<br />
15. Resuspend the cells in 0.75mL of 100mM Lithium Acetate, total volume is roughly 2mL. <br><br />
<br />
16. Qualitatively bring up the volume to 3.5mL by adding 40% Glycerol. <br><br />
<br />
17. Aliquot the cells into 1.5mL centrifuge tubes or 1.7mL cryogenic vials.*** <br><br />
<br />
<br />
*Streak in such a way that there are individual colonies visible on the plate without clumps or satellite colonies. <br><br />
<br />
**Collect only individual visible colonies. Do not collect clumps or satellite colonies. <br><br />
<br />
***The volume of aliquots depends on the number to transformations you intend to do at a time. <br><br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Chemically Competent Transformations </h3><br />
<br />
<br />
<p><br />
<br />
This protocol assumes a 50μL aliquot of yeast competent cells were made. <br><br />
<br />
Furthermore, this protocol prepares enough cells for 6 yeast transformations. <br><br />
<br />
<br><br />
<br />
1. Add the following to 50μL of yeast competent cells: <br><br />
<br />
240μL of Polyethylene Glycol - 3350 (PEG-3350) <br><br />
<br />
36μL of 1M Lithium Acetate <br><br />
<br />
32μL of dH2O <br><br />
<br />
<br />
<br />
2. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
3. Aliquot 59μL of the mixture into a 0.2mL microcentrifuge tube. <br><br />
<br />
4. Add 1uL (~100-200ng) of DNA. <br><br />
<br />
5. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
6. Incubate the mixture at 30C for 30 minutes. <br><br />
<br />
7. Heat shock the mixture at 42C for 20 minutes. <br><br />
<br />
8. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
9. Decant the supernatant. <br><br />
<br />
10. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
11. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
12. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
13. Plate 50-150μL of the mixture onto an appropriate Selective Dropout Media plate. <br><br />
<br />
14. Invert and incubate at 30C for 2 days. <br><br />
<br />
*The exact amount of DNA depends on the transformation efficiency of your competent cells. <br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnight Culturing </h3><br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Swipe 3 individually visible yeast colonies and add them to the culture tube media. <br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%.<br />
<br />
</p><br />
<br />
<br />
<h3> Culture Passaging </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Take 20-50μL from a previous overnight or passage culture and add it to the culture media.<br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%. <br><br />
<br />
Note: The exact amount of culture that you take from a previous culture is irrelevant as long as at least 1 living cell is passaged.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500uL of 40% glycerol and 500μL of Selective Dropout media or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Flow Cytometry"></a><br />
<br />
</html><br />
<br />
<h2> Flow Cytometry </h2><br />
<br />
<br />
<h3> Dilutions </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. From an overnight culture measure the optical density at 660nm by making 1:10 dilutions. <br><br />
<br />
2. Take enough culture to make a 1mL aliquot with OD 0.4. <br><br />
<br />
3. Spin down the aliquot in a 1.5mL centrifuge tube at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 800μL of the appropriate selective dropout media and 200μL of 20% glucose. <br><br />
<br />
6. Transfer the new culture to a 14mL culture tube. <br><br />
<br />
7. Incubate and shake at 30C and 250rpm for at least 6 hours (1.2-1.6 optical density).<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Preparations for Analysis using C6 Accuri Flow Cytometer </h3><br />
<br />
<br />
<p><br />
<br />
<br />
1. From the dilution previously made, measure the optical density, roughly 1.2-1.6. <br><br />
<br />
2. Make an aliquot of 500μL of the dilution culture in a 1.5mL centrifuge tube. <br><br />
<br />
3. Spin down the aliquot at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 500μL of PBS(F). <br><br />
<br />
6. Spin down the resuspension at 3000rpm for 3 minutes. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cell pellet in another 500uL of PBS(F).* <br><br />
<br />
9. Prepare the C6 Accuri Flow Cytometer by running a backflush cycle and a dH2O cycle. <br><br />
<br />
10. Load the sample onto the sip. <br><br />
<br />
11. Run the sample with 100,000 cell count. <br><br />
<br />
12. Repeat for all samples and make sure to change data cells otherwise the old data is erased. <br><br />
<br />
13. Once finished, run a cleaning cycle with Accuri approved cleaning solution, then run a dH2O cycle. <br><br />
<br />
<br />
<br />
*For special cases do not resuspend all samples, instead resuspend immediately before running the sample through the flow cytometer.<br />
<br />
<br />
</p><br />
<br />
<br />
<html><a name="Fluorescence Activated Cell Sorting"></a><br />
<br />
</html><br />
<br />
<h2> Fluorescence Activated Cell Sorting </h2><br />
<br />
<br />
<h3> Final Preparations </h3><br />
<br />
<br />
<br />
<p> <br />
<br />
Sample Prep:<br />
<br />
<br />
Spin down samples and negative control (5000 RPM, 1 min), keeping in mind the library size. Aspirate off supernatant. Resuspend in PBSF. Spin down cells. Aspirate off supernatant. Resuspend in PBSF. <br><br />
<br />
<br><br />
<br />
1. Open the “iGEM Template” file in the FACS Software and change name to current date and sort cycle<br><br />
<br />
2. Make sure the stream is stable (look for green light in bottom right corner). If not, run the Sort Calibration order. <br><br />
<br />
3. Run Negative Control from the PyE1 cells. <br><br />
<br />
a. Load cells onto carrier and into the machine. Press play button on screen. <br><br />
<br />
b. Set gate around lower left quadrant of cells to ensure single cell analysis using Forward Scatter Area and Side Scatter Area as your axes. Make sure oval gate covers around 80% of cell population. <br><br />
<br />
c. Set second gate on the first gated population by double-clicking on the gated population and using Forward Scatter Height and Forward Scatter Width as your axes. You will notice two distinct populations. Try to focus on the single cell portion of the plot. <br><br />
<br />
NOTE: If you see a large portion of the second gated population existing near the upper right edge of the first gate, you may need to enlarge the first gate to fit more of the population. <br><br />
<br />
d. Press record. Record 100,000 events and stop run. Move to Next Tube. <br><br />
<br />
4. Run first control (Gene clone) <br><br />
<br />
a. Follow step 3 to run the first control <br><br />
<br />
5. Run first library sample <br><br />
<br />
a. Follow steps 3b and 3c to set first two gates correctly. <br><br />
<br />
b. Set final gate for sort which includes top 1% of GFP producers from second gated population. <br><br />
<br />
c. Use final gate to set up the sort. <br><br />
<br />
d. Select sort conditions at the bottom of the screen. <br><br />
<br />
e. Insert and load collection tube. <br><br />
<br />
f. Record 100,000 events, and sort 10x the library size <br><br />
<br />
6. Run Bleach and diH2O through FACS to avoid cross-contamination. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Protein Expression"></a><br />
<br />
</html><br />
<br />
<h2> Protein Expression </h2><br />
<br />
<br />
<h3> Overnight Cultures </h3><br />
<br />
<p><br />
<br />
1. Add 25mL TB and 25uL Kan to a 250mL baffled flask <br><br />
<br />
2. Stab a glycerol stock with a p1000 pipette and swirl in the flask of media <br><br />
<br />
3. Put flask in 37C shaker overnight <br />
<br />
</p><br />
<br />
<h3> Protein Expression </h3><br />
<br />
<p> <br />
<br />
1. Add 500μL 1000x Kanamycin to 500mL TB in 2L baffled flask <br><br />
<br />
2. Transfer 10mL overnight culture to TB <br><br />
<br />
3. Shake at 37C (DO WE NEED THE RPM?) <br><br />
<br />
4. Remove flask from shaker when optical density is between 0.5 and 0.8 <br><br />
<br />
5. Allow flask to rest at room temp for 30 min <br><br />
<br />
6. Add 125μL 1M IPTG <br><br />
<br />
7. Shake flask at 18C overnight<br />
<br />
</p><br />
<br />
<h3> Protein Extraction and Purification </h3><br />
<br />
<p><br />
<br />
1. Transfer cell culture to centrifuge flask <br><br />
<br />
2. Centrifuge culture at 4000g for 10 min <br><br />
<br />
3. Discard supernatant <br><br />
<br />
4. Resuspend pellet in 25mL lysis buffer <br><br />
<br />
5. Add 250μL of 100x PMSF, 250μL of 100mg/mL lysozyme, and 250μL of 10mg/mL DNAse <br><br />
<br />
6. Sonicate sample with 0.25inch probe for 5 min at 70% amplitude with 20 sec on and off pulses <br><br />
<br />
7. Take 50μL total sample <br><br />
<br />
8. Transfer lysate to SS-34 centrifuge tube <br><br />
<br />
9. Centrifuge for 30 min at 18000g <br><br />
<br />
10. Take 50μL soluble sample<br />
<br />
</p><br />
<br />
<h3> Nickel Nitrotriacetic Acid Chromatography (Nickel-NTA Chromatography) </h3><br />
<br />
<p><br />
<br />
1. Add 5mL 50%(v/v) nickel resin in ethanol to 25mL column and allow to settle to ~2.5mL(CV) <br><br />
<br />
2. Rinse with 10CV H20 <br><br />
<br />
3. Equilibrate with 10CV lysis buffer <br><br />
<br />
4. Load sample onto column <br><br />
<br />
5. Wash column with 15CV lysis buffer <br><br />
<br />
6. Perform 2 additional wash steps with 15CV <br><br />
<br />
7. Elute sample in 10CV elution buffer and collect eluate <br><br />
<br />
8. Take 50μL pure sample <br />
<br />
</p><br />
<br />
<h3> Size Exclusion Chromatography (S.E.C.) </h3><br />
<p><br />
1. Concentrate sample to as high as possible without inducing protein aggregation<br><br />
2. Pre-equilibrate Superdex 75 column with 48 ml PBS <br><br />
3. Inject 500 ul sample onto column <br><br />
4. Run 36 ml PBS through column at 0.5 ml/min, collecting 1 ml fractions <br><br />
5. Verify presence of protein in fractions by measuring concentration on Nanodrop and running SDS-PAGE (15 kDa protein should elute at ~13 ml)<br><br />
6. Pool fractions containing protein <br />
</p><br />
<br />
<br />
<html><a name="Stability Analysis"></a><br />
<br />
</html><br />
<br />
<h2> Stability Analysis </h2><br />
<br />
<br />
<h3> Circular Dichroism: Wavelength Scan </h3><br />
<p><br />
1. Load 1mm cuvette with 400uL protein solution onto CD <br><br />
2. Take wavelength scan <br><br />
260nm-190nm <br><br />
sample every 1nm <br><br />
averaging time 3sec <br><br />
1 scan <br><br />
step scan <br><br />
25C <br><br />
3. Record wavelength which gives strongest signal (222nm)<br />
</p><br />
<br />
<h3> Circular Dichroism: Guanidine Melt </h3><br />
<p><br />
1. Load 1cm cuvette containing 1.996mL of 0.05mg/mL protein solution and stirrer onto CD <br><br />
2. Prepare 8mL of 0.05mg/mL protein in concentrated guanidine solution <br><br />
3. Set up Automixer with guanidine solution on one syringe and waste tube on other syringe <br><br />
4. Titrate up to 6 M guanidine, taking a CD measurement at 222 nm every 0.15 M interval <br><br />
5. Also measure the fluorescence at 280 nm to ensure the total protein concentration is not changing<br />
</p><br />
<br />
<br />
<br />
<html><br />
<br />
<a href="#linktotop">Back To Top</a><br />
</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2014-10-17T21:19:06Z<p>Kristanguyen: </p>
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<div>{{Template:Team:UW/CSS}}<br />
<br />
<br />
<h1> Protocols </h1><br />
<br />
<br />
<html><a name="Media, Plates, and Sol"></a><br />
<br />
</html><br />
<br />
<br />
<h2> Media, Plates, and Solutions </h2><br />
<br />
<br />
<h3> Competent Cell Media Buffer (CCMB) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
100 g glycerol (liquid) <br><br />
<br />
10 mL x 1 M potassium acetate <br><br />
<br />
11.8 g CaCl2*H2O <br><br />
<br />
4 g MnCl2 <br><br />
<br />
2 g MgCl2 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<h3> Super Optimal Broth (SOB) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
20 g Tryptone <br><br />
<br />
5 g Yeast Extract <br><br />
<br />
10 mL x 1 M NaCl <br><br />
<br />
2.5 mL x 1 M KCl <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Phosphate Buffered Saline (PBS) Solution </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container or 1 L beaker: <br><br />
<br />
8 g NaCl <br><br />
<br />
1.44 g Na2HPO4 <br><br />
<br />
0.8 g KCl <br><br />
<br />
0.24 g KH2PO4 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Buffer to pH 7.4 <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<h3> PSBF (PBS for Flow) </h3> <br />
<br />
<p><br />
<br />
Mix the following in a 1 L beaker: <br><br />
<br />
25 mL 20X PBS, pH 7.4 <br><br />
<br />
574 mL H2O <br><br />
<br />
2.5 g BSA (0.5%)* <br><br />
<br />
Filter and store at 4 °C <br><br />
<br><br />
*Do not need to pH - should be at pH 7.4 like 20X PBS <br><br />
<br />
</p> <br />
<br />
<br />
<h3> Yeast Extract Peptone Dextrose (YPD) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following into 950 mL of dH2O in a 1 L bottle: <br><br />
<br />
20 g peptone <br><br />
<br />
10 g yeast extract <br><br />
<br />
Autoclave (20 min at 121 °C and 20 psi) <br><br />
<br />
Add 50 mL 40% glucose <br><br />
<br />
Sterile filter into a 1 L bottle <br><br />
<br />
<br><br />
<br />
Note: For long-term liquid media storage, do not add 40% glucose. Instead add the glucose directly into cell cultures. <br><br />
<br />
Note: For YPD-plates add 24 g agar to the peptone and yeast extract before autoclaving. <br />
<br />
</p><br />
<br />
<br />
<h3> Selective Dropout Media, C-Uracil and C-Histidine (C-Ura and C-His) </h3><br />
<br />
<br />
<p><br />
<br />
Synthesized by the Yeast Resource Center at the University of Washington's <br />
<br />
Department of Genome Sciences and Department of Biochemistry.<br />
<br />
</p><br />
<br />
<br />
<h3> Guanidinium Hydrogen Chloride </h3><br />
<br />
<br />
<p><br />
<br />
For maximum effectiveness, final concentration should be approximately 8.5 M in PBS.<br><br />
<br />
203 g guanidinium hydrogen chloride <br><br />
<br />
250 mL PBS solution* <br><br />
<br />
Add dilute HCl to pH 7.4 <br><br />
<br />
<br><br />
<br />
*Alternatively add slightly less than 250 mL of PBS in order to buffer the solution to the appropriate volume, then add more dH2O as necessary. <br />
<br />
</p><br />
<br />
<html><a name="Basic Cloning"></a><br />
<br />
</html><br />
<br />
<h2> Basic Cloning </h2><br />
<br />
<br />
<br />
<h3> Polymerase Chain Reaction </h3><br />
<br />
<br />
<p><br />
<br />
All PCRs were done using a standard 50 μL reaction volume with GoTaq® Green Master Mix 2X purchased from PROMEGA Corporation. <br><br />
<br />
Mix the following in a 0.2 mL microcentrifuge tube on ice: <br><br />
<br />
25 μL GoTaq® Green Master Mix 2X <br><br />
<br />
1-5 μL of 10 μM forward primer <br><br />
<br />
1-5 μL of 10 μM reverse primer <br><br />
<br />
<250 ng of DNA template <br><br />
<br />
QS 50 μl nuclease-free H2O <br><br />
<br />
Conduct the reaction in a thermocycler, adjusting anneal temperature and extension times accordingly. See your polymerase supplier protocol for more details on thermocycling.<br />
<br />
</p><br />
<br />
<h3> Error-prone Polymerase Chain Reaction </h3><br />
<br />
<br />
<p> <br />
<br />
<br />
<br />
Prepare 50 μL reaction: <br><br />
<br />
5 μL 10X Mutazyme II Rxn Buffer <br><br />
<br />
1 μL 40 mM dNTP mix (200 μM each final) <br><br />
<br />
1 μL 20 μM forward primer <br><br />
<br />
1 μL 20 μM reverse primer <br><br />
<br />
1 μL Mutazyme II DNA polymerase (2.5 U/μL) <br><br />
<br />
0.01 ng template <br><br />
<br />
QS 50 μL diH2O <br><br />
<br />
<br><br />
<br />
Program thermocycler as follows: <br><br />
<br />
95 °C, 2 min <br><br />
<br />
95 °C, 30 sec <br><br />
<br />
XX °C*, 30 sec <br><br />
<br />
72 °C, X min** <br><br />
<br />
32 cycles <br><br />
<br />
72 °C, 10 min <br><br />
<br />
4 °C, hold<br />
<br />
<br><br />
<br />
*Adjust annealing temperature according to Tm of primer. <br><br />
<br />
**Adjust extension time according to the length of amplified DNA. <br><br />
<br />
<br><br />
<br />
Note: Use 0.01 ng of template (calculate by insert and not by total plasmid). <br><br />
<br />
Calculate amount of template to use as follows: <br><br />
<br />
(bp for amplified region) / (bp in total plasmid) = % amplified region <br><br />
<br />
(conc. of total plasmid) x (% amplified region as a decimal) = conc. of amplified region <br><br />
<br />
<br><br />
<br />
Note: Never pipette less than 0.5 μL. <br><br />
<br />
(0.01 ng of template) / (conc. of amplified region) = vol of template to add to PCR <br><br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Restriction Endonuclease Reaction (Digestion) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
All restriction enzyme reactions were done using a 50 μl reaction volume. Restriction enzymes and buffers were purchased from New England Biolabs® Inc. <br><br />
<br />
Mix the following in a 0.2 mL PCR tube: <br><br />
<br />
<br />
1 μg of DNA <br><br />
<br />
5 μL of the appropriate 10X New England Biolab® Buffer <br><br />
<br />
1 μL of each restriction enzyme (add last) <br><br />
<br />
QS 50μL nuclease-free H2O <br><br />
<br />
Incubate the reaction for 1 hr <br><br />
<br />
Heat inactive the reaction at the appropriate temperature <br><br />
<br><br />
<br />
Note: Thaw the restriction enzyme(s) on ice to improve shelf life <br><br />
<br />
<br />
</p> <br />
<br />
<br />
<h3> Ligation </h3><br />
<br />
<br />
<p><br />
<br />
T4 DNA Ligase and Buffer were purchased from New England Biolabs® Inc. <br><br />
<br />
1. Prepare the following in a 0.2 mL microcentrifuge tube: <br><br />
<br />
50.0 ng vector DNA* <br><br />
<br />
37.5 ng vector DNA* <br><br />
<br />
2 μL 10X T4 DNA Ligase Buffer <br><br />
<br />
1 μL T4 DNA Ligase <br><br />
<br />
QS 20 μL diH2O <br><br />
<br />
2. Incubate the reaction at room temperature for 10-30 minutes or at 16 °C overnight. <br><br />
<br />
3. Heat inactivate at 65 °C for 10 minutes. <br><br />
<br />
4. Chill on ice before starting a transformation reaction. <br><br />
<br />
<br><br />
<br />
*The exact amount of DNA is dependent on the number of base pairs. In order to conduct a proper reaction consult the New England Biolab Ligation Calculator at:<br />
<br />
http://nebiocalculator.neb.com/#!/<br />
<br />
</p><br />
<br />
<br />
<html><a name="Escherichia coli Protocols"></a><br />
<br />
</html><br />
<br />
<br />
<h2> <i> Escherichia coli </i> Protocols (XL1-Blue and XL10-Gold) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<p> <br />
Competent cells take two days to culture and aliquot. <br><br />
Day 1: <br><br />
1. Streak an aliquot of competent cells onto two LB-plates without antibiotics.* <br><br />
2. Incubate at 37 °C overnight. <br><br />
Day 2: <br><br />
1. In two 250 mL baffle flasks add 50 mL of SOB media. <br><br />
2. Scrape as many single colonies into either flask. <br><br />
3. Incubate and shake at 37 °C and 250 rpm for 2-3 hours. <br><br />
4. Check the optical density of the cells at 600 nm after 2 hours. <br><br />
5. Stop incubation when cultures reach approximately 0.5 optical density. <br><br />
6. Add the contents of the flask into separate 50 mL flat bottomed centrifuge tubes. <br><br />
7. Spin down the cells at 2500 rpm at 4 °C for 15 minutes. <br><br />
8. Decant the supernatant. <br><br />
9. Resuspend the cells in 16 mL of CCMB by pipetting or gently vortexing. <br><br />
10. Incubate the cells on ice for 20 minutes. <br><br />
11. Spin down the cells at 2500 rpm at 4 °C for 10 minutes. <br> <br />
12. Decant the supernatant. <br><br />
13. Resuspend the cells in 4 mL of CCMB. <br><br />
14. Quickly aliquot the cells into 1.7 mL cryogenic vials or 1.5 mL centrifuge tubes.** <br><br />
15. Store the competent cell aliquots at -80 °C. <br><br />
<br><br />
*Streak in such a way that there should be individual colony growth and no clumps after the incubation. <br><br />
**We did this in a -20 °C cold room and using an automated repeater pipette. The volume of each aliquot depends on the number of transformations you intend to do at a time. <br><br />
<br><br />
Note: After removing the cells from incubation keep them on ice or as cold as possible. <br><br />
</p><br />
<br />
<br />
<h3> Chemically Competent Cell Transformations </h3><br />
<br />
<br />
<p> <br />
1. Thaw competent <i> E. coli </i> cells on ice (XL1-Blue or XL10-Gold).* <br><br />
2. Add 50 μL of competent cells to sterile 14 mL culture tube. <br><br />
3. Add 1 μL (~100-200 ng)* of the mini-prep to each culture tube. <br><br />
4. Equilibrate the cells on ice for 10 minutes. <br><br />
5. Heat shock the cells at 42 °C for 30-45 seconds.** <br><br />
6. Immediately place the cells back on ice for 3 minutes. <br><br />
7. Add 250 μL LB media without antibiotics and shake at 250 rpm and 37 °C for 30 minutes. <br><br />
8. Spread 10 μL and 290 μL on an appropriate LB-antibiotic plate. <br><br />
9. Invert the plate and incubate at 37 °C overnight.<br />
<br />
*The exact amount of DNA to add depends on your cell's transformation efficiency. However, it is acceptable to add a larger amount to increase the number of transformed cells. <br><br />
**Do not heat shock for an extended duration as this may damage and/or kill your cells.<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnights </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottom tube, add 3-5mL of LB and an appropriate volume of antibiotic(s). <br><br />
<br />
2. Swipe several individual colonies, do not collect satellites or colony clumps, with a pipette tip. <br><br />
<br />
3. Swirl the colony tip in the tube, there should be no visible cell clumps. <br><br />
<br />
4. Incubate and shake the tube at 37C at 250rpm for 12-16 hours and no longer than 20 hours. <br><br />
<br />
</p><br />
<br />
<br />
<h3> DNA-Extraction and mini-preps </h3><br />
<br />
<br />
<p><br />
<br />
All DNA mini-preps were prepared using EPOCH mini-kits and following the supplied protocols.<br />
<br />
</p><br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500μL of 40% Glycerol and 500uL of LB (no antibiotics) or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Saccharomyces cerevisiae"></a><br />
<br />
</html><br />
<br />
<h2> <i> Saccharomyces cerevisiae </i> (PyE1 Yeast) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<br />
<p><br />
<br />
This process take 4 days in lab with a 1 day wait for incubation. <br><br />
<br />
<br />
Day 1: <br><br />
<br />
1. Streak yeast cells onto a YPD plate.* <br><br />
<br />
2. Invert the plate and incubate at 30C for 2 days. <br><br />
<br />
<br />
Day 3: <br><br />
<br />
1. Add 50mL of YPD liquid media into a 250mL baffle flask. <br><br />
<br />
2. Swipe as many individual colonies as you can see into the YPD media.** <br><br />
<br />
3. Incubate and shake the culture at 30C at 250rpm overnight approximately 24 hours. <br><br />
<br />
<br />
Day 4: <br><br />
<br />
1. Take an optical density measurement. <br><br />
<br />
2. In three 250mL baffle flask add the portions of the overnight liquid culture. <br><br />
<br />
3. Dilute each culture to approximately 0.4 optical density with YPD. <br><br />
<br />
4. Incubate and shake the cultures at 30C at 250rpm until the optical density reaches 1.2-1.6. <br><br />
<br />
5. Collect each culture into separate 50ml flat-bottomed centrifuge tubes. <br><br />
<br />
6. Spin down the cells at 4000g for 5 minutes at 4C. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cells in 100mL total for all three culture of dH2O. <br><br />
<br />
9. Combine the suspensions into two 50mL flat-bottomed centrifuge tubes. <br><br />
<br />
10. Spin down the cells as above. <br><br />
<br />
11. Decant the supernatant. <br><br />
<br />
12. Resuspend each in 3mL of 100mM Lithium Acetate. <br><br />
<br />
13. Transfer both cultures into a single 15mL conical centrifuge tube. <br><br />
<br />
14. Spin down the cells at 3000rpm for 5 minutes. <br><br />
<br />
15. Resuspend the cells in 0.75mL of 100mM Lithium Acetate, total volume is roughly 2mL. <br><br />
<br />
16. Qualitatively bring up the volume to 3.5mL by adding 40% Glycerol. <br><br />
<br />
17. Aliquot the cells into 1.5mL centrifuge tubes or 1.7mL cryogenic vials.*** <br><br />
<br />
<br />
*Streak in such a way that there are individual colonies visible on the plate without clumps or satellite colonies. <br><br />
<br />
**Collect only individual visible colonies. Do not collect clumps or satellite colonies. <br><br />
<br />
***The volume of aliquots depends on the number to transformations you intend to do at a time. <br><br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Chemically Competent Transformations </h3><br />
<br />
<br />
<p><br />
<br />
This protocol assumes a 50μL aliquot of yeast competent cells were made. <br><br />
<br />
Furthermore, this protocol prepares enough cells for 6 yeast transformations. <br><br />
<br />
<br><br />
<br />
1. Add the following to 50μL of yeast competent cells: <br><br />
<br />
240μL of Polyethylene Glycol - 3350 (PEG-3350) <br><br />
<br />
36μL of 1M Lithium Acetate <br><br />
<br />
32μL of dH2O <br><br />
<br />
<br />
<br />
2. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
3. Aliquot 59μL of the mixture into a 0.2mL microcentrifuge tube. <br><br />
<br />
4. Add 1uL (~100-200ng) of DNA. <br><br />
<br />
5. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
6. Incubate the mixture at 30C for 30 minutes. <br><br />
<br />
7. Heat shock the mixture at 42C for 20 minutes. <br><br />
<br />
8. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
9. Decant the supernatant. <br><br />
<br />
10. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
11. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
12. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
13. Plate 50-150μL of the mixture onto an appropriate Selective Dropout Media plate. <br><br />
<br />
14. Invert and incubate at 30C for 2 days. <br><br />
<br />
*The exact amount of DNA depends on the transformation efficiency of your competent cells. <br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnight Culturing </h3><br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Swipe 3 individually visible yeast colonies and add them to the culture tube media. <br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%.<br />
<br />
</p><br />
<br />
<br />
<h3> Culture Passaging </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Take 20-50μL from a previous overnight or passage culture and add it to the culture media.<br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%. <br><br />
<br />
Note: The exact amount of culture that you take from a previous culture is irrelevant as long as at least 1 living cell is passaged.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500uL of 40% glycerol and 500μL of Selective Dropout media or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Flow Cytometry"></a><br />
<br />
</html><br />
<br />
<h2> Flow Cytometry </h2><br />
<br />
<br />
<h3> Dilutions </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. From an overnight culture measure the optical density at 660nm by making 1:10 dilutions. <br><br />
<br />
2. Take enough culture to make a 1mL aliquot with OD 0.4. <br><br />
<br />
3. Spin down the aliquot in a 1.5mL centrifuge tube at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 800μL of the appropriate selective dropout media and 200μL of 20% glucose. <br><br />
<br />
6. Transfer the new culture to a 14mL culture tube. <br><br />
<br />
7. Incubate and shake at 30C and 250rpm for at least 6 hours (1.2-1.6 optical density).<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Preparations for Analysis using C6 Accuri Flow Cytometer </h3><br />
<br />
<br />
<p><br />
<br />
<br />
1. From the dilution previously made, measure the optical density, roughly 1.2-1.6. <br><br />
<br />
2. Make an aliquot of 500μL of the dilution culture in a 1.5mL centrifuge tube. <br><br />
<br />
3. Spin down the aliquot at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 500μL of PBS(F). <br><br />
<br />
6. Spin down the resuspension at 3000rpm for 3 minutes. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cell pellet in another 500uL of PBS(F).* <br><br />
<br />
9. Prepare the C6 Accuri Flow Cytometer by running a backflush cycle and a dH2O cycle. <br><br />
<br />
10. Load the sample onto the sip. <br><br />
<br />
11. Run the sample with 100,000 cell count. <br><br />
<br />
12. Repeat for all samples and make sure to change data cells otherwise the old data is erased. <br><br />
<br />
13. Once finished, run a cleaning cycle with Accuri approved cleaning solution, then run a dH2O cycle. <br><br />
<br />
<br />
<br />
*For special cases do not resuspend all samples, instead resuspend immediately before running the sample through the flow cytometer.<br />
<br />
<br />
</p><br />
<br />
<br />
<html><a name="Fluorescence Activated Cell Sorting"></a><br />
<br />
</html><br />
<br />
<h2> Fluorescence Activated Cell Sorting </h2><br />
<br />
<br />
<h3> Final Preparations </h3><br />
<br />
<br />
<br />
<p> <br />
<br />
Sample Prep:<br />
<br />
<br />
Spin down samples and negative control (5000 RPM, 1 min), keeping in mind the library size. Aspirate off supernatant. Resuspend in PBSF. Spin down cells. Aspirate off supernatant. Resuspend in PBSF. <br><br />
<br />
<br><br />
<br />
1. Open the “iGEM Template” file in the FACS Software and change name to current date and sort cycle<br><br />
<br />
2. Make sure the stream is stable (look for green light in bottom right corner). If not, run the Sort Calibration order. <br><br />
<br />
3. Run Negative Control from the PyE1 cells. <br><br />
<br />
a. Load cells onto carrier and into the machine. Press play button on screen. <br><br />
<br />
b. Set gate around lower left quadrant of cells to ensure single cell analysis using Forward Scatter Area and Side Scatter Area as your axes. Make sure oval gate covers around 80% of cell population. <br><br />
<br />
c. Set second gate on the first gated population by double-clicking on the gated population and using Forward Scatter Height and Forward Scatter Width as your axes. You will notice two distinct populations. Try to focus on the single cell portion of the plot. <br><br />
<br />
NOTE: If you see a large portion of the second gated population existing near the upper right edge of the first gate, you may need to enlarge the first gate to fit more of the population. <br><br />
<br />
d. Press record. Record 100,000 events and stop run. Move to Next Tube. <br><br />
<br />
4. Run first control (Gene clone) <br><br />
<br />
a. Follow step 3 to run the first control <br><br />
<br />
5. Run first library sample <br><br />
<br />
a. Follow steps 3b and 3c to set first two gates correctly. <br><br />
<br />
b. Set final gate for sort which includes top 1% of GFP producers from second gated population. <br><br />
<br />
c. Use final gate to set up the sort. <br><br />
<br />
d. Select sort conditions at the bottom of the screen. <br><br />
<br />
e. Insert and load collection tube. <br><br />
<br />
f. Record 100,000 events, and sort 10x the library size <br><br />
<br />
6. Run Bleach and diH2O through FACS to avoid cross-contamination. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Protein Expression"></a><br />
<br />
</html><br />
<br />
<h2> Protein Expression </h2><br />
<br />
<br />
<h3> Overnight Cultures </h3><br />
<br />
<p><br />
<br />
1. Add 25mL TB and 25uL Kan to a 250mL baffled flask <br><br />
<br />
2. Stab a glycerol stock with a p1000 pipette and swirl in the flask of media <br><br />
<br />
3. Put flask in 37C shaker overnight <br />
<br />
</p><br />
<br />
<h3> Protein Expression </h3><br />
<br />
<p> <br />
<br />
1. Add 500μL 1000x Kanamycin to 500mL TB in 2L baffled flask <br><br />
<br />
2. Transfer 10mL overnight culture to TB <br><br />
<br />
3. Shake at 37C (DO WE NEED THE RPM?) <br><br />
<br />
4. Remove flask from shaker when optical density is between 0.5 and 0.8 <br><br />
<br />
5. Allow flask to rest at room temp for 30 min <br><br />
<br />
6. Add 125μL 1M IPTG <br><br />
<br />
7. Shake flask at 18C overnight<br />
<br />
</p><br />
<br />
<h3> Protein Extraction and Purification </h3><br />
<br />
<p><br />
<br />
1. Transfer cell culture to centrifuge flask <br><br />
<br />
2. Centrifuge culture at 4000g for 10 min <br><br />
<br />
3. Discard supernatant <br><br />
<br />
4. Resuspend pellet in 25mL lysis buffer <br><br />
<br />
5. Add 250μL of 100x PMSF, 250μL of 100mg/mL lysozyme, and 250μL of 10mg/mL DNAse <br><br />
<br />
6. Sonicate sample with 0.25inch probe for 5 min at 70% amplitude with 20 sec on and off pulses <br><br />
<br />
7. Take 50μL total sample <br><br />
<br />
8. Transfer lysate to SS-34 centrifuge tube <br><br />
<br />
9. Centrifuge for 30 min at 18000g <br><br />
<br />
10. Take 50μL soluble sample<br />
<br />
</p><br />
<br />
<h3> Nickel Nitrotriacetic Acid Chromatography (Nickel-NTA Chromatography) </h3><br />
<br />
<p><br />
<br />
1. Add 5mL 50%(v/v) nickel resin in ethanol to 25mL column and allow to settle to ~2.5mL(CV) <br><br />
<br />
2. Rinse with 10CV H20 <br><br />
<br />
3. Equilibrate with 10CV lysis buffer <br><br />
<br />
4. Load sample onto column <br><br />
<br />
5. Wash column with 15CV lysis buffer <br><br />
<br />
6. Perform 2 additional wash steps with 15CV <br><br />
<br />
7. Elute sample in 10CV elution buffer and collect eluate <br><br />
<br />
8. Take 50μL pure sample <br />
<br />
</p><br />
<br />
<h3> Size Exclusion Chromatography (S.E.C.) </h3><br />
<p><br />
1. Concentrate sample to as high as possible without inducing protein aggregation<br><br />
2. Pre-equilibrate Superdex 75 column with 48 ml PBS <br><br />
3. Inject 500 ul sample onto column <br><br />
4. Run 36 ml PBS through column at 0.5 ml/min, collecting 1 ml fractions <br><br />
5. Verify presence of protein in fractions by measuring concentration on Nanodrop and running SDS-PAGE (15 kDa protein should elute at ~13 ml)<br><br />
6. Pool fractions containing protein <br />
</p><br />
<br />
<br />
<html><a name="Stability Analysis"></a><br />
<br />
</html><br />
<br />
<h2> Stability Analysis </h2><br />
<br />
<br />
<h3> Circular Dichroism: Wavelength Scan </h3><br />
<p><br />
1. Load 1mm cuvette with 400uL protein solution onto CD <br><br />
2. Take wavelength scan <br><br />
260nm-190nm <br><br />
sample every 1nm <br><br />
averaging time 3sec <br><br />
1 scan <br><br />
step scan <br><br />
25C <br><br />
3. Record wavelength which gives strongest signal (222nm)<br />
</p><br />
<br />
<h3> Circular Dichroism: Guanidine Melt </h3><br />
<p><br />
1. Load 1cm cuvette containing 1.996mL of 0.05mg/mL protein solution and stirrer onto CD <br><br />
2. Prepare 8mL of 0.05mg/mL protein in concentrated guanidine solution <br><br />
3. Set up Automixer with guanidine solution on one syringe and waste tube on other syringe <br><br />
4. Titrate up to 6 M guanidine, taking a CD measurement at 222 nm every 0.15 M interval <br><br />
5. Also measure the fluorescence at 280 nm to ensure the total protein concentration is not changing<br />
</p><br />
<br />
<br />
<br />
<html><br />
<br />
<a href="#linktotop">Back To Top</a><br />
</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2014-10-17T21:17:36Z<p>Kristanguyen: </p>
<hr />
<div>{{Template:Team:UW/CSS}}<br />
<br />
<br />
<h1> Protocols </h1><br />
<br />
<br />
<html><a name="Media, Plates, and Sol"></a><br />
<br />
</html><br />
<br />
<br />
<h2> Media, Plates, and Solutions </h2><br />
<br />
<br />
<h3> Competent Cell Media Buffer (CCMB) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
100 g glycerol (liquid) <br><br />
<br />
10 mL x 1 M potassium acetate <br><br />
<br />
11.8 g CaCl2*H2O <br><br />
<br />
4 g MnCl2 <br><br />
<br />
2 g MgCl2 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<h3> Super Optimal Broth (SOB) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
20 g Tryptone <br><br />
<br />
5 g Yeast Extract <br><br />
<br />
10 mL x 1 M NaCl <br><br />
<br />
2.5 mL x 1 M KCl <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Phosphate Buffered Saline (PBS) Solution </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container or 1 L beaker: <br><br />
<br />
8 g NaCl <br><br />
<br />
1.44 g Na2HPO4 <br><br />
<br />
0.8 g KCl <br><br />
<br />
0.24 g KH2PO4 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Buffer to pH 7.4 <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<h3> PSBF (PBS for Flow) </h3> <br />
<br />
<p><br />
<br />
Mix the following in a 1 L beaker: <br><br />
<br />
25 mL 20X PBS, pH 7.4 <br><br />
<br />
574 mL H2O <br><br />
<br />
2.5 g BSA (0.5%)* <br><br />
<br />
Filter and store at 4 °C <br><br />
<br><br />
*Do not need to pH - should be at pH 7.4 like 20X PBS <br><br />
<br />
</p> <br />
<br />
<br />
<h3> Yeast Extract Peptone Dextrose (YPD) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following into 950 mL of dH2O in a 1 L bottle: <br><br />
<br />
20 g peptone <br><br />
<br />
10 g yeast extract <br><br />
<br />
Autoclave (20 min at 121 °C and 20 psi) <br><br />
<br />
Add 50 mL 40% glucose <br><br />
<br />
Sterile filter into a 1 L bottle <br><br />
<br />
<br><br />
<br />
Note: For long-term liquid media storage, do not add 40% glucose. Instead add the glucose directly into cell cultures. <br><br />
<br />
Note: For YPD-plates add 24 g agar to the peptone and yeast extract before autoclaving. <br />
<br />
</p><br />
<br />
<br />
<h3> Selective Dropout Media, C-Uracil and C-Histidine (C-Ura and C-His) </h3><br />
<br />
<br />
<p><br />
<br />
Synthesized by the Yeast Resource Center at the University of Washington's <br />
<br />
Department of Genome Sciences and Department of Biochemistry.<br />
<br />
</p><br />
<br />
<br />
<h3> Guanidinium Hydrogen Chloride </h3><br />
<br />
<br />
<p><br />
<br />
For maximum effectiveness, final concentration should be approximately 8.5 M in PBS.<br><br />
<br />
203 g guanidinium hydrogen chloride <br><br />
<br />
250 mL PBS solution* <br><br />
<br />
Add dilute HCl to pH 7.4 <br><br />
<br />
<br><br />
<br />
*Alternatively add slightly less than 250 mL of PBS in order to buffer the solution to the appropriate volume, then add more dH2O as necessary. <br />
<br />
</p><br />
<br />
<html><a name="Basic Cloning"></a><br />
<br />
</html><br />
<br />
<h2> Basic Cloning </h2><br />
<br />
<br />
<br />
<h3> Polymerase Chain Reaction </h3><br />
<br />
<br />
<p><br />
<br />
All PCRs were done using a standard 50 μL reaction volume with GoTaq® Green Master Mix 2X purchased from PROMEGA Corporation. <br><br />
<br />
Mix the following in a 0.2 mL microcentrifuge tube on ice: <br><br />
<br />
25 μL GoTaq® Green Master Mix 2X <br><br />
<br />
1-5 μL of 10 μM forward primer <br><br />
<br />
1-5 μL of 10 μM reverse primer <br><br />
<br />
<250 ng of DNA template <br><br />
<br />
QS 50 μl nuclease-free H2O <br><br />
<br />
Conduct the reaction in a thermocycler, adjusting anneal temperature and extension times accordingly. See your polymerase supplier protocol for more details on thermocycling.<br />
<br />
</p><br />
<br />
<h3> Error-prone Polymerase Chain Reaction </h3><br />
<br />
<br />
<p> <br />
<br />
<br />
<br />
Prepare 50 μL reaction: <br><br />
<br />
5 μL 10X Mutazyme II Rxn Buffer <br><br />
<br />
1 μL 40 mM dNTP mix (200 μM each final) <br><br />
<br />
1 μL 20 μM forward primer <br><br />
<br />
1 μL 20 μM reverse primer <br><br />
<br />
1 μL Mutazyme II DNA polymerase (2.5 U/μL) <br><br />
<br />
0.01 ng template <br><br />
<br />
QS 50 μL diH2O <br><br />
<br />
<br><br />
<br />
Program thermocycler as follows: <br><br />
<br />
95 °C, 2 min <br><br />
<br />
95 °C, 30 sec <br><br />
<br />
XX °C*, 30 sec <br><br />
<br />
72 °C, X min** <br><br />
<br />
32 cycles <br><br />
<br />
72 °C, 10 min <br><br />
<br />
4 °C, hold<br />
<br />
<br><br />
<br />
*Adjust annealing temperature according to Tm of primer. <br><br />
<br />
**Adjust extension time according to the length of amplified DNA. <br><br />
<br />
<br><br />
<br />
Note: Use 0.01 ng of template (calculate by insert and not by total plasmid). <br><br />
<br />
Calculate amount of template to use as follows: <br><br />
<br />
(bp for amplified region) / (bp in total plasmid) = % amplified region <br><br />
<br />
(conc. of total plasmid) x (% amplified region as a decimal) = conc. of amplified region <br><br />
<br />
<br><br />
<br />
Note: Never pipette less than 0.5 μL. <br><br />
<br />
(0.01 ng of template) / (conc. of amplified region) = vol of template to add to PCR <br><br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Restriction Endonuclease Reaction (Digestion) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
All restriction enzyme reactions were done using a 50 μl reaction volume. Restriction enzymes and buffers were purchased from New England Biolabs® Inc. <br><br />
<br />
Mix the following in a 0.2 mL PCR tube: <br><br />
<br />
<br />
1 μg of DNA <br><br />
<br />
5 μL of the appropriate 10X New England Biolab® Buffer <br><br />
<br />
1 μL of each restriction enzyme (add last) <br><br />
<br />
QS 50μL nuclease-free H2O <br><br />
<br />
Incubate the reaction for 1 hr <br><br />
<br />
Heat inactive the reaction at the appropriate temperature <br><br />
<br><br />
<br />
Note: Thaw the restriction enzyme(s) on ice to improve shelf life <br><br />
<br />
<br />
</p> <br />
<br />
<br />
<h3> Ligation </h3><br />
<br />
<br />
<p><br />
<br />
T4 DNA Ligase and Buffer were purchased from New England Biolabs® Inc. <br><br />
<br />
1. Prepare the following in a 0.2 mL microcentrifuge tube: <br><br />
<br />
50.0 ng vector DNA* <br><br />
<br />
37.5 ng vector DNA* <br><br />
<br />
2 μL 10X T4 DNA Ligase Buffer <br><br />
<br />
1 μL T4 DNA Ligase <br><br />
<br />
QS 20 μL diH2O <br><br />
<br />
2. Incubate the reaction at room temperature for 10-30 minutes or at 16 °C overnight. <br><br />
<br />
3. Heat inactivate at 65 °C for 10 minutes. <br><br />
<br />
4. Chill on ice before starting a transformation reaction. <br><br />
<br />
<br><br />
<br />
*The exact amount of DNA is dependent on the number of base pairs. In order to conduct a proper reaction consult the New England Biolab Ligation Calculator at:<br />
<br />
http://nebiocalculator.neb.com/#!/<br />
<br />
</p><br />
<br />
<br />
<html><a name="Escherichia coli Protocols"></a><br />
<br />
</html><br />
<br />
<br />
<h2> <i> Escherichia coli </i> Protocols (XL1-Blue and XL10-Gold) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<p> <br />
Competent cells take two days to culture and aliquot. <br><br />
<br><br />
Day 1: <br><br />
1. Streak an aliquot of competent cells onto two LB-plates without antibiotics.* <br><br />
2. Incubate at 37 °C overnight. <br><br />
Day 2: <br><br />
1. In two 250 mL baffle flasks add 50 mL of SOB media. <br><br />
2. Scrape as many single colonies into either flask. <br><br />
3. Incubate and shake at 37 °C and 250 rpm for 2-3 hours. <br><br />
4. Check the optical density of the cells at 600 nm after 2 hours. <br><br />
5. Stop incubation when cultures reach approximately 0.5 optical density. <br><br />
6. Add the contents of the flask into separate 50 mL flat bottomed centrifuge tubes. <br><br />
7. Spin down the cells at 2500 rpm at 4 °C for 15 minutes. <br><br />
8. Decant the supernatant. <br><br />
9. Resuspend the cells in 16 mL of CCMB by pipetting or gently vortexing. <br><br />
10. Incubate the cells on ice for 20 minutes. <br><br />
11. Spin down the cells at 2500 rpm at 4 °C for 10 minutes. <br> <br />
12. Decant the supernatant. <br><br />
13. Resuspend the cells in 4 mL of CCMB. <br><br />
14. Quickly aliquot the cells into 1.7 mL cryogenic vials or 1.5 mL centrifuge tubes.** <br><br />
15. Store the competent cell aliquots at -80 °C. <br><br />
<br><br />
*Streak in such a way that there should be individual colony growth and no clumps after the incubation. <br><br />
**We did this in a -20 °C cold room and using an automated repeater pipette. The volume of each aliquot depends on the number of transformations you intend to do at a time. <br><br />
<br><br />
Note: After removing the cells from incubation keep them on ice or as cold as possible. <br><br />
</p><br />
<br />
<br />
<h3> Chemically Competent Cell Transformations </h3><br />
<br />
<br />
<p> <br />
<br />
1. Thaw competent <i> E.coli </i> cells on ice (XL1-Blue or XL10-Gold)* <br><br />
<br />
2. Add 50μL of competent cells to sterile 15mL conical centrifuge tubes <br><br />
<br />
3. Add 1μL (~100-200ng)* of the mini-prep to each culture tube <br><br />
<br />
4. Equilibrate the cells on ice for 10 min <br><br />
<br />
5. Heat shock the cells at 42C for 30-45 seconds** <br><br />
<br />
6. Immediately place the cells back on ice for 3 min <br><br />
<br />
7. Add 250μL LB media without antibiotics and shake at 250 rpm and 37C for 30 min <br><br />
<br />
8. Spread 10μL and 290μL on an appropriate LB-antibiotic plate <br><br />
<br />
9. Invert the plate and incubate at 37C overnight<br />
<br />
<br />
*The exact amount of DNA to add depends on your cell's transformation efficiency. However, it is acceptable to add a larger amount to increase the number of transformed cells. <br><br />
<br />
** Do not heat shock for an extended duration as this may damage and/or kill your cells.<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnights </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottom tube, add 3-5mL of LB and an appropriate volume of antibiotic(s). <br><br />
<br />
2. Swipe several individual colonies, do not collect satellites or colony clumps, with a pipette tip. <br><br />
<br />
3. Swirl the colony tip in the tube, there should be no visible cell clumps. <br><br />
<br />
4. Incubate and shake the tube at 37C at 250rpm for 12-16 hours and no longer than 20 hours. <br><br />
<br />
</p><br />
<br />
<br />
<h3> DNA-Extraction and mini-preps </h3><br />
<br />
<br />
<p><br />
<br />
All DNA mini-preps were prepared using EPOCH mini-kits and following the supplied protocols.<br />
<br />
</p><br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500μL of 40% Glycerol and 500uL of LB (no antibiotics) or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Saccharomyces cerevisiae"></a><br />
<br />
</html><br />
<br />
<h2> <i> Saccharomyces cerevisiae </i> (PyE1 Yeast) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<br />
<p><br />
<br />
This process take 4 days in lab with a 1 day wait for incubation. <br><br />
<br />
<br />
Day 1: <br><br />
<br />
1. Streak yeast cells onto a YPD plate.* <br><br />
<br />
2. Invert the plate and incubate at 30C for 2 days. <br><br />
<br />
<br />
Day 3: <br><br />
<br />
1. Add 50mL of YPD liquid media into a 250mL baffle flask. <br><br />
<br />
2. Swipe as many individual colonies as you can see into the YPD media.** <br><br />
<br />
3. Incubate and shake the culture at 30C at 250rpm overnight approximately 24 hours. <br><br />
<br />
<br />
Day 4: <br><br />
<br />
1. Take an optical density measurement. <br><br />
<br />
2. In three 250mL baffle flask add the portions of the overnight liquid culture. <br><br />
<br />
3. Dilute each culture to approximately 0.4 optical density with YPD. <br><br />
<br />
4. Incubate and shake the cultures at 30C at 250rpm until the optical density reaches 1.2-1.6. <br><br />
<br />
5. Collect each culture into separate 50ml flat-bottomed centrifuge tubes. <br><br />
<br />
6. Spin down the cells at 4000g for 5 minutes at 4C. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cells in 100mL total for all three culture of dH2O. <br><br />
<br />
9. Combine the suspensions into two 50mL flat-bottomed centrifuge tubes. <br><br />
<br />
10. Spin down the cells as above. <br><br />
<br />
11. Decant the supernatant. <br><br />
<br />
12. Resuspend each in 3mL of 100mM Lithium Acetate. <br><br />
<br />
13. Transfer both cultures into a single 15mL conical centrifuge tube. <br><br />
<br />
14. Spin down the cells at 3000rpm for 5 minutes. <br><br />
<br />
15. Resuspend the cells in 0.75mL of 100mM Lithium Acetate, total volume is roughly 2mL. <br><br />
<br />
16. Qualitatively bring up the volume to 3.5mL by adding 40% Glycerol. <br><br />
<br />
17. Aliquot the cells into 1.5mL centrifuge tubes or 1.7mL cryogenic vials.*** <br><br />
<br />
<br />
*Streak in such a way that there are individual colonies visible on the plate without clumps or satellite colonies. <br><br />
<br />
**Collect only individual visible colonies. Do not collect clumps or satellite colonies. <br><br />
<br />
***The volume of aliquots depends on the number to transformations you intend to do at a time. <br><br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Chemically Competent Transformations </h3><br />
<br />
<br />
<p><br />
<br />
This protocol assumes a 50μL aliquot of yeast competent cells were made. <br><br />
<br />
Furthermore, this protocol prepares enough cells for 6 yeast transformations. <br><br />
<br />
<br><br />
<br />
1. Add the following to 50μL of yeast competent cells: <br><br />
<br />
240μL of Polyethylene Glycol - 3350 (PEG-3350) <br><br />
<br />
36μL of 1M Lithium Acetate <br><br />
<br />
32μL of dH2O <br><br />
<br />
<br />
<br />
2. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
3. Aliquot 59μL of the mixture into a 0.2mL microcentrifuge tube. <br><br />
<br />
4. Add 1uL (~100-200ng) of DNA. <br><br />
<br />
5. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
6. Incubate the mixture at 30C for 30 minutes. <br><br />
<br />
7. Heat shock the mixture at 42C for 20 minutes. <br><br />
<br />
8. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
9. Decant the supernatant. <br><br />
<br />
10. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
11. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
12. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
13. Plate 50-150μL of the mixture onto an appropriate Selective Dropout Media plate. <br><br />
<br />
14. Invert and incubate at 30C for 2 days. <br><br />
<br />
*The exact amount of DNA depends on the transformation efficiency of your competent cells. <br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnight Culturing </h3><br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Swipe 3 individually visible yeast colonies and add them to the culture tube media. <br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%.<br />
<br />
</p><br />
<br />
<br />
<h3> Culture Passaging </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Take 20-50μL from a previous overnight or passage culture and add it to the culture media.<br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%. <br><br />
<br />
Note: The exact amount of culture that you take from a previous culture is irrelevant as long as at least 1 living cell is passaged.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500uL of 40% glycerol and 500μL of Selective Dropout media or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Flow Cytometry"></a><br />
<br />
</html><br />
<br />
<h2> Flow Cytometry </h2><br />
<br />
<br />
<h3> Dilutions </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. From an overnight culture measure the optical density at 660nm by making 1:10 dilutions. <br><br />
<br />
2. Take enough culture to make a 1mL aliquot with OD 0.4. <br><br />
<br />
3. Spin down the aliquot in a 1.5mL centrifuge tube at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 800μL of the appropriate selective dropout media and 200μL of 20% glucose. <br><br />
<br />
6. Transfer the new culture to a 14mL culture tube. <br><br />
<br />
7. Incubate and shake at 30C and 250rpm for at least 6 hours (1.2-1.6 optical density).<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Preparations for Analysis using C6 Accuri Flow Cytometer </h3><br />
<br />
<br />
<p><br />
<br />
<br />
1. From the dilution previously made, measure the optical density, roughly 1.2-1.6. <br><br />
<br />
2. Make an aliquot of 500μL of the dilution culture in a 1.5mL centrifuge tube. <br><br />
<br />
3. Spin down the aliquot at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 500μL of PBS(F). <br><br />
<br />
6. Spin down the resuspension at 3000rpm for 3 minutes. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cell pellet in another 500uL of PBS(F).* <br><br />
<br />
9. Prepare the C6 Accuri Flow Cytometer by running a backflush cycle and a dH2O cycle. <br><br />
<br />
10. Load the sample onto the sip. <br><br />
<br />
11. Run the sample with 100,000 cell count. <br><br />
<br />
12. Repeat for all samples and make sure to change data cells otherwise the old data is erased. <br><br />
<br />
13. Once finished, run a cleaning cycle with Accuri approved cleaning solution, then run a dH2O cycle. <br><br />
<br />
<br />
<br />
*For special cases do not resuspend all samples, instead resuspend immediately before running the sample through the flow cytometer.<br />
<br />
<br />
</p><br />
<br />
<br />
<html><a name="Fluorescence Activated Cell Sorting"></a><br />
<br />
</html><br />
<br />
<h2> Fluorescence Activated Cell Sorting </h2><br />
<br />
<br />
<h3> Final Preparations </h3><br />
<br />
<br />
<br />
<p> <br />
<br />
Sample Prep:<br />
<br />
<br />
Spin down samples and negative control (5000 RPM, 1 min), keeping in mind the library size. Aspirate off supernatant. Resuspend in PBSF. Spin down cells. Aspirate off supernatant. Resuspend in PBSF. <br><br />
<br />
<br><br />
<br />
1. Open the “iGEM Template” file in the FACS Software and change name to current date and sort cycle<br><br />
<br />
2. Make sure the stream is stable (look for green light in bottom right corner). If not, run the Sort Calibration order. <br><br />
<br />
3. Run Negative Control from the PyE1 cells. <br><br />
<br />
a. Load cells onto carrier and into the machine. Press play button on screen. <br><br />
<br />
b. Set gate around lower left quadrant of cells to ensure single cell analysis using Forward Scatter Area and Side Scatter Area as your axes. Make sure oval gate covers around 80% of cell population. <br><br />
<br />
c. Set second gate on the first gated population by double-clicking on the gated population and using Forward Scatter Height and Forward Scatter Width as your axes. You will notice two distinct populations. Try to focus on the single cell portion of the plot. <br><br />
<br />
NOTE: If you see a large portion of the second gated population existing near the upper right edge of the first gate, you may need to enlarge the first gate to fit more of the population. <br><br />
<br />
d. Press record. Record 100,000 events and stop run. Move to Next Tube. <br><br />
<br />
4. Run first control (Gene clone) <br><br />
<br />
a. Follow step 3 to run the first control <br><br />
<br />
5. Run first library sample <br><br />
<br />
a. Follow steps 3b and 3c to set first two gates correctly. <br><br />
<br />
b. Set final gate for sort which includes top 1% of GFP producers from second gated population. <br><br />
<br />
c. Use final gate to set up the sort. <br><br />
<br />
d. Select sort conditions at the bottom of the screen. <br><br />
<br />
e. Insert and load collection tube. <br><br />
<br />
f. Record 100,000 events, and sort 10x the library size <br><br />
<br />
6. Run Bleach and diH2O through FACS to avoid cross-contamination. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Protein Expression"></a><br />
<br />
</html><br />
<br />
<h2> Protein Expression </h2><br />
<br />
<br />
<h3> Overnight Cultures </h3><br />
<br />
<p><br />
<br />
1. Add 25mL TB and 25uL Kan to a 250mL baffled flask <br><br />
<br />
2. Stab a glycerol stock with a p1000 pipette and swirl in the flask of media <br><br />
<br />
3. Put flask in 37C shaker overnight <br />
<br />
</p><br />
<br />
<h3> Protein Expression </h3><br />
<br />
<p> <br />
<br />
1. Add 500μL 1000x Kanamycin to 500mL TB in 2L baffled flask <br><br />
<br />
2. Transfer 10mL overnight culture to TB <br><br />
<br />
3. Shake at 37C (DO WE NEED THE RPM?) <br><br />
<br />
4. Remove flask from shaker when optical density is between 0.5 and 0.8 <br><br />
<br />
5. Allow flask to rest at room temp for 30 min <br><br />
<br />
6. Add 125μL 1M IPTG <br><br />
<br />
7. Shake flask at 18C overnight<br />
<br />
</p><br />
<br />
<h3> Protein Extraction and Purification </h3><br />
<br />
<p><br />
<br />
1. Transfer cell culture to centrifuge flask <br><br />
<br />
2. Centrifuge culture at 4000g for 10 min <br><br />
<br />
3. Discard supernatant <br><br />
<br />
4. Resuspend pellet in 25mL lysis buffer <br><br />
<br />
5. Add 250μL of 100x PMSF, 250μL of 100mg/mL lysozyme, and 250μL of 10mg/mL DNAse <br><br />
<br />
6. Sonicate sample with 0.25inch probe for 5 min at 70% amplitude with 20 sec on and off pulses <br><br />
<br />
7. Take 50μL total sample <br><br />
<br />
8. Transfer lysate to SS-34 centrifuge tube <br><br />
<br />
9. Centrifuge for 30 min at 18000g <br><br />
<br />
10. Take 50μL soluble sample<br />
<br />
</p><br />
<br />
<h3> Nickel Nitrotriacetic Acid Chromatography (Nickel-NTA Chromatography) </h3><br />
<br />
<p><br />
<br />
1. Add 5mL 50%(v/v) nickel resin in ethanol to 25mL column and allow to settle to ~2.5mL(CV) <br><br />
<br />
2. Rinse with 10CV H20 <br><br />
<br />
3. Equilibrate with 10CV lysis buffer <br><br />
<br />
4. Load sample onto column <br><br />
<br />
5. Wash column with 15CV lysis buffer <br><br />
<br />
6. Perform 2 additional wash steps with 15CV <br><br />
<br />
7. Elute sample in 10CV elution buffer and collect eluate <br><br />
<br />
8. Take 50μL pure sample <br />
<br />
</p><br />
<br />
<h3> Size Exclusion Chromatography (S.E.C.) </h3><br />
<p><br />
1. Concentrate sample to as high as possible without inducing protein aggregation<br><br />
2. Pre-equilibrate Superdex 75 column with 48 ml PBS <br><br />
3. Inject 500 ul sample onto column <br><br />
4. Run 36 ml PBS through column at 0.5 ml/min, collecting 1 ml fractions <br><br />
5. Verify presence of protein in fractions by measuring concentration on Nanodrop and running SDS-PAGE (15 kDa protein should elute at ~13 ml)<br><br />
6. Pool fractions containing protein <br />
</p><br />
<br />
<br />
<html><a name="Stability Analysis"></a><br />
<br />
</html><br />
<br />
<h2> Stability Analysis </h2><br />
<br />
<br />
<h3> Circular Dichroism: Wavelength Scan </h3><br />
<p><br />
1. Load 1mm cuvette with 400uL protein solution onto CD <br><br />
2. Take wavelength scan <br><br />
260nm-190nm <br><br />
sample every 1nm <br><br />
averaging time 3sec <br><br />
1 scan <br><br />
step scan <br><br />
25C <br><br />
3. Record wavelength which gives strongest signal (222nm)<br />
</p><br />
<br />
<h3> Circular Dichroism: Guanidine Melt </h3><br />
<p><br />
1. Load 1cm cuvette containing 1.996mL of 0.05mg/mL protein solution and stirrer onto CD <br><br />
2. Prepare 8mL of 0.05mg/mL protein in concentrated guanidine solution <br><br />
3. Set up Automixer with guanidine solution on one syringe and waste tube on other syringe <br><br />
4. Titrate up to 6 M guanidine, taking a CD measurement at 222 nm every 0.15 M interval <br><br />
5. Also measure the fluorescence at 280 nm to ensure the total protein concentration is not changing<br />
</p><br />
<br />
<br />
<br />
<html><br />
<br />
<a href="#linktotop">Back To Top</a><br />
</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2014-10-17T21:16:30Z<p>Kristanguyen: </p>
<hr />
<div>{{Template:Team:UW/CSS}}<br />
<br />
<br />
<h1> Protocols </h1><br />
<br />
<br />
<html><a name="Media, Plates, and Sol"></a><br />
<br />
</html><br />
<br />
<br />
<h2> Media, Plates, and Solutions </h2><br />
<br />
<br />
<h3> Competent Cell Media Buffer (CCMB) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
100 g glycerol (liquid) <br><br />
<br />
10 mL x 1 M potassium acetate <br><br />
<br />
11.8 g CaCl2*H2O <br><br />
<br />
4 g MnCl2 <br><br />
<br />
2 g MgCl2 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<h3> Super Optimal Broth (SOB) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
20 g Tryptone <br><br />
<br />
5 g Yeast Extract <br><br />
<br />
10 mL x 1 M NaCl <br><br />
<br />
2.5 mL x 1 M KCl <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Phosphate Buffered Saline (PBS) Solution </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container or 1 L beaker: <br><br />
<br />
8 g NaCl <br><br />
<br />
1.44 g Na2HPO4 <br><br />
<br />
0.8 g KCl <br><br />
<br />
0.24 g KH2PO4 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Buffer to pH 7.4 <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<h3> PSBF (PBS for Flow) </h3> <br />
<br />
<p><br />
<br />
Mix the following in a 1 L beaker: <br><br />
<br />
25 mL 20X PBS, pH 7.4 <br><br />
<br />
574 mL H2O <br><br />
<br />
2.5 g BSA (0.5%)* <br><br />
<br />
Filter and store at 4 °C <br><br />
<br><br />
*Do not need to pH - should be at pH 7.4 like 20X PBS <br><br />
<br />
</p> <br />
<br />
<br />
<h3> Yeast Extract Peptone Dextrose (YPD) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following into 950 mL of dH2O in a 1 L bottle: <br><br />
<br />
20 g peptone <br><br />
<br />
10 g yeast extract <br><br />
<br />
Autoclave (20 min at 121 °C and 20 psi) <br><br />
<br />
Add 50 mL 40% glucose <br><br />
<br />
Sterile filter into a 1 L bottle <br><br />
<br />
<br><br />
<br />
Note: For long-term liquid media storage, do not add 40% glucose. Instead add the glucose directly into cell cultures. <br><br />
<br />
Note: For YPD-plates add 24 g agar to the peptone and yeast extract before autoclaving. <br />
<br />
</p><br />
<br />
<br />
<h3> Selective Dropout Media, C-Uracil and C-Histidine (C-Ura and C-His) </h3><br />
<br />
<br />
<p><br />
<br />
Synthesized by the Yeast Resource Center at the University of Washington's <br />
<br />
Department of Genome Sciences and Department of Biochemistry.<br />
<br />
</p><br />
<br />
<br />
<h3> Guanidinium Hydrogen Chloride </h3><br />
<br />
<br />
<p><br />
<br />
For maximum effectiveness, final concentration should be approximately 8.5 M in PBS.<br><br />
<br />
203 g guanidinium hydrogen chloride <br><br />
<br />
250 mL PBS solution* <br><br />
<br />
Add dilute HCl to pH 7.4 <br><br />
<br />
<br><br />
<br />
*Alternatively add slightly less than 250 mL of PBS in order to buffer the solution to the appropriate volume, then add more dH2O as necessary. <br />
<br />
</p><br />
<br />
<html><a name="Basic Cloning"></a><br />
<br />
</html><br />
<br />
<h2> Basic Cloning </h2><br />
<br />
<br />
<br />
<h3> Polymerase Chain Reaction </h3><br />
<br />
<br />
<p><br />
<br />
All PCRs were done using a standard 50 μL reaction volume with GoTaq® Green Master Mix 2X purchased from PROMEGA Corporation. <br><br />
<br />
Mix the following in a 0.2 mL microcentrifuge tube on ice: <br><br />
<br />
25 μL GoTaq® Green Master Mix 2X <br><br />
<br />
1-5 μL of 10 μM forward primer <br><br />
<br />
1-5 μL of 10 μM reverse primer <br><br />
<br />
<250 ng of DNA template <br><br />
<br />
QS 50 μl nuclease-free H2O <br><br />
<br />
Conduct the reaction in a thermocycler, adjusting anneal temperature and extension times accordingly. See your polymerase supplier protocol for more details on thermocycling.<br />
<br />
</p><br />
<br />
<h3> Error-prone Polymerase Chain Reaction </h3><br />
<br />
<br />
<p> <br />
<br />
<br />
<br />
Prepare 50 μL reaction: <br><br />
<br />
5 μL 10X Mutazyme II Rxn Buffer <br><br />
<br />
1 μL 40 mM dNTP mix (200 μM each final) <br><br />
<br />
1 μL 20 μM forward primer <br><br />
<br />
1 μL 20 μM reverse primer <br><br />
<br />
1 μL Mutazyme II DNA polymerase (2.5 U/μL) <br><br />
<br />
0.01 ng template <br><br />
<br />
QS 50 μL diH2O <br><br />
<br />
<br><br />
<br />
Program thermocycler as follows: <br><br />
<br />
95 °C, 2 min <br><br />
<br />
95 °C, 30 sec <br><br />
<br />
XX °C*, 30 sec <br><br />
<br />
72 °C, X min** <br><br />
<br />
32 cycles <br><br />
<br />
72 °C, 10 min <br><br />
<br />
4 °C, hold<br />
<br />
<br><br />
<br />
*Adjust annealing temperature according to Tm of primer. <br><br />
<br />
**Adjust extension time according to the length of amplified DNA. <br><br />
<br />
<br><br />
<br />
Note: Use 0.01 ng of template (calculate by insert and not by total plasmid). <br><br />
<br />
Calculate amount of template to use as follows: <br><br />
<br />
(bp for amplified region) / (bp in total plasmid) = % amplified region <br><br />
<br />
(conc. of total plasmid) x (% amplified region as a decimal) = conc. of amplified region <br><br />
<br />
<br><br />
<br />
Note: Never pipette less than 0.5 μL. <br><br />
<br />
(0.01 ng of template) / (conc. of amplified region) = vol of template to add to PCR <br><br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Restriction Endonuclease Reaction (Digestion) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
All restriction enzyme reactions were done using a 50 μl reaction volume. Restriction enzymes and buffers were purchased from New England Biolabs® Inc. <br><br />
<br />
Mix the following in a 0.2 mL PCR tube: <br><br />
<br />
<br />
1 μg of DNA <br><br />
<br />
5 μL of the appropriate 10X New England Biolab® Buffer <br><br />
<br />
1 μL of each restriction enzyme (add last) <br><br />
<br />
QS 50μL nuclease-free H2O <br><br />
<br />
Incubate the reaction for 1 hr <br><br />
<br />
Heat inactive the reaction at the appropriate temperature <br><br />
<br><br />
<br />
Note: Thaw the restriction enzyme(s) on ice to improve shelf life <br><br />
<br />
<br />
</p> <br />
<br />
<br />
<h3> Ligation </h3><br />
<br />
<br />
<p><br />
<br />
T4 DNA Ligase and Buffer were purchased from New England Biolabs® Inc. <br><br />
<br />
1. Prepare the following in a 0.2 mL microcentrifuge tube: <br><br />
<br />
50.0 ng vector DNA* <br><br />
<br />
37.5 ng vector DNA* <br><br />
<br />
2 μL 10X T4 DNA Ligase Buffer <br><br />
<br />
1 μL T4 DNA Ligase <br><br />
<br />
QS 20 μL diH2O <br><br />
<br />
2. Incubate the reaction at room temperature for 10-30 minutes or at 16 °C overnight. <br><br />
<br />
3. Heat inactivate at 65 °C for 10 minutes. <br><br />
<br />
4. Chill on ice before starting a transformation reaction. <br><br />
<br />
<br><br />
<br />
*The exact amount of DNA is dependent on the number of base pairs. In order to conduct a proper reaction consult the New England Biolab Ligation Calculator at:<br />
<br />
http://nebiocalculator.neb.com/#!/<br />
<br />
</p><br />
<br />
<br />
<html><a name="Escherichia coli Protocols"></a><br />
<br />
</html><br />
<br />
<br />
<h2> <i> Escherichia coli </i> Protocols (XL1-Blue and XL10-Gold) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<p> <br />
<br />
Day 1: <br><br />
1. Streak an aliquot of competent cells onto two LB-plates without antibiotics.* <br><br />
2. Incubate at 37 °C overnight. <br><br />
Day 2: <br><br />
1. In two 250 mL baffle flasks add 50 mL of SOB media. <br><br />
2. Scrape as many single colonies into either flask. <br><br />
3. Incubate and shake at 37 °C and 250 rpm for 2-3 hours. <br><br />
4. Check the optical density of the cells at 600 nm after 2 hours. <br><br />
5. Stop incubation when cultures reach approximately 0.5 optical density. <br><br />
6. Add the contents of the flask into separate 50 mL flat bottomed centrifuge tubes. <br><br />
7. Spin down the cells at 2500 rpm at 4 °C for 15 minutes. <br><br />
8. Decant the supernatant. <br><br />
9. Resuspend the cells in 16 mL of CCMB by pipetting or gently vortexing. <br><br />
10. Incubate the cells on ice for 20 minutes. <br><br />
11. Spin down the cells at 2500 rpm at 4 °C for 10 minutes. <br> <br />
12. Decant the supernatant. <br><br />
13. Resuspend the cells in 4 mL of CCMB. <br><br />
14. Quickly aliquot the cells into 1.7 mL cryogenic vials or 1.5 mL centrifuge tubes.** <br><br />
15. Store the competent cell aliquots at -80 °C. <br><br />
<br />
*Streak in such a way that there should be individual colony growth and no clumps after the incubation. <br><br />
**We did this in a -20 °C cold room and using an automated repeater pipette. The volume of each aliquot depends on the number of transformations you intend to do at a time. <br><br />
<br><br />
Note: After removing the cells from incubation keep them on ice or as cold as possible. <br><br />
</p><br />
<br />
<br />
<h3> Chemically Competent Cell Transformations </h3><br />
<br />
<br />
<p> <br />
<br />
1. Thaw competent <i> E.coli </i> cells on ice (XL1-Blue or XL10-Gold)* <br><br />
<br />
2. Add 50μL of competent cells to sterile 15mL conical centrifuge tubes <br><br />
<br />
3. Add 1μL (~100-200ng)* of the mini-prep to each culture tube <br><br />
<br />
4. Equilibrate the cells on ice for 10 min <br><br />
<br />
5. Heat shock the cells at 42C for 30-45 seconds** <br><br />
<br />
6. Immediately place the cells back on ice for 3 min <br><br />
<br />
7. Add 250μL LB media without antibiotics and shake at 250 rpm and 37C for 30 min <br><br />
<br />
8. Spread 10μL and 290μL on an appropriate LB-antibiotic plate <br><br />
<br />
9. Invert the plate and incubate at 37C overnight<br />
<br />
<br />
*The exact amount of DNA to add depends on your cell's transformation efficiency. However, it is acceptable to add a larger amount to increase the number of transformed cells. <br><br />
<br />
** Do not heat shock for an extended duration as this may damage and/or kill your cells.<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnights </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottom tube, add 3-5mL of LB and an appropriate volume of antibiotic(s). <br><br />
<br />
2. Swipe several individual colonies, do not collect satellites or colony clumps, with a pipette tip. <br><br />
<br />
3. Swirl the colony tip in the tube, there should be no visible cell clumps. <br><br />
<br />
4. Incubate and shake the tube at 37C at 250rpm for 12-16 hours and no longer than 20 hours. <br><br />
<br />
</p><br />
<br />
<br />
<h3> DNA-Extraction and mini-preps </h3><br />
<br />
<br />
<p><br />
<br />
All DNA mini-preps were prepared using EPOCH mini-kits and following the supplied protocols.<br />
<br />
</p><br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500μL of 40% Glycerol and 500uL of LB (no antibiotics) or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Saccharomyces cerevisiae"></a><br />
<br />
</html><br />
<br />
<h2> <i> Saccharomyces cerevisiae </i> (PyE1 Yeast) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<br />
<p><br />
<br />
This process take 4 days in lab with a 1 day wait for incubation. <br><br />
<br />
<br />
Day 1: <br><br />
<br />
1. Streak yeast cells onto a YPD plate.* <br><br />
<br />
2. Invert the plate and incubate at 30C for 2 days. <br><br />
<br />
<br />
Day 3: <br><br />
<br />
1. Add 50mL of YPD liquid media into a 250mL baffle flask. <br><br />
<br />
2. Swipe as many individual colonies as you can see into the YPD media.** <br><br />
<br />
3. Incubate and shake the culture at 30C at 250rpm overnight approximately 24 hours. <br><br />
<br />
<br />
Day 4: <br><br />
<br />
1. Take an optical density measurement. <br><br />
<br />
2. In three 250mL baffle flask add the portions of the overnight liquid culture. <br><br />
<br />
3. Dilute each culture to approximately 0.4 optical density with YPD. <br><br />
<br />
4. Incubate and shake the cultures at 30C at 250rpm until the optical density reaches 1.2-1.6. <br><br />
<br />
5. Collect each culture into separate 50ml flat-bottomed centrifuge tubes. <br><br />
<br />
6. Spin down the cells at 4000g for 5 minutes at 4C. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cells in 100mL total for all three culture of dH2O. <br><br />
<br />
9. Combine the suspensions into two 50mL flat-bottomed centrifuge tubes. <br><br />
<br />
10. Spin down the cells as above. <br><br />
<br />
11. Decant the supernatant. <br><br />
<br />
12. Resuspend each in 3mL of 100mM Lithium Acetate. <br><br />
<br />
13. Transfer both cultures into a single 15mL conical centrifuge tube. <br><br />
<br />
14. Spin down the cells at 3000rpm for 5 minutes. <br><br />
<br />
15. Resuspend the cells in 0.75mL of 100mM Lithium Acetate, total volume is roughly 2mL. <br><br />
<br />
16. Qualitatively bring up the volume to 3.5mL by adding 40% Glycerol. <br><br />
<br />
17. Aliquot the cells into 1.5mL centrifuge tubes or 1.7mL cryogenic vials.*** <br><br />
<br />
<br />
*Streak in such a way that there are individual colonies visible on the plate without clumps or satellite colonies. <br><br />
<br />
**Collect only individual visible colonies. Do not collect clumps or satellite colonies. <br><br />
<br />
***The volume of aliquots depends on the number to transformations you intend to do at a time. <br><br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Chemically Competent Transformations </h3><br />
<br />
<br />
<p><br />
<br />
This protocol assumes a 50μL aliquot of yeast competent cells were made. <br><br />
<br />
Furthermore, this protocol prepares enough cells for 6 yeast transformations. <br><br />
<br />
<br><br />
<br />
1. Add the following to 50μL of yeast competent cells: <br><br />
<br />
240μL of Polyethylene Glycol - 3350 (PEG-3350) <br><br />
<br />
36μL of 1M Lithium Acetate <br><br />
<br />
32μL of dH2O <br><br />
<br />
<br />
<br />
2. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
3. Aliquot 59μL of the mixture into a 0.2mL microcentrifuge tube. <br><br />
<br />
4. Add 1uL (~100-200ng) of DNA. <br><br />
<br />
5. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
6. Incubate the mixture at 30C for 30 minutes. <br><br />
<br />
7. Heat shock the mixture at 42C for 20 minutes. <br><br />
<br />
8. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
9. Decant the supernatant. <br><br />
<br />
10. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
11. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
12. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
13. Plate 50-150μL of the mixture onto an appropriate Selective Dropout Media plate. <br><br />
<br />
14. Invert and incubate at 30C for 2 days. <br><br />
<br />
*The exact amount of DNA depends on the transformation efficiency of your competent cells. <br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnight Culturing </h3><br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Swipe 3 individually visible yeast colonies and add them to the culture tube media. <br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%.<br />
<br />
</p><br />
<br />
<br />
<h3> Culture Passaging </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Take 20-50μL from a previous overnight or passage culture and add it to the culture media.<br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%. <br><br />
<br />
Note: The exact amount of culture that you take from a previous culture is irrelevant as long as at least 1 living cell is passaged.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500uL of 40% glycerol and 500μL of Selective Dropout media or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Flow Cytometry"></a><br />
<br />
</html><br />
<br />
<h2> Flow Cytometry </h2><br />
<br />
<br />
<h3> Dilutions </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. From an overnight culture measure the optical density at 660nm by making 1:10 dilutions. <br><br />
<br />
2. Take enough culture to make a 1mL aliquot with OD 0.4. <br><br />
<br />
3. Spin down the aliquot in a 1.5mL centrifuge tube at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 800μL of the appropriate selective dropout media and 200μL of 20% glucose. <br><br />
<br />
6. Transfer the new culture to a 14mL culture tube. <br><br />
<br />
7. Incubate and shake at 30C and 250rpm for at least 6 hours (1.2-1.6 optical density).<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Preparations for Analysis using C6 Accuri Flow Cytometer </h3><br />
<br />
<br />
<p><br />
<br />
<br />
1. From the dilution previously made, measure the optical density, roughly 1.2-1.6. <br><br />
<br />
2. Make an aliquot of 500μL of the dilution culture in a 1.5mL centrifuge tube. <br><br />
<br />
3. Spin down the aliquot at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 500μL of PBS(F). <br><br />
<br />
6. Spin down the resuspension at 3000rpm for 3 minutes. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cell pellet in another 500uL of PBS(F).* <br><br />
<br />
9. Prepare the C6 Accuri Flow Cytometer by running a backflush cycle and a dH2O cycle. <br><br />
<br />
10. Load the sample onto the sip. <br><br />
<br />
11. Run the sample with 100,000 cell count. <br><br />
<br />
12. Repeat for all samples and make sure to change data cells otherwise the old data is erased. <br><br />
<br />
13. Once finished, run a cleaning cycle with Accuri approved cleaning solution, then run a dH2O cycle. <br><br />
<br />
<br />
<br />
*For special cases do not resuspend all samples, instead resuspend immediately before running the sample through the flow cytometer.<br />
<br />
<br />
</p><br />
<br />
<br />
<html><a name="Fluorescence Activated Cell Sorting"></a><br />
<br />
</html><br />
<br />
<h2> Fluorescence Activated Cell Sorting </h2><br />
<br />
<br />
<h3> Final Preparations </h3><br />
<br />
<br />
<br />
<p> <br />
<br />
Sample Prep:<br />
<br />
<br />
Spin down samples and negative control (5000 RPM, 1 min), keeping in mind the library size. Aspirate off supernatant. Resuspend in PBSF. Spin down cells. Aspirate off supernatant. Resuspend in PBSF. <br><br />
<br />
<br><br />
<br />
1. Open the “iGEM Template” file in the FACS Software and change name to current date and sort cycle<br><br />
<br />
2. Make sure the stream is stable (look for green light in bottom right corner). If not, run the Sort Calibration order. <br><br />
<br />
3. Run Negative Control from the PyE1 cells. <br><br />
<br />
a. Load cells onto carrier and into the machine. Press play button on screen. <br><br />
<br />
b. Set gate around lower left quadrant of cells to ensure single cell analysis using Forward Scatter Area and Side Scatter Area as your axes. Make sure oval gate covers around 80% of cell population. <br><br />
<br />
c. Set second gate on the first gated population by double-clicking on the gated population and using Forward Scatter Height and Forward Scatter Width as your axes. You will notice two distinct populations. Try to focus on the single cell portion of the plot. <br><br />
<br />
NOTE: If you see a large portion of the second gated population existing near the upper right edge of the first gate, you may need to enlarge the first gate to fit more of the population. <br><br />
<br />
d. Press record. Record 100,000 events and stop run. Move to Next Tube. <br><br />
<br />
4. Run first control (Gene clone) <br><br />
<br />
a. Follow step 3 to run the first control <br><br />
<br />
5. Run first library sample <br><br />
<br />
a. Follow steps 3b and 3c to set first two gates correctly. <br><br />
<br />
b. Set final gate for sort which includes top 1% of GFP producers from second gated population. <br><br />
<br />
c. Use final gate to set up the sort. <br><br />
<br />
d. Select sort conditions at the bottom of the screen. <br><br />
<br />
e. Insert and load collection tube. <br><br />
<br />
f. Record 100,000 events, and sort 10x the library size <br><br />
<br />
6. Run Bleach and diH2O through FACS to avoid cross-contamination. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Protein Expression"></a><br />
<br />
</html><br />
<br />
<h2> Protein Expression </h2><br />
<br />
<br />
<h3> Overnight Cultures </h3><br />
<br />
<p><br />
<br />
1. Add 25mL TB and 25uL Kan to a 250mL baffled flask <br><br />
<br />
2. Stab a glycerol stock with a p1000 pipette and swirl in the flask of media <br><br />
<br />
3. Put flask in 37C shaker overnight <br />
<br />
</p><br />
<br />
<h3> Protein Expression </h3><br />
<br />
<p> <br />
<br />
1. Add 500μL 1000x Kanamycin to 500mL TB in 2L baffled flask <br><br />
<br />
2. Transfer 10mL overnight culture to TB <br><br />
<br />
3. Shake at 37C (DO WE NEED THE RPM?) <br><br />
<br />
4. Remove flask from shaker when optical density is between 0.5 and 0.8 <br><br />
<br />
5. Allow flask to rest at room temp for 30 min <br><br />
<br />
6. Add 125μL 1M IPTG <br><br />
<br />
7. Shake flask at 18C overnight<br />
<br />
</p><br />
<br />
<h3> Protein Extraction and Purification </h3><br />
<br />
<p><br />
<br />
1. Transfer cell culture to centrifuge flask <br><br />
<br />
2. Centrifuge culture at 4000g for 10 min <br><br />
<br />
3. Discard supernatant <br><br />
<br />
4. Resuspend pellet in 25mL lysis buffer <br><br />
<br />
5. Add 250μL of 100x PMSF, 250μL of 100mg/mL lysozyme, and 250μL of 10mg/mL DNAse <br><br />
<br />
6. Sonicate sample with 0.25inch probe for 5 min at 70% amplitude with 20 sec on and off pulses <br><br />
<br />
7. Take 50μL total sample <br><br />
<br />
8. Transfer lysate to SS-34 centrifuge tube <br><br />
<br />
9. Centrifuge for 30 min at 18000g <br><br />
<br />
10. Take 50μL soluble sample<br />
<br />
</p><br />
<br />
<h3> Nickel Nitrotriacetic Acid Chromatography (Nickel-NTA Chromatography) </h3><br />
<br />
<p><br />
<br />
1. Add 5mL 50%(v/v) nickel resin in ethanol to 25mL column and allow to settle to ~2.5mL(CV) <br><br />
<br />
2. Rinse with 10CV H20 <br><br />
<br />
3. Equilibrate with 10CV lysis buffer <br><br />
<br />
4. Load sample onto column <br><br />
<br />
5. Wash column with 15CV lysis buffer <br><br />
<br />
6. Perform 2 additional wash steps with 15CV <br><br />
<br />
7. Elute sample in 10CV elution buffer and collect eluate <br><br />
<br />
8. Take 50μL pure sample <br />
<br />
</p><br />
<br />
<h3> Size Exclusion Chromatography (S.E.C.) </h3><br />
<p><br />
1. Concentrate sample to as high as possible without inducing protein aggregation<br><br />
2. Pre-equilibrate Superdex 75 column with 48 ml PBS <br><br />
3. Inject 500 ul sample onto column <br><br />
4. Run 36 ml PBS through column at 0.5 ml/min, collecting 1 ml fractions <br><br />
5. Verify presence of protein in fractions by measuring concentration on Nanodrop and running SDS-PAGE (15 kDa protein should elute at ~13 ml)<br><br />
6. Pool fractions containing protein <br />
</p><br />
<br />
<br />
<html><a name="Stability Analysis"></a><br />
<br />
</html><br />
<br />
<h2> Stability Analysis </h2><br />
<br />
<br />
<h3> Circular Dichroism: Wavelength Scan </h3><br />
<p><br />
1. Load 1mm cuvette with 400uL protein solution onto CD <br><br />
2. Take wavelength scan <br><br />
260nm-190nm <br><br />
sample every 1nm <br><br />
averaging time 3sec <br><br />
1 scan <br><br />
step scan <br><br />
25C <br><br />
3. Record wavelength which gives strongest signal (222nm)<br />
</p><br />
<br />
<h3> Circular Dichroism: Guanidine Melt </h3><br />
<p><br />
1. Load 1cm cuvette containing 1.996mL of 0.05mg/mL protein solution and stirrer onto CD <br><br />
2. Prepare 8mL of 0.05mg/mL protein in concentrated guanidine solution <br><br />
3. Set up Automixer with guanidine solution on one syringe and waste tube on other syringe <br><br />
4. Titrate up to 6 M guanidine, taking a CD measurement at 222 nm every 0.15 M interval <br><br />
5. Also measure the fluorescence at 280 nm to ensure the total protein concentration is not changing<br />
</p><br />
<br />
<br />
<br />
<html><br />
<br />
<a href="#linktotop">Back To Top</a><br />
</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2014-10-17T21:12:14Z<p>Kristanguyen: </p>
<hr />
<div>{{Template:Team:UW/CSS}}<br />
<br />
<br />
<h1> Protocols </h1><br />
<br />
<br />
<html><a name="Media, Plates, and Sol"></a><br />
<br />
</html><br />
<br />
<br />
<h2> Media, Plates, and Solutions </h2><br />
<br />
<br />
<h3> Competent Cell Media Buffer (CCMB) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
100 g glycerol (liquid) <br><br />
<br />
10 mL x 1 M potassium acetate <br><br />
<br />
11.8 g CaCl2*H2O <br><br />
<br />
4 g MnCl2 <br><br />
<br />
2 g MgCl2 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<h3> Super Optimal Broth (SOB) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
20 g Tryptone <br><br />
<br />
5 g Yeast Extract <br><br />
<br />
10 mL x 1 M NaCl <br><br />
<br />
2.5 mL x 1 M KCl <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Phosphate Buffered Saline (PBS) Solution </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container or 1 L beaker: <br><br />
<br />
8 g NaCl <br><br />
<br />
1.44 g Na2HPO4 <br><br />
<br />
0.8 g KCl <br><br />
<br />
0.24 g KH2PO4 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Buffer to pH 7.4 <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<h3> PSBF (PBS for Flow) </h3> <br />
<br />
<p><br />
<br />
Mix the following in a 1 L beaker: <br><br />
<br />
25 mL 20X PBS, pH 7.4 <br><br />
<br />
574 mL H2O <br><br />
<br />
2.5 g BSA (0.5%)* <br><br />
<br />
Filter and store at 4 °C <br><br />
<br><br />
*Do not need to pH - should be at pH 7.4 like 20X PBS <br><br />
<br />
</p> <br />
<br />
<br />
<h3> Yeast Extract Peptone Dextrose (YPD) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following into 950 mL of dH2O in a 1 L bottle: <br><br />
<br />
20 g peptone <br><br />
<br />
10 g yeast extract <br><br />
<br />
Autoclave (20 min at 121 °C and 20 psi) <br><br />
<br />
Add 50 mL 40% glucose <br><br />
<br />
Sterile filter into a 1 L bottle <br><br />
<br />
<br><br />
<br />
Note: For long-term liquid media storage, do not add 40% glucose. Instead add the glucose directly into cell cultures. <br><br />
<br />
Note: For YPD-plates add 24 g agar to the peptone and yeast extract before autoclaving. <br />
<br />
</p><br />
<br />
<br />
<h3> Selective Dropout Media, C-Uracil and C-Histidine (C-Ura and C-His) </h3><br />
<br />
<br />
<p><br />
<br />
Synthesized by the Yeast Resource Center at the University of Washington's <br />
<br />
Department of Genome Sciences and Department of Biochemistry.<br />
<br />
</p><br />
<br />
<br />
<h3> Guanidinium Hydrogen Chloride </h3><br />
<br />
<br />
<p><br />
<br />
For maximum effectiveness, final concentration should be approximately 8.5 M in PBS.<br><br />
<br />
203 g guanidinium hydrogen chloride <br><br />
<br />
250 mL PBS solution* <br><br />
<br />
Add dilute HCl to pH 7.4 <br><br />
<br />
<br><br />
<br />
*Alternatively add slightly less than 250 mL of PBS in order to buffer the solution to the appropriate volume, then add more dH2O as necessary. <br />
<br />
</p><br />
<br />
<html><a name="Basic Cloning"></a><br />
<br />
</html><br />
<br />
<h2> Basic Cloning </h2><br />
<br />
<br />
<br />
<h3> Polymerase Chain Reaction </h3><br />
<br />
<br />
<p><br />
<br />
All PCRs were done using a standard 50 μL reaction volume with GoTaq® Green Master Mix 2X purchased from PROMEGA Corporation. <br><br />
<br />
Mix the following in a 0.2 mL microcentrifuge tube on ice: <br><br />
<br />
25 μL GoTaq® Green Master Mix 2X <br><br />
<br />
1-5 μL of 10 μM forward primer <br><br />
<br />
1-5 μL of 10 μM reverse primer <br><br />
<br />
<250 ng of DNA template <br><br />
<br />
QS 50 μl nuclease-free H2O <br><br />
<br />
Conduct the reaction in a thermocycler, adjusting anneal temperature and extension times accordingly. See your polymerase supplier protocol for more details on thermocycling.<br />
<br />
</p><br />
<br />
<h3> Error-prone Polymerase Chain Reaction </h3><br />
<br />
<br />
<p> <br />
<br />
<br />
<br />
Prepare 50 μL reaction: <br><br />
<br />
5 μL 10X Mutazyme II Rxn Buffer <br><br />
<br />
1 μL 40 mM dNTP mix (200 μM each final) <br><br />
<br />
1 μL 20 μM forward primer <br><br />
<br />
1 μL 20 μM reverse primer <br><br />
<br />
1 μL Mutazyme II DNA polymerase (2.5 U/μL) <br><br />
<br />
0.01 ng template <br><br />
<br />
QS 50 μL diH2O <br><br />
<br />
<br><br />
<br />
Program thermocycler as follows: <br><br />
<br />
95 °C, 2 min <br><br />
<br />
95 °C, 30 sec <br><br />
<br />
XX °C*, 30 sec <br><br />
<br />
72 °C, X min** <br><br />
<br />
32 cycles <br><br />
<br />
72 °C, 10 min <br><br />
<br />
4 °C, hold<br />
<br />
<br><br />
<br />
*Adjust annealing temperature according to Tm of primer. <br><br />
<br />
**Adjust extension time according to the length of amplified DNA. <br><br />
<br />
<br><br />
<br />
Note: Use 0.01 ng of template (calculate by insert and not by total plasmid). <br><br />
<br />
Calculate amount of template to use as follows: <br><br />
<br />
(bp for amplified region) / (bp in total plasmid) = % amplified region <br><br />
<br />
(conc. of total plasmid) x (% amplified region as a decimal) = conc. of amplified region <br><br />
<br />
<br><br />
<br />
Note: Never pipette less than 0.5 μL. <br><br />
<br />
(0.01 ng of template) / (conc. of amplified region) = vol of template to add to PCR <br><br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Restriction Endonuclease Reaction (Digestion) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
All restriction enzyme reactions were done using a 50 μl reaction volume. Restriction enzymes and buffers were purchased from New England Biolabs® Inc. <br><br />
<br />
Mix the following in a 0.2 mL PCR tube: <br><br />
<br />
<br />
1 μg of DNA <br><br />
<br />
5 μL of the appropriate 10X New England Biolab® Buffer <br><br />
<br />
1 μL of each restriction enzyme (add last) <br><br />
<br />
QS 50μL nuclease-free H2O <br><br />
<br />
Incubate the reaction for 1 hr <br><br />
<br />
Heat inactive the reaction at the appropriate temperature <br><br />
<br><br />
<br />
Note: Thaw the restriction enzyme(s) on ice to improve shelf life <br><br />
<br />
<br />
</p> <br />
<br />
<br />
<h3> Ligation </h3><br />
<br />
<br />
<p><br />
<br />
T4 DNA Ligase and Buffer were purchased from New England Biolabs® Inc. <br><br />
<br />
1. Prepare the following in a 0.2 mL microcentrifuge tube: <br><br />
<br />
50.0 ng vector DNA* <br><br />
<br />
37.5 ng vector DNA* <br><br />
<br />
2 μL 10X T4 DNA Ligase Buffer <br><br />
<br />
1 μL T4 DNA Ligase <br><br />
<br />
QS 20 μL diH2O <br><br />
<br />
2. Incubate the reaction at room temperature for 10-30 minutes or at 16 °C overnight. <br><br />
<br />
3. Heat inactivate at 65 °C for 10 minutes. <br><br />
<br />
4. Chill on ice before starting a transformation reaction. <br><br />
<br />
<br><br />
<br />
*The exact amount of DNA is dependent on the number of base pairs. In order to conduct a proper reaction consult the New England Biolab Ligation Calculator at:<br />
<br />
http://nebiocalculator.neb.com/#!/<br />
<br />
</p><br />
<br />
<br />
<html><a name="Escherichia coli Protocols"></a><br />
<br />
</html><br />
<br />
<br />
<h2> <i> Escherichia coli </i> Protocols (XL1-Blue and XL10-Gold) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<p> <br />
<br />
1. Plate 10 ul of cells onto an antibiotic free LB-Agar plate. <br><br />
<br />
2. Incubate overnight at 37OC <br><br />
<br />
3. Inoculate lots of colonies into 50ml SOB-Mg media in a 250ml flask. <br><br />
4. Incubate at 37 OC, 200rpm, until OD at 550nm reaches 0.3, takes about 3 hours. I do my first check at 2 hours.<br><br />
<br />
5.Transfer the cell culture into a sterile 50ml Falcon tube and chill on ice for 10 minutes. <br><br />
<br />
6. Pellet the cells at 2500rpm for 15 minutes at 4 OC.<br><br />
<br />
7. Decant supernatant and invert tubes to remove excess culture medium. <br><br />
<br />
8. Resuspend the cells in 16ml CCMB by gentle vortexing or pipetting (I shake the falcon tube).<br />
(16ml CCMB per 50ml culture) <br><br />
<br />
9. Incubate on ice for 20 minutes. <br><br />
<br />
10. Centrifuge at 2500rpm for 10 minutes at 4 OC. <br><br />
<br />
11. Decant supernatant and invert tubes to remove excess liquid. <br><br />
<br />
12. Resuspend the cells in 4ml CCMB by gentle vortexing or pipetting.<br />
(4ml CCMB per culture) <br><br />
<br />
13. If making multiple 50ml cultures, combine all of the cells and mix before aliquoting. <br> <br />
<br />
14. In cold room: make 205ul aliquots, flash freeze in liquid nitrogen, and store at -80 OC.<br><br />
<br />
<br />
<br />
<br />
NOTE: Make SOB-Mg media and autoclave the 250ml flasks with water in them the same day the cells are plated (step 1) <br><br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Chemically Competent Cell Transformations </h3><br />
<br />
<br />
<p> <br />
<br />
1. Thaw competent <i> E.coli </i> cells on ice (XL1-Blue or XL10-Gold)* <br><br />
<br />
2. Add 50μL of competent cells to sterile 15mL conical centrifuge tubes <br><br />
<br />
3. Add 1μL (~100-200ng)* of the mini-prep to each culture tube <br><br />
<br />
4. Equilibrate the cells on ice for 10 min <br><br />
<br />
5. Heat shock the cells at 42C for 30-45 seconds** <br><br />
<br />
6. Immediately place the cells back on ice for 3 min <br><br />
<br />
7. Add 250μL LB media without antibiotics and shake at 250 rpm and 37C for 30 min <br><br />
<br />
8. Spread 10μL and 290μL on an appropriate LB-antibiotic plate <br><br />
<br />
9. Invert the plate and incubate at 37C overnight<br />
<br />
<br />
*The exact amount of DNA to add depends on your cell's transformation efficiency. However, it is acceptable to add a larger amount to increase the number of transformed cells. <br><br />
<br />
** Do not heat shock for an extended duration as this may damage and/or kill your cells.<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnights </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottom tube, add 3-5mL of LB and an appropriate volume of antibiotic(s). <br><br />
<br />
2. Swipe several individual colonies, do not collect satellites or colony clumps, with a pipette tip. <br><br />
<br />
3. Swirl the colony tip in the tube, there should be no visible cell clumps. <br><br />
<br />
4. Incubate and shake the tube at 37C at 250rpm for 12-16 hours and no longer than 20 hours. <br><br />
<br />
</p><br />
<br />
<br />
<h3> DNA-Extraction and mini-preps </h3><br />
<br />
<br />
<p><br />
<br />
All DNA mini-preps were prepared using EPOCH mini-kits and following the supplied protocols.<br />
<br />
</p><br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500μL of 40% Glycerol and 500uL of LB (no antibiotics) or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Saccharomyces cerevisiae"></a><br />
<br />
</html><br />
<br />
<h2> <i> Saccharomyces cerevisiae </i> (PyE1 Yeast) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<br />
<p><br />
<br />
This process take 4 days in lab with a 1 day wait for incubation. <br><br />
<br />
<br />
Day 1: <br><br />
<br />
1. Streak yeast cells onto a YPD plate.* <br><br />
<br />
2. Invert the plate and incubate at 30C for 2 days. <br><br />
<br />
<br />
Day 3: <br><br />
<br />
1. Add 50mL of YPD liquid media into a 250mL baffle flask. <br><br />
<br />
2. Swipe as many individual colonies as you can see into the YPD media.** <br><br />
<br />
3. Incubate and shake the culture at 30C at 250rpm overnight approximately 24 hours. <br><br />
<br />
<br />
Day 4: <br><br />
<br />
1. Take an optical density measurement. <br><br />
<br />
2. In three 250mL baffle flask add the portions of the overnight liquid culture. <br><br />
<br />
3. Dilute each culture to approximately 0.4 optical density with YPD. <br><br />
<br />
4. Incubate and shake the cultures at 30C at 250rpm until the optical density reaches 1.2-1.6. <br><br />
<br />
5. Collect each culture into separate 50ml flat-bottomed centrifuge tubes. <br><br />
<br />
6. Spin down the cells at 4000g for 5 minutes at 4C. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cells in 100mL total for all three culture of dH2O. <br><br />
<br />
9. Combine the suspensions into two 50mL flat-bottomed centrifuge tubes. <br><br />
<br />
10. Spin down the cells as above. <br><br />
<br />
11. Decant the supernatant. <br><br />
<br />
12. Resuspend each in 3mL of 100mM Lithium Acetate. <br><br />
<br />
13. Transfer both cultures into a single 15mL conical centrifuge tube. <br><br />
<br />
14. Spin down the cells at 3000rpm for 5 minutes. <br><br />
<br />
15. Resuspend the cells in 0.75mL of 100mM Lithium Acetate, total volume is roughly 2mL. <br><br />
<br />
16. Qualitatively bring up the volume to 3.5mL by adding 40% Glycerol. <br><br />
<br />
17. Aliquot the cells into 1.5mL centrifuge tubes or 1.7mL cryogenic vials.*** <br><br />
<br />
<br />
*Streak in such a way that there are individual colonies visible on the plate without clumps or satellite colonies. <br><br />
<br />
**Collect only individual visible colonies. Do not collect clumps or satellite colonies. <br><br />
<br />
***The volume of aliquots depends on the number to transformations you intend to do at a time. <br><br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Chemically Competent Transformations </h3><br />
<br />
<br />
<p><br />
<br />
This protocol assumes a 50μL aliquot of yeast competent cells were made. <br><br />
<br />
Furthermore, this protocol prepares enough cells for 6 yeast transformations. <br><br />
<br />
<br><br />
<br />
1. Add the following to 50μL of yeast competent cells: <br><br />
<br />
240μL of Polyethylene Glycol - 3350 (PEG-3350) <br><br />
<br />
36μL of 1M Lithium Acetate <br><br />
<br />
32μL of dH2O <br><br />
<br />
<br />
<br />
2. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
3. Aliquot 59μL of the mixture into a 0.2mL microcentrifuge tube. <br><br />
<br />
4. Add 1uL (~100-200ng) of DNA. <br><br />
<br />
5. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
6. Incubate the mixture at 30C for 30 minutes. <br><br />
<br />
7. Heat shock the mixture at 42C for 20 minutes. <br><br />
<br />
8. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
9. Decant the supernatant. <br><br />
<br />
10. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
11. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
12. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
13. Plate 50-150μL of the mixture onto an appropriate Selective Dropout Media plate. <br><br />
<br />
14. Invert and incubate at 30C for 2 days. <br><br />
<br />
*The exact amount of DNA depends on the transformation efficiency of your competent cells. <br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnight Culturing </h3><br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Swipe 3 individually visible yeast colonies and add them to the culture tube media. <br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%.<br />
<br />
</p><br />
<br />
<br />
<h3> Culture Passaging </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Take 20-50μL from a previous overnight or passage culture and add it to the culture media.<br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%. <br><br />
<br />
Note: The exact amount of culture that you take from a previous culture is irrelevant as long as at least 1 living cell is passaged.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500uL of 40% glycerol and 500μL of Selective Dropout media or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Flow Cytometry"></a><br />
<br />
</html><br />
<br />
<h2> Flow Cytometry </h2><br />
<br />
<br />
<h3> Dilutions </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. From an overnight culture measure the optical density at 660nm by making 1:10 dilutions. <br><br />
<br />
2. Take enough culture to make a 1mL aliquot with OD 0.4. <br><br />
<br />
3. Spin down the aliquot in a 1.5mL centrifuge tube at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 800μL of the appropriate selective dropout media and 200μL of 20% glucose. <br><br />
<br />
6. Transfer the new culture to a 14mL culture tube. <br><br />
<br />
7. Incubate and shake at 30C and 250rpm for at least 6 hours (1.2-1.6 optical density).<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Preparations for Analysis using C6 Accuri Flow Cytometer </h3><br />
<br />
<br />
<p><br />
<br />
<br />
1. From the dilution previously made, measure the optical density, roughly 1.2-1.6. <br><br />
<br />
2. Make an aliquot of 500μL of the dilution culture in a 1.5mL centrifuge tube. <br><br />
<br />
3. Spin down the aliquot at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 500μL of PBS(F). <br><br />
<br />
6. Spin down the resuspension at 3000rpm for 3 minutes. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cell pellet in another 500uL of PBS(F).* <br><br />
<br />
9. Prepare the C6 Accuri Flow Cytometer by running a backflush cycle and a dH2O cycle. <br><br />
<br />
10. Load the sample onto the sip. <br><br />
<br />
11. Run the sample with 100,000 cell count. <br><br />
<br />
12. Repeat for all samples and make sure to change data cells otherwise the old data is erased. <br><br />
<br />
13. Once finished, run a cleaning cycle with Accuri approved cleaning solution, then run a dH2O cycle. <br><br />
<br />
<br />
<br />
*For special cases do not resuspend all samples, instead resuspend immediately before running the sample through the flow cytometer.<br />
<br />
<br />
</p><br />
<br />
<br />
<html><a name="Fluorescence Activated Cell Sorting"></a><br />
<br />
</html><br />
<br />
<h2> Fluorescence Activated Cell Sorting </h2><br />
<br />
<br />
<h3> Final Preparations </h3><br />
<br />
<br />
<br />
<p> <br />
<br />
Sample Prep:<br />
<br />
<br />
Spin down samples and negative control (5000 RPM, 1 min), keeping in mind the library size. Aspirate off supernatant. Resuspend in PBSF. Spin down cells. Aspirate off supernatant. Resuspend in PBSF. <br><br />
<br />
<br><br />
<br />
1. Open the “iGEM Template” file in the FACS Software and change name to current date and sort cycle<br><br />
<br />
2. Make sure the stream is stable (look for green light in bottom right corner). If not, run the Sort Calibration order. <br><br />
<br />
3. Run Negative Control from the PyE1 cells. <br><br />
<br />
a. Load cells onto carrier and into the machine. Press play button on screen. <br><br />
<br />
b. Set gate around lower left quadrant of cells to ensure single cell analysis using Forward Scatter Area and Side Scatter Area as your axes. Make sure oval gate covers around 80% of cell population. <br><br />
<br />
c. Set second gate on the first gated population by double-clicking on the gated population and using Forward Scatter Height and Forward Scatter Width as your axes. You will notice two distinct populations. Try to focus on the single cell portion of the plot. <br><br />
<br />
NOTE: If you see a large portion of the second gated population existing near the upper right edge of the first gate, you may need to enlarge the first gate to fit more of the population. <br><br />
<br />
d. Press record. Record 100,000 events and stop run. Move to Next Tube. <br><br />
<br />
4. Run first control (Gene clone) <br><br />
<br />
a. Follow step 3 to run the first control <br><br />
<br />
5. Run first library sample <br><br />
<br />
a. Follow steps 3b and 3c to set first two gates correctly. <br><br />
<br />
b. Set final gate for sort which includes top 1% of GFP producers from second gated population. <br><br />
<br />
c. Use final gate to set up the sort. <br><br />
<br />
d. Select sort conditions at the bottom of the screen. <br><br />
<br />
e. Insert and load collection tube. <br><br />
<br />
f. Record 100,000 events, and sort 10x the library size <br><br />
<br />
6. Run Bleach and diH2O through FACS to avoid cross-contamination. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Protein Expression"></a><br />
<br />
</html><br />
<br />
<h2> Protein Expression </h2><br />
<br />
<br />
<h3> Overnight Cultures </h3><br />
<br />
<p><br />
<br />
1. Add 25mL TB and 25uL Kan to a 250mL baffled flask <br><br />
<br />
2. Stab a glycerol stock with a p1000 pipette and swirl in the flask of media <br><br />
<br />
3. Put flask in 37C shaker overnight <br />
<br />
</p><br />
<br />
<h3> Protein Expression </h3><br />
<br />
<p> <br />
<br />
1. Add 500μL 1000x Kanamycin to 500mL TB in 2L baffled flask <br><br />
<br />
2. Transfer 10mL overnight culture to TB <br><br />
<br />
3. Shake at 37C (DO WE NEED THE RPM?) <br><br />
<br />
4. Remove flask from shaker when optical density is between 0.5 and 0.8 <br><br />
<br />
5. Allow flask to rest at room temp for 30 min <br><br />
<br />
6. Add 125μL 1M IPTG <br><br />
<br />
7. Shake flask at 18C overnight<br />
<br />
</p><br />
<br />
<h3> Protein Extraction and Purification </h3><br />
<br />
<p><br />
<br />
1. Transfer cell culture to centrifuge flask <br><br />
<br />
2. Centrifuge culture at 4000g for 10 min <br><br />
<br />
3. Discard supernatant <br><br />
<br />
4. Resuspend pellet in 25mL lysis buffer <br><br />
<br />
5. Add 250μL of 100x PMSF, 250μL of 100mg/mL lysozyme, and 250μL of 10mg/mL DNAse <br><br />
<br />
6. Sonicate sample with 0.25inch probe for 5 min at 70% amplitude with 20 sec on and off pulses <br><br />
<br />
7. Take 50μL total sample <br><br />
<br />
8. Transfer lysate to SS-34 centrifuge tube <br><br />
<br />
9. Centrifuge for 30 min at 18000g <br><br />
<br />
10. Take 50μL soluble sample<br />
<br />
</p><br />
<br />
<h3> Nickel Nitrotriacetic Acid Chromatography (Nickel-NTA Chromatography) </h3><br />
<br />
<p><br />
<br />
1. Add 5mL 50%(v/v) nickel resin in ethanol to 25mL column and allow to settle to ~2.5mL(CV) <br><br />
<br />
2. Rinse with 10CV H20 <br><br />
<br />
3. Equilibrate with 10CV lysis buffer <br><br />
<br />
4. Load sample onto column <br><br />
<br />
5. Wash column with 15CV lysis buffer <br><br />
<br />
6. Perform 2 additional wash steps with 15CV <br><br />
<br />
7. Elute sample in 10CV elution buffer and collect eluate <br><br />
<br />
8. Take 50μL pure sample <br />
<br />
</p><br />
<br />
<h3> Size Exclusion Chromatography (S.E.C.) </h3><br />
<p><br />
1. Concentrate sample to as high as possible without inducing protein aggregation<br><br />
2. Pre-equilibrate Superdex 75 column with 48 ml PBS <br><br />
3. Inject 500 ul sample onto column <br><br />
4. Run 36 ml PBS through column at 0.5 ml/min, collecting 1 ml fractions <br><br />
5. Verify presence of protein in fractions by measuring concentration on Nanodrop and running SDS-PAGE (15 kDa protein should elute at ~13 ml)<br><br />
6. Pool fractions containing protein <br />
</p><br />
<br />
<br />
<html><a name="Stability Analysis"></a><br />
<br />
</html><br />
<br />
<h2> Stability Analysis </h2><br />
<br />
<br />
<h3> Circular Dichroism: Wavelength Scan </h3><br />
<p><br />
1. Load 1mm cuvette with 400uL protein solution onto CD <br><br />
2. Take wavelength scan <br><br />
260nm-190nm <br><br />
sample every 1nm <br><br />
averaging time 3sec <br><br />
1 scan <br><br />
step scan <br><br />
25C <br><br />
3. Record wavelength which gives strongest signal (222nm)<br />
</p><br />
<br />
<h3> Circular Dichroism: Guanidine Melt </h3><br />
<p><br />
1. Load 1cm cuvette containing 1.996mL of 0.05mg/mL protein solution and stirrer onto CD <br><br />
2. Prepare 8mL of 0.05mg/mL protein in concentrated guanidine solution <br><br />
3. Set up Automixer with guanidine solution on one syringe and waste tube on other syringe <br><br />
4. Titrate up to 6 M guanidine, taking a CD measurement at 222 nm every 0.15 M interval <br><br />
5. Also measure the fluorescence at 280 nm to ensure the total protein concentration is not changing<br />
</p><br />
<br />
<br />
<br />
<html><br />
<br />
<a href="#linktotop">Back To Top</a><br />
</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2014-10-17T20:04:29Z<p>Kristanguyen: </p>
<hr />
<div>{{Template:Team:UW/CSS}}<br />
<br />
<br />
<h1> Protocols </h1><br />
<br />
<br />
<html><a name="Media, Plates, and Sol"></a><br />
<br />
</html><br />
<br />
<br />
<h2> Media, Plates, and Solutions </h2><br />
<br />
<br />
<h3> Competent Cell Media Buffer (CCMB) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
100 g glycerol (liquid) <br><br />
<br />
10 mL x 1 M potassium acetate <br><br />
<br />
11.8 g CaCl2*H2O <br><br />
<br />
4 g MnCl2 <br><br />
<br />
2 g MgCl2 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<h3> Super Optimal Broth (SOB) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
20 g Tryptone <br><br />
<br />
5 g Yeast Extract <br><br />
<br />
10 mL x 1 M NaCl <br><br />
<br />
2.5 mL x 1 M KCl <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Phosphate Buffered Saline (PBS) Solution </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container or 1 L beaker: <br><br />
<br />
8 g NaCl <br><br />
<br />
1.44 g Na2HPO4 <br><br />
<br />
0.8 g KCl <br><br />
<br />
0.24 g KH2PO4 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Buffer to pH 7.4 <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<h3> PSBF (PBS for Flow) </h3> <br />
<br />
<p><br />
<br />
Mix the following in a 1 L beaker: <br><br />
<br />
25 mL 20X PBS, pH 7.4 <br><br />
<br />
574 mL H2O <br><br />
<br />
2.5 g BSA (0.5%)* <br><br />
<br />
Filter and store at 4 °C <br><br />
<br><br />
*Do not need to pH - should be at pH 7.4 like 20X PBS <br><br />
<br />
</p> <br />
<br />
<br />
<h3> Yeast Extract Peptone Dextrose (YPD) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following into 950 mL of dH2O in a 1 L bottle: <br><br />
<br />
20 g peptone <br><br />
<br />
10 g yeast extract <br><br />
<br />
Autoclave (20 min at 121 °C and 20 psi) <br><br />
<br />
Add 50 mL 40% glucose <br><br />
<br />
Sterile filter into a 1 L bottle <br><br />
<br />
<br><br />
<br />
Note: For long-term liquid media storage, do not add 40% glucose. Instead add the glucose directly into cell cultures. <br><br />
<br />
Note: For YPD-plates add 24 g agar to the peptone and yeast extract before autoclaving. <br />
<br />
</p><br />
<br />
<br />
<h3> Selective Dropout Media, C-Uracil and C-Histidine (C-Ura and C-His) </h3><br />
<br />
<br />
<p><br />
<br />
Synthesized by the Yeast Resource Center at the University of Washington's <br />
<br />
Department of Genome Sciences and Department of Biochemistry.<br />
<br />
</p><br />
<br />
<br />
<h3> Guanidinium Hydrogen Chloride </h3><br />
<br />
<br />
<p><br />
<br />
For maximum effectiveness, final concentration should be approximately 8.5 M in PBS.<br><br />
<br />
203 g guanidinium hydrogen chloride <br><br />
<br />
250 mL PBS solution* <br><br />
<br />
Add dilute HCl to pH 7.4 <br><br />
<br />
<br><br />
<br />
*Alternatively add slightly less than 250 mL of PBS in order to buffer the solution to the appropriate volume, then add more dH2O as necessary. <br />
<br />
</p><br />
<br />
<html><a name="Basic Cloning"></a><br />
<br />
</html><br />
<br />
<h2> Basic Cloning </h2><br />
<br />
<br />
<br />
<h3> Polymerase Chain Reaction </h3><br />
<br />
<br />
<p><br />
<br />
All PCRs were done using a standard 50 μL reaction volume with GoTaq® Green Master Mix 2X purchased from PROMEGA Corporation. <br><br />
<br />
Mix the following in a 0.2 mL microcentrifuge tube on ice: <br><br />
<br />
25 μL GoTaq® Green Master Mix 2X <br><br />
<br />
1-5 μL of 10 μM forward primer <br><br />
<br />
1-5 μL of 10 μM reverse primer <br><br />
<br />
<250 ng of DNA template <br><br />
<br />
QS 50 μl nuclease-free H2O <br><br />
<br />
Conduct the reaction in a thermocycler, adjusting anneal temperature and extension times accordingly. See your polymerase supplier protocol for more details on thermocycling.<br />
<br />
</p><br />
<br />
<h3> Error-prone Polymerase Chain Reaction </h3><br />
<br />
<br />
<p> <br />
<br />
<br />
<br />
Prepare 50 μL reaction: <br><br />
<br />
5 μL 10X Mutazyme II Rxn Buffer <br><br />
<br />
1 μL 40 mM dNTP mix (200 μM each final) <br><br />
<br />
1 μL 20 μM forward primer <br><br />
<br />
1 μL 20 μM reverse primer <br><br />
<br />
1 μL Mutazyme II DNA polymerase (2.5 U/μL) <br><br />
<br />
0.01 ng template <br><br />
<br />
QS 50 μL diH2O <br><br />
<br />
<br><br />
<br />
Program thermocycler as follows: <br><br />
<br />
95 °C, 2 min <br><br />
<br />
95 °C, 30 sec <br><br />
<br />
XX °C*, 30 sec <br><br />
<br />
72 °C, X min** <br><br />
<br />
32 cycles <br><br />
<br />
72 °C, 10 min <br><br />
<br />
4 °C, hold<br />
<br />
<br><br />
<br />
*Adjust annealing temperature according to Tm of primer. <br><br />
<br />
**Adjust extension time according to the length of amplified DNA. <br><br />
<br />
<br><br />
<br />
Note: Use 0.01 ng of template (calculate by insert and not by total plasmid). <br><br />
<br />
Calculate amount of template to use as follows: <br><br />
<br />
(bp for amplified region) / (bp in total plasmid) = % amplified region <br><br />
<br />
(conc. of total plasmid) x (% amplified region as a decimal) = conc. of amplified region <br><br />
<br />
<br><br />
<br />
Note: Never pipette less than 0.5 μL. <br><br />
<br />
(0.01 ng of template) / (conc. of amplified region) = vol of template to add to PCR <br><br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Restriction Endonuclease Reaction (Digestion) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
All restriction enzyme reactions were done using a 50 μl reaction volume. Restriction enzymes and buffers were purchased from New England Biolabs® Inc. <br><br />
<br />
Mix the following in a 0.2 mL PCR tube: <br><br />
<br />
<br />
1 μg of DNA <br><br />
<br />
5 μL of the appropriate 10X New England Biolab® Buffer <br><br />
<br />
1 μL of each restriction enzyme (add last) <br><br />
<br />
QS 50μL nuclease-free H2O <br><br />
<br />
Incubate the reaction for 1 hr <br><br />
<br />
Heat inactive the reaction at the appropriate temperature <br><br />
<br><br />
<br />
Note: Thaw the restriction enzyme(s) on ice to improve shelf life <br><br />
<br />
<br />
</p> <br />
<br />
<br />
<h3> Ligation </h3><br />
<br />
<br />
<p><br />
<br />
T4 DNA Ligase and Buffer were purchased from New England Biolabs® Inc. <br><br />
<br />
1. Prepare the following in a 0.2 mL microcentrifuge tube: <br><br />
<br />
50.0 ng vector DNA* <br><br />
<br />
37.5 ng vector DNA* <br><br />
<br />
2 μL 10X T4 DNA Ligase Buffer <br><br />
<br />
1 μL T4 DNA Ligase <br><br />
<br />
QS 20 μL diH2O <br><br />
<br />
2. Incubate the reaction at room temperature for 10-30 minutes or at 16 °C overnight. <br><br />
<br />
3. Heat inactivate at 65 °C for 10 minutes. <br><br />
<br />
4. Chill on ice before starting a transformation reaction. <br><br />
<br />
<br><br />
<br />
*The exact amount of DNA is dependent on the number of base pairs. In order to conduct a proper reaction consult the New England Biolab Ligation Calculator at:<br />
<br />
http://nebiocalculator.neb.com/#!/<br />
<br />
</p><br />
<br />
<br />
<html><a name="Escherichia coli Protocols"></a><br />
<br />
</html><br />
<br />
<br />
<h2> <i> Escherichia coli </i> Protocols (XL1-Blue and XL10-Gold) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<p> <br />
<br />
1. Plate 10 ul of cells onto an antibiotic free LB/Agar plate..* <br><br />
<br />
2. Incubate overnight at 37OC <br><br />
<br />
3. Inoculate lots of colonies into 50ml SOB-Mg media in a 250ml flask. <br><br />
4. Incubate at 37 OC, 200rpm, until OD at 550nm reaches 0.3, takes about 3 hours. I do my first check at 2 hours.<br><br />
<br />
5.Transfer the cell culture into a sterile 50ml Falcon tube and chill on ice for 10 minutes. <br><br />
<br />
6. Pellet the cells at 2500rpm for 15 minutes at 4 OC.<br><br />
<br />
7. Decant supernatant and invert tubes to remove excess culture medium. <br><br />
<br />
8. Resuspend the cells in 16ml CCMB by gentle vortexing or pipetting (I shake the falcon tube).<br />
(16ml CCMB per 50ml culture) <br><br />
<br />
9. Incubate on ice for 20 minutes. <br><br />
<br />
10. Centrifuge at 2500rpm for 10 minutes at 4 OC. <br><br />
<br />
11. Decant supernatant and invert tubes to remove excess liquid. <br><br />
<br />
12. Resuspend the cells in 4ml CCMB by gentle vortexing or pipetting.<br />
(4ml CCMB per culture) <br><br />
<br />
13. If making multiple 50ml cultures, combine all of the cells and mix before aliquoting. <br> <br />
<br />
14. In cold room: make 205ul aliquots, flash freeze in liquid nitrogen, and store at -80 OC.<br><br />
<br />
<br />
<br />
<br />
NOTE: Make SOB-Mg media and autoclave the 250ml flasks with water in them the same day the cells are plated (step 1) <br><br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Chemically Competent Cell Transformations </h3><br />
<br />
<br />
<p> <br />
<br />
1. Thaw competent <i> E.coli </i> cells on ice (XL1-Blue or XL10-Gold)* <br><br />
<br />
2. Add 50μL of competent cells to sterile 15mL conical centrifuge tubes <br><br />
<br />
3. Add 1μL (~100-200ng)* of the mini-prep to each culture tube <br><br />
<br />
4. Equilibrate the cells on ice for 10 min <br><br />
<br />
5. Heat shock the cells at 42C for 30-45 seconds** <br><br />
<br />
6. Immediately place the cells back on ice for 3 min <br><br />
<br />
7. Add 250μL LB media without antibiotics and shake at 250 rpm and 37C for 30 min <br><br />
<br />
8. Spread 10μL and 290μL on an appropriate LB-antibiotic plate <br><br />
<br />
9. Invert the plate and incubate at 37C overnight<br />
<br />
<br />
*The exact amount of DNA to add depends on your cell's transformation efficiency. However, it is acceptable to add a larger amount to increase the number of transformed cells. <br><br />
<br />
** Do not heat shock for an extended duration as this may damage and/or kill your cells.<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnights </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottom tube, add 3-5mL of LB and an appropriate volume of antibiotic(s). <br><br />
<br />
2. Swipe several individual colonies, do not collect satellites or colony clumps, with a pipette tip. <br><br />
<br />
3. Swirl the colony tip in the tube, there should be no visible cell clumps. <br><br />
<br />
4. Incubate and shake the tube at 37C at 250rpm for 12-16 hours and no longer than 20 hours. <br><br />
<br />
</p><br />
<br />
<br />
<h3> DNA-Extraction and mini-preps </h3><br />
<br />
<br />
<p><br />
<br />
All DNA mini-preps were prepared using EPOCH mini-kits and following the supplied protocols.<br />
<br />
</p><br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500μL of 40% Glycerol and 500uL of LB (no antibiotics) or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Saccharomyces cerevisiae"></a><br />
<br />
</html><br />
<br />
<h2> <i> Saccharomyces cerevisiae </i> (PyE1 Yeast) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Culturing </h3><br />
<br />
<br />
<br />
<p><br />
<br />
This process take 4 days in lab with a 1 day wait for incubation. <br><br />
<br />
<br />
Day 1: <br><br />
<br />
1. Streak yeast cells onto a YPD plate.* <br><br />
<br />
2. Invert the plate and incubate at 30C for 2 days. <br><br />
<br />
<br />
Day 3: <br><br />
<br />
1. Add 50mL of YPD liquid media into a 250mL baffle flask. <br><br />
<br />
2. Swipe as many individual colonies as you can see into the YPD media.** <br><br />
<br />
3. Incubate and shake the culture at 30C at 250rpm overnight approximately 24 hours. <br><br />
<br />
<br />
Day 4: <br><br />
<br />
1. Take an optical density measurement. <br><br />
<br />
2. In three 250mL baffle flask add the portions of the overnight liquid culture. <br><br />
<br />
3. Dilute each culture to approximately 0.4 optical density with YPD. <br><br />
<br />
4. Incubate and shake the cultures at 30C at 250rpm until the optical density reaches 1.2-1.6. <br><br />
<br />
5. Collect each culture into separate 50ml flat-bottomed centrifuge tubes. <br><br />
<br />
6. Spin down the cells at 4000g for 5 minutes at 4C. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cells in 100mL total for all three culture of dH2O. <br><br />
<br />
9. Combine the suspensions into two 50mL flat-bottomed centrifuge tubes. <br><br />
<br />
10. Spin down the cells as above. <br><br />
<br />
11. Decant the supernatant. <br><br />
<br />
12. Resuspend each in 3mL of 100mM Lithium Acetate. <br><br />
<br />
13. Transfer both cultures into a single 15mL conical centrifuge tube. <br><br />
<br />
14. Spin down the cells at 3000rpm for 5 minutes. <br><br />
<br />
15. Resuspend the cells in 0.75mL of 100mM Lithium Acetate, total volume is roughly 2mL. <br><br />
<br />
16. Qualitatively bring up the volume to 3.5mL by adding 40% Glycerol. <br><br />
<br />
17. Aliquot the cells into 1.5mL centrifuge tubes or 1.7mL cryogenic vials.*** <br><br />
<br />
<br />
*Streak in such a way that there are individual colonies visible on the plate without clumps or satellite colonies. <br><br />
<br />
**Collect only individual visible colonies. Do not collect clumps or satellite colonies. <br><br />
<br />
***The volume of aliquots depends on the number to transformations you intend to do at a time. <br><br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Chemically Competent Transformations </h3><br />
<br />
<br />
<p><br />
<br />
This protocol assumes a 50μL aliquot of yeast competent cells were made. <br><br />
<br />
Furthermore, this protocol prepares enough cells for 6 yeast transformations. <br><br />
<br />
<br><br />
<br />
1. Add the following to 50μL of yeast competent cells: <br><br />
<br />
240μL of Polyethylene Glycol - 3350 (PEG-3350) <br><br />
<br />
36μL of 1M Lithium Acetate <br><br />
<br />
32μL of dH2O <br><br />
<br />
<br />
<br />
2. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
3. Aliquot 59μL of the mixture into a 0.2mL microcentrifuge tube. <br><br />
<br />
4. Add 1uL (~100-200ng) of DNA. <br><br />
<br />
5. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
6. Incubate the mixture at 30C for 30 minutes. <br><br />
<br />
7. Heat shock the mixture at 42C for 20 minutes. <br><br />
<br />
8. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
9. Decant the supernatant. <br><br />
<br />
10. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
11. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
12. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
13. Plate 50-150μL of the mixture onto an appropriate Selective Dropout Media plate. <br><br />
<br />
14. Invert and incubate at 30C for 2 days. <br><br />
<br />
*The exact amount of DNA depends on the transformation efficiency of your competent cells. <br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnight Culturing </h3><br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Swipe 3 individually visible yeast colonies and add them to the culture tube media. <br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%.<br />
<br />
</p><br />
<br />
<br />
<h3> Culture Passaging </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Take 20-50μL from a previous overnight or passage culture and add it to the culture media.<br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%. <br><br />
<br />
Note: The exact amount of culture that you take from a previous culture is irrelevant as long as at least 1 living cell is passaged.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500uL of 40% glycerol and 500μL of Selective Dropout media or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Flow Cytometry"></a><br />
<br />
</html><br />
<br />
<h2> Flow Cytometry </h2><br />
<br />
<br />
<h3> Dilutions </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. From an overnight culture measure the optical density at 660nm by making 1:10 dilutions. <br><br />
<br />
2. Take enough culture to make a 1mL aliquot with OD 0.4. <br><br />
<br />
3. Spin down the aliquot in a 1.5mL centrifuge tube at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 800μL of the appropriate selective dropout media and 200μL of 20% glucose. <br><br />
<br />
6. Transfer the new culture to a 14mL culture tube. <br><br />
<br />
7. Incubate and shake at 30C and 250rpm for at least 6 hours (1.2-1.6 optical density).<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Preparations for Analysis using C6 Accuri Flow Cytometer </h3><br />
<br />
<br />
<p><br />
<br />
<br />
1. From the dilution previously made, measure the optical density, roughly 1.2-1.6. <br><br />
<br />
2. Make an aliquot of 500μL of the dilution culture in a 1.5mL centrifuge tube. <br><br />
<br />
3. Spin down the aliquot at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 500μL of PBS(F). <br><br />
<br />
6. Spin down the resuspension at 3000rpm for 3 minutes. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cell pellet in another 500uL of PBS(F).* <br><br />
<br />
9. Prepare the C6 Accuri Flow Cytometer by running a backflush cycle and a dH2O cycle. <br><br />
<br />
10. Load the sample onto the sip. <br><br />
<br />
11. Run the sample with 100,000 cell count. <br><br />
<br />
12. Repeat for all samples and make sure to change data cells otherwise the old data is erased. <br><br />
<br />
13. Once finished, run a cleaning cycle with Accuri approved cleaning solution, then run a dH2O cycle. <br><br />
<br />
<br />
<br />
*For special cases do not resuspend all samples, instead resuspend immediately before running the sample through the flow cytometer.<br />
<br />
<br />
</p><br />
<br />
<br />
<html><a name="Fluorescence Activated Cell Sorting"></a><br />
<br />
</html><br />
<br />
<h2> Fluorescence Activated Cell Sorting </h2><br />
<br />
<br />
<h3> Final Preparations </h3><br />
<br />
<br />
<br />
<p> <br />
<br />
Sample Prep:<br />
<br />
<br />
Spin down samples and negative control (5000 RPM, 1 min), keeping in mind the library size. Aspirate off supernatant. Resuspend in PBSF. Spin down cells. Aspirate off supernatant. Resuspend in PBSF. <br><br />
<br />
<br><br />
<br />
1. Open the “iGEM Template” file in the FACS Software and change name to current date and sort cycle<br><br />
<br />
2. Make sure the stream is stable (look for green light in bottom right corner). If not, run the Sort Calibration order. <br><br />
<br />
3. Run Negative Control from the PyE1 cells. <br><br />
<br />
a. Load cells onto carrier and into the machine. Press play button on screen. <br><br />
<br />
b. Set gate around lower left quadrant of cells to ensure single cell analysis using Forward Scatter Area and Side Scatter Area as your axes. Make sure oval gate covers around 80% of cell population. <br><br />
<br />
c. Set second gate on the first gated population by double-clicking on the gated population and using Forward Scatter Height and Forward Scatter Width as your axes. You will notice two distinct populations. Try to focus on the single cell portion of the plot. <br><br />
<br />
NOTE: If you see a large portion of the second gated population existing near the upper right edge of the first gate, you may need to enlarge the first gate to fit more of the population. <br><br />
<br />
d. Press record. Record 100,000 events and stop run. Move to Next Tube. <br><br />
<br />
4. Run first control (Gene clone) <br><br />
<br />
a. Follow step 3 to run the first control <br><br />
<br />
5. Run first library sample <br><br />
<br />
a. Follow steps 3b and 3c to set first two gates correctly. <br><br />
<br />
b. Set final gate for sort which includes top 1% of GFP producers from second gated population. <br><br />
<br />
c. Use final gate to set up the sort. <br><br />
<br />
d. Select sort conditions at the bottom of the screen. <br><br />
<br />
e. Insert and load collection tube. <br><br />
<br />
f. Record 100,000 events, and sort 10x the library size <br><br />
<br />
6. Run Bleach and diH2O through FACS to avoid cross-contamination. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Protein Expression"></a><br />
<br />
</html><br />
<br />
<h2> Protein Expression </h2><br />
<br />
<br />
<h3> Overnight Cultures </h3><br />
<br />
<p><br />
<br />
1. Add 25mL TB and 25uL Kan to a 250mL baffled flask <br><br />
<br />
2. Stab a glycerol stock with a p1000 pipette and swirl in the flask of media <br><br />
<br />
3. Put flask in 37C shaker overnight <br />
<br />
</p><br />
<br />
<h3> Protein Expression </h3><br />
<br />
<p> <br />
<br />
1. Add 500μL 1000x Kanamycin to 500mL TB in 2L baffled flask <br><br />
<br />
2. Transfer 10mL overnight culture to TB <br><br />
<br />
3. Shake at 37C (DO WE NEED THE RPM?) <br><br />
<br />
4. Remove flask from shaker when optical density is between 0.5 and 0.8 <br><br />
<br />
5. Allow flask to rest at room temp for 30 min <br><br />
<br />
6. Add 125μL 1M IPTG <br><br />
<br />
7. Shake flask at 18C overnight<br />
<br />
</p><br />
<br />
<h3> Protein Extraction and Purification </h3><br />
<br />
<p><br />
<br />
1. Transfer cell culture to centrifuge flask <br><br />
<br />
2. Centrifuge culture at 4000g for 10 min <br><br />
<br />
3. Discard supernatant <br><br />
<br />
4. Resuspend pellet in 25mL lysis buffer <br><br />
<br />
5. Add 250μL of 100x PMSF, 250μL of 100mg/mL lysozyme, and 250μL of 10mg/mL DNAse <br><br />
<br />
6. Sonicate sample with 0.25inch probe for 5 min at 70% amplitude with 20 sec on and off pulses <br><br />
<br />
7. Take 50μL total sample <br><br />
<br />
8. Transfer lysate to SS-34 centrifuge tube <br><br />
<br />
9. Centrifuge for 30 min at 18000g <br><br />
<br />
10. Take 50μL soluble sample<br />
<br />
</p><br />
<br />
<h3> Nickel Nitrotriacetic Acid Chromatography (Nickel-NTA Chromatography) </h3><br />
<br />
<p><br />
<br />
1. Add 5mL 50%(v/v) nickel resin in ethanol to 25mL column and allow to settle to ~2.5mL(CV) <br><br />
<br />
2. Rinse with 10CV H20 <br><br />
<br />
3. Equilibrate with 10CV lysis buffer <br><br />
<br />
4. Load sample onto column <br><br />
<br />
5. Wash column with 15CV lysis buffer <br><br />
<br />
6. Perform 2 additional wash steps with 15CV <br><br />
<br />
7. Elute sample in 10CV elution buffer and collect eluate <br><br />
<br />
8. Take 50μL pure sample <br />
<br />
</p><br />
<br />
<h3> Size Exclusion Chromatography (S.E.C.) </h3><br />
<p><br />
1. Concentrate sample to as high as possible without inducing protein aggregation<br><br />
2. Pre-equilibrate Superdex 75 column with 48 ml PBS <br><br />
3. Inject 500 ul sample onto column <br><br />
4. Run 36 ml PBS through column at 0.5 ml/min, collecting 1 ml fractions <br><br />
5. Verify presence of protein in fractions by measuring concentration on Nanodrop and running SDS-PAGE (15 kDa protein should elute at ~13 ml)<br><br />
6. Pool fractions containing protein <br />
</p><br />
<br />
<br />
<html><a name="Stability Analysis"></a><br />
<br />
</html><br />
<br />
<h2> Stability Analysis </h2><br />
<br />
<br />
<h3> Circular Dichroism: Wavelength Scan </h3><br />
<p><br />
1. Load 1mm cuvette with 400uL protein solution onto CD <br><br />
2. Take wavelength scan <br><br />
260nm-190nm <br><br />
sample every 1nm <br><br />
averaging time 3sec <br><br />
1 scan <br><br />
step scan <br><br />
25C <br><br />
3. Record wavelength which gives strongest signal (222nm)<br />
</p><br />
<br />
<h3> Circular Dichroism: Guanidine Melt </h3><br />
<p><br />
1. Load 1cm cuvette containing 1.996mL of 0.05mg/mL protein solution and stirrer onto CD <br><br />
2. Prepare 8mL of 0.05mg/mL protein in concentrated guanidine solution <br><br />
3. Set up Automixer with guanidine solution on one syringe and waste tube on other syringe <br><br />
4. Titrate up to 6 M guanidine, taking a CD measurement at 222 nm every 0.15 M interval <br><br />
5. Also measure the fluorescence at 280 nm to ensure the total protein concentration is not changing<br />
</p><br />
<br />
<br />
<br />
<html><br />
<br />
<a href="#linktotop">Back To Top</a><br />
</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2014-10-17T20:03:41Z<p>Kristanguyen: </p>
<hr />
<div>{{Template:Team:UW/CSS}}<br />
<br />
<br />
<h1> Protocols </h1><br />
<br />
<br />
<html><a name="Media, Plates, and Sol"></a><br />
<br />
</html><br />
<br />
<br />
<h2> Media, Plates, and Solutions </h2><br />
<br />
<br />
<h3> Competent Cell Media Buffer (CCMB) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
100 g glycerol (liquid) <br><br />
<br />
10 mL x 1 M potassium acetate <br><br />
<br />
11.8 g CaCl2*H2O <br><br />
<br />
4 g MnCl2 <br><br />
<br />
2 g MgCl2 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<h3> Super Optimal Broth (SOB) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
20 g Tryptone <br><br />
<br />
5 g Yeast Extract <br><br />
<br />
10 mL x 1 M NaCl <br><br />
<br />
2.5 mL x 1 M KCl <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Phosphate Buffered Saline (PBS) Solution </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container or 1 L beaker: <br><br />
<br />
8 g NaCl <br><br />
<br />
1.44 g Na2HPO4 <br><br />
<br />
0.8 g KCl <br><br />
<br />
0.24 g KH2PO4 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Buffer to pH 7.4 <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<h3> PSBF (PBS for Flow) </h3> <br />
<br />
<p><br />
<br />
Mix the following in a 1 L beaker: <br><br />
<br />
25 mL 20X PBS, pH 7.4 <br><br />
<br />
574 mL H2O <br><br />
<br />
2.5 g BSA (0.5%)* <br><br />
<br />
Filter and store at 4 °C <br><br />
<br><br />
*Do not need to pH - should be at pH 7.4 like 20X PBS <br><br />
<br />
</p> <br />
<br />
<br />
<h3> Yeast Extract Peptone Dextrose (YPD) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following into 950 mL of dH2O in a 1 L bottle: <br><br />
<br />
20 g peptone <br><br />
<br />
10 g yeast extract <br><br />
<br />
Autoclave (20 min at 121 °C and 20 psi) <br><br />
<br />
Add 50 mL 40% glucose <br><br />
<br />
Sterile filter into a 1 L bottle <br><br />
<br />
<br><br />
<br />
Note: For long-term liquid media storage, do not add 40% glucose. Instead add the glucose directly into cell cultures. <br><br />
<br />
Note: For YPD-plates add 24 g agar to the peptone and yeast extract before autoclaving. <br />
<br />
</p><br />
<br />
<br />
<h3> Selective Dropout Media, C-Uracil and C-Histidine (C-Ura and C-His) </h3><br />
<br />
<br />
<p><br />
<br />
Synthesized by the Yeast Resource Center at the University of Washington's <br />
<br />
Department of Genome Sciences and Department of Biochemistry.<br />
<br />
</p><br />
<br />
<br />
<h3> Guanidinium Hydrogen Chloride </h3><br />
<br />
<br />
<p><br />
<br />
For maximum effectiveness, final concentration should be approximately 8.5 M in PBS.<br><br />
<br />
203 g guanidinium hydrogen chloride <br><br />
<br />
250 mL PBS solution* <br><br />
<br />
Add dilute HCl to pH 7.4 <br><br />
<br />
<br><br />
<br />
*Alternatively add slightly less than 250 mL of PBS in order to buffer the solution to the appropriate volume, then add more dH2O as necessary. <br />
<br />
</p><br />
<br />
<html><a name="Basic Cloning"></a><br />
<br />
</html><br />
<br />
<h2> Basic Cloning </h2><br />
<br />
<br />
<br />
<h3> Polymerase Chain Reaction </h3><br />
<br />
<br />
<p><br />
<br />
All PCRs were done using a standard 50 μL reaction volume with GoTaq® Green Master Mix 2X purchased from PROMEGA Corporation. <br><br />
<br />
Mix the following in a 0.2 mL microcentrifuge tube on ice: <br><br />
<br />
25 μL GoTaq® Green Master Mix 2X <br><br />
<br />
1-5 μL of 10 μM forward primer <br><br />
<br />
1-5 μL of 10 μM reverse primer <br><br />
<br />
<250 ng of DNA template <br><br />
<br />
QS 50 μl nuclease-free H2O <br><br />
<br />
Conduct the reaction in a thermocycler, adjusting anneal temperature and extension times accordingly. See your polymerase supplier protocol for more details on thermocycling.<br />
<br />
</p><br />
<br />
<h3> Error-prone Polymerase Chain Reaction </h3><br />
<br />
<br />
<p> <br />
<br />
<br />
<br />
Prepare 50 μL reaction: <br><br />
<br />
5 μL 10X Mutazyme II Rxn Buffer <br><br />
<br />
1 μL 40 mM dNTP mix (200 μM each final) <br><br />
<br />
1 μL 20 μM forward primer <br><br />
<br />
1 μL 20 μM reverse primer <br><br />
<br />
1 μL Mutazyme II DNA polymerase (2.5 U/μL) <br><br />
<br />
0.01 ng template <br><br />
<br />
QS 50 μL diH2O <br><br />
<br />
<br><br />
<br />
Program thermocycler as follows: <br><br />
<br />
95 °C, 2 min <br><br />
<br />
95 °C, 30 sec <br><br />
<br />
XX °C*, 30 sec <br><br />
<br />
72 °C, X min** <br><br />
<br />
32 cycles <br><br />
<br />
72 °C, 10 min <br><br />
<br />
4 °C, hold<br />
<br />
<br><br />
<br />
*Adjust annealing temperature according to Tm of primer. <br><br />
<br />
**Adjust extension time according to the length of amplified DNA. <br><br />
<br />
<br><br />
<br />
Note: Use 0.01 ng of template (calculate by insert and not by total plasmid). <br><br />
<br />
Calculate amount of template to use as follows: <br><br />
<br />
(bp for amplified region) / (bp in total plasmid) = % amplified region <br><br />
<br />
(conc. of total plasmid) x (% amplified region as a decimal) = conc. of amplified region <br><br />
<br />
<br><br />
<br />
Note: Never pipette less than 0.5 μL. <br><br />
<br />
(0.01 ng of template) / (conc. of amplified region) = vol of template to add to PCR <br><br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Restriction Endonuclease Reaction (Digestion) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
All restriction enzyme reactions were done using a 50 μl reaction volume. Restriction enzymes and buffers were purchased from New England Biolabs® Inc. <br><br />
<br />
Mix the following in a 0.2 mL PCR tube: <br><br />
<br />
<br />
1 μg of DNA <br><br />
<br />
5 μL of the appropriate 10X New England Biolab® Buffer <br><br />
<br />
1 μL of each restriction enzyme (add last) <br><br />
<br />
QS 50μL nuclease-free H2O <br><br />
<br />
Incubate the reaction for 1 hr <br><br />
<br />
Heat inactive the reaction at the appropriate temperature <br><br />
<br><br />
<br />
Note: Thaw the restriction enzyme(s) on ice to improve shelf life <br><br />
<br />
<br />
</p> <br />
<br />
<br />
<h3> Ligation </h3><br />
<br />
<br />
<p><br />
<br />
T4 DNA Ligase and Buffer were purchased from New England Biolabs® Inc. <br><br />
<br />
1. Prepare the following in a 0.2 mL microcentrifuge tube: <br><br />
<br />
50.0 ng vector DNA* <br><br />
<br />
37.5 ng vector DNA* <br><br />
<br />
2 μL 10X T4 DNA Ligase Buffer <br><br />
<br />
1 μL T4 DNA Ligase <br><br />
<br />
QS 20 μL diH2O <br><br />
<br />
2. Incubate the reaction at room temperature for 10-30 minutes or at 16 °C overnight. <br><br />
<br />
3. Heat inactivate at 65 °C for 10 minutes. <br><br />
<br />
4. Chill on ice before starting a transformation reaction. <br><br />
<br />
*The exact amount of DNA is dependent on the number of base pairs. In order to conduct a proper reaction consult the New England Biolab Ligation Calculator at:<br />
<br />
http://nebiocalculator.neb.com/#!/<br />
<br />
</p><br />
<br />
<br />
<html><a name="Escherichia coli Protocols"></a><br />
<br />
</html><br />
<br />
<br />
<h2> <i> Escherichia coli </i> Protocols (XL1-Blue and XL10-Gold) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<p> <br />
<br />
1. Plate 10 ul of cells onto an antibiotic free LB/Agar plate..* <br><br />
<br />
2. Incubate overnight at 37OC <br><br />
<br />
3. Inoculate lots of colonies into 50ml SOB-Mg media in a 250ml flask. <br><br />
4. Incubate at 37 OC, 200rpm, until OD at 550nm reaches 0.3, takes about 3 hours. I do my first check at 2 hours.<br><br />
<br />
5.Transfer the cell culture into a sterile 50ml Falcon tube and chill on ice for 10 minutes. <br><br />
<br />
6. Pellet the cells at 2500rpm for 15 minutes at 4 OC.<br><br />
<br />
7. Decant supernatant and invert tubes to remove excess culture medium. <br><br />
<br />
8. Resuspend the cells in 16ml CCMB by gentle vortexing or pipetting (I shake the falcon tube).<br />
(16ml CCMB per 50ml culture) <br><br />
<br />
9. Incubate on ice for 20 minutes. <br><br />
<br />
10. Centrifuge at 2500rpm for 10 minutes at 4 OC. <br><br />
<br />
11. Decant supernatant and invert tubes to remove excess liquid. <br><br />
<br />
12. Resuspend the cells in 4ml CCMB by gentle vortexing or pipetting.<br />
(4ml CCMB per culture) <br><br />
<br />
13. If making multiple 50ml cultures, combine all of the cells and mix before aliquoting. <br> <br />
<br />
14. In cold room: make 205ul aliquots, flash freeze in liquid nitrogen, and store at -80 OC.<br><br />
<br />
<br />
<br />
<br />
NOTE: Make SOB-Mg media and autoclave the 250ml flasks with water in them the same day the cells are plated (step 1) <br><br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Chemically Competent Cell Transformations </h3><br />
<br />
<br />
<p> <br />
<br />
1. Thaw competent <i> E.coli </i> cells on ice (XL1-Blue or XL10-Gold)* <br><br />
<br />
2. Add 50μL of competent cells to sterile 15mL conical centrifuge tubes <br><br />
<br />
3. Add 1μL (~100-200ng)* of the mini-prep to each culture tube <br><br />
<br />
4. Equilibrate the cells on ice for 10 min <br><br />
<br />
5. Heat shock the cells at 42C for 30-45 seconds** <br><br />
<br />
6. Immediately place the cells back on ice for 3 min <br><br />
<br />
7. Add 250μL LB media without antibiotics and shake at 250 rpm and 37C for 30 min <br><br />
<br />
8. Spread 10μL and 290μL on an appropriate LB-antibiotic plate <br><br />
<br />
9. Invert the plate and incubate at 37C overnight<br />
<br />
<br />
*The exact amount of DNA to add depends on your cell's transformation efficiency. However, it is acceptable to add a larger amount to increase the number of transformed cells. <br><br />
<br />
** Do not heat shock for an extended duration as this may damage and/or kill your cells.<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnights </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottom tube, add 3-5mL of LB and an appropriate volume of antibiotic(s). <br><br />
<br />
2. Swipe several individual colonies, do not collect satellites or colony clumps, with a pipette tip. <br><br />
<br />
3. Swirl the colony tip in the tube, there should be no visible cell clumps. <br><br />
<br />
4. Incubate and shake the tube at 37C at 250rpm for 12-16 hours and no longer than 20 hours. <br><br />
<br />
</p><br />
<br />
<br />
<h3> DNA-Extraction and mini-preps </h3><br />
<br />
<br />
<p><br />
<br />
All DNA mini-preps were prepared using EPOCH mini-kits and following the supplied protocols.<br />
<br />
</p><br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500μL of 40% Glycerol and 500uL of LB (no antibiotics) or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Saccharomyces cerevisiae"></a><br />
<br />
</html><br />
<br />
<h2> <i> Saccharomyces cerevisiae </i> (PyE1 Yeast) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Culturing </h3><br />
<br />
<br />
<br />
<p><br />
<br />
This process take 4 days in lab with a 1 day wait for incubation. <br><br />
<br />
<br />
Day 1: <br><br />
<br />
1. Streak yeast cells onto a YPD plate.* <br><br />
<br />
2. Invert the plate and incubate at 30C for 2 days. <br><br />
<br />
<br />
Day 3: <br><br />
<br />
1. Add 50mL of YPD liquid media into a 250mL baffle flask. <br><br />
<br />
2. Swipe as many individual colonies as you can see into the YPD media.** <br><br />
<br />
3. Incubate and shake the culture at 30C at 250rpm overnight approximately 24 hours. <br><br />
<br />
<br />
Day 4: <br><br />
<br />
1. Take an optical density measurement. <br><br />
<br />
2. In three 250mL baffle flask add the portions of the overnight liquid culture. <br><br />
<br />
3. Dilute each culture to approximately 0.4 optical density with YPD. <br><br />
<br />
4. Incubate and shake the cultures at 30C at 250rpm until the optical density reaches 1.2-1.6. <br><br />
<br />
5. Collect each culture into separate 50ml flat-bottomed centrifuge tubes. <br><br />
<br />
6. Spin down the cells at 4000g for 5 minutes at 4C. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cells in 100mL total for all three culture of dH2O. <br><br />
<br />
9. Combine the suspensions into two 50mL flat-bottomed centrifuge tubes. <br><br />
<br />
10. Spin down the cells as above. <br><br />
<br />
11. Decant the supernatant. <br><br />
<br />
12. Resuspend each in 3mL of 100mM Lithium Acetate. <br><br />
<br />
13. Transfer both cultures into a single 15mL conical centrifuge tube. <br><br />
<br />
14. Spin down the cells at 3000rpm for 5 minutes. <br><br />
<br />
15. Resuspend the cells in 0.75mL of 100mM Lithium Acetate, total volume is roughly 2mL. <br><br />
<br />
16. Qualitatively bring up the volume to 3.5mL by adding 40% Glycerol. <br><br />
<br />
17. Aliquot the cells into 1.5mL centrifuge tubes or 1.7mL cryogenic vials.*** <br><br />
<br />
<br />
*Streak in such a way that there are individual colonies visible on the plate without clumps or satellite colonies. <br><br />
<br />
**Collect only individual visible colonies. Do not collect clumps or satellite colonies. <br><br />
<br />
***The volume of aliquots depends on the number to transformations you intend to do at a time. <br><br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Chemically Competent Transformations </h3><br />
<br />
<br />
<p><br />
<br />
This protocol assumes a 50μL aliquot of yeast competent cells were made. <br><br />
<br />
Furthermore, this protocol prepares enough cells for 6 yeast transformations. <br><br />
<br />
<br><br />
<br />
1. Add the following to 50μL of yeast competent cells: <br><br />
<br />
240μL of Polyethylene Glycol - 3350 (PEG-3350) <br><br />
<br />
36μL of 1M Lithium Acetate <br><br />
<br />
32μL of dH2O <br><br />
<br />
<br />
<br />
2. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
3. Aliquot 59μL of the mixture into a 0.2mL microcentrifuge tube. <br><br />
<br />
4. Add 1uL (~100-200ng) of DNA. <br><br />
<br />
5. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
6. Incubate the mixture at 30C for 30 minutes. <br><br />
<br />
7. Heat shock the mixture at 42C for 20 minutes. <br><br />
<br />
8. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
9. Decant the supernatant. <br><br />
<br />
10. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
11. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
12. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
13. Plate 50-150μL of the mixture onto an appropriate Selective Dropout Media plate. <br><br />
<br />
14. Invert and incubate at 30C for 2 days. <br><br />
<br />
*The exact amount of DNA depends on the transformation efficiency of your competent cells. <br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnight Culturing </h3><br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Swipe 3 individually visible yeast colonies and add them to the culture tube media. <br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%.<br />
<br />
</p><br />
<br />
<br />
<h3> Culture Passaging </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Take 20-50μL from a previous overnight or passage culture and add it to the culture media.<br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%. <br><br />
<br />
Note: The exact amount of culture that you take from a previous culture is irrelevant as long as at least 1 living cell is passaged.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500uL of 40% glycerol and 500μL of Selective Dropout media or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Flow Cytometry"></a><br />
<br />
</html><br />
<br />
<h2> Flow Cytometry </h2><br />
<br />
<br />
<h3> Dilutions </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. From an overnight culture measure the optical density at 660nm by making 1:10 dilutions. <br><br />
<br />
2. Take enough culture to make a 1mL aliquot with OD 0.4. <br><br />
<br />
3. Spin down the aliquot in a 1.5mL centrifuge tube at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 800μL of the appropriate selective dropout media and 200μL of 20% glucose. <br><br />
<br />
6. Transfer the new culture to a 14mL culture tube. <br><br />
<br />
7. Incubate and shake at 30C and 250rpm for at least 6 hours (1.2-1.6 optical density).<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Preparations for Analysis using C6 Accuri Flow Cytometer </h3><br />
<br />
<br />
<p><br />
<br />
<br />
1. From the dilution previously made, measure the optical density, roughly 1.2-1.6. <br><br />
<br />
2. Make an aliquot of 500μL of the dilution culture in a 1.5mL centrifuge tube. <br><br />
<br />
3. Spin down the aliquot at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 500μL of PBS(F). <br><br />
<br />
6. Spin down the resuspension at 3000rpm for 3 minutes. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cell pellet in another 500uL of PBS(F).* <br><br />
<br />
9. Prepare the C6 Accuri Flow Cytometer by running a backflush cycle and a dH2O cycle. <br><br />
<br />
10. Load the sample onto the sip. <br><br />
<br />
11. Run the sample with 100,000 cell count. <br><br />
<br />
12. Repeat for all samples and make sure to change data cells otherwise the old data is erased. <br><br />
<br />
13. Once finished, run a cleaning cycle with Accuri approved cleaning solution, then run a dH2O cycle. <br><br />
<br />
<br />
<br />
*For special cases do not resuspend all samples, instead resuspend immediately before running the sample through the flow cytometer.<br />
<br />
<br />
</p><br />
<br />
<br />
<html><a name="Fluorescence Activated Cell Sorting"></a><br />
<br />
</html><br />
<br />
<h2> Fluorescence Activated Cell Sorting </h2><br />
<br />
<br />
<h3> Final Preparations </h3><br />
<br />
<br />
<br />
<p> <br />
<br />
Sample Prep:<br />
<br />
<br />
Spin down samples and negative control (5000 RPM, 1 min), keeping in mind the library size. Aspirate off supernatant. Resuspend in PBSF. Spin down cells. Aspirate off supernatant. Resuspend in PBSF. <br><br />
<br />
<br><br />
<br />
1. Open the “iGEM Template” file in the FACS Software and change name to current date and sort cycle<br><br />
<br />
2. Make sure the stream is stable (look for green light in bottom right corner). If not, run the Sort Calibration order. <br><br />
<br />
3. Run Negative Control from the PyE1 cells. <br><br />
<br />
a. Load cells onto carrier and into the machine. Press play button on screen. <br><br />
<br />
b. Set gate around lower left quadrant of cells to ensure single cell analysis using Forward Scatter Area and Side Scatter Area as your axes. Make sure oval gate covers around 80% of cell population. <br><br />
<br />
c. Set second gate on the first gated population by double-clicking on the gated population and using Forward Scatter Height and Forward Scatter Width as your axes. You will notice two distinct populations. Try to focus on the single cell portion of the plot. <br><br />
<br />
NOTE: If you see a large portion of the second gated population existing near the upper right edge of the first gate, you may need to enlarge the first gate to fit more of the population. <br><br />
<br />
d. Press record. Record 100,000 events and stop run. Move to Next Tube. <br><br />
<br />
4. Run first control (Gene clone) <br><br />
<br />
a. Follow step 3 to run the first control <br><br />
<br />
5. Run first library sample <br><br />
<br />
a. Follow steps 3b and 3c to set first two gates correctly. <br><br />
<br />
b. Set final gate for sort which includes top 1% of GFP producers from second gated population. <br><br />
<br />
c. Use final gate to set up the sort. <br><br />
<br />
d. Select sort conditions at the bottom of the screen. <br><br />
<br />
e. Insert and load collection tube. <br><br />
<br />
f. Record 100,000 events, and sort 10x the library size <br><br />
<br />
6. Run Bleach and diH2O through FACS to avoid cross-contamination. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Protein Expression"></a><br />
<br />
</html><br />
<br />
<h2> Protein Expression </h2><br />
<br />
<br />
<h3> Overnight Cultures </h3><br />
<br />
<p><br />
<br />
1. Add 25mL TB and 25uL Kan to a 250mL baffled flask <br><br />
<br />
2. Stab a glycerol stock with a p1000 pipette and swirl in the flask of media <br><br />
<br />
3. Put flask in 37C shaker overnight <br />
<br />
</p><br />
<br />
<h3> Protein Expression </h3><br />
<br />
<p> <br />
<br />
1. Add 500μL 1000x Kanamycin to 500mL TB in 2L baffled flask <br><br />
<br />
2. Transfer 10mL overnight culture to TB <br><br />
<br />
3. Shake at 37C (DO WE NEED THE RPM?) <br><br />
<br />
4. Remove flask from shaker when optical density is between 0.5 and 0.8 <br><br />
<br />
5. Allow flask to rest at room temp for 30 min <br><br />
<br />
6. Add 125μL 1M IPTG <br><br />
<br />
7. Shake flask at 18C overnight<br />
<br />
</p><br />
<br />
<h3> Protein Extraction and Purification </h3><br />
<br />
<p><br />
<br />
1. Transfer cell culture to centrifuge flask <br><br />
<br />
2. Centrifuge culture at 4000g for 10 min <br><br />
<br />
3. Discard supernatant <br><br />
<br />
4. Resuspend pellet in 25mL lysis buffer <br><br />
<br />
5. Add 250μL of 100x PMSF, 250μL of 100mg/mL lysozyme, and 250μL of 10mg/mL DNAse <br><br />
<br />
6. Sonicate sample with 0.25inch probe for 5 min at 70% amplitude with 20 sec on and off pulses <br><br />
<br />
7. Take 50μL total sample <br><br />
<br />
8. Transfer lysate to SS-34 centrifuge tube <br><br />
<br />
9. Centrifuge for 30 min at 18000g <br><br />
<br />
10. Take 50μL soluble sample<br />
<br />
</p><br />
<br />
<h3> Nickel Nitrotriacetic Acid Chromatography (Nickel-NTA Chromatography) </h3><br />
<br />
<p><br />
<br />
1. Add 5mL 50%(v/v) nickel resin in ethanol to 25mL column and allow to settle to ~2.5mL(CV) <br><br />
<br />
2. Rinse with 10CV H20 <br><br />
<br />
3. Equilibrate with 10CV lysis buffer <br><br />
<br />
4. Load sample onto column <br><br />
<br />
5. Wash column with 15CV lysis buffer <br><br />
<br />
6. Perform 2 additional wash steps with 15CV <br><br />
<br />
7. Elute sample in 10CV elution buffer and collect eluate <br><br />
<br />
8. Take 50μL pure sample <br />
<br />
</p><br />
<br />
<h3> Size Exclusion Chromatography (S.E.C.) </h3><br />
<p><br />
1. Concentrate sample to as high as possible without inducing protein aggregation<br><br />
2. Pre-equilibrate Superdex 75 column with 48 ml PBS <br><br />
3. Inject 500 ul sample onto column <br><br />
4. Run 36 ml PBS through column at 0.5 ml/min, collecting 1 ml fractions <br><br />
5. Verify presence of protein in fractions by measuring concentration on Nanodrop and running SDS-PAGE (15 kDa protein should elute at ~13 ml)<br><br />
6. Pool fractions containing protein <br />
</p><br />
<br />
<br />
<html><a name="Stability Analysis"></a><br />
<br />
</html><br />
<br />
<h2> Stability Analysis </h2><br />
<br />
<br />
<h3> Circular Dichroism: Wavelength Scan </h3><br />
<p><br />
1. Load 1mm cuvette with 400uL protein solution onto CD <br><br />
2. Take wavelength scan <br><br />
260nm-190nm <br><br />
sample every 1nm <br><br />
averaging time 3sec <br><br />
1 scan <br><br />
step scan <br><br />
25C <br><br />
3. Record wavelength which gives strongest signal (222nm)<br />
</p><br />
<br />
<h3> Circular Dichroism: Guanidine Melt </h3><br />
<p><br />
1. Load 1cm cuvette containing 1.996mL of 0.05mg/mL protein solution and stirrer onto CD <br><br />
2. Prepare 8mL of 0.05mg/mL protein in concentrated guanidine solution <br><br />
3. Set up Automixer with guanidine solution on one syringe and waste tube on other syringe <br><br />
4. Titrate up to 6 M guanidine, taking a CD measurement at 222 nm every 0.15 M interval <br><br />
5. Also measure the fluorescence at 280 nm to ensure the total protein concentration is not changing<br />
</p><br />
<br />
<br />
<br />
<html><br />
<br />
<a href="#linktotop">Back To Top</a><br />
</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2014-10-17T20:02:07Z<p>Kristanguyen: </p>
<hr />
<div>{{Template:Team:UW/CSS}}<br />
<br />
<br />
<h1> Protocols </h1><br />
<br />
<br />
<html><a name="Media, Plates, and Sol"></a><br />
<br />
</html><br />
<br />
<br />
<h2> Media, Plates, and Solutions </h2><br />
<br />
<br />
<h3> Competent Cell Media Buffer (CCMB) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
100 g glycerol (liquid) <br><br />
<br />
10 mL x 1 M potassium acetate <br><br />
<br />
11.8 g CaCl2*H2O <br><br />
<br />
4 g MnCl2 <br><br />
<br />
2 g MgCl2 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<h3> Super Optimal Broth (SOB) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
20 g Tryptone <br><br />
<br />
5 g Yeast Extract <br><br />
<br />
10 mL x 1 M NaCl <br><br />
<br />
2.5 mL x 1 M KCl <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Phosphate Buffered Saline (PBS) Solution </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container or 1 L beaker: <br><br />
<br />
8 g NaCl <br><br />
<br />
1.44 g Na2HPO4 <br><br />
<br />
0.8 g KCl <br><br />
<br />
0.24 g KH2PO4 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Buffer to pH 7.4 <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<h3> PSBF (PBS for Flow) </h3> <br />
<br />
<p><br />
<br />
Mix the following in a 1 L beaker: <br><br />
<br />
25 mL 20X PBS, pH 7.4 <br><br />
<br />
574 mL H2O <br><br />
<br />
2.5 g BSA (0.5%)* <br><br />
<br />
Filter and store at 4 °C <br><br />
<br><br />
*Do not need to pH - should be at pH 7.4 like 20X PBS <br><br />
<br />
</p> <br />
<br />
<br />
<h3> Yeast Extract Peptone Dextrose (YPD) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following into 950 mL of dH2O in a 1 L bottle: <br><br />
<br />
20 g peptone <br><br />
<br />
10 g yeast extract <br><br />
<br />
Autoclave (20 min at 121 °C and 20 psi) <br><br />
<br />
Add 50 mL 40% glucose <br><br />
<br />
Sterile filter into a 1 L bottle <br><br />
<br />
<br><br />
<br />
Note: For long-term liquid media storage, do not add 40% glucose. Instead add the glucose directly into cell cultures. <br><br />
<br />
Note: For YPD-plates add 24 g agar to the peptone and yeast extract before autoclaving. <br />
<br />
</p><br />
<br />
<br />
<h3> Selective Dropout Media, C-Uracil and C-Histidine (C-Ura and C-His) </h3><br />
<br />
<br />
<p><br />
<br />
Synthesized by the Yeast Resource Center at the University of Washington's <br />
<br />
Department of Genome Sciences and Department of Biochemistry.<br />
<br />
</p><br />
<br />
<br />
<h3> Guanidinium Hydrogen Chloride </h3><br />
<br />
<br />
<p><br />
<br />
For maximum effectiveness, final concentration should be approximately 8.5 M in PBS.<br><br />
<br />
203 g guanidinium hydrogen chloride <br><br />
<br />
250 mL PBS solution* <br><br />
<br />
Add dilute HCl to pH 7.4 <br><br />
<br />
<br><br />
<br />
*Alternatively add slightly less than 250 mL of PBS in order to buffer the solution to the appropriate volume, then add more dH2O as necessary. <br />
<br />
</p><br />
<br />
<html><a name="Basic Cloning"></a><br />
<br />
</html><br />
<br />
<h2> Basic Cloning </h2><br />
<br />
<br />
<br />
<h3> Polymerase Chain Reaction </h3><br />
<br />
<br />
<p><br />
<br />
All PCRs were done using a standard 50 μL reaction volume with GoTaq® Green Master Mix 2X purchased from PROMEGA Corporation. <br><br />
<br />
Mix the following in a 0.2 mL microcentrifuge tube on ice: <br><br />
<br />
25 μL GoTaq® Green Master Mix 2X <br><br />
<br />
1-5 μL of 10 μM forward primer <br><br />
<br />
1-5 μL of 10 μM reverse primer <br><br />
<br />
<250 ng of DNA template <br><br />
<br />
QS 50 μl nuclease-free H2O <br><br />
<br />
Conduct the reaction in a thermocycler, adjusting anneal temperature and extension times accordingly. See your polymerase supplier protocol for more details on thermocycling.<br />
<br />
</p><br />
<br />
<h3> Error-prone Polymerase Chain Reaction </h3><br />
<br />
<br />
<p> <br />
<br />
<br />
<br />
Prepare 50 μL reaction: <br><br />
<br />
5 μL 10X Mutazyme II Rxn Buffer <br><br />
<br />
1 μL 40 mM dNTP mix (200 μM each final) <br><br />
<br />
1 μL 20 μM forward primer <br><br />
<br />
1 μL 20 μM reverse primer <br><br />
<br />
1 μL Mutazyme II DNA polymerase (2.5 U/μL) <br><br />
<br />
0.01 ng template <br><br />
<br />
QS 50 μL diH2O <br><br />
<br />
<br><br />
<br />
Program thermocycler as follows: <br><br />
<br />
95 °C, 2 min <br><br />
<br />
95 °C, 30 sec <br><br />
<br />
XX °C*, 30 sec <br><br />
<br />
72 °C, X min** <br><br />
<br />
32 cycles <br><br />
<br />
72 °C, 10 min <br><br />
<br />
4 °C, hold<br />
<br />
<br><br />
<br />
*Adjust annealing temperature according to Tm of primer. <br><br />
<br />
**Adjust extension time according to the length of amplified DNA. <br><br />
<br />
<br><br />
<br />
Note: Use 0.01 ng of template (calculate by insert and not by total plasmid). <br><br />
<br />
Calculate amount of template to use as follows: <br><br />
<br />
(bp for amplified region) / (bp in total plasmid) = % amplified region <br><br />
<br />
(conc. of total plasmid) x (% amplified region as a decimal) = conc. of amplified region <br><br />
<br />
<br><br />
<br />
Note: Never pipette less than 0.5 μL. <br><br />
<br />
(0.01 ng of template) / (conc. of amplified region) = vol of template to add to PCR <br><br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Restriction Endonuclease Reaction (Digestion) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
All restriction enzyme reactions were done using a 50 μl reaction volume. Restriction enzymes and buffers were purchased from New England Biolabs® Inc. <br><br />
<br />
Mix the following in a 0.2 mL PCR tube: <br><br />
<br />
<br />
1 μg of DNA <br><br />
<br />
5 μL of the appropriate 10X New England Biolab® Buffer <br><br />
<br />
1 μL of each restriction enzyme (add last) <br><br />
<br />
QS 50μL nuclease-free H2O <br><br />
<br />
Incubate the reaction for 1 hr <br><br />
<br />
Heat inactive the reaction at the appropriate temperature <br><br />
<br />
<br />
Note: Thaw the restriction enzyme(s) on ice to improve shelf life <br><br />
<br />
<br />
</p> <br />
<br />
<br />
<h3> Ligation </h3><br />
<br />
<br />
<p><br />
<br />
T4 DNA Ligase and Buffer were purchased from New England Biolabs® Inc. <br><br />
<br />
1. Prepare the following in a 0.2 mL microcentrifuge tube: <br><br />
<br />
50.0 ng vector DNA* <br><br />
<br />
37.5 ng vector DNA* <br><br />
<br />
2 μL 10X T4 DNA Ligase Buffer <br><br />
<br />
1 μL T4 DNA Ligase <br><br />
<br />
QS 20 μL diH2O <br><br />
<br />
2. Incubate the reaction at room temperature for 10-30 minutes or at 16 °C overnight. <br><br />
<br />
3. Heat inactivate at 65 °C for 10 minutes. <br><br />
<br />
4. Chill on ice before starting a transformation reaction. <br><br />
<br />
*The exact amount of DNA is dependent on the number of base pairs. In order to conduct a proper reaction consult the New England Biolab Ligation Calculator at:<br />
<br />
http://nebiocalculator.neb.com/#!/<br />
<br />
</p><br />
<br />
<br />
<html><a name="Escherichia coli Protocols"></a><br />
<br />
</html><br />
<br />
<br />
<h2> <i> Escherichia coli </i> Protocols (XL1-Blue and XL10-Gold) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<p> <br />
<br />
1. Plate 10 ul of cells onto an antibiotic free LB/Agar plate..* <br><br />
<br />
2. Incubate overnight at 37OC <br><br />
<br />
3. Inoculate lots of colonies into 50ml SOB-Mg media in a 250ml flask. <br><br />
4. Incubate at 37 OC, 200rpm, until OD at 550nm reaches 0.3, takes about 3 hours. I do my first check at 2 hours.<br><br />
<br />
5.Transfer the cell culture into a sterile 50ml Falcon tube and chill on ice for 10 minutes. <br><br />
<br />
6. Pellet the cells at 2500rpm for 15 minutes at 4 OC.<br><br />
<br />
7. Decant supernatant and invert tubes to remove excess culture medium. <br><br />
<br />
8. Resuspend the cells in 16ml CCMB by gentle vortexing or pipetting (I shake the falcon tube).<br />
(16ml CCMB per 50ml culture) <br><br />
<br />
9. Incubate on ice for 20 minutes. <br><br />
<br />
10. Centrifuge at 2500rpm for 10 minutes at 4 OC. <br><br />
<br />
11. Decant supernatant and invert tubes to remove excess liquid. <br><br />
<br />
12. Resuspend the cells in 4ml CCMB by gentle vortexing or pipetting.<br />
(4ml CCMB per culture) <br><br />
<br />
13. If making multiple 50ml cultures, combine all of the cells and mix before aliquoting. <br> <br />
<br />
14. In cold room: make 205ul aliquots, flash freeze in liquid nitrogen, and store at -80 OC.<br><br />
<br />
<br />
<br />
<br />
NOTE: Make SOB-Mg media and autoclave the 250ml flasks with water in them the same day the cells are plated (step 1) <br><br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Chemically Competent Cell Transformations </h3><br />
<br />
<br />
<p> <br />
<br />
1. Thaw competent <i> E.coli </i> cells on ice (XL1-Blue or XL10-Gold)* <br><br />
<br />
2. Add 50μL of competent cells to sterile 15mL conical centrifuge tubes <br><br />
<br />
3. Add 1μL (~100-200ng)* of the mini-prep to each culture tube <br><br />
<br />
4. Equilibrate the cells on ice for 10 min <br><br />
<br />
5. Heat shock the cells at 42C for 30-45 seconds** <br><br />
<br />
6. Immediately place the cells back on ice for 3 min <br><br />
<br />
7. Add 250μL LB media without antibiotics and shake at 250 rpm and 37C for 30 min <br><br />
<br />
8. Spread 10μL and 290μL on an appropriate LB-antibiotic plate <br><br />
<br />
9. Invert the plate and incubate at 37C overnight<br />
<br />
<br />
*The exact amount of DNA to add depends on your cell's transformation efficiency. However, it is acceptable to add a larger amount to increase the number of transformed cells. <br><br />
<br />
** Do not heat shock for an extended duration as this may damage and/or kill your cells.<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnights </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottom tube, add 3-5mL of LB and an appropriate volume of antibiotic(s). <br><br />
<br />
2. Swipe several individual colonies, do not collect satellites or colony clumps, with a pipette tip. <br><br />
<br />
3. Swirl the colony tip in the tube, there should be no visible cell clumps. <br><br />
<br />
4. Incubate and shake the tube at 37C at 250rpm for 12-16 hours and no longer than 20 hours. <br><br />
<br />
</p><br />
<br />
<br />
<h3> DNA-Extraction and mini-preps </h3><br />
<br />
<br />
<p><br />
<br />
All DNA mini-preps were prepared using EPOCH mini-kits and following the supplied protocols.<br />
<br />
</p><br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500μL of 40% Glycerol and 500uL of LB (no antibiotics) or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Saccharomyces cerevisiae"></a><br />
<br />
</html><br />
<br />
<h2> <i> Saccharomyces cerevisiae </i> (PyE1 Yeast) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Culturing </h3><br />
<br />
<br />
<br />
<p><br />
<br />
This process take 4 days in lab with a 1 day wait for incubation. <br><br />
<br />
<br />
Day 1: <br><br />
<br />
1. Streak yeast cells onto a YPD plate.* <br><br />
<br />
2. Invert the plate and incubate at 30C for 2 days. <br><br />
<br />
<br />
Day 3: <br><br />
<br />
1. Add 50mL of YPD liquid media into a 250mL baffle flask. <br><br />
<br />
2. Swipe as many individual colonies as you can see into the YPD media.** <br><br />
<br />
3. Incubate and shake the culture at 30C at 250rpm overnight approximately 24 hours. <br><br />
<br />
<br />
Day 4: <br><br />
<br />
1. Take an optical density measurement. <br><br />
<br />
2. In three 250mL baffle flask add the portions of the overnight liquid culture. <br><br />
<br />
3. Dilute each culture to approximately 0.4 optical density with YPD. <br><br />
<br />
4. Incubate and shake the cultures at 30C at 250rpm until the optical density reaches 1.2-1.6. <br><br />
<br />
5. Collect each culture into separate 50ml flat-bottomed centrifuge tubes. <br><br />
<br />
6. Spin down the cells at 4000g for 5 minutes at 4C. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cells in 100mL total for all three culture of dH2O. <br><br />
<br />
9. Combine the suspensions into two 50mL flat-bottomed centrifuge tubes. <br><br />
<br />
10. Spin down the cells as above. <br><br />
<br />
11. Decant the supernatant. <br><br />
<br />
12. Resuspend each in 3mL of 100mM Lithium Acetate. <br><br />
<br />
13. Transfer both cultures into a single 15mL conical centrifuge tube. <br><br />
<br />
14. Spin down the cells at 3000rpm for 5 minutes. <br><br />
<br />
15. Resuspend the cells in 0.75mL of 100mM Lithium Acetate, total volume is roughly 2mL. <br><br />
<br />
16. Qualitatively bring up the volume to 3.5mL by adding 40% Glycerol. <br><br />
<br />
17. Aliquot the cells into 1.5mL centrifuge tubes or 1.7mL cryogenic vials.*** <br><br />
<br />
<br />
*Streak in such a way that there are individual colonies visible on the plate without clumps or satellite colonies. <br><br />
<br />
**Collect only individual visible colonies. Do not collect clumps or satellite colonies. <br><br />
<br />
***The volume of aliquots depends on the number to transformations you intend to do at a time. <br><br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Chemically Competent Transformations </h3><br />
<br />
<br />
<p><br />
<br />
This protocol assumes a 50μL aliquot of yeast competent cells were made. <br><br />
<br />
Furthermore, this protocol prepares enough cells for 6 yeast transformations. <br><br />
<br />
<br><br />
<br />
1. Add the following to 50μL of yeast competent cells: <br><br />
<br />
240μL of Polyethylene Glycol - 3350 (PEG-3350) <br><br />
<br />
36μL of 1M Lithium Acetate <br><br />
<br />
32μL of dH2O <br><br />
<br />
<br />
<br />
2. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
3. Aliquot 59μL of the mixture into a 0.2mL microcentrifuge tube. <br><br />
<br />
4. Add 1uL (~100-200ng) of DNA. <br><br />
<br />
5. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
6. Incubate the mixture at 30C for 30 minutes. <br><br />
<br />
7. Heat shock the mixture at 42C for 20 minutes. <br><br />
<br />
8. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
9. Decant the supernatant. <br><br />
<br />
10. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
11. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
12. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
13. Plate 50-150μL of the mixture onto an appropriate Selective Dropout Media plate. <br><br />
<br />
14. Invert and incubate at 30C for 2 days. <br><br />
<br />
*The exact amount of DNA depends on the transformation efficiency of your competent cells. <br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnight Culturing </h3><br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Swipe 3 individually visible yeast colonies and add them to the culture tube media. <br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%.<br />
<br />
</p><br />
<br />
<br />
<h3> Culture Passaging </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Take 20-50μL from a previous overnight or passage culture and add it to the culture media.<br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%. <br><br />
<br />
Note: The exact amount of culture that you take from a previous culture is irrelevant as long as at least 1 living cell is passaged.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500uL of 40% glycerol and 500μL of Selective Dropout media or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Flow Cytometry"></a><br />
<br />
</html><br />
<br />
<h2> Flow Cytometry </h2><br />
<br />
<br />
<h3> Dilutions </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. From an overnight culture measure the optical density at 660nm by making 1:10 dilutions. <br><br />
<br />
2. Take enough culture to make a 1mL aliquot with OD 0.4. <br><br />
<br />
3. Spin down the aliquot in a 1.5mL centrifuge tube at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 800μL of the appropriate selective dropout media and 200μL of 20% glucose. <br><br />
<br />
6. Transfer the new culture to a 14mL culture tube. <br><br />
<br />
7. Incubate and shake at 30C and 250rpm for at least 6 hours (1.2-1.6 optical density).<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Preparations for Analysis using C6 Accuri Flow Cytometer </h3><br />
<br />
<br />
<p><br />
<br />
<br />
1. From the dilution previously made, measure the optical density, roughly 1.2-1.6. <br><br />
<br />
2. Make an aliquot of 500μL of the dilution culture in a 1.5mL centrifuge tube. <br><br />
<br />
3. Spin down the aliquot at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 500μL of PBS(F). <br><br />
<br />
6. Spin down the resuspension at 3000rpm for 3 minutes. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cell pellet in another 500uL of PBS(F).* <br><br />
<br />
9. Prepare the C6 Accuri Flow Cytometer by running a backflush cycle and a dH2O cycle. <br><br />
<br />
10. Load the sample onto the sip. <br><br />
<br />
11. Run the sample with 100,000 cell count. <br><br />
<br />
12. Repeat for all samples and make sure to change data cells otherwise the old data is erased. <br><br />
<br />
13. Once finished, run a cleaning cycle with Accuri approved cleaning solution, then run a dH2O cycle. <br><br />
<br />
<br />
<br />
*For special cases do not resuspend all samples, instead resuspend immediately before running the sample through the flow cytometer.<br />
<br />
<br />
</p><br />
<br />
<br />
<html><a name="Fluorescence Activated Cell Sorting"></a><br />
<br />
</html><br />
<br />
<h2> Fluorescence Activated Cell Sorting </h2><br />
<br />
<br />
<h3> Final Preparations </h3><br />
<br />
<br />
<br />
<p> <br />
<br />
Sample Prep:<br />
<br />
<br />
Spin down samples and negative control (5000 RPM, 1 min), keeping in mind the library size. Aspirate off supernatant. Resuspend in PBSF. Spin down cells. Aspirate off supernatant. Resuspend in PBSF. <br><br />
<br />
<br><br />
<br />
1. Open the “iGEM Template” file in the FACS Software and change name to current date and sort cycle<br><br />
<br />
2. Make sure the stream is stable (look for green light in bottom right corner). If not, run the Sort Calibration order. <br><br />
<br />
3. Run Negative Control from the PyE1 cells. <br><br />
<br />
a. Load cells onto carrier and into the machine. Press play button on screen. <br><br />
<br />
b. Set gate around lower left quadrant of cells to ensure single cell analysis using Forward Scatter Area and Side Scatter Area as your axes. Make sure oval gate covers around 80% of cell population. <br><br />
<br />
c. Set second gate on the first gated population by double-clicking on the gated population and using Forward Scatter Height and Forward Scatter Width as your axes. You will notice two distinct populations. Try to focus on the single cell portion of the plot. <br><br />
<br />
NOTE: If you see a large portion of the second gated population existing near the upper right edge of the first gate, you may need to enlarge the first gate to fit more of the population. <br><br />
<br />
d. Press record. Record 100,000 events and stop run. Move to Next Tube. <br><br />
<br />
4. Run first control (Gene clone) <br><br />
<br />
a. Follow step 3 to run the first control <br><br />
<br />
5. Run first library sample <br><br />
<br />
a. Follow steps 3b and 3c to set first two gates correctly. <br><br />
<br />
b. Set final gate for sort which includes top 1% of GFP producers from second gated population. <br><br />
<br />
c. Use final gate to set up the sort. <br><br />
<br />
d. Select sort conditions at the bottom of the screen. <br><br />
<br />
e. Insert and load collection tube. <br><br />
<br />
f. Record 100,000 events, and sort 10x the library size <br><br />
<br />
6. Run Bleach and diH2O through FACS to avoid cross-contamination. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Protein Expression"></a><br />
<br />
</html><br />
<br />
<h2> Protein Expression </h2><br />
<br />
<br />
<h3> Overnight Cultures </h3><br />
<br />
<p><br />
<br />
1. Add 25mL TB and 25uL Kan to a 250mL baffled flask <br><br />
<br />
2. Stab a glycerol stock with a p1000 pipette and swirl in the flask of media <br><br />
<br />
3. Put flask in 37C shaker overnight <br />
<br />
</p><br />
<br />
<h3> Protein Expression </h3><br />
<br />
<p> <br />
<br />
1. Add 500μL 1000x Kanamycin to 500mL TB in 2L baffled flask <br><br />
<br />
2. Transfer 10mL overnight culture to TB <br><br />
<br />
3. Shake at 37C (DO WE NEED THE RPM?) <br><br />
<br />
4. Remove flask from shaker when optical density is between 0.5 and 0.8 <br><br />
<br />
5. Allow flask to rest at room temp for 30 min <br><br />
<br />
6. Add 125μL 1M IPTG <br><br />
<br />
7. Shake flask at 18C overnight<br />
<br />
</p><br />
<br />
<h3> Protein Extraction and Purification </h3><br />
<br />
<p><br />
<br />
1. Transfer cell culture to centrifuge flask <br><br />
<br />
2. Centrifuge culture at 4000g for 10 min <br><br />
<br />
3. Discard supernatant <br><br />
<br />
4. Resuspend pellet in 25mL lysis buffer <br><br />
<br />
5. Add 250μL of 100x PMSF, 250μL of 100mg/mL lysozyme, and 250μL of 10mg/mL DNAse <br><br />
<br />
6. Sonicate sample with 0.25inch probe for 5 min at 70% amplitude with 20 sec on and off pulses <br><br />
<br />
7. Take 50μL total sample <br><br />
<br />
8. Transfer lysate to SS-34 centrifuge tube <br><br />
<br />
9. Centrifuge for 30 min at 18000g <br><br />
<br />
10. Take 50μL soluble sample<br />
<br />
</p><br />
<br />
<h3> Nickel Nitrotriacetic Acid Chromatography (Nickel-NTA Chromatography) </h3><br />
<br />
<p><br />
<br />
1. Add 5mL 50%(v/v) nickel resin in ethanol to 25mL column and allow to settle to ~2.5mL(CV) <br><br />
<br />
2. Rinse with 10CV H20 <br><br />
<br />
3. Equilibrate with 10CV lysis buffer <br><br />
<br />
4. Load sample onto column <br><br />
<br />
5. Wash column with 15CV lysis buffer <br><br />
<br />
6. Perform 2 additional wash steps with 15CV <br><br />
<br />
7. Elute sample in 10CV elution buffer and collect eluate <br><br />
<br />
8. Take 50μL pure sample <br />
<br />
</p><br />
<br />
<h3> Size Exclusion Chromatography (S.E.C.) </h3><br />
<p><br />
1. Concentrate sample to as high as possible without inducing protein aggregation<br><br />
2. Pre-equilibrate Superdex 75 column with 48 ml PBS <br><br />
3. Inject 500 ul sample onto column <br><br />
4. Run 36 ml PBS through column at 0.5 ml/min, collecting 1 ml fractions <br><br />
5. Verify presence of protein in fractions by measuring concentration on Nanodrop and running SDS-PAGE (15 kDa protein should elute at ~13 ml)<br><br />
6. Pool fractions containing protein <br />
</p><br />
<br />
<br />
<html><a name="Stability Analysis"></a><br />
<br />
</html><br />
<br />
<h2> Stability Analysis </h2><br />
<br />
<br />
<h3> Circular Dichroism: Wavelength Scan </h3><br />
<p><br />
1. Load 1mm cuvette with 400uL protein solution onto CD <br><br />
2. Take wavelength scan <br><br />
260nm-190nm <br><br />
sample every 1nm <br><br />
averaging time 3sec <br><br />
1 scan <br><br />
step scan <br><br />
25C <br><br />
3. Record wavelength which gives strongest signal (222nm)<br />
</p><br />
<br />
<h3> Circular Dichroism: Guanidine Melt </h3><br />
<p><br />
1. Load 1cm cuvette containing 1.996mL of 0.05mg/mL protein solution and stirrer onto CD <br><br />
2. Prepare 8mL of 0.05mg/mL protein in concentrated guanidine solution <br><br />
3. Set up Automixer with guanidine solution on one syringe and waste tube on other syringe <br><br />
4. Titrate up to 6 M guanidine, taking a CD measurement at 222 nm every 0.15 M interval <br><br />
5. Also measure the fluorescence at 280 nm to ensure the total protein concentration is not changing<br />
</p><br />
<br />
<br />
<br />
<html><br />
<br />
<a href="#linktotop">Back To Top</a><br />
</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2014-10-17T20:01:43Z<p>Kristanguyen: </p>
<hr />
<div>{{Template:Team:UW/CSS}}<br />
<br />
<br />
<h1> Protocols </h1><br />
<br />
<br />
<html><a name="Media, Plates, and Sol"></a><br />
<br />
</html><br />
<br />
<br />
<h2> Media, Plates, and Solutions </h2><br />
<br />
<br />
<h3> Competent Cell Media Buffer (CCMB) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
100 g glycerol (liquid) <br><br />
<br />
10 mL x 1 M potassium acetate <br><br />
<br />
11.8 g CaCl2*H2O <br><br />
<br />
4 g MnCl2 <br><br />
<br />
2 g MgCl2 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<h3> Super Optimal Broth (SOB) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
20 g Tryptone <br><br />
<br />
5 g Yeast Extract <br><br />
<br />
10 mL x 1 M NaCl <br><br />
<br />
2.5 mL x 1 M KCl <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Phosphate Buffered Saline (PBS) Solution </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container or 1 L beaker: <br><br />
<br />
8 g NaCl <br><br />
<br />
1.44 g Na2HPO4 <br><br />
<br />
0.8 g KCl <br><br />
<br />
0.24 g KH2PO4 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Buffer to pH 7.4 <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<h3> PSBF (PBS for Flow) </h3> <br />
<br />
<p><br />
<br />
Mix the following in a 1 L beaker: <br><br />
<br />
25 mL 20X PBS, pH 7.4 <br><br />
<br />
574 mL H2O <br><br />
<br />
2.5 g BSA (0.5%)* <br><br />
<br />
Filter and store at 4 °C <br><br />
<br><br />
*Do not need to pH - should be at pH 7.4 like 20X PBS <br><br />
<br />
</p> <br />
<br />
<br />
<h3> Yeast Extract Peptone Dextrose (YPD) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following into 950 mL of dH2O in a 1 L bottle: <br><br />
<br />
20 g peptone <br><br />
<br />
10 g yeast extract <br><br />
<br />
Autoclave (20 min at 121 °C and 20 psi) <br><br />
<br />
Add 50 mL 40% glucose <br><br />
<br />
Sterile filter into a 1 L bottle <br><br />
<br />
<br><br />
<br />
Note: For long-term liquid media storage, do not add 40% glucose. Instead add the glucose directly into cell cultures. <br><br />
<br />
Note: For YPD-plates add 24 g agar to the peptone and yeast extract before autoclaving. <br />
<br />
</p><br />
<br />
<br />
<h3> Selective Dropout Media, C-Uracil and C-Histidine (C-Ura and C-His) </h3><br />
<br />
<br />
<p><br />
<br />
Synthesized by the Yeast Resource Center at the University of Washington's <br />
<br />
Department of Genome Sciences and Department of Biochemistry.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<h3> Guanidinium Hydrogen Chloride </h3><br />
<br />
<br />
<p><br />
<br />
For maximum effectiveness, final concentration should be approximately 8.5 M in PBS.<br><br />
<br />
203 g guanidinium hydrogen chloride <br><br />
<br />
250 mL PBS solution* <br><br />
<br />
Add dilute HCl to pH 7.4 <br><br />
<br />
<br><br />
<br />
*Alternatively add slightly less than 250 mL of PBS in order to buffer the solution to the appropriate volume, then add more dH2O as necessary. <br />
<br />
</p><br />
<br />
<html><a name="Basic Cloning"></a><br />
<br />
</html><br />
<br />
<h2> Basic Cloning </h2><br />
<br />
<br />
<br />
<h3> Polymerase Chain Reaction </h3><br />
<br />
<br />
<p><br />
<br />
All PCRs were done using a standard 50 μL reaction volume with GoTaq® Green Master Mix 2X purchased from PROMEGA Corporation. <br><br />
<br />
Mix the following in a 0.2 mL microcentrifuge tube on ice: <br><br />
<br />
25 μL GoTaq® Green Master Mix 2X <br><br />
<br />
1-5 μL of 10 μM forward primer <br><br />
<br />
1-5 μL of 10 μM reverse primer <br><br />
<br />
<250 ng of DNA template <br><br />
<br />
QS 50 μl nuclease-free H2O <br><br />
<br />
Conduct the reaction in a thermocycler, adjusting anneal temperature and extension times accordingly. See your polymerase supplier protocol for more details on thermocycling.<br />
<br />
</p><br />
<br />
<h3> Error-prone Polymerase Chain Reaction </h3><br />
<br />
<br />
<p> <br />
<br />
<br />
<br />
Prepare 50 μL reaction: <br><br />
<br />
5 μL 10X Mutazyme II Rxn Buffer <br><br />
<br />
1 μL 40 mM dNTP mix (200 μM each final) <br><br />
<br />
1 μL 20 μM forward primer <br><br />
<br />
1 μL 20 μM reverse primer <br><br />
<br />
1 μL Mutazyme II DNA polymerase (2.5 U/μL) <br><br />
<br />
0.01 ng template <br><br />
<br />
QS 50 μL diH2O <br><br />
<br />
<br><br />
<br />
Program thermocycler as follows: <br><br />
<br />
95 °C, 2 min <br><br />
<br />
95 °C, 30 sec <br><br />
<br />
XX °C*, 30 sec <br><br />
<br />
72 °C, X min** <br><br />
<br />
32 cycles <br><br />
<br />
72 °C, 10 min <br><br />
<br />
4 °C, hold<br />
<br />
<br><br />
<br />
*Adjust annealing temperature according to Tm of primer. <br><br />
<br />
**Adjust extension time according to the length of amplified DNA. <br><br />
<br />
<br><br />
<br />
Note: Use 0.01 ng of template (calculate by insert and not by total plasmid). <br><br />
<br />
Calculate amount of template to use as follows: <br><br />
<br />
(bp for amplified region) / (bp in total plasmid) = % amplified region <br><br />
<br />
(conc. of total plasmid) x (% amplified region as a decimal) = conc. of amplified region <br><br />
<br />
<br><br />
<br />
Note: Never pipette less than 0.5 μL. <br><br />
<br />
(0.01 ng of template) / (conc. of amplified region) = vol of template to add to PCR <br><br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Restriction Endonuclease Reaction (Digestion) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
All restriction enzyme reactions were done using a 50 μl reaction volume. Restriction enzymes and buffers were purchased from New England Biolabs® Inc. <br><br />
<br />
Mix the following in a 0.2 mL PCR tube: <br><br />
<br />
<br />
1 μg of DNA <br><br />
<br />
5 μL of the appropriate 10X New England Biolab® Buffer <br><br />
<br />
1 μL of each restriction enzyme (add last) <br><br />
<br />
QS 50μL nuclease-free H2O <br><br />
<br />
Incubate the reaction for 1 hr <br><br />
<br />
Heat inactive the reaction at the appropriate temperature <br><br />
<br />
<br />
Note: Thaw the restriction enzyme(s) on ice to improve shelf life <br><br />
<br />
<br />
</p> <br />
<br />
<br />
<h3> Ligation </h3><br />
<br />
<br />
<p><br />
<br />
T4 DNA Ligase and Buffer were purchased from New England Biolabs® Inc. <br><br />
<br />
1. Prepare the following in a 0.2 mL microcentrifuge tube: <br><br />
<br />
50.0 ng vector DNA* <br><br />
<br />
37.5 ng vector DNA* <br><br />
<br />
2 μL 10X T4 DNA Ligase Buffer <br><br />
<br />
1 μL T4 DNA Ligase <br><br />
<br />
QS 20 μL diH2O <br><br />
<br />
2. Incubate the reaction at room temperature for 10-30 minutes or at 16 °C overnight. <br><br />
<br />
3. Heat inactivate at 65 °C for 10 minutes. <br><br />
<br />
4. Chill on ice before starting a transformation reaction. <br><br />
<br />
*The exact amount of DNA is dependent on the number of base pairs. In order to conduct a proper reaction consult the New England Biolab Ligation Calculator at:<br />
<br />
http://nebiocalculator.neb.com/#!/<br />
<br />
</p><br />
<br />
<br />
<html><a name="Escherichia coli Protocols"></a><br />
<br />
</html><br />
<br />
<br />
<h2> <i> Escherichia coli </i> Protocols (XL1-Blue and XL10-Gold) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<p> <br />
<br />
1. Plate 10 ul of cells onto an antibiotic free LB/Agar plate..* <br><br />
<br />
2. Incubate overnight at 37OC <br><br />
<br />
3. Inoculate lots of colonies into 50ml SOB-Mg media in a 250ml flask. <br><br />
4. Incubate at 37 OC, 200rpm, until OD at 550nm reaches 0.3, takes about 3 hours. I do my first check at 2 hours.<br><br />
<br />
5.Transfer the cell culture into a sterile 50ml Falcon tube and chill on ice for 10 minutes. <br><br />
<br />
6. Pellet the cells at 2500rpm for 15 minutes at 4 OC.<br><br />
<br />
7. Decant supernatant and invert tubes to remove excess culture medium. <br><br />
<br />
8. Resuspend the cells in 16ml CCMB by gentle vortexing or pipetting (I shake the falcon tube).<br />
(16ml CCMB per 50ml culture) <br><br />
<br />
9. Incubate on ice for 20 minutes. <br><br />
<br />
10. Centrifuge at 2500rpm for 10 minutes at 4 OC. <br><br />
<br />
11. Decant supernatant and invert tubes to remove excess liquid. <br><br />
<br />
12. Resuspend the cells in 4ml CCMB by gentle vortexing or pipetting.<br />
(4ml CCMB per culture) <br><br />
<br />
13. If making multiple 50ml cultures, combine all of the cells and mix before aliquoting. <br> <br />
<br />
14. In cold room: make 205ul aliquots, flash freeze in liquid nitrogen, and store at -80 OC.<br><br />
<br />
<br />
<br />
<br />
NOTE: Make SOB-Mg media and autoclave the 250ml flasks with water in them the same day the cells are plated (step 1) <br><br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Chemically Competent Cell Transformations </h3><br />
<br />
<br />
<p> <br />
<br />
1. Thaw competent <i> E.coli </i> cells on ice (XL1-Blue or XL10-Gold)* <br><br />
<br />
2. Add 50μL of competent cells to sterile 15mL conical centrifuge tubes <br><br />
<br />
3. Add 1μL (~100-200ng)* of the mini-prep to each culture tube <br><br />
<br />
4. Equilibrate the cells on ice for 10 min <br><br />
<br />
5. Heat shock the cells at 42C for 30-45 seconds** <br><br />
<br />
6. Immediately place the cells back on ice for 3 min <br><br />
<br />
7. Add 250μL LB media without antibiotics and shake at 250 rpm and 37C for 30 min <br><br />
<br />
8. Spread 10μL and 290μL on an appropriate LB-antibiotic plate <br><br />
<br />
9. Invert the plate and incubate at 37C overnight<br />
<br />
<br />
*The exact amount of DNA to add depends on your cell's transformation efficiency. However, it is acceptable to add a larger amount to increase the number of transformed cells. <br><br />
<br />
** Do not heat shock for an extended duration as this may damage and/or kill your cells.<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnights </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottom tube, add 3-5mL of LB and an appropriate volume of antibiotic(s). <br><br />
<br />
2. Swipe several individual colonies, do not collect satellites or colony clumps, with a pipette tip. <br><br />
<br />
3. Swirl the colony tip in the tube, there should be no visible cell clumps. <br><br />
<br />
4. Incubate and shake the tube at 37C at 250rpm for 12-16 hours and no longer than 20 hours. <br><br />
<br />
</p><br />
<br />
<br />
<h3> DNA-Extraction and mini-preps </h3><br />
<br />
<br />
<p><br />
<br />
All DNA mini-preps were prepared using EPOCH mini-kits and following the supplied protocols.<br />
<br />
</p><br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500μL of 40% Glycerol and 500uL of LB (no antibiotics) or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Saccharomyces cerevisiae"></a><br />
<br />
</html><br />
<br />
<h2> <i> Saccharomyces cerevisiae </i> (PyE1 Yeast) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Culturing </h3><br />
<br />
<br />
<br />
<p><br />
<br />
This process take 4 days in lab with a 1 day wait for incubation. <br><br />
<br />
<br />
Day 1: <br><br />
<br />
1. Streak yeast cells onto a YPD plate.* <br><br />
<br />
2. Invert the plate and incubate at 30C for 2 days. <br><br />
<br />
<br />
Day 3: <br><br />
<br />
1. Add 50mL of YPD liquid media into a 250mL baffle flask. <br><br />
<br />
2. Swipe as many individual colonies as you can see into the YPD media.** <br><br />
<br />
3. Incubate and shake the culture at 30C at 250rpm overnight approximately 24 hours. <br><br />
<br />
<br />
Day 4: <br><br />
<br />
1. Take an optical density measurement. <br><br />
<br />
2. In three 250mL baffle flask add the portions of the overnight liquid culture. <br><br />
<br />
3. Dilute each culture to approximately 0.4 optical density with YPD. <br><br />
<br />
4. Incubate and shake the cultures at 30C at 250rpm until the optical density reaches 1.2-1.6. <br><br />
<br />
5. Collect each culture into separate 50ml flat-bottomed centrifuge tubes. <br><br />
<br />
6. Spin down the cells at 4000g for 5 minutes at 4C. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cells in 100mL total for all three culture of dH2O. <br><br />
<br />
9. Combine the suspensions into two 50mL flat-bottomed centrifuge tubes. <br><br />
<br />
10. Spin down the cells as above. <br><br />
<br />
11. Decant the supernatant. <br><br />
<br />
12. Resuspend each in 3mL of 100mM Lithium Acetate. <br><br />
<br />
13. Transfer both cultures into a single 15mL conical centrifuge tube. <br><br />
<br />
14. Spin down the cells at 3000rpm for 5 minutes. <br><br />
<br />
15. Resuspend the cells in 0.75mL of 100mM Lithium Acetate, total volume is roughly 2mL. <br><br />
<br />
16. Qualitatively bring up the volume to 3.5mL by adding 40% Glycerol. <br><br />
<br />
17. Aliquot the cells into 1.5mL centrifuge tubes or 1.7mL cryogenic vials.*** <br><br />
<br />
<br />
*Streak in such a way that there are individual colonies visible on the plate without clumps or satellite colonies. <br><br />
<br />
**Collect only individual visible colonies. Do not collect clumps or satellite colonies. <br><br />
<br />
***The volume of aliquots depends on the number to transformations you intend to do at a time. <br><br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Chemically Competent Transformations </h3><br />
<br />
<br />
<p><br />
<br />
This protocol assumes a 50μL aliquot of yeast competent cells were made. <br><br />
<br />
Furthermore, this protocol prepares enough cells for 6 yeast transformations. <br><br />
<br />
<br><br />
<br />
1. Add the following to 50μL of yeast competent cells: <br><br />
<br />
240μL of Polyethylene Glycol - 3350 (PEG-3350) <br><br />
<br />
36μL of 1M Lithium Acetate <br><br />
<br />
32μL of dH2O <br><br />
<br />
<br />
<br />
2. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
3. Aliquot 59μL of the mixture into a 0.2mL microcentrifuge tube. <br><br />
<br />
4. Add 1uL (~100-200ng) of DNA. <br><br />
<br />
5. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
6. Incubate the mixture at 30C for 30 minutes. <br><br />
<br />
7. Heat shock the mixture at 42C for 20 minutes. <br><br />
<br />
8. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
9. Decant the supernatant. <br><br />
<br />
10. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
11. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
12. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
13. Plate 50-150μL of the mixture onto an appropriate Selective Dropout Media plate. <br><br />
<br />
14. Invert and incubate at 30C for 2 days. <br><br />
<br />
*The exact amount of DNA depends on the transformation efficiency of your competent cells. <br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnight Culturing </h3><br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Swipe 3 individually visible yeast colonies and add them to the culture tube media. <br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%.<br />
<br />
</p><br />
<br />
<br />
<h3> Culture Passaging </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Take 20-50μL from a previous overnight or passage culture and add it to the culture media.<br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%. <br><br />
<br />
Note: The exact amount of culture that you take from a previous culture is irrelevant as long as at least 1 living cell is passaged.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500uL of 40% glycerol and 500μL of Selective Dropout media or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Flow Cytometry"></a><br />
<br />
</html><br />
<br />
<h2> Flow Cytometry </h2><br />
<br />
<br />
<h3> Dilutions </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. From an overnight culture measure the optical density at 660nm by making 1:10 dilutions. <br><br />
<br />
2. Take enough culture to make a 1mL aliquot with OD 0.4. <br><br />
<br />
3. Spin down the aliquot in a 1.5mL centrifuge tube at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 800μL of the appropriate selective dropout media and 200μL of 20% glucose. <br><br />
<br />
6. Transfer the new culture to a 14mL culture tube. <br><br />
<br />
7. Incubate and shake at 30C and 250rpm for at least 6 hours (1.2-1.6 optical density).<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Preparations for Analysis using C6 Accuri Flow Cytometer </h3><br />
<br />
<br />
<p><br />
<br />
<br />
1. From the dilution previously made, measure the optical density, roughly 1.2-1.6. <br><br />
<br />
2. Make an aliquot of 500μL of the dilution culture in a 1.5mL centrifuge tube. <br><br />
<br />
3. Spin down the aliquot at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 500μL of PBS(F). <br><br />
<br />
6. Spin down the resuspension at 3000rpm for 3 minutes. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cell pellet in another 500uL of PBS(F).* <br><br />
<br />
9. Prepare the C6 Accuri Flow Cytometer by running a backflush cycle and a dH2O cycle. <br><br />
<br />
10. Load the sample onto the sip. <br><br />
<br />
11. Run the sample with 100,000 cell count. <br><br />
<br />
12. Repeat for all samples and make sure to change data cells otherwise the old data is erased. <br><br />
<br />
13. Once finished, run a cleaning cycle with Accuri approved cleaning solution, then run a dH2O cycle. <br><br />
<br />
<br />
<br />
*For special cases do not resuspend all samples, instead resuspend immediately before running the sample through the flow cytometer.<br />
<br />
<br />
</p><br />
<br />
<br />
<html><a name="Fluorescence Activated Cell Sorting"></a><br />
<br />
</html><br />
<br />
<h2> Fluorescence Activated Cell Sorting </h2><br />
<br />
<br />
<h3> Final Preparations </h3><br />
<br />
<br />
<br />
<p> <br />
<br />
Sample Prep:<br />
<br />
<br />
Spin down samples and negative control (5000 RPM, 1 min), keeping in mind the library size. Aspirate off supernatant. Resuspend in PBSF. Spin down cells. Aspirate off supernatant. Resuspend in PBSF. <br><br />
<br />
<br><br />
<br />
1. Open the “iGEM Template” file in the FACS Software and change name to current date and sort cycle<br><br />
<br />
2. Make sure the stream is stable (look for green light in bottom right corner). If not, run the Sort Calibration order. <br><br />
<br />
3. Run Negative Control from the PyE1 cells. <br><br />
<br />
a. Load cells onto carrier and into the machine. Press play button on screen. <br><br />
<br />
b. Set gate around lower left quadrant of cells to ensure single cell analysis using Forward Scatter Area and Side Scatter Area as your axes. Make sure oval gate covers around 80% of cell population. <br><br />
<br />
c. Set second gate on the first gated population by double-clicking on the gated population and using Forward Scatter Height and Forward Scatter Width as your axes. You will notice two distinct populations. Try to focus on the single cell portion of the plot. <br><br />
<br />
NOTE: If you see a large portion of the second gated population existing near the upper right edge of the first gate, you may need to enlarge the first gate to fit more of the population. <br><br />
<br />
d. Press record. Record 100,000 events and stop run. Move to Next Tube. <br><br />
<br />
4. Run first control (Gene clone) <br><br />
<br />
a. Follow step 3 to run the first control <br><br />
<br />
5. Run first library sample <br><br />
<br />
a. Follow steps 3b and 3c to set first two gates correctly. <br><br />
<br />
b. Set final gate for sort which includes top 1% of GFP producers from second gated population. <br><br />
<br />
c. Use final gate to set up the sort. <br><br />
<br />
d. Select sort conditions at the bottom of the screen. <br><br />
<br />
e. Insert and load collection tube. <br><br />
<br />
f. Record 100,000 events, and sort 10x the library size <br><br />
<br />
6. Run Bleach and diH2O through FACS to avoid cross-contamination. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Protein Expression"></a><br />
<br />
</html><br />
<br />
<h2> Protein Expression </h2><br />
<br />
<br />
<h3> Overnight Cultures </h3><br />
<br />
<p><br />
<br />
1. Add 25mL TB and 25uL Kan to a 250mL baffled flask <br><br />
<br />
2. Stab a glycerol stock with a p1000 pipette and swirl in the flask of media <br><br />
<br />
3. Put flask in 37C shaker overnight <br />
<br />
</p><br />
<br />
<h3> Protein Expression </h3><br />
<br />
<p> <br />
<br />
1. Add 500μL 1000x Kanamycin to 500mL TB in 2L baffled flask <br><br />
<br />
2. Transfer 10mL overnight culture to TB <br><br />
<br />
3. Shake at 37C (DO WE NEED THE RPM?) <br><br />
<br />
4. Remove flask from shaker when optical density is between 0.5 and 0.8 <br><br />
<br />
5. Allow flask to rest at room temp for 30 min <br><br />
<br />
6. Add 125μL 1M IPTG <br><br />
<br />
7. Shake flask at 18C overnight<br />
<br />
</p><br />
<br />
<h3> Protein Extraction and Purification </h3><br />
<br />
<p><br />
<br />
1. Transfer cell culture to centrifuge flask <br><br />
<br />
2. Centrifuge culture at 4000g for 10 min <br><br />
<br />
3. Discard supernatant <br><br />
<br />
4. Resuspend pellet in 25mL lysis buffer <br><br />
<br />
5. Add 250μL of 100x PMSF, 250μL of 100mg/mL lysozyme, and 250μL of 10mg/mL DNAse <br><br />
<br />
6. Sonicate sample with 0.25inch probe for 5 min at 70% amplitude with 20 sec on and off pulses <br><br />
<br />
7. Take 50μL total sample <br><br />
<br />
8. Transfer lysate to SS-34 centrifuge tube <br><br />
<br />
9. Centrifuge for 30 min at 18000g <br><br />
<br />
10. Take 50μL soluble sample<br />
<br />
</p><br />
<br />
<h3> Nickel Nitrotriacetic Acid Chromatography (Nickel-NTA Chromatography) </h3><br />
<br />
<p><br />
<br />
1. Add 5mL 50%(v/v) nickel resin in ethanol to 25mL column and allow to settle to ~2.5mL(CV) <br><br />
<br />
2. Rinse with 10CV H20 <br><br />
<br />
3. Equilibrate with 10CV lysis buffer <br><br />
<br />
4. Load sample onto column <br><br />
<br />
5. Wash column with 15CV lysis buffer <br><br />
<br />
6. Perform 2 additional wash steps with 15CV <br><br />
<br />
7. Elute sample in 10CV elution buffer and collect eluate <br><br />
<br />
8. Take 50μL pure sample <br />
<br />
</p><br />
<br />
<h3> Size Exclusion Chromatography (S.E.C.) </h3><br />
<p><br />
1. Concentrate sample to as high as possible without inducing protein aggregation<br><br />
2. Pre-equilibrate Superdex 75 column with 48 ml PBS <br><br />
3. Inject 500 ul sample onto column <br><br />
4. Run 36 ml PBS through column at 0.5 ml/min, collecting 1 ml fractions <br><br />
5. Verify presence of protein in fractions by measuring concentration on Nanodrop and running SDS-PAGE (15 kDa protein should elute at ~13 ml)<br><br />
6. Pool fractions containing protein <br />
</p><br />
<br />
<br />
<html><a name="Stability Analysis"></a><br />
<br />
</html><br />
<br />
<h2> Stability Analysis </h2><br />
<br />
<br />
<h3> Circular Dichroism: Wavelength Scan </h3><br />
<p><br />
1. Load 1mm cuvette with 400uL protein solution onto CD <br><br />
2. Take wavelength scan <br><br />
260nm-190nm <br><br />
sample every 1nm <br><br />
averaging time 3sec <br><br />
1 scan <br><br />
step scan <br><br />
25C <br><br />
3. Record wavelength which gives strongest signal (222nm)<br />
</p><br />
<br />
<h3> Circular Dichroism: Guanidine Melt </h3><br />
<p><br />
1. Load 1cm cuvette containing 1.996mL of 0.05mg/mL protein solution and stirrer onto CD <br><br />
2. Prepare 8mL of 0.05mg/mL protein in concentrated guanidine solution <br><br />
3. Set up Automixer with guanidine solution on one syringe and waste tube on other syringe <br><br />
4. Titrate up to 6 M guanidine, taking a CD measurement at 222 nm every 0.15 M interval <br><br />
5. Also measure the fluorescence at 280 nm to ensure the total protein concentration is not changing<br />
</p><br />
<br />
<br />
<br />
<html><br />
<br />
<a href="#linktotop">Back To Top</a><br />
</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2014-10-17T18:09:23Z<p>Kristanguyen: </p>
<hr />
<div>{{Template:Team:UW/CSS}}<br />
<br />
<br />
<h1> Protocols </h1><br />
<br />
<br />
<html><a name="Media, Plates, and Sol"></a><br />
<br />
</html><br />
<br />
<br />
<h2> Media, Plates, and Solutions </h2><br />
<br />
<br />
<h3> Competent Cell Media Buffer (CCMB) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
100 g glycerol (liquid) <br><br />
<br />
10 mL x 1 M potassium acetate <br><br />
<br />
11.8 g CaCl2*H2O <br><br />
<br />
4 g MnCl2 <br><br />
<br />
2 g MgCl2 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<h3> Super Optimal Broth (SOB) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
20 g Tryptone <br><br />
<br />
5 g Yeast Extract <br><br />
<br />
10 mL x 1 M NaCl <br><br />
<br />
2.5 mL x 1 M KCl <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Phosphate Buffered Saline (PBS) Solution </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container or 1 L beaker: <br><br />
<br />
8 g NaCl <br><br />
<br />
1.44 g Na2HPO4 <br><br />
<br />
0.8 g KCl <br><br />
<br />
0.24 g KH2PO4 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Buffer to pH 7.4 <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<h3> PSBF (PBS for Flow) </h3> <br />
<br />
<p><br />
<br />
Mix the following in a 1 L beaker: <br><br />
<br />
25 mL 20X PBS, pH 7.4 <br><br />
<br />
574 mL H2O <br><br />
<br />
2.5 g BSA (0.5%)* <br><br />
<br />
Filter and store at 4 °C <br><br />
<br><br />
*Do not need to pH - should be at pH 7.4 like 20X PBS <br><br />
<br />
</p> <br />
<br />
<br />
<h3> Yeast Extract Peptone Dextrose (YPD) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following into 950 mL of dH2O in a 1 L bottle: <br><br />
<br />
20 g peptone <br><br />
<br />
10 g yeast extract <br><br />
<br />
Autoclave (20 min at 121 °C and 20 psi) <br><br />
<br />
Add 50 mL 40% glucose <br><br />
<br />
Sterile filter into a 1 L bottle <br><br />
<br />
<br />
<br />
Note: For long-term liquid media storage, do not add 40% glucose. Instead add the glucose directly into cell cultures. <br><br />
<br />
Note: For YPD-plates add 24 g agar to the peptone and yeast extract before autoclaving. <br />
<br />
</p><br />
<br />
<br />
<h3> Selective Dropout Media, C-Uracil and C-Histidine (C-Ura and C-His) </h3><br />
<br />
<br />
<p><br />
<br />
Synthesized by the Yeast Resource Center at the University of Washington's <br />
<br />
Department of Genome Sciences and Department of Biochemistry.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<h3> Guanidinium Hydrogen Chloride </h3><br />
<br />
<br />
<p><br />
<br />
For maximum effectiveness, final concentration should be approximately 8.5 M in PBS.<br><br />
<br />
203 g guanidinium hydrogen chloride <br><br />
<br />
250 mL PBS solution* <br><br />
<br />
Add dilute HCl to pH 7.4 <br><br />
<br />
<br><br />
<br />
*Alternatively add slightly less than 250 mL of PBS in order to buffer the solution to the appropriate volume, then add more dH2O as necessary. <br />
<br />
</p><br />
<br />
<html><a name="Basic Cloning"></a><br />
<br />
</html><br />
<br />
<h2> Basic Cloning </h2><br />
<br />
<br />
<br />
<h3> Polymerase Chain Reaction </h3><br />
<br />
<br />
<p><br />
<br />
All PCRs were done using a standard 50 μL reaction volume with GoTaq® Green Master Mix 2X purchased from PROMEGA Corporation. <br><br />
<br />
Mix the following in a 0.2 mL microcentrifuge tube on ice: <br><br />
<br />
25 μL GoTaq® Green Master Mix 2X <br><br />
<br />
1-5 μL of 10 μM forward primer <br><br />
<br />
1-5 μL of 10 μM reverse primer <br><br />
<br />
<250 ng of DNA template <br><br />
<br />
QS 50 μl nuclease-free H2O <br><br />
<br />
Conduct the reaction in a thermocycler, adjusting anneal temperature and extension times accordingly. See your polymerase supplier protocol for more details on thermocycling.<br />
<br />
</p><br />
<br />
<h3> Error-prone Polymerase Chain Reaction </h3><br />
<br />
<br />
<p> <br />
<br />
<br />
<br />
Prepare 50 μL reaction: <br><br />
<br />
5 μL 10X Mutazyme II Rxn Buffer <br><br />
<br />
1 μL 40 mM dNTP mix (200 μM each final) <br><br />
<br />
1 μL 20 μM forward primer <br><br />
<br />
1 μL 20 μM reverse primer <br><br />
<br />
1 μL Mutazyme II DNA polymerase (2.5 U/μL) <br><br />
<br />
0.01 ng template <br><br />
<br />
QS 50 μL diH2O <br><br />
<br />
<br><br />
<br />
Program thermocycler as follows: <br><br />
<br />
95 °C, 2 min <br><br />
<br />
95 °C, 30 sec <br><br />
<br />
XX °C*, 30 sec <br><br />
<br />
72 °C, X min** <br><br />
<br />
32 cycles <br><br />
<br />
72 °C, 10 min <br><br />
<br />
4 °C, hold<br />
<br />
<br><br />
<br />
*Adjust annealing temperature according to Tm of primer. <br><br />
<br />
**Adjust extension time according to the length of amplified DNA. <br><br />
<br />
<br><br />
<br />
Note: Use 0.01 ng of template (calculate by insert and not by total plasmid). <br><br />
<br />
Calculate amount of template to use as follows: <br><br />
<br />
(bp for amplified region) / (bp in total plasmid) = % amplified region <br><br />
<br />
(conc. of total plasmid) x (% amplified region as a decimal) = conc. of amplified region <br><br />
<br />
<br><br />
<br />
Note: Never pipette less than 0.5 μL. <br><br />
<br />
(0.01 ng of template) / (conc. of amplified region) = vol of template to add to PCR <br><br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Restriction Endonuclease Reaction (Digestion) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
All restriction enzyme reactions were done using a 50 μl reaction volume. Restriction enzymes and buffers were purchased from New England Biolabs® Inc. <br><br />
<br />
Mix the following in a 0.2 mL PCR tube: <br><br />
<br />
<br />
1 μg of DNA <br><br />
<br />
5 μL of the appropriate 10X New England Biolab® Buffer <br><br />
<br />
1 μL of each restriction enzyme (add last) <br><br />
<br />
QS 50μL nuclease-free H2O <br><br />
<br />
Incubate the reaction for 1 hr <br><br />
<br />
Heat inactive the reaction at the appropriate temperature <br><br />
<br />
<br />
Note: Thaw the restriction enzyme(s) on ice to improve shelf life <br><br />
<br />
<br />
</p> <br />
<br />
<br />
<h3> Ligation </h3><br />
<br />
<br />
<p><br />
<br />
T4 DNA Ligase and Buffer were purchased from New England Biolabs® Inc. <br><br />
<br />
1. Prepare the following in a 0.2 mL microcentrifuge tube: <br><br />
<br />
50.0 ng vector DNA* <br><br />
<br />
37.5 ng vector DNA* <br><br />
<br />
2 μL 10X T4 DNA Ligase Buffer <br><br />
<br />
1 μL T4 DNA Ligase <br><br />
<br />
QS 20 μL diH2O <br><br />
<br />
2. Incubate the reaction at room temperature for 10-30 minutes or at 16 °C overnight. <br><br />
<br />
3. Heat inactivate at 65 °C for 10 minutes. <br><br />
<br />
4. Chill on ice before starting a transformation reaction. <br><br />
<br />
*The exact amount of DNA is dependent on the number of base pairs. In order to conduct a proper reaction consult the New England Biolab Ligation Calculator at:<br />
<br />
http://nebiocalculator.neb.com/#!/<br />
<br />
</p><br />
<br />
<br />
<html><a name="Escherichia coli Protocols"></a><br />
<br />
</html><br />
<br />
<br />
<h2> <i> Escherichia coli </i> Protocols (XL1-Blue and XL10-Gold) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<p> <br />
<br />
1. Plate 10 ul of cells onto an antibiotic free LB/Agar plate..* <br><br />
<br />
2. Incubate overnight at 37OC <br><br />
<br />
3. Inoculate lots of colonies into 50ml SOB-Mg media in a 250ml flask. <br><br />
4. Incubate at 37 OC, 200rpm, until OD at 550nm reaches 0.3, takes about 3 hours. I do my first check at 2 hours.<br><br />
<br />
5.Transfer the cell culture into a sterile 50ml Falcon tube and chill on ice for 10 minutes. <br><br />
<br />
6. Pellet the cells at 2500rpm for 15 minutes at 4 OC.<br><br />
<br />
7. Decant supernatant and invert tubes to remove excess culture medium. <br><br />
<br />
8. Resuspend the cells in 16ml CCMB by gentle vortexing or pipetting (I shake the falcon tube).<br />
(16ml CCMB per 50ml culture) <br><br />
<br />
9. Incubate on ice for 20 minutes. <br><br />
<br />
10. Centrifuge at 2500rpm for 10 minutes at 4 OC. <br><br />
<br />
11. Decant supernatant and invert tubes to remove excess liquid. <br><br />
<br />
12. Resuspend the cells in 4ml CCMB by gentle vortexing or pipetting.<br />
(4ml CCMB per culture) <br><br />
<br />
13. If making multiple 50ml cultures, combine all of the cells and mix before aliquoting. <br> <br />
<br />
14. In cold room: make 205ul aliquots, flash freeze in liquid nitrogen, and store at -80 OC.<br><br />
<br />
<br />
<br />
<br />
NOTE: Make SOB-Mg media and autoclave the 250ml flasks with water in them the same day the cells are plated (step 1) <br><br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Chemically Competent Cell Transformations </h3><br />
<br />
<br />
<p> <br />
<br />
1. Thaw competent <i> E.coli </i> cells on ice (XL1-Blue or XL10-Gold)* <br><br />
<br />
2. Add 50μL of competent cells to sterile 15mL conical centrifuge tubes <br><br />
<br />
3. Add 1μL (~100-200ng)* of the mini-prep to each culture tube <br><br />
<br />
4. Equilibrate the cells on ice for 10 min <br><br />
<br />
5. Heat shock the cells at 42C for 30-45 seconds** <br><br />
<br />
6. Immediately place the cells back on ice for 3 min <br><br />
<br />
7. Add 250μL LB media without antibiotics and shake at 250 rpm and 37C for 30 min <br><br />
<br />
8. Spread 10μL and 290μL on an appropriate LB-antibiotic plate <br><br />
<br />
9. Invert the plate and incubate at 37C overnight<br />
<br />
<br />
*The exact amount of DNA to add depends on your cell's transformation efficiency. However, it is acceptable to add a larger amount to increase the number of transformed cells. <br><br />
<br />
** Do not heat shock for an extended duration as this may damage and/or kill your cells.<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnights </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottom tube, add 3-5mL of LB and an appropriate volume of antibiotic(s). <br><br />
<br />
2. Swipe several individual colonies, do not collect satellites or colony clumps, with a pipette tip. <br><br />
<br />
3. Swirl the colony tip in the tube, there should be no visible cell clumps. <br><br />
<br />
4. Incubate and shake the tube at 37C at 250rpm for 12-16 hours and no longer than 20 hours. <br><br />
<br />
</p><br />
<br />
<br />
<h3> DNA-Extraction and mini-preps </h3><br />
<br />
<br />
<p><br />
<br />
All DNA mini-preps were prepared using EPOCH mini-kits and following the supplied protocols.<br />
<br />
</p><br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500μL of 40% Glycerol and 500uL of LB (no antibiotics) or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Saccharomyces cerevisiae"></a><br />
<br />
</html><br />
<br />
<h2> <i> Saccharomyces cerevisiae </i> (PyE1 Yeast) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Culturing </h3><br />
<br />
<br />
<br />
<p><br />
<br />
This process take 4 days in lab with a 1 day wait for incubation. <br><br />
<br />
<br />
Day 1: <br><br />
<br />
1. Streak yeast cells onto a YPD plate.* <br><br />
<br />
2. Invert the plate and incubate at 30C for 2 days. <br><br />
<br />
<br />
Day 3: <br><br />
<br />
1. Add 50mL of YPD liquid media into a 250mL baffle flask. <br><br />
<br />
2. Swipe as many individual colonies as you can see into the YPD media.** <br><br />
<br />
3. Incubate and shake the culture at 30C at 250rpm overnight approximately 24 hours. <br><br />
<br />
<br />
Day 4: <br><br />
<br />
1. Take an optical density measurement. <br><br />
<br />
2. In three 250mL baffle flask add the portions of the overnight liquid culture. <br><br />
<br />
3. Dilute each culture to approximately 0.4 optical density with YPD. <br><br />
<br />
4. Incubate and shake the cultures at 30C at 250rpm until the optical density reaches 1.2-1.6. <br><br />
<br />
5. Collect each culture into separate 50ml flat-bottomed centrifuge tubes. <br><br />
<br />
6. Spin down the cells at 4000g for 5 minutes at 4C. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cells in 100mL total for all three culture of dH2O. <br><br />
<br />
9. Combine the suspensions into two 50mL flat-bottomed centrifuge tubes. <br><br />
<br />
10. Spin down the cells as above. <br><br />
<br />
11. Decant the supernatant. <br><br />
<br />
12. Resuspend each in 3mL of 100mM Lithium Acetate. <br><br />
<br />
13. Transfer both cultures into a single 15mL conical centrifuge tube. <br><br />
<br />
14. Spin down the cells at 3000rpm for 5 minutes. <br><br />
<br />
15. Resuspend the cells in 0.75mL of 100mM Lithium Acetate, total volume is roughly 2mL. <br><br />
<br />
16. Qualitatively bring up the volume to 3.5mL by adding 40% Glycerol. <br><br />
<br />
17. Aliquot the cells into 1.5mL centrifuge tubes or 1.7mL cryogenic vials.*** <br><br />
<br />
<br />
*Streak in such a way that there are individual colonies visible on the plate without clumps or satellite colonies. <br><br />
<br />
**Collect only individual visible colonies. Do not collect clumps or satellite colonies. <br><br />
<br />
***The volume of aliquots depends on the number to transformations you intend to do at a time. <br><br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Chemically Competent Transformations </h3><br />
<br />
<br />
<p><br />
<br />
This protocol assumes a 50μL aliquot of yeast competent cells were made. <br><br />
<br />
Furthermore, this protocol prepares enough cells for 6 yeast transformations. <br><br />
<br />
<br><br />
<br />
1. Add the following to 50μL of yeast competent cells: <br><br />
<br />
240μL of Polyethylene Glycol - 3350 (PEG-3350) <br><br />
<br />
36μL of 1M Lithium Acetate <br><br />
<br />
32μL of dH2O <br><br />
<br />
<br />
<br />
2. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
3. Aliquot 59μL of the mixture into a 0.2mL microcentrifuge tube. <br><br />
<br />
4. Add 1uL (~100-200ng) of DNA. <br><br />
<br />
5. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
6. Incubate the mixture at 30C for 30 minutes. <br><br />
<br />
7. Heat shock the mixture at 42C for 20 minutes. <br><br />
<br />
8. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
9. Decant the supernatant. <br><br />
<br />
10. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
11. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
12. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
13. Plate 50-150μL of the mixture onto an appropriate Selective Dropout Media plate. <br><br />
<br />
14. Invert and incubate at 30C for 2 days. <br><br />
<br />
*The exact amount of DNA depends on the transformation efficiency of your competent cells. <br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnight Culturing </h3><br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Swipe 3 individually visible yeast colonies and add them to the culture tube media. <br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%.<br />
<br />
</p><br />
<br />
<br />
<h3> Culture Passaging </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Take 20-50μL from a previous overnight or passage culture and add it to the culture media.<br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%. <br><br />
<br />
Note: The exact amount of culture that you take from a previous culture is irrelevant as long as at least 1 living cell is passaged.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500uL of 40% glycerol and 500μL of Selective Dropout media or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Flow Cytometry"></a><br />
<br />
</html><br />
<br />
<h2> Flow Cytometry </h2><br />
<br />
<br />
<h3> Dilutions </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. From an overnight culture measure the optical density at 660nm by making 1:10 dilutions. <br><br />
<br />
2. Take enough culture to make a 1mL aliquot with OD 0.4. <br><br />
<br />
3. Spin down the aliquot in a 1.5mL centrifuge tube at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 800μL of the appropriate selective dropout media and 200μL of 20% glucose. <br><br />
<br />
6. Transfer the new culture to a 14mL culture tube. <br><br />
<br />
7. Incubate and shake at 30C and 250rpm for at least 6 hours (1.2-1.6 optical density).<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Preparations for Analysis using C6 Accuri Flow Cytometer </h3><br />
<br />
<br />
<p><br />
<br />
<br />
1. From the dilution previously made, measure the optical density, roughly 1.2-1.6. <br><br />
<br />
2. Make an aliquot of 500μL of the dilution culture in a 1.5mL centrifuge tube. <br><br />
<br />
3. Spin down the aliquot at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 500μL of PBS(F). <br><br />
<br />
6. Spin down the resuspension at 3000rpm for 3 minutes. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cell pellet in another 500uL of PBS(F).* <br><br />
<br />
9. Prepare the C6 Accuri Flow Cytometer by running a backflush cycle and a dH2O cycle. <br><br />
<br />
10. Load the sample onto the sip. <br><br />
<br />
11. Run the sample with 100,000 cell count. <br><br />
<br />
12. Repeat for all samples and make sure to change data cells otherwise the old data is erased. <br><br />
<br />
13. Once finished, run a cleaning cycle with Accuri approved cleaning solution, then run a dH2O cycle. <br><br />
<br />
<br />
<br />
*For special cases do not resuspend all samples, instead resuspend immediately before running the sample through the flow cytometer.<br />
<br />
<br />
</p><br />
<br />
<br />
<html><a name="Fluorescence Activated Cell Sorting"></a><br />
<br />
</html><br />
<br />
<h2> Fluorescence Activated Cell Sorting </h2><br />
<br />
<br />
<h3> Final Preparations </h3><br />
<br />
<br />
<br />
<p> <br />
<br />
Sample Prep:<br />
<br />
<br />
Spin down samples and negative control (5000 RPM, 1 min), keeping in mind the library size. Aspirate off supernatant. Resuspend in PBSF. Spin down cells. Aspirate off supernatant. Resuspend in PBSF. <br><br />
<br />
<br><br />
<br />
1. Open the “iGEM Template” file in the FACS Software and change name to current date and sort cycle<br><br />
<br />
2. Make sure the stream is stable (look for green light in bottom right corner). If not, run the Sort Calibration order. <br><br />
<br />
3. Run Negative Control from the PyE1 cells. <br><br />
<br />
a. Load cells onto carrier and into the machine. Press play button on screen. <br><br />
<br />
b. Set gate around lower left quadrant of cells to ensure single cell analysis using Forward Scatter Area and Side Scatter Area as your axes. Make sure oval gate covers around 80% of cell population. <br><br />
<br />
c. Set second gate on the first gated population by double-clicking on the gated population and using Forward Scatter Height and Forward Scatter Width as your axes. You will notice two distinct populations. Try to focus on the single cell portion of the plot. <br><br />
<br />
NOTE: If you see a large portion of the second gated population existing near the upper right edge of the first gate, you may need to enlarge the first gate to fit more of the population. <br><br />
<br />
d. Press record. Record 100,000 events and stop run. Move to Next Tube. <br><br />
<br />
4. Run first control (Gene clone) <br><br />
<br />
a. Follow step 3 to run the first control <br><br />
<br />
5. Run first library sample <br><br />
<br />
a. Follow steps 3b and 3c to set first two gates correctly. <br><br />
<br />
b. Set final gate for sort which includes top 1% of GFP producers from second gated population. <br><br />
<br />
c. Use final gate to set up the sort. <br><br />
<br />
d. Select sort conditions at the bottom of the screen. <br><br />
<br />
e. Insert and load collection tube. <br><br />
<br />
f. Record 100,000 events, and sort 10x the library size <br><br />
<br />
6. Run Bleach and diH2O through FACS to avoid cross-contamination. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Protein Expression"></a><br />
<br />
</html><br />
<br />
<h2> Protein Expression </h2><br />
<br />
<br />
<h3> Overnight Cultures </h3><br />
<br />
<p><br />
<br />
1. Add 25mL TB and 25uL Kan to a 250mL baffled flask <br><br />
<br />
2. Stab a glycerol stock with a p1000 pipette and swirl in the flask of media <br><br />
<br />
3. Put flask in 37C shaker overnight <br />
<br />
</p><br />
<br />
<h3> Protein Expression </h3><br />
<br />
<p> <br />
<br />
1. Add 500μL 1000x Kanamycin to 500mL TB in 2L baffled flask <br><br />
<br />
2. Transfer 10mL overnight culture to TB <br><br />
<br />
3. Shake at 37C (DO WE NEED THE RPM?) <br><br />
<br />
4. Remove flask from shaker when optical density is between 0.5 and 0.8 <br><br />
<br />
5. Allow flask to rest at room temp for 30 min <br><br />
<br />
6. Add 125μL 1M IPTG <br><br />
<br />
7. Shake flask at 18C overnight<br />
<br />
</p><br />
<br />
<h3> Protein Extraction and Purification </h3><br />
<br />
<p><br />
<br />
1. Transfer cell culture to centrifuge flask <br><br />
<br />
2. Centrifuge culture at 4000g for 10 min <br><br />
<br />
3. Discard supernatant <br><br />
<br />
4. Resuspend pellet in 25mL lysis buffer <br><br />
<br />
5. Add 250μL of 100x PMSF, 250μL of 100mg/mL lysozyme, and 250μL of 10mg/mL DNAse <br><br />
<br />
6. Sonicate sample with 0.25inch probe for 5 min at 70% amplitude with 20 sec on and off pulses <br><br />
<br />
7. Take 50μL total sample <br><br />
<br />
8. Transfer lysate to SS-34 centrifuge tube <br><br />
<br />
9. Centrifuge for 30 min at 18000g <br><br />
<br />
10. Take 50μL soluble sample<br />
<br />
</p><br />
<br />
<h3> Nickel Nitrotriacetic Acid Chromatography (Nickel-NTA Chromatography) </h3><br />
<br />
<p><br />
<br />
1. Add 5mL 50%(v/v) nickel resin in ethanol to 25mL column and allow to settle to ~2.5mL(CV) <br><br />
<br />
2. Rinse with 10CV H20 <br><br />
<br />
3. Equilibrate with 10CV lysis buffer <br><br />
<br />
4. Load sample onto column <br><br />
<br />
5. Wash column with 15CV lysis buffer <br><br />
<br />
6. Perform 2 additional wash steps with 15CV <br><br />
<br />
7. Elute sample in 10CV elution buffer and collect eluate <br><br />
<br />
8. Take 50μL pure sample <br />
<br />
</p><br />
<br />
<h3> Size Exclusion Chromatography (S.E.C.) </h3><br />
<p><br />
1. Concentrate sample to as high as possible without inducing protein aggregation<br><br />
2. Pre-equilibrate Superdex 75 column with 48 ml PBS <br><br />
3. Inject 500 ul sample onto column <br><br />
4. Run 36 ml PBS through column at 0.5 ml/min, collecting 1 ml fractions <br><br />
5. Verify presence of protein in fractions by measuring concentration on Nanodrop and running SDS-PAGE (15 kDa protein should elute at ~13 ml)<br><br />
6. Pool fractions containing protein <br />
</p><br />
<br />
<br />
<html><a name="Stability Analysis"></a><br />
<br />
</html><br />
<br />
<h2> Stability Analysis </h2><br />
<br />
<br />
<h3> Circular Dichroism: Wavelength Scan </h3><br />
<p><br />
1. Load 1mm cuvette with 400uL protein solution onto CD <br><br />
2. Take wavelength scan <br><br />
260nm-190nm <br><br />
sample every 1nm <br><br />
averaging time 3sec <br><br />
1 scan <br><br />
step scan <br><br />
25C <br><br />
3. Record wavelength which gives strongest signal (222nm)<br />
</p><br />
<br />
<h3> Circular Dichroism: Guanidine Melt </h3><br />
<p><br />
1. Load 1cm cuvette containing 1.996mL of 0.05mg/mL protein solution and stirrer onto CD <br><br />
2. Prepare 8mL of 0.05mg/mL protein in concentrated guanidine solution <br><br />
3. Set up Automixer with guanidine solution on one syringe and waste tube on other syringe <br><br />
4. Titrate up to 6 M guanidine, taking a CD measurement at 222 nm every 0.15 M interval <br><br />
5. Also measure the fluorescence at 280 nm to ensure the total protein concentration is not changing<br />
</p><br />
<br />
<br />
<br />
<html><br />
<br />
<a href="#linktotop">Back To Top</a><br />
</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2014-10-17T18:00:24Z<p>Kristanguyen: </p>
<hr />
<div>{{Template:Team:UW/CSS}}<br />
<br />
<br />
<h1> Protocols </h1><br />
<br />
<br />
<html><a name="Media, Plates, and Sol"></a><br />
<br />
</html><br />
<br />
<br />
<h2> Media, Plates, and Solutions </h2><br />
<br />
<br />
<h3> Competent Cell Media Buffer (CCMB) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
100 g glycerol (liquid) <br><br />
<br />
10 mL x 1 M potassium acetate <br><br />
<br />
11.8 g CaCl2*H2O <br><br />
<br />
4 g MnCl2 <br><br />
<br />
2 g MgCl2 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<h3> Super Optimal Broth (SOB) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
20 g Tryptone <br><br />
<br />
5 g Yeast Extract <br><br />
<br />
10 mL x 1 M NaCl <br><br />
<br />
2.5 mL x 1 M KCl <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Phosphate Buffered Saline (PBS) Solution </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container or 1 L beaker: <br><br />
<br />
8 g NaCl <br><br />
<br />
1.44 g Na2HPO4 <br><br />
<br />
0.8 g KCl <br><br />
<br />
0.24 g KH2PO4 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Buffer to pH 7.4 <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<h3> PSBF (PBS for Flow) </h3> <br />
<br />
<p><br />
<br />
Mix the following in a 1 L beaker: <br><br />
<br />
25 mL 20X PBS, pH 7.4 <br><br />
<br />
574 mL H2O <br><br />
<br />
2.5 g BSA (0.5%)* <br><br />
<br />
Filter and store at 4 °C <br><br />
<br><br />
*Do not need to pH - should be at pH 7.4 like 20X PBS <br><br />
<br />
</p> <br />
<br />
<br />
<h3> Yeast Extract Peptone Dextrose (YPD) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following into 950 mL of dH2O in a 1 L bottle: <br><br />
<br />
20 g peptone <br><br />
<br />
10 g yeast extract <br><br />
<br />
Autoclave (20 min at 121 °C and 20 psi) <br><br />
<br />
Add 50 mL 40% glucose <br><br />
<br />
Sterile filter into a 1 L bottle <br><br />
<br />
<br />
<br />
Note: For long-term liquid media storage, do not add 40% glucose. Instead add the glucose directly into cell cultures. <br><br />
<br />
Note: For YPD-plates add 24 g agar to the peptone and yeast extract before autoclaving. <br />
<br />
</p><br />
<br />
<br />
<h3> Selective Dropout Media, C-Uracil and C-Histidine (C-Ura and C-His) </h3><br />
<br />
<br />
<p><br />
<br />
Synthesized by the Yeast Resource Center at the University of Washington's <br />
<br />
Department of Genome Sciences and Department of Biochemistry.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<h3> Guanidinium Hydrogen Chloride </h3><br />
<br />
<br />
<p><br />
<br />
For maximum effectiveness, final concentration should be approximately 8.5 M in PBS.<br><br />
<br />
203 g guanidinium hydrogen chloride <br><br />
<br />
250 mL PBS solution* <br><br />
<br />
Add dilute HCl to pH 7.4 <br><br />
<br />
<br><br />
<br />
*Alternatively add slightly less than 250 mL of PBS in order to buffer the solution to the appropriate volume, then add more dH2O as necessary. <br />
<br />
</p><br />
<br />
<html><a name="Basic Cloning"></a><br />
<br />
</html><br />
<br />
<h2> Basic Cloning </h2><br />
<br />
<br />
<br />
<h3> Polymerase Chain Reaction </h3><br />
<br />
<br />
<p><br />
<br />
All PCRs were done using a standard 50 μL reaction volume with GoTaq® Green Master Mix 2X purchased from PROMEGA Corporation. <br><br />
<br />
Mix the following in a 0.2 mL microcentrifuge tube on ice: <br><br />
<br />
25 μL GoTaq® Green Master Mix 2X <br><br />
<br />
1-5 μL of 10 μM forward primer <br><br />
<br />
1-5 μL of 10 μM reverse primer <br><br />
<br />
<250 ng of DNA template <br><br />
<br />
QS 50 μl nuclease-free H2O <br><br />
<br />
Conduct the reaction in a thermocycler, adjusting anneal temperature and extension times accordingly. See your polymerase supplier protocol for more details on thermocycling.<br />
<br />
</p><br />
<br />
<h3> Error-prone Polymerase Chain Reaction </h3><br />
<br />
<br />
<p> <br />
<br />
<br />
<br />
Prepare 50 μL reaction: <br><br />
<br />
5 μL 10X Mutazyme II Rxn Buffer <br><br />
<br />
1 μL 40 mM dNTP mix (200 μM each final) <br><br />
<br />
1 μL 20 μM forward primer <br><br />
<br />
1 μL 20 μM reverse primer <br><br />
<br />
1 μL Mutazyme II DNA polymerase (2.5 U/μL) <br><br />
<br />
0.01 ng template <br><br />
<br />
QS 50 μL diH2O <br><br />
<br />
<br><br />
<br />
Program thermocycler as follows: <br><br />
<br />
95 °C, 2 min <br><br />
<br />
95 °C, 30 sec <br><br />
<br />
XX °C*, 30 sec <br><br />
<br />
72 °C, X min** <br><br />
<br />
32 cycles <br><br />
<br />
72 °C, 10 min <br><br />
<br />
4 °C, hold<br />
<br />
<br><br />
<br />
*Adjust annealing temperature according to Tm of primer. <br><br />
<br />
**Adjust extension time according to the length of amplified DNA. <br><br />
<br />
<br><br />
<br />
Note: Use 0.01 ng of template (calculate by insert and not by total plasmid). <br><br />
<br />
Calculate amount of template to use as follows: <br><br />
<br />
(bp for amplified region) / (bp in total plasmid) = % amplified region <br><br />
<br />
(conc. of total plasmid) x (% amplified region as a decimal) = conc. of amplified region <br><br />
<br />
<br><br />
<br />
Note: Never pipette less than 0.5 μL. <br><br />
<br />
(0.01 ng of template) / (conc. of amplified region) = vol of template to add to PCR <br><br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Restriction Endonuclease Reaction (Digestion) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
All restriction enzyme reactions were done using a 50 μl reaction volume. Restriction enzymes and buffers were purchased from New England Biolabs® Inc. <br><br />
<br />
Mix the following in a 0.2 mL PCR tube: <br><br />
<br />
<br />
1 μg of DNA <br><br />
<br />
5 μL of the appropriate 10X New England Biolab® Buffer <br><br />
<br />
1 μL of each restriction enzyme (add last) <br><br />
<br />
QS 50μL nuclease-free H2O <br><br />
<br />
Incubate the reaction for 1 hr <br><br />
<br />
Heat inactive the reaction at the appropriate temperature <br><br />
<br />
<br />
Note: Thaw the restriction enzyme(s) on ice to improve shelf life <br><br />
<br />
<br />
</p> <br />
<br />
<br />
<h3> Ligation </h3><br />
<br />
<br />
<p><br />
<br />
T4 DNA Ligase and Buffer were purchased from New England Biolabs® Inc. <br><br />
<br />
1. Prepare the following in a 0.2 mL microcentrifuge tube: <br><br />
<br />
50.0 ng vector DNA* <br><br />
<br />
37.5 ng vector DNA* <br><br />
<br />
2 μL 10X T4 DNA Ligase Buffer <br><br />
<br />
1 μL T4 DNA Ligase <br><br />
<br />
QS 20 μL diH2O <br><br />
<br />
2. Incubate the reaction at room temperature for 10-30 minutes or at 16 °C overnight. <br><br />
<br />
3. Heat inactivate at 65 °C for 10 minutes. <br><br />
<br />
4. Chill on ice before starting a transformation reaction. <br><br />
<br />
*The exact amount of DNA is dependent on the number of base pairs. In order to conduct a proper reaction consult the New England Biolab Ligation Calculator at:<br />
<br />
http://nebiocalculator.neb.com/#!/<br />
<br />
</p><br />
<br />
<br />
<html><a name="Escherichia coli Protocols"></a><br />
<br />
</html><br />
<br />
<br />
<h2> <i> Escherichia coli </i> Protocols (XL1-Blue and XL10-Gold) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Cultures </h3><br />
<br />
<br />
<p> <br />
<br />
1. Plate 10 ul of cells onto an antibiotic free LB/Agar plate..* <br><br />
<br />
2. Incubate overnight at 37OC <br><br />
<br />
3. Inoculate lots of colonies into 50ml SOB-Mg media in a 250ml flask. <br><br />
4. Incubate at 37 OC, 200rpm, until OD at 550nm reaches 0.3, takes about 3 hours. I do my first check at 2 hours.<br><br />
<br />
5.Transfer the cell culture into a sterile 50ml Falcon tube and chill on ice for 10 minutes. <br><br />
<br />
6. Pellet the cells at 2500rpm for 15 minutes at 4 OC.<br><br />
<br />
7. Decant supernatant and invert tubes to remove excess culture medium. <br><br />
<br />
8. Resuspend the cells in 16ml CCMB by gentle vortexing or pipetting (I shake the falcon tube).<br />
(16ml CCMB per 50ml culture) <br><br />
<br />
9. Incubate on ice for 20 minutes. <br><br />
<br />
10. Centrifuge at 2500rpm for 10 minutes at 4 OC. <br><br />
<br />
11. Decant supernatant and invert tubes to remove excess liquid. <br><br />
<br />
12. Resuspend the cells in 4ml CCMB by gentle vortexing or pipetting.<br />
(4ml CCMB per culture) <br><br />
<br />
13. If making multiple 50ml cultures, combine all of the cells and mix before aliquoting. <br> <br />
<br />
14. In cold room: make 205ul aliquots, flash freeze in liquid nitrogen, and store at -80 OC.<br><br />
<br />
<br />
<br />
<br />
NOTE: Make SOB-Mg media and autoclave the 250ml flasks with water in them the same day the cells are plated (step 1) <br><br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Chemically Competent Cell Transformations </h3><br />
<br />
<br />
<p> <br />
<br />
1. Thaw competent <i> E.coli </i> cells on ice (XL1-Blue or XL10-Gold)* <br><br />
<br />
2. Add 50μL of competent cells to sterile 15mL conical centrifuge tubes <br><br />
<br />
3. Add 1μL (~100-200ng)* of the mini-prep to each culture tube <br><br />
<br />
4. Equilibrate the cells on ice for 10 min <br><br />
<br />
5. Heat shock the cells at 42C for 30-45 seconds** <br><br />
<br />
6. Immediately place the cells back on ice for 3 min <br><br />
<br />
7. Add 250μL LB media without antibiotics and shake at 250 rpm and 37C for 30 min <br><br />
<br />
8. Spread 10μL and 290μL on an appropriate LB-antibiotic plate <br><br />
<br />
9. Invert the plate and incubate at 37C overnight<br />
<br />
<br />
*The exact amount of DNA to add depends on your cell's transformation efficiency. However, it is acceptable to add a larger amount to increase the number of transformed cells. <br><br />
<br />
** Do not heat shock for an extended duration as this may damage and/or kill your cells.<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnights </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottom tube, add 3-5mL of LB and an appropriate volume of antibiotic(s). <br><br />
<br />
2. Swipe several individual colonies, do not collect satellites or colony clumps, with a pipette tip. <br><br />
<br />
3. Swirl the colony tip in the tube, there should be no visible cell clumps. <br><br />
<br />
4. Incubate and shake the tube at 37C at 250rpm for 12-16 hours and no longer than 20 hours. <br><br />
<br />
</p><br />
<br />
<br />
<h3> DNA-Extraction and mini-preps </h3><br />
<br />
<br />
<p><br />
<br />
All DNA mini-preps were prepared using EPOCH mini-kits and following the supplied protocols.<br />
<br />
</p><br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500μL of 40% Glycerol and 500uL of LB (no antibiotics) or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Saccharomyces cerevisiae"></a><br />
<br />
</html><br />
<br />
<h2> <i> Saccharomyces cerevisiae </i> (PYE1 Yeast) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Culturing </h3><br />
<br />
<br />
<br />
<p><br />
<br />
This process take 4 days in lab with a 1 day wait for incubation. <br><br />
<br />
<br />
Day 1: <br><br />
<br />
1. Streak yeast cells onto a YPD plate.* <br><br />
<br />
2. Invert the plate and incubate at 30C for 2 days. <br><br />
<br />
<br />
Day 3: <br><br />
<br />
1. Add 50mL of YPD liquid media into a 250mL baffle flask. <br><br />
<br />
2. Swipe as many individual colonies as you can see into the YPD media.** <br><br />
<br />
3. Incubate and shake the culture at 30C at 250rpm overnight approximately 24 hours. <br><br />
<br />
<br />
Day 4: <br><br />
<br />
1. Take an optical density measurement. <br><br />
<br />
2. In three 250mL baffle flask add the portions of the overnight liquid culture. <br><br />
<br />
3. Dilute each culture to approximately 0.4 optical density with YPD. <br><br />
<br />
4. Incubate and shake the cultures at 30C at 250rpm until the optical density reaches 1.2-1.6. <br><br />
<br />
5. Collect each culture into separate 50ml flat-bottomed centrifuge tubes. <br><br />
<br />
6. Spin down the cells at 4000g for 5 minutes at 4C. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cells in 100mL total for all three culture of dH2O. <br><br />
<br />
9. Combine the suspensions into two 50mL flat-bottomed centrifuge tubes. <br><br />
<br />
10. Spin down the cells as above. <br><br />
<br />
11. Decant the supernatant. <br><br />
<br />
12. Resuspend each in 3mL of 100mM Lithium Acetate. <br><br />
<br />
13. Transfer both cultures into a single 15mL conical centrifuge tube. <br><br />
<br />
14. Spin down the cells at 3000rpm for 5 minutes. <br><br />
<br />
15. Resuspend the cells in 0.75mL of 100mM Lithium Acetate, total volume is roughly 2mL. <br><br />
<br />
16. Qualitatively bring up the volume to 3.5mL by adding 40% Glycerol. <br><br />
<br />
17. Aliquot the cells into 1.5mL centrifuge tubes or 1.7mL cryogenic vials.*** <br><br />
<br />
<br />
*Streak in such a way that there are individual colonies visible on the plate without clumps or satellite colonies. <br><br />
<br />
**Collect only individual visible colonies. Do not collect clumps or satellite colonies. <br><br />
<br />
***The volume of aliquots depends on the number to transformations you intend to do at a time. <br><br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Chemically Competent Transformations </h3><br />
<br />
<br />
<p><br />
<br />
This protocol assumes a 50μL aliquot of yeast competent cells were made. <br><br />
<br />
Furthermore, this protocol prepares enough cells for 6 yeast transformations. <br><br />
<br />
<br><br />
<br />
1. Add the following to 50μL of yeast competent cells: <br><br />
<br />
240μL of Polyethylene Glycol - 3350 (PEG-3350) <br><br />
<br />
36μL of 1M Lithium Acetate <br><br />
<br />
32μL of dH2O <br><br />
<br />
<br />
<br />
2. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
3. Aliquot 59μL of the mixture into a 0.2mL microcentrifuge tube. <br><br />
<br />
4. Add 1uL (~100-200ng) of DNA. <br><br />
<br />
5. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
6. Incubate the mixture at 30C for 30 minutes. <br><br />
<br />
7. Heat shock the mixture at 42C for 20 minutes. <br><br />
<br />
8. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
9. Decant the supernatant. <br><br />
<br />
10. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
11. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
12. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
13. Plate 50-150μL of the mixture onto an appropriate Selective Dropout Media plate. <br><br />
<br />
14. Invert and incubate at 30C for 2 days. <br><br />
<br />
*The exact amount of DNA depends on the transformation efficiency of your competent cells. <br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnight Culturing </h3><br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Swipe 3 individually visible yeast colonies and add them to the culture tube media. <br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%.<br />
<br />
</p><br />
<br />
<br />
<h3> Culture Passaging </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Take 20-50μL from a previous overnight or passage culture and add it to the culture media.<br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%. <br><br />
<br />
Note: The exact amount of culture that you take from a previous culture is irrelevant as long as at least 1 living cell is passaged.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500uL of 40% glycerol and 500μL of Selective Dropout media or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Flow Cytometry"></a><br />
<br />
</html><br />
<br />
<h2> Flow Cytometry </h2><br />
<br />
<br />
<h3> Dilutions </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. From an overnight culture measure the optical density at 660nm by making 1:10 dilutions. <br><br />
<br />
2. Take enough culture to make a 1mL aliquot with OD 0.4. <br><br />
<br />
3. Spin down the aliquot in a 1.5mL centrifuge tube at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 800μL of the appropriate selective dropout media and 200μL of 20% glucose. <br><br />
<br />
6. Transfer the new culture to a 14mL culture tube. <br><br />
<br />
7. Incubate and shake at 30C and 250rpm for at least 6 hours (1.2-1.6 optical density).<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Preparations for Analysis using C6 Accuri Flow Cytometer </h3><br />
<br />
<br />
<p><br />
<br />
<br />
1. From the dilution previously made, measure the optical density, roughly 1.2-1.6. <br><br />
<br />
2. Make an aliquot of 500μL of the dilution culture in a 1.5mL centrifuge tube. <br><br />
<br />
3. Spin down the aliquot at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 500μL of PBS(F). <br><br />
<br />
6. Spin down the resuspension at 3000rpm for 3 minutes. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cell pellet in another 500uL of PBS(F).* <br><br />
<br />
9. Prepare the C6 Accuri Flow Cytometer by running a backflush cycle and a dH2O cycle. <br><br />
<br />
10. Load the sample onto the sip. <br><br />
<br />
11. Run the sample with 100,000 cell count. <br><br />
<br />
12. Repeat for all samples and make sure to change data cells otherwise the old data is erased. <br><br />
<br />
13. Once finished, run a cleaning cycle with Accuri approved cleaning solution, then run a dH2O cycle. <br><br />
<br />
<br />
<br />
*For special cases do not resuspend all samples, instead resuspend immediately before running the sample through the flow cytometer.<br />
<br />
<br />
</p><br />
<br />
<br />
<html><a name="Fluorescence Activated Cell Sorting"></a><br />
<br />
</html><br />
<br />
<h2> Fluorescence Activated Cell Sorting </h2><br />
<br />
<br />
<h3> Final Preparations </h3><br />
<br />
<br />
<br />
<p> <br />
<br />
Sample Prep:<br />
<br />
<br />
Spin down samples and negative control (5000 RPM, 1 min), keeping in mind the library size. Aspirate off supernatant. Resuspend in PBSF. Spin down cells. Aspirate off supernatant. Resuspend in PBSF. <br><br />
<br />
<br><br />
<br />
1. Open the “iGEM Template” file in the FACS Software and change name to current date and sort cycle<br><br />
<br />
2. Make sure the stream is stable (look for green light in bottom right corner). If not, run the Sort Calibration order. <br><br />
<br />
3. Run Negative Control from the PyE1 cells. <br><br />
<br />
a. Load cells onto carrier and into the machine. Press play button on screen. <br><br />
<br />
b. Set gate around lower left quadrant of cells to ensure single cell analysis using Forward Scatter Area and Side Scatter Area as your axes. Make sure oval gate covers around 80% of cell population. <br><br />
<br />
c. Set second gate on the first gated population by double-clicking on the gated population and using Forward Scatter Height and Forward Scatter Width as your axes. You will notice two distinct populations. Try to focus on the single cell portion of the plot. <br><br />
<br />
NOTE: If you see a large portion of the second gated population existing near the upper right edge of the first gate, you may need to enlarge the first gate to fit more of the population. <br><br />
<br />
d. Press record. Record 100,000 events and stop run. Move to Next Tube. <br><br />
<br />
4. Run first control (Gene clone) <br><br />
<br />
a. Follow step 3 to run the first control <br><br />
<br />
5. Run first library sample <br><br />
<br />
a. Follow steps 3b and 3c to set first two gates correctly. <br><br />
<br />
b. Set final gate for sort which includes top 1% of GFP producers from second gated population. <br><br />
<br />
c. Use final gate to set up the sort. <br><br />
<br />
d. Select sort conditions at the bottom of the screen. <br><br />
<br />
e. Insert and load collection tube. <br><br />
<br />
f. Record 100,000 events, and sort 10x the library size <br><br />
<br />
6. Run Bleach and diH2O through FACS to avoid cross-contamination. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Protein Expression"></a><br />
<br />
</html><br />
<br />
<h2> Protein Expression </h2><br />
<br />
<br />
<h3> Overnight Cultures </h3><br />
<br />
<p><br />
<br />
1. Add 25mL TB and 25uL Kan to a 250mL baffled flask <br><br />
<br />
2. Stab a glycerol stock with a p1000 pipette and swirl in the flask of media <br><br />
<br />
3. Put flask in 37C shaker overnight <br />
<br />
</p><br />
<br />
<h3> Protein Expression </h3><br />
<br />
<p> <br />
<br />
1. Add 500μL 1000x Kanamycin to 500mL TB in 2L baffled flask <br><br />
<br />
2. Transfer 10mL overnight culture to TB <br><br />
<br />
3. Shake at 37C (DO WE NEED THE RPM?) <br><br />
<br />
4. Remove flask from shaker when optical density is between 0.5 and 0.8 <br><br />
<br />
5. Allow flask to rest at room temp for 30 min <br><br />
<br />
6. Add 125μL 1M IPTG <br><br />
<br />
7. Shake flask at 18C overnight<br />
<br />
</p><br />
<br />
<h3> Protein Extraction and Purification </h3><br />
<br />
<p><br />
<br />
1. Transfer cell culture to centrifuge flask <br><br />
<br />
2. Centrifuge culture at 4000g for 10 min <br><br />
<br />
3. Discard supernatant <br><br />
<br />
4. Resuspend pellet in 25mL lysis buffer <br><br />
<br />
5. Add 250μL of 100x PMSF, 250μL of 100mg/mL lysozyme, and 250μL of 10mg/mL DNAse <br><br />
<br />
6. Sonicate sample with 0.25inch probe for 5 min at 70% amplitude with 20 sec on and off pulses <br><br />
<br />
7. Take 50μL total sample <br><br />
<br />
8. Transfer lysate to SS-34 centrifuge tube <br><br />
<br />
9. Centrifuge for 30 min at 18000g <br><br />
<br />
10. Take 50μL soluble sample<br />
<br />
</p><br />
<br />
<h3> Nickel Nitrotriacetic Acid Chromatography (Nickel-NTA Chromatography) </h3><br />
<br />
<p><br />
<br />
1. Add 5mL 50%(v/v) nickel resin in ethanol to 25mL column and allow to settle to ~2.5mL(CV) <br><br />
<br />
2. Rinse with 10CV H20 <br><br />
<br />
3. Equilibrate with 10CV lysis buffer <br><br />
<br />
4. Load sample onto column <br><br />
<br />
5. Wash column with 15CV lysis buffer <br><br />
<br />
6. Perform 2 additional wash steps with 15CV <br><br />
<br />
7. Elute sample in 10CV elution buffer and collect eluate <br><br />
<br />
8. Take 50μL pure sample <br />
<br />
</p><br />
<br />
<h3> Size Exclusion Chromatography (S.E.C.) </h3><br />
<p><br />
1. Concentrate sample to as high as possible without inducing protein aggregation<br><br />
2. Pre-equilibrate Superdex 75 column with 48 ml PBS <br><br />
3. Inject 500 ul sample onto column <br><br />
4. Run 36 ml PBS through column at 0.5 ml/min, collecting 1 ml fractions <br><br />
5. Verify presence of protein in fractions by measuring concentration on Nanodrop and running SDS-PAGE (15 kDa protein should elute at ~13 ml)<br><br />
6. Pool fractions containing protein <br />
</p><br />
<br />
<br />
<html><a name="Stability Analysis"></a><br />
<br />
</html><br />
<br />
<h2> Stability Analysis </h2><br />
<br />
<br />
<h3> Circular Dichroism: Wavelength Scan </h3><br />
<p><br />
1. Load 1mm cuvette with 400uL protein solution onto CD <br><br />
2. Take wavelength scan <br><br />
260nm-190nm <br><br />
sample every 1nm <br><br />
averaging time 3sec <br><br />
1 scan <br><br />
step scan <br><br />
25C <br><br />
3. Record wavelength which gives strongest signal (222nm)<br />
</p><br />
<br />
<h3> Circular Dichroism: Guanidine Melt </h3><br />
<p><br />
1. Load 1cm cuvette containing 1.996mL of 0.05mg/mL protein solution and stirrer onto CD <br><br />
2. Prepare 8mL of 0.05mg/mL protein in concentrated guanidine solution <br><br />
3. Set up Automixer with guanidine solution on one syringe and waste tube on other syringe <br><br />
4. Titrate up to 6 M guanidine, taking a CD measurement at 222 nm every 0.15 M interval <br><br />
5. Also measure the fluorescence at 280 nm to ensure the total protein concentration is not changing<br />
</p><br />
<br />
<br />
<br />
<html><br />
<br />
<a href="#linktotop">Back To Top</a><br />
</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2014-10-17T17:37:46Z<p>Kristanguyen: </p>
<hr />
<div>{{Template:Team:UW/CSS}}<br />
<br />
<br />
<h1> Protocols </h1><br />
<br />
<br />
<html><a name="Media, Plates, and Sol"></a><br />
<br />
</html><br />
<br />
<br />
<h2> Media, Plates, and Solutions </h2><br />
<br />
<br />
<h3> Competent Cell Media Buffer (CCMB) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
100 g glycerol (liquid) <br><br />
<br />
10 mL x 1 M potassium acetate <br><br />
<br />
11.8 g CaCl2*H2O <br><br />
<br />
4 g MnCl2 <br><br />
<br />
2 g MgCl2 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<h3> Super Optimal Broth (SOB) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
20 g Tryptone <br><br />
<br />
5 g Yeast Extract <br><br />
<br />
10 mL x 1 M NaCl <br><br />
<br />
2.5 mL x 1 M KCl <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Phosphate Buffered Saline (PBS) Solution </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container or 1 L beaker: <br><br />
<br />
8 g NaCl <br><br />
<br />
1.44 g Na2HPO4 <br><br />
<br />
0.8 g KCl <br><br />
<br />
0.24 g KH2PO4 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Buffer to pH 7.4 <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<h3> PSBF (PBS for Flow) </h3> <br />
<br />
<p><br />
<br />
Mix the following in a 1 L beaker: <br><br />
<br />
25 mL 20X PBS, pH 7.4 <br><br />
<br />
574 mL H2O <br><br />
<br />
2.5 g BSA (0.5%)* <br><br />
<br />
Filter and store at 4 °C <br><br />
<br><br />
*Do not need to pH - should be at pH 7.4 like 20X PBS <br><br />
<br />
</p> <br />
<br />
<br />
<h3> Yeast Extract Peptone Dextrose (YPD) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following into 950mL of dH2O in a 1L bottle: <br><br />
<br />
20g Bacto Peptone <br><br />
<br />
10g Yeast Extract <br><br />
<br />
Autoclave (20min at 121C and 20psi) <br><br />
<br />
Add 50mL 40% Glucose <br><br />
<br />
Sterile filter into a 1L bottle <br><br />
<br />
<br />
<br />
For long-term liquid media storage, do not add 40% Glucose instead add the glucose directly into cell cultures. <br><br />
<br />
For YPD-plates add 24g Bacto Agar to the Bacto Peptone and Yeast Extract before autoclaving. <br />
<br />
</p><br />
<br />
<br />
<h3> Selective Dropout media, C-Uracil and C-Histidine (C-Ura and C-His) </h3><br />
<br />
<br />
<p><br />
<br />
Synthesized by the Yeast Resource Center at the University of Washington's <br />
<br />
Department of Genome Sciences and Department of Biochemistry.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<h3> Guanidinium Hydrogen Chloride </h3><br />
<br />
<br />
<p><br />
<br />
For maximum effectiveness, final concentration should be approximately 8.5M in PBS<br><br />
<br />
203g Guanidinium Hydrogen Chloride <br><br />
<br />
250mL PBS solution <br><br />
<br />
Add dilute HCl to 7.4pH <br><br />
<br />
<br />
*Alternatively add slightly less than 250mL of PBS in order to buffer the solution to the appropriate volume then add more dH2O as neccessary. <br />
<br />
</p><br />
<br />
<html><a name="Basic Cloning"></a><br />
<br />
</html><br />
<br />
<h2> Basic Cloning </h2><br />
<br />
<br />
<br />
<h3> Polymerase Chain Reaction </h3><br />
<br />
<br />
<p><br />
<br />
All PCRs were done using a standard 50μL reaction volume. <br><br />
<br />
PCRs were done using GoTaq Green Master Mix 2X purchased from PROMEGA Corporation. <br><br />
<br />
Protocols for the PROMEGA GoTaq Green Master Mix 2X: <br><br />
<br />
Mix the following in a 0.2mL microcentrifuge tube on ice: <br><br />
<br />
25μL GoTaq® Green Master Mix 2X <br><br />
<br />
1-5μL of 10μM forward primer <br><br />
<br />
1-5μL of 10μM reverse primer <br><br />
<br />
<250ng of DNA template <br><br />
<br />
Nuclease-free water to 50μl <br><br />
<br />
Conduct the reaction in a Thermocycler, adjusting anneal temperature and extension times accordingly. See your polymerase supplier protocol for more details on thermocycling.<br />
<br />
</p><br />
<br />
<h3> Error-prone Polymerase Chain Reaction </h3><br />
<br />
<br />
<p> <br />
<br />
<br />
<br />
Prepare 50μL reaction: <br><br />
<br />
5μL 10X Mutazyme II Rxn Buffer <br><br />
<br />
1μL 40mM dNTP mix (200μM each final) <br><br />
<br />
1μL 20μM forward primer <br><br />
<br />
1μL 20μM reverse primer <br><br />
<br />
1μL Mutazyme II DNA polymerase (2.5U/μL) <br><br />
<br />
0.01ng template <br><br />
<br />
QS 50μL diH2O <br><br />
<br />
<br><br />
<br />
Thermocycler: <br><br />
<br />
95C, 2min <br><br />
<br />
95C, 30sec <br><br />
<br />
XXC*, 30sec <br><br />
<br />
72C, Xmin** <br><br />
<br />
32 cycles <br><br />
<br />
72C, 10min <br><br />
<br />
4C, hold<br />
<br />
<br><br />
<br />
*Adjust annealing temperature according to Tm of primer. <br><br />
<br />
**Adjust extension time according to the length of amplified DNA. <br><br />
<br />
<br><br />
<br />
Note: Use 0.01ng (calculate by insert and not by total plasmid). <br><br />
<br />
Calculate amount of template to use. <br><br />
<br />
(bp for amplified region) / (bp in total plasmid) = % amplified region <br><br />
<br />
(conc of total plasmid) x (% amplified region as a decimal) = conc of amplified region <br><br />
<br />
<br><br />
<br />
Note: Will probably need to dilute. Never pipette less than 0.5μL. <br><br />
<br />
(0.01ng) / (conc of amplified region) = vol to add to PCR <br><br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Restriction Endonuclease Reaction (Digestion) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
All restriction enzyme reactions were done using a 50ul reaction volume. <br><br />
<br />
Restriction enzymes and buffers were purchased from New England Biolabs Incorporated. <br><br />
<br />
Protocols for various New England Biolab restriction enzyme reactions: <br><br />
<br />
Mix the following in a 0.2mL PCR tube: <br><br />
<br />
1μL of each Restriction Enzyme, add the RE last <br><br />
<br />
1μg of DNA <br><br />
<br />
5μL of the appropriate 10X New Englan Biolab Buffer <br><br />
<br />
Nuclease-free water to 50μL <br><br />
<br />
Incubate the reaction for 1hr <br><br />
<br />
Heat inactive the the reaction at the appropriate temperature <br><br />
<br />
<br />
Notes: Add the restriction enzyme(s) to the reaction last <br><br />
<br />
Thaw the restriction enzyme(s) on ice to improve shelf life <br><br />
<br />
<br />
<br />
</p> <br />
<br />
<br />
<h3> Ligation </h3><br />
<br />
<br />
<p><br />
<br />
T4 DNA Ligase and Buffer was purchased from New England BioLabs Corporation. <br><br />
<br />
1. Prepare the following in a 0.2mL microcentrifuge tube: <br><br />
<br />
50.0ng Vector DNA* <br><br />
<br />
37.5ng Vector DNA* <br><br />
<br />
2μL 10X T4 DNA Ligase Buffer <br><br />
<br />
1μL T4 DNA Ligase <br><br />
<br />
Add diH2O to 20μL <br><br />
<br />
2. Incubate the reaction at room temperature for 10-30 minutes or at 16C overnight. <br><br />
<br />
3. Heat inactivate at 65C for 10 minutes. <br><br />
<br />
4. Chill on ice before starting a transformation reaction. <br><br />
<br />
*The exact amount of DNA is dependent on the number of base pairs. In order to conduct a proper reaction consult the New England Biolab Ligation Calculator at:<br />
<br />
http://nebiocalculator.neb.com/#!/<br />
<br />
</p><br />
<br />
<br />
<html><a name="Escherichia coli Protocols"></a><br />
<br />
</html><br />
<br />
<br />
<h2> <i> Escherichia coli Protocols </i> (XL1-Blue and XL10-Gold) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Culturing </h3><br />
<br />
<br />
<br />
<p> <br />
<br />
<br />
Chemically Competent Cell Stocks – CCMB Transformation <br />
For BL21*, CJ236, etc. Cell Lines. <br><br />
<br />
NOTE: Make SOB-Mg media and autoclave the 250ml flasks with water in them the same day the cells are plated (step 1) <br><br />
<br><br />
<br />
1. Plate 10ul of cells onto an antibiotic free LB/Agar plate..* <br><br />
<br />
2. Incubate overnight at 37OC <br><br />
<br />
3. Inoculate lots of colonies into 50ml SOB-Mg media in a 250ml flask. <br><br />
4. Incubate at 37 OC, 200rpm, until OD at 550nm reaches 0.3, takes about 3 hours. I do my first check at 2 hours.<br><br />
<br />
5.Transfer the cell culture into a sterile 50ml Falcon tube and chill on ice for 10 minutes. <br><br />
<br />
6. Pellet the cells at 2500rpm for 15 minutes at 4 OC.<br><br />
<br />
7. Decant supernatant and invert tubes to remove excess culture medium. <br><br />
<br />
8. Resuspend the cells in 16ml CCMB by gentle vortexing or pipetting (I shake the falcon tube).<br />
(16ml CCMB per 50ml culture) <br><br />
<br />
9. Incubate on ice for 20 minutes. <br><br />
<br />
10. Centrifuge at 2500rpm for 10 minutes at 4 OC. <br><br />
<br />
11. Decant supernatant and invert tubes to remove excess liquid. <br><br />
<br />
12. Resuspend the cells in 4ml CCMB by gentle vortexing or pipetting.<br />
(4ml CCMB per culture) <br><br />
<br />
13. If making multiple 50ml cultures, combine all of the cells and mix before aliquoting. <br> <br />
<br />
14. In cold room: make 205ul aliquots, flash freeze in liquid nitrogen, and store at -80 OC.<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Chemically Competent Cell Transformations </h3><br />
<br />
<br />
<p> <br />
<br />
1. Thaw competent <i> E.coli </i> cells on ice (XL1-Blue or XL10-Gold)* <br><br />
<br />
2. Add 50μL of competent cells to sterile 15mL conical centrifuge tubes <br><br />
<br />
3. Add 1μL (~100-200ng)* of the mini-prep to each culture tube <br><br />
<br />
4. Equilibrate the cells on ice for 10 min <br><br />
<br />
5. Heat shock the cells at 42C for 30-45 seconds** <br><br />
<br />
6. Immediately place the cells back on ice for 3 min <br><br />
<br />
7. Add 250μL LB media without antibiotics and shake at 250 rpm and 37C for 30 min <br><br />
<br />
8. Spread 10μL and 290μL on an appropriate LB-antibiotic plate <br><br />
<br />
9. Invert the plate and incubate at 37C overnight<br />
<br />
<br />
*The exact amount of DNA to add depends on your cell's transformation efficiency. However, it is acceptable to add a larger amount to increase the number of transformed cells. <br><br />
<br />
** Do not heat shock for an extended duration as this may damage and/or kill your cells.<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnights </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottom tube, add 3-5mL of LB and an appropriate volume of antibiotic(s). <br><br />
<br />
2. Swipe several individual colonies, do not collect satellites or colony clumps, with a pipette tip. <br><br />
<br />
3. Swirl the colony tip in the tube, there should be no visible cell clumps. <br><br />
<br />
4. Incubate and shake the tube at 37C at 250rpm for 12-16 hours and no longer than 20 hours. <br><br />
<br />
</p><br />
<br />
<br />
<h3> DNA-Extraction and mini-preps </h3><br />
<br />
<br />
<p><br />
<br />
All DNA mini-preps were prepared using EPOCH mini-kits and following the supplied protocols.<br />
<br />
</p><br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500μL of 40% Glycerol and 500uL of LB (no antibiotics) or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Saccharomyces cerevisiae"></a><br />
<br />
</html><br />
<br />
<h2> <i> Saccharomyces cerevisiae </i> (PYE1 Yeast) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Culturing </h3><br />
<br />
<br />
<br />
<p><br />
<br />
This process take 4 days in lab with a 1 day wait for incubation. <br><br />
<br />
<br />
Day 1: <br><br />
<br />
1. Streak yeast cells onto a YPD plate.* <br><br />
<br />
2. Invert the plate and incubate at 30C for 2 days. <br><br />
<br />
<br />
Day 3: <br><br />
<br />
1. Add 50mL of YPD liquid media into a 250mL baffle flask. <br><br />
<br />
2. Swipe as many individual colonies as you can see into the YPD media.** <br><br />
<br />
3. Incubate and shake the culture at 30C at 250rpm overnight approximately 24 hours. <br><br />
<br />
<br />
Day 4: <br><br />
<br />
1. Take an optical density measurement. <br><br />
<br />
2. In three 250mL baffle flask add the portions of the overnight liquid culture. <br><br />
<br />
3. Dilute each culture to approximately 0.4 optical density with YPD. <br><br />
<br />
4. Incubate and shake the cultures at 30C at 250rpm until the optical density reaches 1.2-1.6. <br><br />
<br />
5. Collect each culture into separate 50ml flat-bottomed centrifuge tubes. <br><br />
<br />
6. Spin down the cells at 4000g for 5 minutes at 4C. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cells in 100mL total for all three culture of dH2O. <br><br />
<br />
9. Combine the suspensions into two 50mL flat-bottomed centrifuge tubes. <br><br />
<br />
10. Spin down the cells as above. <br><br />
<br />
11. Decant the supernatant. <br><br />
<br />
12. Resuspend each in 3mL of 100mM Lithium Acetate. <br><br />
<br />
13. Transfer both cultures into a single 15mL conical centrifuge tube. <br><br />
<br />
14. Spin down the cells at 3000rpm for 5 minutes. <br><br />
<br />
15. Resuspend the cells in 0.75mL of 100mM Lithium Acetate, total volume is roughly 2mL. <br><br />
<br />
16. Qualitatively bring up the volume to 3.5mL by adding 40% Glycerol. <br><br />
<br />
17. Aliquot the cells into 1.5mL centrifuge tubes or 1.7mL cryogenic vials.*** <br><br />
<br />
<br />
*Streak in such a way that there are individual colonies visible on the plate without clumps or satellite colonies. <br><br />
<br />
**Collect only individual visible colonies. Do not collect clumps or satellite colonies. <br><br />
<br />
***The volume of aliquots depends on the number to transformations you intend to do at a time. <br><br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Chemically Competent Transformations </h3><br />
<br />
<br />
<p><br />
<br />
This protocol assumes a 50μL aliquot of yeast competent cells were made. <br><br />
<br />
Furthermore, this protocol prepares enough cells for 6 yeast transformations. <br><br />
<br />
<br><br />
<br />
1. Add the following to 50μL of yeast competent cells: <br><br />
<br />
240μL of Polyethylene Glycol - 3350 (PEG-3350) <br><br />
<br />
36μL of 1M Lithium Acetate <br><br />
<br />
32μL of dH2O <br><br />
<br />
<br />
<br />
2. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
3. Aliquot 59μL of the mixture into a 0.2mL microcentrifuge tube. <br><br />
<br />
4. Add 1uL (~100-200ng) of DNA. <br><br />
<br />
5. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
6. Incubate the mixture at 30C for 30 minutes. <br><br />
<br />
7. Heat shock the mixture at 42C for 20 minutes. <br><br />
<br />
8. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
9. Decant the supernatant. <br><br />
<br />
10. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
11. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
12. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
13. Plate 50-150μL of the mixture onto an appropriate Selective Dropout Media plate. <br><br />
<br />
14. Invert and incubate at 30C for 2 days. <br><br />
<br />
*The exact amount of DNA depends on the transformation efficiency of your competent cells. <br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnight Culturing </h3><br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Swipe 3 individually visible yeast colonies and add them to the culture tube media. <br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%.<br />
<br />
</p><br />
<br />
<br />
<h3> Culture Passaging </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Take 20-50μL from a previous overnight or passage culture and add it to the culture media.<br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%. <br><br />
<br />
Note: The exact amount of culture that you take from a previous culture is irrelevant as long as at least 1 living cell is passaged.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500uL of 40% glycerol and 500μL of Selective Dropout media or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Flow Cytometry"></a><br />
<br />
</html><br />
<br />
<h2> Flow Cytometry </h2><br />
<br />
<br />
<h3> Dilutions </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. From an overnight culture measure the optical density at 660nm by making 1:10 dilutions. <br><br />
<br />
2. Take enough culture to make a 1mL aliquot with OD 0.4. <br><br />
<br />
3. Spin down the aliquot in a 1.5mL centrifuge tube at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 800μL of the appropriate selective dropout media and 200μL of 20% glucose. <br><br />
<br />
6. Transfer the new culture to a 14mL culture tube. <br><br />
<br />
7. Incubate and shake at 30C and 250rpm for at least 6 hours (1.2-1.6 optical density).<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Preparations for Analysis using C6 Accuri Flow Cytometer </h3><br />
<br />
<br />
<p><br />
<br />
<br />
1. From the dilution previously made, measure the optical density, roughly 1.2-1.6. <br><br />
<br />
2. Make an aliquot of 500μL of the dilution culture in a 1.5mL centrifuge tube. <br><br />
<br />
3. Spin down the aliquot at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 500μL of PBS(F). <br><br />
<br />
6. Spin down the resuspension at 3000rpm for 3 minutes. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cell pellet in another 500uL of PBS(F).* <br><br />
<br />
9. Prepare the C6 Accuri Flow Cytometer by running a backflush cycle and a dH2O cycle. <br><br />
<br />
10. Load the sample onto the sip. <br><br />
<br />
11. Run the sample with 100,000 cell count. <br><br />
<br />
12. Repeat for all samples and make sure to change data cells otherwise the old data is erased. <br><br />
<br />
13. Once finished, run a cleaning cycle with Accuri approved cleaning solution, then run a dH2O cycle. <br><br />
<br />
<br />
<br />
*For special cases do not resuspend all samples, instead resuspend immediately before running the sample through the flow cytometer.<br />
<br />
<br />
</p><br />
<br />
<br />
<html><a name="Fluorescence Activated Cell Sorting"></a><br />
<br />
</html><br />
<br />
<h2> Fluorescence Activated Cell Sorting </h2><br />
<br />
<br />
<h3> Final Preparations </h3><br />
<br />
<br />
<br />
<p> <br />
<br />
Sample Prep:<br />
<br />
<br />
Spin down samples and negative control (5000 RPM, 1 min), keeping in mind the library size. Aspirate off supernatant. Resuspend in PBSF. Spin down cells. Aspirate off supernatant. Resuspend in PBSF. <br><br />
<br />
<br><br />
<br />
1. Open the “iGEM Template” file in the FACS Software and change name to current date and sort cycle<br><br />
<br />
2. Make sure the stream is stable (look for green light in bottom right corner). If not, run the Sort Calibration order. <br><br />
<br />
3. Run Negative Control from the PyE1 cells. <br><br />
<br />
a. Load cells onto carrier and into the machine. Press play button on screen. <br><br />
<br />
b. Set gate around lower left quadrant of cells to ensure single cell analysis using Forward Scatter Area and Side Scatter Area as your axes. Make sure oval gate covers around 80% of cell population. <br><br />
<br />
c. Set second gate on the first gated population by double-clicking on the gated population and using Forward Scatter Height and Forward Scatter Width as your axes. You will notice two distinct populations. Try to focus on the single cell portion of the plot. <br><br />
<br />
NOTE: If you see a large portion of the second gated population existing near the upper right edge of the first gate, you may need to enlarge the first gate to fit more of the population. <br><br />
<br />
d. Press record. Record 100,000 events and stop run. Move to Next Tube. <br><br />
<br />
4. Run first control (Gene clone) <br><br />
<br />
a. Follow step 3 to run the first control <br><br />
<br />
5. Run first library sample <br><br />
<br />
a. Follow steps 3b and 3c to set first two gates correctly. <br><br />
<br />
b. Set final gate for sort which includes top 1% of GFP producers from second gated population. <br><br />
<br />
c. Use final gate to set up the sort. <br><br />
<br />
d. Select sort conditions at the bottom of the screen. <br><br />
<br />
e. Insert and load collection tube. <br><br />
<br />
f. Record 100,000 events, and sort 10x the library size <br><br />
<br />
6. Run Bleach and diH2O through FACS to avoid cross-contamination. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Protein Expression"></a><br />
<br />
</html><br />
<br />
<h2> Protein Expression </h2><br />
<br />
<br />
<h3> Overnight Cultures </h3><br />
<br />
<p><br />
<br />
1. Add 25mL TB and 25uL Kan to a 250mL baffled flask <br><br />
<br />
2. Stab a glycerol stock with a p1000 pipette and swirl in the flask of media <br><br />
<br />
3. Put flask in 37C shaker overnight <br />
<br />
</p><br />
<br />
<h3> Protein Expression </h3><br />
<br />
<p> <br />
<br />
1. Add 500μL 1000x Kanamycin to 500mL TB in 2L baffled flask <br><br />
<br />
2. Transfer 10mL overnight culture to TB <br><br />
<br />
3. Shake at 37C (DO WE NEED THE RPM?) <br><br />
<br />
4. Remove flask from shaker when optical density is between 0.5 and 0.8 <br><br />
<br />
5. Allow flask to rest at room temp for 30 min <br><br />
<br />
6. Add 125μL 1M IPTG <br><br />
<br />
7. Shake flask at 18C overnight<br />
<br />
</p><br />
<br />
<h3> Protein Extraction and Purification </h3><br />
<br />
<p><br />
<br />
1. Transfer cell culture to centrifuge flask <br><br />
<br />
2. Centrifuge culture at 4000g for 10 min <br><br />
<br />
3. Discard supernatant <br><br />
<br />
4. Resuspend pellet in 25mL lysis buffer <br><br />
<br />
5. Add 250μL of 100x PMSF, 250μL of 100mg/mL lysozyme, and 250μL of 10mg/mL DNAse <br><br />
<br />
6. Sonicate sample with 0.25inch probe for 5 min at 70% amplitude with 20 sec on and off pulses <br><br />
<br />
7. Take 50μL total sample <br><br />
<br />
8. Transfer lysate to SS-34 centrifuge tube <br><br />
<br />
9. Centrifuge for 30 min at 18000g <br><br />
<br />
10. Take 50μL soluble sample<br />
<br />
</p><br />
<br />
<h3> Nickel Nitrotriacetic Acid Chromatography (Nickel-NTA Chromatography) </h3><br />
<br />
<p><br />
<br />
1. Add 5mL 50%(v/v) nickel resin in ethanol to 25mL column and allow to settle to ~2.5mL(CV) <br><br />
<br />
2. Rinse with 10CV H20 <br><br />
<br />
3. Equilibrate with 10CV lysis buffer <br><br />
<br />
4. Load sample onto column <br><br />
<br />
5. Wash column with 15CV lysis buffer <br><br />
<br />
6. Perform 2 additional wash steps with 15CV <br><br />
<br />
7. Elute sample in 10CV elution buffer and collect eluate <br><br />
<br />
8. Take 50μL pure sample <br />
<br />
</p><br />
<br />
<h3> Size Exclusion Chromatography (S.E.C.) </h3><br />
<p><br />
1. Concentrate sample to as high as possible without inducing protein aggregation<br><br />
2. Pre-equilibrate Superdex 75 column with 48 ml PBS <br><br />
3. Inject 500 ul sample onto column <br><br />
4. Run 36 ml PBS through column at 0.5 ml/min, collecting 1 ml fractions <br><br />
5. Verify presence of protein in fractions by measuring concentration on Nanodrop and running SDS-PAGE (15 kDa protein should elute at ~13 ml)<br><br />
6. Pool fractions containing protein <br />
</p><br />
<br />
<br />
<html><a name="Stability Analysis"></a><br />
<br />
</html><br />
<br />
<h2> Stability Analysis </h2><br />
<br />
<br />
<h3> Circular Dichroism: Wavelength Scan </h3><br />
<p><br />
1. Load 1mm cuvette with 400uL protein solution onto CD <br><br />
2. Take wavelength scan <br><br />
260nm-190nm <br><br />
sample every 1nm <br><br />
averaging time 3sec <br><br />
1 scan <br><br />
step scan <br><br />
25C <br><br />
3. Record wavelength which gives strongest signal (222nm)<br />
</p><br />
<br />
<h3> Circular Dichroism: Guanidine Melt </h3><br />
<p><br />
1. Load 1cm cuvette containing 1.996mL of 0.05mg/mL protein solution and stirrer onto CD <br><br />
2. Prepare 8mL of 0.05mg/mL protein in concentrated guanidine solution <br><br />
3. Set up Automixer with guanidine solution on one syringe and waste tube on other syringe <br><br />
4. Titrate up to 6 M guanidine, taking a CD measurement at 222 nm every 0.15 M interval <br><br />
5. Also measure the fluorescence at 280 nm to ensure the total protein concentration is not changing<br />
</p><br />
<br />
<br />
<br />
<html><br />
<br />
<a href="#linktotop">Back To Top</a><br />
</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2014-10-17T17:33:54Z<p>Kristanguyen: </p>
<hr />
<div>{{Template:Team:UW/CSS}}<br />
<br />
<br />
<h1> Protocols </h1><br />
<br />
<br />
<html><a name="Media, Plates, and Sol"></a><br />
<br />
</html><br />
<br />
<br />
<h2> Media, Plates, and Solutions </h2><br />
<br />
<br />
<h3> Competent Cell Media Buffer (CCMB) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
100 g glycerol (liquid) <br><br />
<br />
10 mL x 1 M potassium acetate <br><br />
<br />
11.8 g CaCl2*H2O <br><br />
<br />
4 g MnCl2 <br><br />
<br />
2 g MgCl2 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<h3> Super Optimal Broth (SOB) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
20 g Tryptone <br><br />
<br />
5 g Yeast Extract <br><br />
<br />
10 mL x 1 M NaCl <br><br />
<br />
2.5 mL x 1 M KCl <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Phosphate Buffered Saline (PBS) Solution </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container or 1 L beaker: <br><br />
<br />
8 g NaCl <br><br />
<br />
1.44 g Na2HPO4 <br><br />
<br />
0.8 g KCl <br><br />
<br />
0.24 g KH2PO4 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Buffer to pH 7.4 <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Yeast Extract Peptone Dextrose (YPD) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following into 950mL of dH2O in a 1L bottle: <br><br />
<br />
20g Bacto Peptone <br><br />
<br />
10g Yeast Extract <br><br />
<br />
Autoclave (20min at 121C and 20psi) <br><br />
<br />
Add 50mL 40% Glucose <br><br />
<br />
Sterile filter into a 1L bottle <br><br />
<br />
<br />
<br />
For long-term liquid media storage, do not add 40% Glucose instead add the glucose directly into cell cultures. <br><br />
<br />
For YPD-plates add 24g Bacto Agar to the Bacto Peptone and Yeast Extract before autoclaving. <br />
<br />
</p><br />
<br />
<br />
<h3> Selective Dropout media, C-Uracil and C-Histidine (C-Ura and C-His) </h3><br />
<br />
<br />
<p><br />
<br />
Synthesized by the Yeast Resource Center at the University of Washington's <br />
<br />
Department of Genome Sciences and Department of Biochemistry.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<h3> Guanidinium Hydrogen Chloride </h3><br />
<br />
<br />
<p><br />
<br />
For maximum effectiveness, final concentration should be approximately 8.5M in PBS<br><br />
<br />
203g Guanidinium Hydrogen Chloride <br><br />
<br />
250mL PBS solution <br><br />
<br />
Add dilute HCl to 7.4pH <br><br />
<br />
<br />
*Alternatively add slightly less than 250mL of PBS in order to buffer the solution to the appropriate volume then add more dH2O as neccessary. <br />
<br />
</p><br />
<br />
<html><a name="Basic Cloning"></a><br />
<br />
</html><br />
<br />
<h2> Basic Cloning </h2><br />
<br />
<br />
<br />
<h3> Polymerase Chain Reaction </h3><br />
<br />
<br />
<p><br />
<br />
All PCRs were done using a standard 50μL reaction volume. <br><br />
<br />
PCRs were done using GoTaq Green Master Mix 2X purchased from PROMEGA Corporation. <br><br />
<br />
Protocols for the PROMEGA GoTaq Green Master Mix 2X: <br><br />
<br />
Mix the following in a 0.2mL microcentrifuge tube on ice: <br><br />
<br />
25μL GoTaq® Green Master Mix 2X <br><br />
<br />
1-5μL of 10μM forward primer <br><br />
<br />
1-5μL of 10μM reverse primer <br><br />
<br />
<250ng of DNA template <br><br />
<br />
Nuclease-free water to 50μl <br><br />
<br />
Conduct the reaction in a Thermocycler, adjusting anneal temperature and extension times accordingly. See your polymerase supplier protocol for more details on thermocycling.<br />
<br />
</p><br />
<br />
<h3> Error-prone Polymerase Chain Reaction </h3><br />
<br />
<br />
<p> <br />
<br />
<br />
<br />
Prepare 50μL reaction: <br><br />
<br />
5μL 10X Mutazyme II Rxn Buffer <br><br />
<br />
1μL 40mM dNTP mix (200μM each final) <br><br />
<br />
1μL 20μM forward primer <br><br />
<br />
1μL 20μM reverse primer <br><br />
<br />
1μL Mutazyme II DNA polymerase (2.5U/μL) <br><br />
<br />
0.01ng template <br><br />
<br />
QS 50μL diH2O <br><br />
<br />
<br><br />
<br />
Thermocycler: <br><br />
<br />
95C, 2min <br><br />
<br />
95C, 30sec <br><br />
<br />
XXC*, 30sec <br><br />
<br />
72C, Xmin** <br><br />
<br />
32 cycles <br><br />
<br />
72C, 10min <br><br />
<br />
4C, hold<br />
<br />
<br><br />
<br />
*Adjust annealing temperature according to Tm of primer. <br><br />
<br />
**Adjust extension time according to the length of amplified DNA. <br><br />
<br />
<br><br />
<br />
Note: Use 0.01ng (calculate by insert and not by total plasmid). <br><br />
<br />
Calculate amount of template to use. <br><br />
<br />
(bp for amplified region) / (bp in total plasmid) = % amplified region <br><br />
<br />
(conc of total plasmid) x (% amplified region as a decimal) = conc of amplified region <br><br />
<br />
<br><br />
<br />
Note: Will probably need to dilute. Never pipette less than 0.5μL. <br><br />
<br />
(0.01ng) / (conc of amplified region) = vol to add to PCR <br><br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Restriction Endonuclease Reaction (Digestion) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
All restriction enzyme reactions were done using a 50ul reaction volume. <br><br />
<br />
Restriction enzymes and buffers were purchased from New England Biolabs Incorporated. <br><br />
<br />
Protocols for various New England Biolab restriction enzyme reactions: <br><br />
<br />
Mix the following in a 0.2mL PCR tube: <br><br />
<br />
1μL of each Restriction Enzyme, add the RE last <br><br />
<br />
1μg of DNA <br><br />
<br />
5μL of the appropriate 10X New Englan Biolab Buffer <br><br />
<br />
Nuclease-free water to 50μL <br><br />
<br />
Incubate the reaction for 1hr <br><br />
<br />
Heat inactive the the reaction at the appropriate temperature <br><br />
<br />
<br />
Notes: Add the restriction enzyme(s) to the reaction last <br><br />
<br />
Thaw the restriction enzyme(s) on ice to improve shelf life <br><br />
<br />
<br />
<br />
</p> <br />
<br />
<br />
<h3> Ligation </h3><br />
<br />
<br />
<p><br />
<br />
T4 DNA Ligase and Buffer was purchased from New England BioLabs Corporation. <br><br />
<br />
1. Prepare the following in a 0.2mL microcentrifuge tube: <br><br />
<br />
50.0ng Vector DNA* <br><br />
<br />
37.5ng Vector DNA* <br><br />
<br />
2μL 10X T4 DNA Ligase Buffer <br><br />
<br />
1μL T4 DNA Ligase <br><br />
<br />
Add diH2O to 20μL <br><br />
<br />
2. Incubate the reaction at room temperature for 10-30 minutes or at 16C overnight. <br><br />
<br />
3. Heat inactivate at 65C for 10 minutes. <br><br />
<br />
4. Chill on ice before starting a transformation reaction. <br><br />
<br />
*The exact amount of DNA is dependent on the number of base pairs. In order to conduct a proper reaction consult the New England Biolab Ligation Calculator at:<br />
<br />
http://nebiocalculator.neb.com/#!/<br />
<br />
</p><br />
<br />
<br />
<html><a name="Escherichia coli Protocols"></a><br />
<br />
</html><br />
<br />
<br />
<h2> <i> Escherichia coli Protocols </i> (XL1-Blue and XL10-Gold) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Culturing </h3><br />
<br />
<br />
<br />
<p> <br />
<br />
<br />
Chemically Competent Cell Stocks – CCMB Transformation <br />
For BL21*, CJ236, etc. Cell Lines. <br><br />
<br />
NOTE: Make SOB-Mg media and autoclave the 250ml flasks with water in them the same day the cells are plated (step 1) <br><br />
<br><br />
<br />
1. Plate 10ul of cells onto an antibiotic free LB/Agar plate..* <br><br />
<br />
2. Incubate overnight at 37OC <br><br />
<br />
3. Inoculate lots of colonies into 50ml SOB-Mg media in a 250ml flask. <br><br />
4. Incubate at 37 OC, 200rpm, until OD at 550nm reaches 0.3, takes about 3 hours. I do my first check at 2 hours.<br><br />
<br />
5.Transfer the cell culture into a sterile 50ml Falcon tube and chill on ice for 10 minutes. <br><br />
<br />
6. Pellet the cells at 2500rpm for 15 minutes at 4 OC.<br><br />
<br />
7. Decant supernatant and invert tubes to remove excess culture medium. <br><br />
<br />
8. Resuspend the cells in 16ml CCMB by gentle vortexing or pipetting (I shake the falcon tube).<br />
(16ml CCMB per 50ml culture) <br><br />
<br />
9. Incubate on ice for 20 minutes. <br><br />
<br />
10. Centrifuge at 2500rpm for 10 minutes at 4 OC. <br><br />
<br />
11. Decant supernatant and invert tubes to remove excess liquid. <br><br />
<br />
12. Resuspend the cells in 4ml CCMB by gentle vortexing or pipetting.<br />
(4ml CCMB per culture) <br><br />
<br />
13. If making multiple 50ml cultures, combine all of the cells and mix before aliquoting. <br> <br />
<br />
14. In cold room: make 205ul aliquots, flash freeze in liquid nitrogen, and store at -80 OC.<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Chemically Competent Cell Transformations </h3><br />
<br />
<br />
<p> <br />
<br />
1. Thaw competent <i> E.coli </i> cells on ice (XL1-Blue or XL10-Gold)* <br><br />
<br />
2. Add 50μL of competent cells to sterile 15mL conical centrifuge tubes <br><br />
<br />
3. Add 1μL (~100-200ng)* of the mini-prep to each culture tube <br><br />
<br />
4. Equilibrate the cells on ice for 10 min <br><br />
<br />
5. Heat shock the cells at 42C for 30-45 seconds** <br><br />
<br />
6. Immediately place the cells back on ice for 3 min <br><br />
<br />
7. Add 250μL LB media without antibiotics and shake at 250 rpm and 37C for 30 min <br><br />
<br />
8. Spread 10μL and 290μL on an appropriate LB-antibiotic plate <br><br />
<br />
9. Invert the plate and incubate at 37C overnight<br />
<br />
<br />
*The exact amount of DNA to add depends on your cell's transformation efficiency. However, it is acceptable to add a larger amount to increase the number of transformed cells. <br><br />
<br />
** Do not heat shock for an extended duration as this may damage and/or kill your cells.<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnights </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottom tube, add 3-5mL of LB and an appropriate volume of antibiotic(s). <br><br />
<br />
2. Swipe several individual colonies, do not collect satellites or colony clumps, with a pipette tip. <br><br />
<br />
3. Swirl the colony tip in the tube, there should be no visible cell clumps. <br><br />
<br />
4. Incubate and shake the tube at 37C at 250rpm for 12-16 hours and no longer than 20 hours. <br><br />
<br />
</p><br />
<br />
<br />
<h3> DNA-Extraction and mini-preps </h3><br />
<br />
<br />
<p><br />
<br />
All DNA mini-preps were prepared using EPOCH mini-kits and following the supplied protocols.<br />
<br />
</p><br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500μL of 40% Glycerol and 500uL of LB (no antibiotics) or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Saccharomyces cerevisiae"></a><br />
<br />
</html><br />
<br />
<h2> <i> Saccharomyces cerevisiae </i> (PYE1 Yeast) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Culturing </h3><br />
<br />
<br />
<br />
<p><br />
<br />
This process take 4 days in lab with a 1 day wait for incubation. <br><br />
<br />
<br />
Day 1: <br><br />
<br />
1. Streak yeast cells onto a YPD plate.* <br><br />
<br />
2. Invert the plate and incubate at 30C for 2 days. <br><br />
<br />
<br />
Day 3: <br><br />
<br />
1. Add 50mL of YPD liquid media into a 250mL baffle flask. <br><br />
<br />
2. Swipe as many individual colonies as you can see into the YPD media.** <br><br />
<br />
3. Incubate and shake the culture at 30C at 250rpm overnight approximately 24 hours. <br><br />
<br />
<br />
Day 4: <br><br />
<br />
1. Take an optical density measurement. <br><br />
<br />
2. In three 250mL baffle flask add the portions of the overnight liquid culture. <br><br />
<br />
3. Dilute each culture to approximately 0.4 optical density with YPD. <br><br />
<br />
4. Incubate and shake the cultures at 30C at 250rpm until the optical density reaches 1.2-1.6. <br><br />
<br />
5. Collect each culture into separate 50ml flat-bottomed centrifuge tubes. <br><br />
<br />
6. Spin down the cells at 4000g for 5 minutes at 4C. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cells in 100mL total for all three culture of dH2O. <br><br />
<br />
9. Combine the suspensions into two 50mL flat-bottomed centrifuge tubes. <br><br />
<br />
10. Spin down the cells as above. <br><br />
<br />
11. Decant the supernatant. <br><br />
<br />
12. Resuspend each in 3mL of 100mM Lithium Acetate. <br><br />
<br />
13. Transfer both cultures into a single 15mL conical centrifuge tube. <br><br />
<br />
14. Spin down the cells at 3000rpm for 5 minutes. <br><br />
<br />
15. Resuspend the cells in 0.75mL of 100mM Lithium Acetate, total volume is roughly 2mL. <br><br />
<br />
16. Qualitatively bring up the volume to 3.5mL by adding 40% Glycerol. <br><br />
<br />
17. Aliquot the cells into 1.5mL centrifuge tubes or 1.7mL cryogenic vials.*** <br><br />
<br />
<br />
*Streak in such a way that there are individual colonies visible on the plate without clumps or satellite colonies. <br><br />
<br />
**Collect only individual visible colonies. Do not collect clumps or satellite colonies. <br><br />
<br />
***The volume of aliquots depends on the number to transformations you intend to do at a time. <br><br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Chemically Competent Transformations </h3><br />
<br />
<br />
<p><br />
<br />
This protocol assumes a 50μL aliquot of yeast competent cells were made. <br><br />
<br />
Furthermore, this protocol prepares enough cells for 6 yeast transformations. <br><br />
<br />
<br><br />
<br />
1. Add the following to 50μL of yeast competent cells: <br><br />
<br />
240μL of Polyethylene Glycol - 3350 (PEG-3350) <br><br />
<br />
36μL of 1M Lithium Acetate <br><br />
<br />
32μL of dH2O <br><br />
<br />
<br />
<br />
2. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
3. Aliquot 59μL of the mixture into a 0.2mL microcentrifuge tube. <br><br />
<br />
4. Add 1uL (~100-200ng) of DNA. <br><br />
<br />
5. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
6. Incubate the mixture at 30C for 30 minutes. <br><br />
<br />
7. Heat shock the mixture at 42C for 20 minutes. <br><br />
<br />
8. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
9. Decant the supernatant. <br><br />
<br />
10. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
11. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
12. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
13. Plate 50-150μL of the mixture onto an appropriate Selective Dropout Media plate. <br><br />
<br />
14. Invert and incubate at 30C for 2 days. <br><br />
<br />
*The exact amount of DNA depends on the transformation efficiency of your competent cells. <br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnight Culturing </h3><br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Swipe 3 individually visible yeast colonies and add them to the culture tube media. <br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%.<br />
<br />
</p><br />
<br />
<br />
<h3> Culture Passaging </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Take 20-50μL from a previous overnight or passage culture and add it to the culture media.<br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%. <br><br />
<br />
Note: The exact amount of culture that you take from a previous culture is irrelevant as long as at least 1 living cell is passaged.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500uL of 40% glycerol and 500μL of Selective Dropout media or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Flow Cytometry"></a><br />
<br />
</html><br />
<br />
<h2> Flow Cytometry </h2><br />
<br />
<br />
<h3> Dilutions </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. From an overnight culture measure the optical density at 660nm by making 1:10 dilutions. <br><br />
<br />
2. Take enough culture to make a 1mL aliquot with OD 0.4. <br><br />
<br />
3. Spin down the aliquot in a 1.5mL centrifuge tube at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 800μL of the appropriate selective dropout media and 200μL of 20% glucose. <br><br />
<br />
6. Transfer the new culture to a 14mL culture tube. <br><br />
<br />
7. Incubate and shake at 30C and 250rpm for at least 6 hours (1.2-1.6 optical density).<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Preparations for Analysis using C6 Accuri Flow Cytometer </h3><br />
<br />
<br />
<p><br />
<br />
<br />
1. From the dilution previously made, measure the optical density, roughly 1.2-1.6. <br><br />
<br />
2. Make an aliquot of 500μL of the dilution culture in a 1.5mL centrifuge tube. <br><br />
<br />
3. Spin down the aliquot at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 500μL of PBS(F). <br><br />
<br />
6. Spin down the resuspension at 3000rpm for 3 minutes. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cell pellet in another 500uL of PBS(F).* <br><br />
<br />
9. Prepare the C6 Accuri Flow Cytometer by running a backflush cycle and a dH2O cycle. <br><br />
<br />
10. Load the sample onto the sip. <br><br />
<br />
11. Run the sample with 100,000 cell count. <br><br />
<br />
12. Repeat for all samples and make sure to change data cells otherwise the old data is erased. <br><br />
<br />
13. Once finished, run a cleaning cycle with Accuri approved cleaning solution, then run a dH2O cycle. <br><br />
<br />
<br />
<br />
*For special cases do not resuspend all samples, instead resuspend immediately before running the sample through the flow cytometer.<br />
<br />
<br />
</p><br />
<br />
<br />
<html><a name="Fluorescence Activated Cell Sorting"></a><br />
<br />
</html><br />
<br />
<h2> Fluorescence Activated Cell Sorting </h2><br />
<br />
<br />
<h3> Final Preparations </h3><br />
<br />
<br />
<br />
<p> <br />
<br />
Sample Prep:<br />
<br />
<br />
Spin down samples and negative control (5000 RPM, 1 min), keeping in mind the library size. Aspirate off supernatant. Resuspend in PBSF. Spin down cells. Aspirate off supernatant. Resuspend in PBSF. <br><br />
<br />
<br><br />
<br />
1. Open the “iGEM Template” file in the FACS Software and change name to current date and sort cycle<br><br />
<br />
2. Make sure the stream is stable (look for green light in bottom right corner). If not, run the Sort Calibration order. <br><br />
<br />
3. Run Negative Control from the PyE1 cells. <br><br />
<br />
a. Load cells onto carrier and into the machine. Press play button on screen. <br><br />
<br />
b. Set gate around lower left quadrant of cells to ensure single cell analysis using Forward Scatter Area and Side Scatter Area as your axes. Make sure oval gate covers around 80% of cell population. <br><br />
<br />
c. Set second gate on the first gated population by double-clicking on the gated population and using Forward Scatter Height and Forward Scatter Width as your axes. You will notice two distinct populations. Try to focus on the single cell portion of the plot. <br><br />
<br />
NOTE: If you see a large portion of the second gated population existing near the upper right edge of the first gate, you may need to enlarge the first gate to fit more of the population. <br><br />
<br />
d. Press record. Record 100,000 events and stop run. Move to Next Tube. <br><br />
<br />
4. Run first control (Gene clone) <br><br />
<br />
a. Follow step 3 to run the first control <br><br />
<br />
5. Run first library sample <br><br />
<br />
a. Follow steps 3b and 3c to set first two gates correctly. <br><br />
<br />
b. Set final gate for sort which includes top 1% of GFP producers from second gated population. <br><br />
<br />
c. Use final gate to set up the sort. <br><br />
<br />
d. Select sort conditions at the bottom of the screen. <br><br />
<br />
e. Insert and load collection tube. <br><br />
<br />
f. Record 100,000 events, and sort 10x the library size <br><br />
<br />
6. Run Bleach and diH2O through FACS to avoid cross-contamination. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Protein Expression"></a><br />
<br />
</html><br />
<br />
<h2> Protein Expression </h2><br />
<br />
<br />
<h3> Overnight Cultures </h3><br />
<br />
<p><br />
<br />
1. Add 25mL TB and 25uL Kan to a 250mL baffled flask <br><br />
<br />
2. Stab a glycerol stock with a p1000 pipette and swirl in the flask of media <br><br />
<br />
3. Put flask in 37C shaker overnight <br />
<br />
</p><br />
<br />
<h3> Protein Expression </h3><br />
<br />
<p> <br />
<br />
1. Add 500μL 1000x Kanamycin to 500mL TB in 2L baffled flask <br><br />
<br />
2. Transfer 10mL overnight culture to TB <br><br />
<br />
3. Shake at 37C (DO WE NEED THE RPM?) <br><br />
<br />
4. Remove flask from shaker when optical density is between 0.5 and 0.8 <br><br />
<br />
5. Allow flask to rest at room temp for 30 min <br><br />
<br />
6. Add 125μL 1M IPTG <br><br />
<br />
7. Shake flask at 18C overnight<br />
<br />
</p><br />
<br />
<h3> Protein Extraction and Purification </h3><br />
<br />
<p><br />
<br />
1. Transfer cell culture to centrifuge flask <br><br />
<br />
2. Centrifuge culture at 4000g for 10 min <br><br />
<br />
3. Discard supernatant <br><br />
<br />
4. Resuspend pellet in 25mL lysis buffer <br><br />
<br />
5. Add 250μL of 100x PMSF, 250μL of 100mg/mL lysozyme, and 250μL of 10mg/mL DNAse <br><br />
<br />
6. Sonicate sample with 0.25inch probe for 5 min at 70% amplitude with 20 sec on and off pulses <br><br />
<br />
7. Take 50μL total sample <br><br />
<br />
8. Transfer lysate to SS-34 centrifuge tube <br><br />
<br />
9. Centrifuge for 30 min at 18000g <br><br />
<br />
10. Take 50μL soluble sample<br />
<br />
</p><br />
<br />
<h3> Nickel Nitrotriacetic Acid Chromatography (Nickel-NTA Chromatography) </h3><br />
<br />
<p><br />
<br />
1. Add 5mL 50%(v/v) nickel resin in ethanol to 25mL column and allow to settle to ~2.5mL(CV) <br><br />
<br />
2. Rinse with 10CV H20 <br><br />
<br />
3. Equilibrate with 10CV lysis buffer <br><br />
<br />
4. Load sample onto column <br><br />
<br />
5. Wash column with 15CV lysis buffer <br><br />
<br />
6. Perform 2 additional wash steps with 15CV <br><br />
<br />
7. Elute sample in 10CV elution buffer and collect eluate <br><br />
<br />
8. Take 50μL pure sample <br />
<br />
</p><br />
<br />
<h3> Size Exclusion Chromatography (S.E.C.) </h3><br />
<p><br />
1. Concentrate sample to as high as possible without inducing protein aggregation<br><br />
2. Pre-equilibrate Superdex 75 column with 48 ml PBS <br><br />
3. Inject 500 ul sample onto column <br><br />
4. Run 36 ml PBS through column at 0.5 ml/min, collecting 1 ml fractions <br><br />
5. Verify presence of protein in fractions by measuring concentration on Nanodrop and running SDS-PAGE (15 kDa protein should elute at ~13 ml)<br><br />
6. Pool fractions containing protein <br />
</p><br />
<br />
<br />
<html><a name="Stability Analysis"></a><br />
<br />
</html><br />
<br />
<h2> Stability Analysis </h2><br />
<br />
<br />
<h3> Circular Dichroism: Wavelength Scan </h3><br />
<p><br />
1. Load 1mm cuvette with 400uL protein solution onto CD <br><br />
2. Take wavelength scan <br><br />
260nm-190nm <br><br />
sample every 1nm <br><br />
averaging time 3sec <br><br />
1 scan <br><br />
step scan <br><br />
25C <br><br />
3. Record wavelength which gives strongest signal (222nm)<br />
</p><br />
<br />
<h3> Circular Dichroism: Guanidine Melt </h3><br />
<p><br />
1. Load 1cm cuvette containing 1.996mL of 0.05mg/mL protein solution and stirrer onto CD <br><br />
2. Prepare 8mL of 0.05mg/mL protein in concentrated guanidine solution <br><br />
3. Set up Automixer with guanidine solution on one syringe and waste tube on other syringe <br><br />
4. Titrate up to 6 M guanidine, taking a CD measurement at 222 nm every 0.15 M interval <br><br />
5. Also measure the fluorescence at 280 nm to ensure the total protein concentration is not changing<br />
</p><br />
<br />
<br />
<br />
<html><br />
<br />
<a href="#linktotop">Back To Top</a><br />
</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2014-10-17T17:33:14Z<p>Kristanguyen: </p>
<hr />
<div>{{Template:Team:UW/CSS}}<br />
<br />
<br />
<h1> Protocols </h1><br />
<br />
<br />
<html><a name="Media, Plates, and Sol"></a><br />
<br />
</html><br />
<br />
<br />
<h2> Media, Plates, and Solutions </h2><br />
<br />
<br />
<h3> Competent Cell Media Buffer (CCMB) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
100 g glycerol (liquid) <br><br />
<br />
10 mL x 1 M potassium acetate <br><br />
<br />
11.8 g CaCl2*H2O <br><br />
<br />
4 g MnCl2 <br><br />
<br />
2 g MgCl2 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<h3> Super Optimal Broth (SOB) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
20 g Tryptone <br><br />
<br />
5 g Yeast Extract <br><br />
<br />
10 mL x 1 M NaCl <br><br />
<br />
2.5 mL x 1 M KCl <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Phosphate Buffered Saline (PBS) Solution </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container or 1 L beaker: <br><br />
<br />
8 g NaCl <br><br />
<br />
1.44 g Na2HPO4 <br><br />
<br />
0.8 g KCl <br><br />
<br />
0.24 g KH2PO4 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Buffer to pH 7.4 <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<h3>PBSF (PBS for Flow)</h3><br />
<br />
</p><br />
Mix the following in a 1 L beaker: <br><br />
<br />
25 mL 20X PBS, pH 7.4 <br><br />
<br />
475 mL H2O <br><br />
<br />
2.5 g BSA (0.5%)* <br> <br />
<br />
Filter and store at 4 °C <br><br />
<br />
<br><br />
*Do not need to pH - should be at pH 7.4 like 20X PBS<br><br />
<br />
</p><br />
<br />
<br />
<h3> Yeast Extract Peptone Dextrose (YPD) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following into 950mL of dH2O in a 1L bottle: <br><br />
<br />
20g Bacto Peptone <br><br />
<br />
10g Yeast Extract <br><br />
<br />
Autoclave (20min at 121C and 20psi) <br><br />
<br />
Add 50mL 40% Glucose <br><br />
<br />
Sterile filter into a 1L bottle <br><br />
<br />
<br />
<br />
For long-term liquid media storage, do not add 40% Glucose instead add the glucose directly into cell cultures. <br><br />
<br />
For YPD-plates add 24g Bacto Agar to the Bacto Peptone and Yeast Extract before autoclaving. <br />
<br />
</p><br />
<br />
<br />
<h3> Selective Dropout media, C-Uracil and C-Histidine (C-Ura and C-His) </h3><br />
<br />
<br />
<p><br />
<br />
Synthesized by the Yeast Resource Center at the University of Washington's <br />
<br />
Department of Genome Sciences and Department of Biochemistry.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<h3> Guanidinium Hydrogen Chloride </h3><br />
<br />
<br />
<p><br />
<br />
For maximum effectiveness, final concentration should be approximately 8.5M in PBS<br><br />
<br />
203g Guanidinium Hydrogen Chloride <br><br />
<br />
250mL PBS solution <br><br />
<br />
Add dilute HCl to 7.4pH <br><br />
<br />
<br />
*Alternatively add slightly less than 250mL of PBS in order to buffer the solution to the appropriate volume then add more dH2O as neccessary. <br />
<br />
</p><br />
<br />
<html><a name="Basic Cloning"></a><br />
<br />
</html><br />
<br />
<h2> Basic Cloning </h2><br />
<br />
<br />
<br />
<h3> Polymerase Chain Reaction </h3><br />
<br />
<br />
<p><br />
<br />
All PCRs were done using a standard 50μL reaction volume. <br><br />
<br />
PCRs were done using GoTaq Green Master Mix 2X purchased from PROMEGA Corporation. <br><br />
<br />
Protocols for the PROMEGA GoTaq Green Master Mix 2X: <br><br />
<br />
Mix the following in a 0.2mL microcentrifuge tube on ice: <br><br />
<br />
25μL GoTaq® Green Master Mix 2X <br><br />
<br />
1-5μL of 10μM forward primer <br><br />
<br />
1-5μL of 10μM reverse primer <br><br />
<br />
<250ng of DNA template <br><br />
<br />
Nuclease-free water to 50μl <br><br />
<br />
Conduct the reaction in a Thermocycler, adjusting anneal temperature and extension times accordingly. See your polymerase supplier protocol for more details on thermocycling.<br />
<br />
</p><br />
<br />
<h3> Error-prone Polymerase Chain Reaction </h3><br />
<br />
<br />
<p> <br />
<br />
<br />
<br />
Prepare 50μL reaction: <br><br />
<br />
5μL 10X Mutazyme II Rxn Buffer <br><br />
<br />
1μL 40mM dNTP mix (200μM each final) <br><br />
<br />
1μL 20μM forward primer <br><br />
<br />
1μL 20μM reverse primer <br><br />
<br />
1μL Mutazyme II DNA polymerase (2.5U/μL) <br><br />
<br />
0.01ng template <br><br />
<br />
QS 50μL diH2O <br><br />
<br />
<br><br />
<br />
Thermocycler: <br><br />
<br />
95C, 2min <br><br />
<br />
95C, 30sec <br><br />
<br />
XXC*, 30sec <br><br />
<br />
72C, Xmin** <br><br />
<br />
32 cycles <br><br />
<br />
72C, 10min <br><br />
<br />
4C, hold<br />
<br />
<br><br />
<br />
*Adjust annealing temperature according to Tm of primer. <br><br />
<br />
**Adjust extension time according to the length of amplified DNA. <br><br />
<br />
<br><br />
<br />
Note: Use 0.01ng (calculate by insert and not by total plasmid). <br><br />
<br />
Calculate amount of template to use. <br><br />
<br />
(bp for amplified region) / (bp in total plasmid) = % amplified region <br><br />
<br />
(conc of total plasmid) x (% amplified region as a decimal) = conc of amplified region <br><br />
<br />
<br><br />
<br />
Note: Will probably need to dilute. Never pipette less than 0.5μL. <br><br />
<br />
(0.01ng) / (conc of amplified region) = vol to add to PCR <br><br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Restriction Endonuclease Reaction (Digestion) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
All restriction enzyme reactions were done using a 50ul reaction volume. <br><br />
<br />
Restriction enzymes and buffers were purchased from New England Biolabs Incorporated. <br><br />
<br />
Protocols for various New England Biolab restriction enzyme reactions: <br><br />
<br />
Mix the following in a 0.2mL PCR tube: <br><br />
<br />
1μL of each Restriction Enzyme, add the RE last <br><br />
<br />
1μg of DNA <br><br />
<br />
5μL of the appropriate 10X New Englan Biolab Buffer <br><br />
<br />
Nuclease-free water to 50μL <br><br />
<br />
Incubate the reaction for 1hr <br><br />
<br />
Heat inactive the the reaction at the appropriate temperature <br><br />
<br />
<br />
Notes: Add the restriction enzyme(s) to the reaction last <br><br />
<br />
Thaw the restriction enzyme(s) on ice to improve shelf life <br><br />
<br />
<br />
<br />
</p> <br />
<br />
<br />
<h3> Ligation </h3><br />
<br />
<br />
<p><br />
<br />
T4 DNA Ligase and Buffer was purchased from New England BioLabs Corporation. <br><br />
<br />
1. Prepare the following in a 0.2mL microcentrifuge tube: <br><br />
<br />
50.0ng Vector DNA* <br><br />
<br />
37.5ng Vector DNA* <br><br />
<br />
2μL 10X T4 DNA Ligase Buffer <br><br />
<br />
1μL T4 DNA Ligase <br><br />
<br />
Add diH2O to 20μL <br><br />
<br />
2. Incubate the reaction at room temperature for 10-30 minutes or at 16C overnight. <br><br />
<br />
3. Heat inactivate at 65C for 10 minutes. <br><br />
<br />
4. Chill on ice before starting a transformation reaction. <br><br />
<br />
*The exact amount of DNA is dependent on the number of base pairs. In order to conduct a proper reaction consult the New England Biolab Ligation Calculator at:<br />
<br />
http://nebiocalculator.neb.com/#!/<br />
<br />
</p><br />
<br />
<br />
<html><a name="Escherichia coli Protocols"></a><br />
<br />
</html><br />
<br />
<br />
<h2> <i> Escherichia coli Protocols </i> (XL1-Blue and XL10-Gold) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Culturing </h3><br />
<br />
<br />
<br />
<p> <br />
<br />
<br />
Chemically Competent Cell Stocks – CCMB Transformation <br />
For BL21*, CJ236, etc. Cell Lines. <br><br />
<br />
NOTE: Make SOB-Mg media and autoclave the 250ml flasks with water in them the same day the cells are plated (step 1) <br><br />
<br><br />
<br />
1. Plate 10ul of cells onto an antibiotic free LB/Agar plate..* <br><br />
<br />
2. Incubate overnight at 37OC <br><br />
<br />
3. Inoculate lots of colonies into 50ml SOB-Mg media in a 250ml flask. <br><br />
4. Incubate at 37 OC, 200rpm, until OD at 550nm reaches 0.3, takes about 3 hours. I do my first check at 2 hours.<br><br />
<br />
5.Transfer the cell culture into a sterile 50ml Falcon tube and chill on ice for 10 minutes. <br><br />
<br />
6. Pellet the cells at 2500rpm for 15 minutes at 4 OC.<br><br />
<br />
7. Decant supernatant and invert tubes to remove excess culture medium. <br><br />
<br />
8. Resuspend the cells in 16ml CCMB by gentle vortexing or pipetting (I shake the falcon tube).<br />
(16ml CCMB per 50ml culture) <br><br />
<br />
9. Incubate on ice for 20 minutes. <br><br />
<br />
10. Centrifuge at 2500rpm for 10 minutes at 4 OC. <br><br />
<br />
11. Decant supernatant and invert tubes to remove excess liquid. <br><br />
<br />
12. Resuspend the cells in 4ml CCMB by gentle vortexing or pipetting.<br />
(4ml CCMB per culture) <br><br />
<br />
13. If making multiple 50ml cultures, combine all of the cells and mix before aliquoting. <br> <br />
<br />
14. In cold room: make 205ul aliquots, flash freeze in liquid nitrogen, and store at -80 OC.<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Chemically Competent Cell Transformations </h3><br />
<br />
<br />
<p> <br />
<br />
1. Thaw competent <i> E.coli </i> cells on ice (XL1-Blue or XL10-Gold)* <br><br />
<br />
2. Add 50μL of competent cells to sterile 15mL conical centrifuge tubes <br><br />
<br />
3. Add 1μL (~100-200ng)* of the mini-prep to each culture tube <br><br />
<br />
4. Equilibrate the cells on ice for 10 min <br><br />
<br />
5. Heat shock the cells at 42C for 30-45 seconds** <br><br />
<br />
6. Immediately place the cells back on ice for 3 min <br><br />
<br />
7. Add 250μL LB media without antibiotics and shake at 250 rpm and 37C for 30 min <br><br />
<br />
8. Spread 10μL and 290μL on an appropriate LB-antibiotic plate <br><br />
<br />
9. Invert the plate and incubate at 37C overnight<br />
<br />
<br />
*The exact amount of DNA to add depends on your cell's transformation efficiency. However, it is acceptable to add a larger amount to increase the number of transformed cells. <br><br />
<br />
** Do not heat shock for an extended duration as this may damage and/or kill your cells.<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnights </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottom tube, add 3-5mL of LB and an appropriate volume of antibiotic(s). <br><br />
<br />
2. Swipe several individual colonies, do not collect satellites or colony clumps, with a pipette tip. <br><br />
<br />
3. Swirl the colony tip in the tube, there should be no visible cell clumps. <br><br />
<br />
4. Incubate and shake the tube at 37C at 250rpm for 12-16 hours and no longer than 20 hours. <br><br />
<br />
</p><br />
<br />
<br />
<h3> DNA-Extraction and mini-preps </h3><br />
<br />
<br />
<p><br />
<br />
All DNA mini-preps were prepared using EPOCH mini-kits and following the supplied protocols.<br />
<br />
</p><br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500μL of 40% Glycerol and 500uL of LB (no antibiotics) or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Saccharomyces cerevisiae"></a><br />
<br />
</html><br />
<br />
<h2> <i> Saccharomyces cerevisiae </i> (PYE1 Yeast) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Culturing </h3><br />
<br />
<br />
<br />
<p><br />
<br />
This process take 4 days in lab with a 1 day wait for incubation. <br><br />
<br />
<br />
Day 1: <br><br />
<br />
1. Streak yeast cells onto a YPD plate.* <br><br />
<br />
2. Invert the plate and incubate at 30C for 2 days. <br><br />
<br />
<br />
Day 3: <br><br />
<br />
1. Add 50mL of YPD liquid media into a 250mL baffle flask. <br><br />
<br />
2. Swipe as many individual colonies as you can see into the YPD media.** <br><br />
<br />
3. Incubate and shake the culture at 30C at 250rpm overnight approximately 24 hours. <br><br />
<br />
<br />
Day 4: <br><br />
<br />
1. Take an optical density measurement. <br><br />
<br />
2. In three 250mL baffle flask add the portions of the overnight liquid culture. <br><br />
<br />
3. Dilute each culture to approximately 0.4 optical density with YPD. <br><br />
<br />
4. Incubate and shake the cultures at 30C at 250rpm until the optical density reaches 1.2-1.6. <br><br />
<br />
5. Collect each culture into separate 50ml flat-bottomed centrifuge tubes. <br><br />
<br />
6. Spin down the cells at 4000g for 5 minutes at 4C. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cells in 100mL total for all three culture of dH2O. <br><br />
<br />
9. Combine the suspensions into two 50mL flat-bottomed centrifuge tubes. <br><br />
<br />
10. Spin down the cells as above. <br><br />
<br />
11. Decant the supernatant. <br><br />
<br />
12. Resuspend each in 3mL of 100mM Lithium Acetate. <br><br />
<br />
13. Transfer both cultures into a single 15mL conical centrifuge tube. <br><br />
<br />
14. Spin down the cells at 3000rpm for 5 minutes. <br><br />
<br />
15. Resuspend the cells in 0.75mL of 100mM Lithium Acetate, total volume is roughly 2mL. <br><br />
<br />
16. Qualitatively bring up the volume to 3.5mL by adding 40% Glycerol. <br><br />
<br />
17. Aliquot the cells into 1.5mL centrifuge tubes or 1.7mL cryogenic vials.*** <br><br />
<br />
<br />
*Streak in such a way that there are individual colonies visible on the plate without clumps or satellite colonies. <br><br />
<br />
**Collect only individual visible colonies. Do not collect clumps or satellite colonies. <br><br />
<br />
***The volume of aliquots depends on the number to transformations you intend to do at a time. <br><br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Chemically Competent Transformations </h3><br />
<br />
<br />
<p><br />
<br />
This protocol assumes a 50μL aliquot of yeast competent cells were made. <br><br />
<br />
Furthermore, this protocol prepares enough cells for 6 yeast transformations. <br><br />
<br />
<br><br />
<br />
1. Add the following to 50μL of yeast competent cells: <br><br />
<br />
240μL of Polyethylene Glycol - 3350 (PEG-3350) <br><br />
<br />
36μL of 1M Lithium Acetate <br><br />
<br />
32μL of dH2O <br><br />
<br />
<br />
<br />
2. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
3. Aliquot 59μL of the mixture into a 0.2mL microcentrifuge tube. <br><br />
<br />
4. Add 1uL (~100-200ng) of DNA. <br><br />
<br />
5. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
6. Incubate the mixture at 30C for 30 minutes. <br><br />
<br />
7. Heat shock the mixture at 42C for 20 minutes. <br><br />
<br />
8. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
9. Decant the supernatant. <br><br />
<br />
10. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
11. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
12. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
13. Plate 50-150μL of the mixture onto an appropriate Selective Dropout Media plate. <br><br />
<br />
14. Invert and incubate at 30C for 2 days. <br><br />
<br />
*The exact amount of DNA depends on the transformation efficiency of your competent cells. <br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnight Culturing </h3><br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Swipe 3 individually visible yeast colonies and add them to the culture tube media. <br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%.<br />
<br />
</p><br />
<br />
<br />
<h3> Culture Passaging </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Take 20-50μL from a previous overnight or passage culture and add it to the culture media.<br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%. <br><br />
<br />
Note: The exact amount of culture that you take from a previous culture is irrelevant as long as at least 1 living cell is passaged.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500uL of 40% glycerol and 500μL of Selective Dropout media or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Flow Cytometry"></a><br />
<br />
</html><br />
<br />
<h2> Flow Cytometry </h2><br />
<br />
<br />
<h3> Dilutions </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. From an overnight culture measure the optical density at 660nm by making 1:10 dilutions. <br><br />
<br />
2. Take enough culture to make a 1mL aliquot with OD 0.4. <br><br />
<br />
3. Spin down the aliquot in a 1.5mL centrifuge tube at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 800μL of the appropriate selective dropout media and 200μL of 20% glucose. <br><br />
<br />
6. Transfer the new culture to a 14mL culture tube. <br><br />
<br />
7. Incubate and shake at 30C and 250rpm for at least 6 hours (1.2-1.6 optical density).<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Preparations for Analysis using C6 Accuri Flow Cytometer </h3><br />
<br />
<br />
<p><br />
<br />
<br />
1. From the dilution previously made, measure the optical density, roughly 1.2-1.6. <br><br />
<br />
2. Make an aliquot of 500μL of the dilution culture in a 1.5mL centrifuge tube. <br><br />
<br />
3. Spin down the aliquot at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 500μL of PBS(F). <br><br />
<br />
6. Spin down the resuspension at 3000rpm for 3 minutes. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cell pellet in another 500uL of PBS(F).* <br><br />
<br />
9. Prepare the C6 Accuri Flow Cytometer by running a backflush cycle and a dH2O cycle. <br><br />
<br />
10. Load the sample onto the sip. <br><br />
<br />
11. Run the sample with 100,000 cell count. <br><br />
<br />
12. Repeat for all samples and make sure to change data cells otherwise the old data is erased. <br><br />
<br />
13. Once finished, run a cleaning cycle with Accuri approved cleaning solution, then run a dH2O cycle. <br><br />
<br />
<br />
<br />
*For special cases do not resuspend all samples, instead resuspend immediately before running the sample through the flow cytometer.<br />
<br />
<br />
</p><br />
<br />
<br />
<html><a name="Fluorescence Activated Cell Sorting"></a><br />
<br />
</html><br />
<br />
<h2> Fluorescence Activated Cell Sorting </h2><br />
<br />
<br />
<h3> Final Preparations </h3><br />
<br />
<br />
<br />
<p> <br />
<br />
Sample Prep:<br />
<br />
<br />
Spin down samples and negative control (5000 RPM, 1 min), keeping in mind the library size. Aspirate off supernatant. Resuspend in PBSF. Spin down cells. Aspirate off supernatant. Resuspend in PBSF. <br><br />
<br />
<br><br />
<br />
1. Open the “iGEM Template” file in the FACS Software and change name to current date and sort cycle<br><br />
<br />
2. Make sure the stream is stable (look for green light in bottom right corner). If not, run the Sort Calibration order. <br><br />
<br />
3. Run Negative Control from the PyE1 cells. <br><br />
<br />
a. Load cells onto carrier and into the machine. Press play button on screen. <br><br />
<br />
b. Set gate around lower left quadrant of cells to ensure single cell analysis using Forward Scatter Area and Side Scatter Area as your axes. Make sure oval gate covers around 80% of cell population. <br><br />
<br />
c. Set second gate on the first gated population by double-clicking on the gated population and using Forward Scatter Height and Forward Scatter Width as your axes. You will notice two distinct populations. Try to focus on the single cell portion of the plot. <br><br />
<br />
NOTE: If you see a large portion of the second gated population existing near the upper right edge of the first gate, you may need to enlarge the first gate to fit more of the population. <br><br />
<br />
d. Press record. Record 100,000 events and stop run. Move to Next Tube. <br><br />
<br />
4. Run first control (Gene clone) <br><br />
<br />
a. Follow step 3 to run the first control <br><br />
<br />
5. Run first library sample <br><br />
<br />
a. Follow steps 3b and 3c to set first two gates correctly. <br><br />
<br />
b. Set final gate for sort which includes top 1% of GFP producers from second gated population. <br><br />
<br />
c. Use final gate to set up the sort. <br><br />
<br />
d. Select sort conditions at the bottom of the screen. <br><br />
<br />
e. Insert and load collection tube. <br><br />
<br />
f. Record 100,000 events, and sort 10x the library size <br><br />
<br />
6. Run Bleach and diH2O through FACS to avoid cross-contamination. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Protein Expression"></a><br />
<br />
</html><br />
<br />
<h2> Protein Expression </h2><br />
<br />
<br />
<h3> Overnight Cultures </h3><br />
<br />
<p><br />
<br />
1. Add 25mL TB and 25uL Kan to a 250mL baffled flask <br><br />
<br />
2. Stab a glycerol stock with a p1000 pipette and swirl in the flask of media <br><br />
<br />
3. Put flask in 37C shaker overnight <br />
<br />
</p><br />
<br />
<h3> Protein Expression </h3><br />
<br />
<p> <br />
<br />
1. Add 500μL 1000x Kanamycin to 500mL TB in 2L baffled flask <br><br />
<br />
2. Transfer 10mL overnight culture to TB <br><br />
<br />
3. Shake at 37C (DO WE NEED THE RPM?) <br><br />
<br />
4. Remove flask from shaker when optical density is between 0.5 and 0.8 <br><br />
<br />
5. Allow flask to rest at room temp for 30 min <br><br />
<br />
6. Add 125μL 1M IPTG <br><br />
<br />
7. Shake flask at 18C overnight<br />
<br />
</p><br />
<br />
<h3> Protein Extraction and Purification </h3><br />
<br />
<p><br />
<br />
1. Transfer cell culture to centrifuge flask <br><br />
<br />
2. Centrifuge culture at 4000g for 10 min <br><br />
<br />
3. Discard supernatant <br><br />
<br />
4. Resuspend pellet in 25mL lysis buffer <br><br />
<br />
5. Add 250μL of 100x PMSF, 250μL of 100mg/mL lysozyme, and 250μL of 10mg/mL DNAse <br><br />
<br />
6. Sonicate sample with 0.25inch probe for 5 min at 70% amplitude with 20 sec on and off pulses <br><br />
<br />
7. Take 50μL total sample <br><br />
<br />
8. Transfer lysate to SS-34 centrifuge tube <br><br />
<br />
9. Centrifuge for 30 min at 18000g <br><br />
<br />
10. Take 50μL soluble sample<br />
<br />
</p><br />
<br />
<h3> Nickel Nitrotriacetic Acid Chromatography (Nickel-NTA Chromatography) </h3><br />
<br />
<p><br />
<br />
1. Add 5mL 50%(v/v) nickel resin in ethanol to 25mL column and allow to settle to ~2.5mL(CV) <br><br />
<br />
2. Rinse with 10CV H20 <br><br />
<br />
3. Equilibrate with 10CV lysis buffer <br><br />
<br />
4. Load sample onto column <br><br />
<br />
5. Wash column with 15CV lysis buffer <br><br />
<br />
6. Perform 2 additional wash steps with 15CV <br><br />
<br />
7. Elute sample in 10CV elution buffer and collect eluate <br><br />
<br />
8. Take 50μL pure sample <br />
<br />
</p><br />
<br />
<h3> Size Exclusion Chromatography (S.E.C.) </h3><br />
<p><br />
1. Concentrate sample to as high as possible without inducing protein aggregation<br><br />
2. Pre-equilibrate Superdex 75 column with 48 ml PBS <br><br />
3. Inject 500 ul sample onto column <br><br />
4. Run 36 ml PBS through column at 0.5 ml/min, collecting 1 ml fractions <br><br />
5. Verify presence of protein in fractions by measuring concentration on Nanodrop and running SDS-PAGE (15 kDa protein should elute at ~13 ml)<br><br />
6. Pool fractions containing protein <br />
</p><br />
<br />
<br />
<html><a name="Stability Analysis"></a><br />
<br />
</html><br />
<br />
<h2> Stability Analysis </h2><br />
<br />
<br />
<h3> Circular Dichroism: Wavelength Scan </h3><br />
<p><br />
1. Load 1mm cuvette with 400uL protein solution onto CD <br><br />
2. Take wavelength scan <br><br />
260nm-190nm <br><br />
sample every 1nm <br><br />
averaging time 3sec <br><br />
1 scan <br><br />
step scan <br><br />
25C <br><br />
3. Record wavelength which gives strongest signal (222nm)<br />
</p><br />
<br />
<h3> Circular Dichroism: Guanidine Melt </h3><br />
<p><br />
1. Load 1cm cuvette containing 1.996mL of 0.05mg/mL protein solution and stirrer onto CD <br><br />
2. Prepare 8mL of 0.05mg/mL protein in concentrated guanidine solution <br><br />
3. Set up Automixer with guanidine solution on one syringe and waste tube on other syringe <br><br />
4. Titrate up to 6 M guanidine, taking a CD measurement at 222 nm every 0.15 M interval <br><br />
5. Also measure the fluorescence at 280 nm to ensure the total protein concentration is not changing<br />
</p><br />
<br />
<br />
<br />
<html><br />
<br />
<a href="#linktotop">Back To Top</a><br />
</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2014-10-17T17:29:15Z<p>Kristanguyen: </p>
<hr />
<div>{{Template:Team:UW/CSS}}<br />
<br />
<br />
<h1> Protocols </h1><br />
<br />
<br />
<html><a name="Media, Plates, and Sol"></a><br />
<br />
</html><br />
<br />
<br />
<h2> Media, Plates, and Solutions </h2><br />
<br />
<br />
<h3> Competent Cell Media Buffer (CCMB) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
100 g glycerol (liquid) <br><br />
<br />
10 mL x 1 M potassium acetate <br><br />
<br />
11.8 g CaCl2*H2O <br><br />
<br />
4 g MnCl2 <br><br />
<br />
2 g MgCl2 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121 °C and 20 psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Luria Broth (LB) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2L container: <br><br />
<br />
10g tryptone <br><br />
<br />
5g yeast extract <br><br />
<br />
10g NaCl <br> <br />
<br />
1L of dH2O <br><br />
<br />
Autoclave in two 500 ml bottle (20 min at 121C and 20psi) <br><br />
<br />
<br />
*If using antibiotics create a separate aliquot<br />
<br />
</p><br />
<br />
<br />
<h3> LB-Agar </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2L container: <br><br />
<br />
1000ml LB as above <br><br />
<br />
15g agar <br><br />
<br />
1L of dH2O <br><br />
<br />
Autoclave in two 500mL bottles (20 min at 121C at 20psi) <br> <br />
<br />
<br />
<br />
*If using antibiotics create a separate aliquot<br />
<br />
<br />
</p><br />
<br />
<br />
<h3> Super Optimal Broth (SOB) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2L container: <br><br />
<br />
20g BactoTryptone <br><br />
<br />
5g BactoYeast Extract <br><br />
<br />
10mL x 1M NaCl <br><br />
<br />
2.5mL x 1M KCl <br><br />
<br />
1L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121C and 20psi) in a 1L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Phosphate Buffered Saline (PBS) Solution </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2L container or 1L beaker: <br><br />
<br />
8g NaCl <br><br />
<br />
1.44g Na2HPO4 <br><br />
<br />
0.8g KCl <br><br />
<br />
0.24g KH2PO4 <br><br />
<br />
1L of dH2O <br><br />
<br />
Buffer to pH 7.4 <br><br />
<br />
Sterile filter or autoclave (20 min at 121C and 20psi) in a 1L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Terrific Broth (TB) </h3><br />
<br />
<br />
<p><br />
<br />
<br />
<br />
Mix the following in a 1L bottle: <br><br />
<br />
6g Tryptone <br><br />
<br />
12g Yeast Extract <br><br />
<br />
2mL Glycerol <br><br />
<br />
500mL of dH2O <br><br />
<br />
Autoclave (20min at 121C and 20psi) <br><br />
<br />
Cool and add 5mL of 100X Potassium Phosphate Salts (17mM KH2PO4 and 72mM K2HPO4)<br />
<br />
</p> <br />
<br />
<br />
<h3> Yeast Extract Peptone Dextrose (YPD) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following into 950mL of dH2O in a 1L bottle: <br><br />
<br />
20g Bacto Peptone <br><br />
<br />
10g Yeast Extract <br><br />
<br />
Autoclave (20min at 121C and 20psi) <br><br />
<br />
Add 50mL 40% Glucose <br><br />
<br />
Sterile filter into a 1L bottle <br><br />
<br />
<br />
<br />
For long-term liquid media storage, do not add 40% Glucose instead add the glucose directly into cell cultures. <br><br />
<br />
For YPD-plates add 24g Bacto Agar to the Bacto Peptone and Yeast Extract before autoclaving. <br />
<br />
</p><br />
<br />
<br />
<h3> Selective Dropout media, C-Uracil and C-Histidine (C-Ura and C-His) </h3><br />
<br />
<br />
<p><br />
<br />
Synthesized by the Yeast Resource Center at the University of Washington's <br />
<br />
Department of Genome Sciences and Department of Biochemistry.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<h3> Guanidinium Hydrogen Chloride </h3><br />
<br />
<br />
<p><br />
<br />
For maximum effectiveness, final concentration should be approximately 8.5M in PBS<br><br />
<br />
203g Guanidinium Hydrogen Chloride <br><br />
<br />
250mL PBS solution <br><br />
<br />
Add dilute HCl to 7.4pH <br><br />
<br />
<br />
*Alternatively add slightly less than 250mL of PBS in order to buffer the solution to the appropriate volume then add more dH2O as neccessary. <br />
<br />
</p><br />
<br />
<html><a name="Basic Cloning"></a><br />
<br />
</html><br />
<br />
<h2> Basic Cloning </h2><br />
<br />
<br />
<br />
<h3> Polymerase Chain Reaction </h3><br />
<br />
<br />
<p><br />
<br />
All PCRs were done using a standard 50μL reaction volume. <br><br />
<br />
PCRs were done using GoTaq Green Master Mix 2X purchased from PROMEGA Corporation. <br><br />
<br />
Protocols for the PROMEGA GoTaq Green Master Mix 2X: <br><br />
<br />
Mix the following in a 0.2mL microcentrifuge tube on ice: <br><br />
<br />
25μL GoTaq® Green Master Mix 2X <br><br />
<br />
1-5μL of 10μM forward primer <br><br />
<br />
1-5μL of 10μM reverse primer <br><br />
<br />
<250ng of DNA template <br><br />
<br />
Nuclease-free water to 50μl <br><br />
<br />
Conduct the reaction in a Thermocycler, adjusting anneal temperature and extension times accordingly. See your polymerase supplier protocol for more details on thermocycling.<br />
<br />
</p><br />
<br />
<h3> Error-prone Polymerase Chain Reaction </h3><br />
<br />
<br />
<p> <br />
<br />
<br />
<br />
Prepare 50μL reaction: <br><br />
<br />
5μL 10X Mutazyme II Rxn Buffer <br><br />
<br />
1μL 40mM dNTP mix (200μM each final) <br><br />
<br />
1μL 20μM forward primer <br><br />
<br />
1μL 20μM reverse primer <br><br />
<br />
1μL Mutazyme II DNA polymerase (2.5U/μL) <br><br />
<br />
0.01ng template <br><br />
<br />
QS 50μL diH2O <br><br />
<br />
<br><br />
<br />
Thermocycler: <br><br />
<br />
95C, 2min <br><br />
<br />
95C, 30sec <br><br />
<br />
XXC*, 30sec <br><br />
<br />
72C, Xmin** <br><br />
<br />
32 cycles <br><br />
<br />
72C, 10min <br><br />
<br />
4C, hold<br />
<br />
<br><br />
<br />
*Adjust annealing temperature according to Tm of primer. <br><br />
<br />
**Adjust extension time according to the length of amplified DNA. <br><br />
<br />
<br><br />
<br />
Note: Use 0.01ng (calculate by insert and not by total plasmid). <br><br />
<br />
Calculate amount of template to use. <br><br />
<br />
(bp for amplified region) / (bp in total plasmid) = % amplified region <br><br />
<br />
(conc of total plasmid) x (% amplified region as a decimal) = conc of amplified region <br><br />
<br />
<br><br />
<br />
Note: Will probably need to dilute. Never pipette less than 0.5μL. <br><br />
<br />
(0.01ng) / (conc of amplified region) = vol to add to PCR <br><br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Restriction Endonuclease Reaction (Digestion) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
All restriction enzyme reactions were done using a 50ul reaction volume. <br><br />
<br />
Restriction enzymes and buffers were purchased from New England Biolabs Incorporated. <br><br />
<br />
Protocols for various New England Biolab restriction enzyme reactions: <br><br />
<br />
Mix the following in a 0.2mL PCR tube: <br><br />
<br />
1μL of each Restriction Enzyme, add the RE last <br><br />
<br />
1μg of DNA <br><br />
<br />
5μL of the appropriate 10X New Englan Biolab Buffer <br><br />
<br />
Nuclease-free water to 50μL <br><br />
<br />
Incubate the reaction for 1hr <br><br />
<br />
Heat inactive the the reaction at the appropriate temperature <br><br />
<br />
<br />
Notes: Add the restriction enzyme(s) to the reaction last <br><br />
<br />
Thaw the restriction enzyme(s) on ice to improve shelf life <br><br />
<br />
<br />
<br />
</p> <br />
<br />
<br />
<h3> Ligation </h3><br />
<br />
<br />
<p><br />
<br />
T4 DNA Ligase and Buffer was purchased from New England BioLabs Corporation. <br><br />
<br />
1. Prepare the following in a 0.2mL microcentrifuge tube: <br><br />
<br />
50.0ng Vector DNA* <br><br />
<br />
37.5ng Vector DNA* <br><br />
<br />
2μL 10X T4 DNA Ligase Buffer <br><br />
<br />
1μL T4 DNA Ligase <br><br />
<br />
Add diH2O to 20μL <br><br />
<br />
2. Incubate the reaction at room temperature for 10-30 minutes or at 16C overnight. <br><br />
<br />
3. Heat inactivate at 65C for 10 minutes. <br><br />
<br />
4. Chill on ice before starting a transformation reaction. <br><br />
<br />
*The exact amount of DNA is dependent on the number of base pairs. In order to conduct a proper reaction consult the New England Biolab Ligation Calculator at:<br />
<br />
http://nebiocalculator.neb.com/#!/<br />
<br />
</p><br />
<br />
<br />
<html><a name="Escherichia coli Protocols"></a><br />
<br />
</html><br />
<br />
<br />
<h2> <i> Escherichia coli Protocols </i> (XL1-Blue and XL10-Gold) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Culturing </h3><br />
<br />
<br />
<br />
<p> <br />
<br />
<br />
Chemically Competent Cell Stocks – CCMB Transformation <br />
For BL21*, CJ236, etc. Cell Lines. <br><br />
<br />
NOTE: Make SOB-Mg media and autoclave the 250ml flasks with water in them the same day the cells are plated (step 1) <br><br />
<br><br />
<br />
1. Plate 10ul of cells onto an antibiotic free LB/Agar plate..* <br><br />
<br />
2. Incubate overnight at 37OC <br><br />
<br />
3. Inoculate lots of colonies into 50ml SOB-Mg media in a 250ml flask. <br><br />
4. Incubate at 37 OC, 200rpm, until OD at 550nm reaches 0.3, takes about 3 hours. I do my first check at 2 hours.<br><br />
<br />
5.Transfer the cell culture into a sterile 50ml Falcon tube and chill on ice for 10 minutes. <br><br />
<br />
6. Pellet the cells at 2500rpm for 15 minutes at 4 OC.<br><br />
<br />
7. Decant supernatant and invert tubes to remove excess culture medium. <br><br />
<br />
8. Resuspend the cells in 16ml CCMB by gentle vortexing or pipetting (I shake the falcon tube).<br />
(16ml CCMB per 50ml culture) <br><br />
<br />
9. Incubate on ice for 20 minutes. <br><br />
<br />
10. Centrifuge at 2500rpm for 10 minutes at 4 OC. <br><br />
<br />
11. Decant supernatant and invert tubes to remove excess liquid. <br><br />
<br />
12. Resuspend the cells in 4ml CCMB by gentle vortexing or pipetting.<br />
(4ml CCMB per culture) <br><br />
<br />
13. If making multiple 50ml cultures, combine all of the cells and mix before aliquoting. <br> <br />
<br />
14. In cold room: make 205ul aliquots, flash freeze in liquid nitrogen, and store at -80 OC.<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Chemically Competent Cell Transformations </h3><br />
<br />
<br />
<p> <br />
<br />
1. Thaw competent <i> E.coli </i> cells on ice (XL1-Blue or XL10-Gold)* <br><br />
<br />
2. Add 50μL of competent cells to sterile 15mL conical centrifuge tubes <br><br />
<br />
3. Add 1μL (~100-200ng)* of the mini-prep to each culture tube <br><br />
<br />
4. Equilibrate the cells on ice for 10 min <br><br />
<br />
5. Heat shock the cells at 42C for 30-45 seconds** <br><br />
<br />
6. Immediately place the cells back on ice for 3 min <br><br />
<br />
7. Add 250μL LB media without antibiotics and shake at 250 rpm and 37C for 30 min <br><br />
<br />
8. Spread 10μL and 290μL on an appropriate LB-antibiotic plate <br><br />
<br />
9. Invert the plate and incubate at 37C overnight<br />
<br />
<br />
*The exact amount of DNA to add depends on your cell's transformation efficiency. However, it is acceptable to add a larger amount to increase the number of transformed cells. <br><br />
<br />
** Do not heat shock for an extended duration as this may damage and/or kill your cells.<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnights </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottom tube, add 3-5mL of LB and an appropriate volume of antibiotic(s). <br><br />
<br />
2. Swipe several individual colonies, do not collect satellites or colony clumps, with a pipette tip. <br><br />
<br />
3. Swirl the colony tip in the tube, there should be no visible cell clumps. <br><br />
<br />
4. Incubate and shake the tube at 37C at 250rpm for 12-16 hours and no longer than 20 hours. <br><br />
<br />
</p><br />
<br />
<br />
<h3> DNA-Extraction and mini-preps </h3><br />
<br />
<br />
<p><br />
<br />
All DNA mini-preps were prepared using EPOCH mini-kits and following the supplied protocols.<br />
<br />
</p><br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500μL of 40% Glycerol and 500uL of LB (no antibiotics) or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Saccharomyces cerevisiae"></a><br />
<br />
</html><br />
<br />
<h2> <i> Saccharomyces cerevisiae </i> (PYE1 Yeast) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Culturing </h3><br />
<br />
<br />
<br />
<p><br />
<br />
This process take 4 days in lab with a 1 day wait for incubation. <br><br />
<br />
<br />
Day 1: <br><br />
<br />
1. Streak yeast cells onto a YPD plate.* <br><br />
<br />
2. Invert the plate and incubate at 30C for 2 days. <br><br />
<br />
<br />
Day 3: <br><br />
<br />
1. Add 50mL of YPD liquid media into a 250mL baffle flask. <br><br />
<br />
2. Swipe as many individual colonies as you can see into the YPD media.** <br><br />
<br />
3. Incubate and shake the culture at 30C at 250rpm overnight approximately 24 hours. <br><br />
<br />
<br />
Day 4: <br><br />
<br />
1. Take an optical density measurement. <br><br />
<br />
2. In three 250mL baffle flask add the portions of the overnight liquid culture. <br><br />
<br />
3. Dilute each culture to approximately 0.4 optical density with YPD. <br><br />
<br />
4. Incubate and shake the cultures at 30C at 250rpm until the optical density reaches 1.2-1.6. <br><br />
<br />
5. Collect each culture into separate 50ml flat-bottomed centrifuge tubes. <br><br />
<br />
6. Spin down the cells at 4000g for 5 minutes at 4C. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cells in 100mL total for all three culture of dH2O. <br><br />
<br />
9. Combine the suspensions into two 50mL flat-bottomed centrifuge tubes. <br><br />
<br />
10. Spin down the cells as above. <br><br />
<br />
11. Decant the supernatant. <br><br />
<br />
12. Resuspend each in 3mL of 100mM Lithium Acetate. <br><br />
<br />
13. Transfer both cultures into a single 15mL conical centrifuge tube. <br><br />
<br />
14. Spin down the cells at 3000rpm for 5 minutes. <br><br />
<br />
15. Resuspend the cells in 0.75mL of 100mM Lithium Acetate, total volume is roughly 2mL. <br><br />
<br />
16. Qualitatively bring up the volume to 3.5mL by adding 40% Glycerol. <br><br />
<br />
17. Aliquot the cells into 1.5mL centrifuge tubes or 1.7mL cryogenic vials.*** <br><br />
<br />
<br />
*Streak in such a way that there are individual colonies visible on the plate without clumps or satellite colonies. <br><br />
<br />
**Collect only individual visible colonies. Do not collect clumps or satellite colonies. <br><br />
<br />
***The volume of aliquots depends on the number to transformations you intend to do at a time. <br><br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Chemically Competent Transformations </h3><br />
<br />
<br />
<p><br />
<br />
This protocol assumes a 50μL aliquot of yeast competent cells were made. <br><br />
<br />
Furthermore, this protocol prepares enough cells for 6 yeast transformations. <br><br />
<br />
<br><br />
<br />
1. Add the following to 50μL of yeast competent cells: <br><br />
<br />
240μL of Polyethylene Glycol - 3350 (PEG-3350) <br><br />
<br />
36μL of 1M Lithium Acetate <br><br />
<br />
32μL of dH2O <br><br />
<br />
<br />
<br />
2. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
3. Aliquot 59μL of the mixture into a 0.2mL microcentrifuge tube. <br><br />
<br />
4. Add 1uL (~100-200ng) of DNA. <br><br />
<br />
5. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
6. Incubate the mixture at 30C for 30 minutes. <br><br />
<br />
7. Heat shock the mixture at 42C for 20 minutes. <br><br />
<br />
8. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
9. Decant the supernatant. <br><br />
<br />
10. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
11. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
12. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
13. Plate 50-150μL of the mixture onto an appropriate Selective Dropout Media plate. <br><br />
<br />
14. Invert and incubate at 30C for 2 days. <br><br />
<br />
*The exact amount of DNA depends on the transformation efficiency of your competent cells. <br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnight Culturing </h3><br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Swipe 3 individually visible yeast colonies and add them to the culture tube media. <br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%.<br />
<br />
</p><br />
<br />
<br />
<h3> Culture Passaging </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Take 20-50μL from a previous overnight or passage culture and add it to the culture media.<br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%. <br><br />
<br />
Note: The exact amount of culture that you take from a previous culture is irrelevant as long as at least 1 living cell is passaged.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500uL of 40% glycerol and 500μL of Selective Dropout media or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Flow Cytometry"></a><br />
<br />
</html><br />
<br />
<h2> Flow Cytometry </h2><br />
<br />
<br />
<h3> Dilutions </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. From an overnight culture measure the optical density at 660nm by making 1:10 dilutions. <br><br />
<br />
2. Take enough culture to make a 1mL aliquot with OD 0.4. <br><br />
<br />
3. Spin down the aliquot in a 1.5mL centrifuge tube at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 800μL of the appropriate selective dropout media and 200μL of 20% glucose. <br><br />
<br />
6. Transfer the new culture to a 14mL culture tube. <br><br />
<br />
7. Incubate and shake at 30C and 250rpm for at least 6 hours (1.2-1.6 optical density).<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Preparations for Analysis using C6 Accuri Flow Cytometer </h3><br />
<br />
<br />
<p><br />
<br />
<br />
1. From the dilution previously made, measure the optical density, roughly 1.2-1.6. <br><br />
<br />
2. Make an aliquot of 500μL of the dilution culture in a 1.5mL centrifuge tube. <br><br />
<br />
3. Spin down the aliquot at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 500μL of PBS(F). <br><br />
<br />
6. Spin down the resuspension at 3000rpm for 3 minutes. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cell pellet in another 500uL of PBS(F).* <br><br />
<br />
9. Prepare the C6 Accuri Flow Cytometer by running a backflush cycle and a dH2O cycle. <br><br />
<br />
10. Load the sample onto the sip. <br><br />
<br />
11. Run the sample with 100,000 cell count. <br><br />
<br />
12. Repeat for all samples and make sure to change data cells otherwise the old data is erased. <br><br />
<br />
13. Once finished, run a cleaning cycle with Accuri approved cleaning solution, then run a dH2O cycle. <br><br />
<br />
<br />
<br />
*For special cases do not resuspend all samples, instead resuspend immediately before running the sample through the flow cytometer.<br />
<br />
<br />
</p><br />
<br />
<br />
<html><a name="Fluorescence Activated Cell Sorting"></a><br />
<br />
</html><br />
<br />
<h2> Fluorescence Activated Cell Sorting </h2><br />
<br />
<br />
<h3> Final Preparations </h3><br />
<br />
<br />
<br />
<p> <br />
<br />
Sample Prep:<br />
<br />
<br />
Spin down samples and negative control (5000 RPM, 1 min), keeping in mind the library size. Aspirate off supernatant. Resuspend in PBSF. Spin down cells. Aspirate off supernatant. Resuspend in PBSF. <br><br />
<br />
<br><br />
<br />
1. Open the “iGEM Template” file in the FACS Software and change name to current date and sort cycle<br><br />
<br />
2. Make sure the stream is stable (look for green light in bottom right corner). If not, run the Sort Calibration order. <br><br />
<br />
3. Run Negative Control from the PyE1 cells. <br><br />
<br />
a. Load cells onto carrier and into the machine. Press play button on screen. <br><br />
<br />
b. Set gate around lower left quadrant of cells to ensure single cell analysis using Forward Scatter Area and Side Scatter Area as your axes. Make sure oval gate covers around 80% of cell population. <br><br />
<br />
c. Set second gate on the first gated population by double-clicking on the gated population and using Forward Scatter Height and Forward Scatter Width as your axes. You will notice two distinct populations. Try to focus on the single cell portion of the plot. <br><br />
<br />
NOTE: If you see a large portion of the second gated population existing near the upper right edge of the first gate, you may need to enlarge the first gate to fit more of the population. <br><br />
<br />
d. Press record. Record 100,000 events and stop run. Move to Next Tube. <br><br />
<br />
4. Run first control (Gene clone) <br><br />
<br />
a. Follow step 3 to run the first control <br><br />
<br />
5. Run first library sample <br><br />
<br />
a. Follow steps 3b and 3c to set first two gates correctly. <br><br />
<br />
b. Set final gate for sort which includes top 1% of GFP producers from second gated population. <br><br />
<br />
c. Use final gate to set up the sort. <br><br />
<br />
d. Select sort conditions at the bottom of the screen. <br><br />
<br />
e. Insert and load collection tube. <br><br />
<br />
f. Record 100,000 events, and sort 10x the library size <br><br />
<br />
6. Run Bleach and diH2O through FACS to avoid cross-contamination. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Protein Expression"></a><br />
<br />
</html><br />
<br />
<h2> Protein Expression </h2><br />
<br />
<br />
<h3> Overnight Cultures </h3><br />
<br />
<p><br />
<br />
1. Add 25mL TB and 25uL Kan to a 250mL baffled flask <br><br />
<br />
2. Stab a glycerol stock with a p1000 pipette and swirl in the flask of media <br><br />
<br />
3. Put flask in 37C shaker overnight <br />
<br />
</p><br />
<br />
<h3> Protein Expression </h3><br />
<br />
<p> <br />
<br />
1. Add 500μL 1000x Kanamycin to 500mL TB in 2L baffled flask <br><br />
<br />
2. Transfer 10mL overnight culture to TB <br><br />
<br />
3. Shake at 37C (DO WE NEED THE RPM?) <br><br />
<br />
4. Remove flask from shaker when optical density is between 0.5 and 0.8 <br><br />
<br />
5. Allow flask to rest at room temp for 30 min <br><br />
<br />
6. Add 125μL 1M IPTG <br><br />
<br />
7. Shake flask at 18C overnight<br />
<br />
</p><br />
<br />
<h3> Protein Extraction and Purification </h3><br />
<br />
<p><br />
<br />
1. Transfer cell culture to centrifuge flask <br><br />
<br />
2. Centrifuge culture at 4000g for 10 min <br><br />
<br />
3. Discard supernatant <br><br />
<br />
4. Resuspend pellet in 25mL lysis buffer <br><br />
<br />
5. Add 250μL of 100x PMSF, 250μL of 100mg/mL lysozyme, and 250μL of 10mg/mL DNAse <br><br />
<br />
6. Sonicate sample with 0.25inch probe for 5 min at 70% amplitude with 20 sec on and off pulses <br><br />
<br />
7. Take 50μL total sample <br><br />
<br />
8. Transfer lysate to SS-34 centrifuge tube <br><br />
<br />
9. Centrifuge for 30 min at 18000g <br><br />
<br />
10. Take 50μL soluble sample<br />
<br />
</p><br />
<br />
<h3> Nickel Nitrotriacetic Acid Chromatography (Nickel-NTA Chromatography) </h3><br />
<br />
<p><br />
<br />
1. Add 5mL 50%(v/v) nickel resin in ethanol to 25mL column and allow to settle to ~2.5mL(CV) <br><br />
<br />
2. Rinse with 10CV H20 <br><br />
<br />
3. Equilibrate with 10CV lysis buffer <br><br />
<br />
4. Load sample onto column <br><br />
<br />
5. Wash column with 15CV lysis buffer <br><br />
<br />
6. Perform 2 additional wash steps with 15CV <br><br />
<br />
7. Elute sample in 10CV elution buffer and collect eluate <br><br />
<br />
8. Take 50μL pure sample <br />
<br />
</p><br />
<br />
<h3> Size Exclusion Chromatography (S.E.C.) </h3><br />
<p><br />
1. Concentrate sample to as high as possible without inducing protein aggregation<br><br />
2. Pre-equilibrate Superdex 75 column with 48 ml PBS <br><br />
3. Inject 500 ul sample onto column <br><br />
4. Run 36 ml PBS through column at 0.5 ml/min, collecting 1 ml fractions <br><br />
5. Verify presence of protein in fractions by measuring concentration on Nanodrop and running SDS-PAGE (15 kDa protein should elute at ~13 ml)<br><br />
6. Pool fractions containing protein <br />
</p><br />
<br />
<br />
<html><a name="Stability Analysis"></a><br />
<br />
</html><br />
<br />
<h2> Stability Analysis </h2><br />
<br />
<br />
<h3> Circular Dichroism: Wavelength Scan </h3><br />
<p><br />
1. Load 1mm cuvette with 400uL protein solution onto CD <br><br />
2. Take wavelength scan <br><br />
260nm-190nm <br><br />
sample every 1nm <br><br />
averaging time 3sec <br><br />
1 scan <br><br />
step scan <br><br />
25C <br><br />
3. Record wavelength which gives strongest signal (222nm)<br />
</p><br />
<br />
<h3> Circular Dichroism: Guanidine Melt </h3><br />
<p><br />
1. Load 1cm cuvette containing 1.996mL of 0.05mg/mL protein solution and stirrer onto CD <br><br />
2. Prepare 8mL of 0.05mg/mL protein in concentrated guanidine solution <br><br />
3. Set up Automixer with guanidine solution on one syringe and waste tube on other syringe <br><br />
4. Titrate up to 6 M guanidine, taking a CD measurement at 222 nm every 0.15 M interval <br><br />
5. Also measure the fluorescence at 280 nm to ensure the total protein concentration is not changing<br />
</p><br />
<br />
<br />
<br />
<html><br />
<br />
<a href="#linktotop">Back To Top</a><br />
</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2014-10-17T17:28:10Z<p>Kristanguyen: </p>
<hr />
<div>{{Template:Team:UW/CSS}}<br />
<br />
<br />
<h1> Protocols </h1><br />
<br />
<br />
<html><a name="Media, Plates, and Sol"></a><br />
<br />
</html><br />
<br />
<br />
<h2> Media, Plates, and Solutions </h2><br />
<br />
<br />
<h3> 40% and 20% Glucose </h3><br />
<br />
<br />
<p><br />
<br />
40g for 40% or 20g for 20% of Glucose <br><br />
<br />
Mix in 100mL dH2O <br><br />
<br />
Sterile filter into a 150mL bottle <br><br />
<br />
</p><br />
<br />
<br />
<h3> 20% Glycerol </h3><br />
<br />
<br />
<p><br />
<br />
20g Glycerol (Liquid) <br><br />
<br />
Mix in 100mL dH2O <br><br />
<br />
Sterile filter into a 150mL bottle <br><br />
<br />
</p><br />
<br />
<br />
<h3> Competent Cell Media Buffer (CCMB) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
100 g glycerol (liquid) <br><br />
<br />
10 mL x 1 M potassium acetate <br><br />
<br />
11.8 g CaCl2*H2O <br><br />
<br />
4 g MnCl2 <br><br />
<br />
2 g MgCl2 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20min at 121 °C and 20psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Luria Broth (LB) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2L container: <br><br />
<br />
10g tryptone <br><br />
<br />
5g yeast extract <br><br />
<br />
10g NaCl <br> <br />
<br />
1L of dH2O <br><br />
<br />
Autoclave in two 500 ml bottle (20 min at 121C and 20psi) <br><br />
<br />
<br />
*If using antibiotics create a separate aliquot<br />
<br />
</p><br />
<br />
<br />
<h3> LB-Agar </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2L container: <br><br />
<br />
1000ml LB as above <br><br />
<br />
15g agar <br><br />
<br />
1L of dH2O <br><br />
<br />
Autoclave in two 500mL bottles (20 min at 121C at 20psi) <br> <br />
<br />
<br />
<br />
*If using antibiotics create a separate aliquot<br />
<br />
<br />
</p><br />
<br />
<br />
<h3> Super Optimal Broth (SOB) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2L container: <br><br />
<br />
20g BactoTryptone <br><br />
<br />
5g BactoYeast Extract <br><br />
<br />
10mL x 1M NaCl <br><br />
<br />
2.5mL x 1M KCl <br><br />
<br />
1L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121C and 20psi) in a 1L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Phosphate Buffered Saline (PBS) Solution </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2L container or 1L beaker: <br><br />
<br />
8g NaCl <br><br />
<br />
1.44g Na2HPO4 <br><br />
<br />
0.8g KCl <br><br />
<br />
0.24g KH2PO4 <br><br />
<br />
1L of dH2O <br><br />
<br />
Buffer to pH 7.4 <br><br />
<br />
Sterile filter or autoclave (20 min at 121C and 20psi) in a 1L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Terrific Broth (TB) </h3><br />
<br />
<br />
<p><br />
<br />
<br />
<br />
Mix the following in a 1L bottle: <br><br />
<br />
6g Tryptone <br><br />
<br />
12g Yeast Extract <br><br />
<br />
2mL Glycerol <br><br />
<br />
500mL of dH2O <br><br />
<br />
Autoclave (20min at 121C and 20psi) <br><br />
<br />
Cool and add 5mL of 100X Potassium Phosphate Salts (17mM KH2PO4 and 72mM K2HPO4)<br />
<br />
</p> <br />
<br />
<br />
<h3> Yeast Extract Peptone Dextrose (YPD) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following into 950mL of dH2O in a 1L bottle: <br><br />
<br />
20g Bacto Peptone <br><br />
<br />
10g Yeast Extract <br><br />
<br />
Autoclave (20min at 121C and 20psi) <br><br />
<br />
Add 50mL 40% Glucose <br><br />
<br />
Sterile filter into a 1L bottle <br><br />
<br />
<br />
<br />
For long-term liquid media storage, do not add 40% Glucose instead add the glucose directly into cell cultures. <br><br />
<br />
For YPD-plates add 24g Bacto Agar to the Bacto Peptone and Yeast Extract before autoclaving. <br />
<br />
</p><br />
<br />
<br />
<h3> Selective Dropout media, C-Uracil and C-Histidine (C-Ura and C-His) </h3><br />
<br />
<br />
<p><br />
<br />
Synthesized by the Yeast Resource Center at the University of Washington's <br />
<br />
Department of Genome Sciences and Department of Biochemistry.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<h3> Guanidinium Hydrogen Chloride </h3><br />
<br />
<br />
<p><br />
<br />
For maximum effectiveness, final concentration should be approximately 8.5M in PBS<br><br />
<br />
203g Guanidinium Hydrogen Chloride <br><br />
<br />
250mL PBS solution <br><br />
<br />
Add dilute HCl to 7.4pH <br><br />
<br />
<br />
*Alternatively add slightly less than 250mL of PBS in order to buffer the solution to the appropriate volume then add more dH2O as neccessary. <br />
<br />
</p><br />
<br />
<html><a name="Basic Cloning"></a><br />
<br />
</html><br />
<br />
<h2> Basic Cloning </h2><br />
<br />
<br />
<br />
<h3> Polymerase Chain Reaction </h3><br />
<br />
<br />
<p><br />
<br />
All PCRs were done using a standard 50μL reaction volume. <br><br />
<br />
PCRs were done using GoTaq Green Master Mix 2X purchased from PROMEGA Corporation. <br><br />
<br />
Protocols for the PROMEGA GoTaq Green Master Mix 2X: <br><br />
<br />
Mix the following in a 0.2mL microcentrifuge tube on ice: <br><br />
<br />
25μL GoTaq® Green Master Mix 2X <br><br />
<br />
1-5μL of 10μM forward primer <br><br />
<br />
1-5μL of 10μM reverse primer <br><br />
<br />
<250ng of DNA template <br><br />
<br />
Nuclease-free water to 50μl <br><br />
<br />
Conduct the reaction in a Thermocycler, adjusting anneal temperature and extension times accordingly. See your polymerase supplier protocol for more details on thermocycling.<br />
<br />
</p><br />
<br />
<h3> Error-prone Polymerase Chain Reaction </h3><br />
<br />
<br />
<p> <br />
<br />
<br />
<br />
Prepare 50μL reaction: <br><br />
<br />
5μL 10X Mutazyme II Rxn Buffer <br><br />
<br />
1μL 40mM dNTP mix (200μM each final) <br><br />
<br />
1μL 20μM forward primer <br><br />
<br />
1μL 20μM reverse primer <br><br />
<br />
1μL Mutazyme II DNA polymerase (2.5U/μL) <br><br />
<br />
0.01ng template <br><br />
<br />
QS 50μL diH2O <br><br />
<br />
<br><br />
<br />
Thermocycler: <br><br />
<br />
95C, 2min <br><br />
<br />
95C, 30sec <br><br />
<br />
XXC*, 30sec <br><br />
<br />
72C, Xmin** <br><br />
<br />
32 cycles <br><br />
<br />
72C, 10min <br><br />
<br />
4C, hold<br />
<br />
<br><br />
<br />
*Adjust annealing temperature according to Tm of primer. <br><br />
<br />
**Adjust extension time according to the length of amplified DNA. <br><br />
<br />
<br><br />
<br />
Note: Use 0.01ng (calculate by insert and not by total plasmid). <br><br />
<br />
Calculate amount of template to use. <br><br />
<br />
(bp for amplified region) / (bp in total plasmid) = % amplified region <br><br />
<br />
(conc of total plasmid) x (% amplified region as a decimal) = conc of amplified region <br><br />
<br />
<br><br />
<br />
Note: Will probably need to dilute. Never pipette less than 0.5μL. <br><br />
<br />
(0.01ng) / (conc of amplified region) = vol to add to PCR <br><br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Restriction Endonuclease Reaction (Digestion) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
All restriction enzyme reactions were done using a 50ul reaction volume. <br><br />
<br />
Restriction enzymes and buffers were purchased from New England Biolabs Incorporated. <br><br />
<br />
Protocols for various New England Biolab restriction enzyme reactions: <br><br />
<br />
Mix the following in a 0.2mL PCR tube: <br><br />
<br />
1μL of each Restriction Enzyme, add the RE last <br><br />
<br />
1μg of DNA <br><br />
<br />
5μL of the appropriate 10X New Englan Biolab Buffer <br><br />
<br />
Nuclease-free water to 50μL <br><br />
<br />
Incubate the reaction for 1hr <br><br />
<br />
Heat inactive the the reaction at the appropriate temperature <br><br />
<br />
<br />
Notes: Add the restriction enzyme(s) to the reaction last <br><br />
<br />
Thaw the restriction enzyme(s) on ice to improve shelf life <br><br />
<br />
<br />
<br />
</p> <br />
<br />
<br />
<h3> Ligation </h3><br />
<br />
<br />
<p><br />
<br />
T4 DNA Ligase and Buffer was purchased from New England BioLabs Corporation. <br><br />
<br />
1. Prepare the following in a 0.2mL microcentrifuge tube: <br><br />
<br />
50.0ng Vector DNA* <br><br />
<br />
37.5ng Vector DNA* <br><br />
<br />
2μL 10X T4 DNA Ligase Buffer <br><br />
<br />
1μL T4 DNA Ligase <br><br />
<br />
Add diH2O to 20μL <br><br />
<br />
2. Incubate the reaction at room temperature for 10-30 minutes or at 16C overnight. <br><br />
<br />
3. Heat inactivate at 65C for 10 minutes. <br><br />
<br />
4. Chill on ice before starting a transformation reaction. <br><br />
<br />
*The exact amount of DNA is dependent on the number of base pairs. In order to conduct a proper reaction consult the New England Biolab Ligation Calculator at:<br />
<br />
http://nebiocalculator.neb.com/#!/<br />
<br />
</p><br />
<br />
<br />
<html><a name="Escherichia coli Protocols"></a><br />
<br />
</html><br />
<br />
<br />
<h2> <i> Escherichia coli Protocols </i> (XL1-Blue and XL10-Gold) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Culturing </h3><br />
<br />
<br />
<br />
<p> <br />
<br />
<br />
Chemically Competent Cell Stocks – CCMB Transformation <br />
For BL21*, CJ236, etc. Cell Lines. <br><br />
<br />
NOTE: Make SOB-Mg media and autoclave the 250ml flasks with water in them the same day the cells are plated (step 1) <br><br />
<br><br />
<br />
1. Plate 10ul of cells onto an antibiotic free LB/Agar plate..* <br><br />
<br />
2. Incubate overnight at 37OC <br><br />
<br />
3. Inoculate lots of colonies into 50ml SOB-Mg media in a 250ml flask. <br><br />
4. Incubate at 37 OC, 200rpm, until OD at 550nm reaches 0.3, takes about 3 hours. I do my first check at 2 hours.<br><br />
<br />
5.Transfer the cell culture into a sterile 50ml Falcon tube and chill on ice for 10 minutes. <br><br />
<br />
6. Pellet the cells at 2500rpm for 15 minutes at 4 OC.<br><br />
<br />
7. Decant supernatant and invert tubes to remove excess culture medium. <br><br />
<br />
8. Resuspend the cells in 16ml CCMB by gentle vortexing or pipetting (I shake the falcon tube).<br />
(16ml CCMB per 50ml culture) <br><br />
<br />
9. Incubate on ice for 20 minutes. <br><br />
<br />
10. Centrifuge at 2500rpm for 10 minutes at 4 OC. <br><br />
<br />
11. Decant supernatant and invert tubes to remove excess liquid. <br><br />
<br />
12. Resuspend the cells in 4ml CCMB by gentle vortexing or pipetting.<br />
(4ml CCMB per culture) <br><br />
<br />
13. If making multiple 50ml cultures, combine all of the cells and mix before aliquoting. <br> <br />
<br />
14. In cold room: make 205ul aliquots, flash freeze in liquid nitrogen, and store at -80 OC.<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Chemically Competent Cell Transformations </h3><br />
<br />
<br />
<p> <br />
<br />
1. Thaw competent <i> E.coli </i> cells on ice (XL1-Blue or XL10-Gold)* <br><br />
<br />
2. Add 50μL of competent cells to sterile 15mL conical centrifuge tubes <br><br />
<br />
3. Add 1μL (~100-200ng)* of the mini-prep to each culture tube <br><br />
<br />
4. Equilibrate the cells on ice for 10 min <br><br />
<br />
5. Heat shock the cells at 42C for 30-45 seconds** <br><br />
<br />
6. Immediately place the cells back on ice for 3 min <br><br />
<br />
7. Add 250μL LB media without antibiotics and shake at 250 rpm and 37C for 30 min <br><br />
<br />
8. Spread 10μL and 290μL on an appropriate LB-antibiotic plate <br><br />
<br />
9. Invert the plate and incubate at 37C overnight<br />
<br />
<br />
*The exact amount of DNA to add depends on your cell's transformation efficiency. However, it is acceptable to add a larger amount to increase the number of transformed cells. <br><br />
<br />
** Do not heat shock for an extended duration as this may damage and/or kill your cells.<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnights </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottom tube, add 3-5mL of LB and an appropriate volume of antibiotic(s). <br><br />
<br />
2. Swipe several individual colonies, do not collect satellites or colony clumps, with a pipette tip. <br><br />
<br />
3. Swirl the colony tip in the tube, there should be no visible cell clumps. <br><br />
<br />
4. Incubate and shake the tube at 37C at 250rpm for 12-16 hours and no longer than 20 hours. <br><br />
<br />
</p><br />
<br />
<br />
<h3> DNA-Extraction and mini-preps </h3><br />
<br />
<br />
<p><br />
<br />
All DNA mini-preps were prepared using EPOCH mini-kits and following the supplied protocols.<br />
<br />
</p><br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500μL of 40% Glycerol and 500uL of LB (no antibiotics) or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Saccharomyces cerevisiae"></a><br />
<br />
</html><br />
<br />
<h2> <i> Saccharomyces cerevisiae </i> (PYE1 Yeast) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Culturing </h3><br />
<br />
<br />
<br />
<p><br />
<br />
This process take 4 days in lab with a 1 day wait for incubation. <br><br />
<br />
<br />
Day 1: <br><br />
<br />
1. Streak yeast cells onto a YPD plate.* <br><br />
<br />
2. Invert the plate and incubate at 30C for 2 days. <br><br />
<br />
<br />
Day 3: <br><br />
<br />
1. Add 50mL of YPD liquid media into a 250mL baffle flask. <br><br />
<br />
2. Swipe as many individual colonies as you can see into the YPD media.** <br><br />
<br />
3. Incubate and shake the culture at 30C at 250rpm overnight approximately 24 hours. <br><br />
<br />
<br />
Day 4: <br><br />
<br />
1. Take an optical density measurement. <br><br />
<br />
2. In three 250mL baffle flask add the portions of the overnight liquid culture. <br><br />
<br />
3. Dilute each culture to approximately 0.4 optical density with YPD. <br><br />
<br />
4. Incubate and shake the cultures at 30C at 250rpm until the optical density reaches 1.2-1.6. <br><br />
<br />
5. Collect each culture into separate 50ml flat-bottomed centrifuge tubes. <br><br />
<br />
6. Spin down the cells at 4000g for 5 minutes at 4C. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cells in 100mL total for all three culture of dH2O. <br><br />
<br />
9. Combine the suspensions into two 50mL flat-bottomed centrifuge tubes. <br><br />
<br />
10. Spin down the cells as above. <br><br />
<br />
11. Decant the supernatant. <br><br />
<br />
12. Resuspend each in 3mL of 100mM Lithium Acetate. <br><br />
<br />
13. Transfer both cultures into a single 15mL conical centrifuge tube. <br><br />
<br />
14. Spin down the cells at 3000rpm for 5 minutes. <br><br />
<br />
15. Resuspend the cells in 0.75mL of 100mM Lithium Acetate, total volume is roughly 2mL. <br><br />
<br />
16. Qualitatively bring up the volume to 3.5mL by adding 40% Glycerol. <br><br />
<br />
17. Aliquot the cells into 1.5mL centrifuge tubes or 1.7mL cryogenic vials.*** <br><br />
<br />
<br />
*Streak in such a way that there are individual colonies visible on the plate without clumps or satellite colonies. <br><br />
<br />
**Collect only individual visible colonies. Do not collect clumps or satellite colonies. <br><br />
<br />
***The volume of aliquots depends on the number to transformations you intend to do at a time. <br><br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Chemically Competent Transformations </h3><br />
<br />
<br />
<p><br />
<br />
This protocol assumes a 50μL aliquot of yeast competent cells were made. <br><br />
<br />
Furthermore, this protocol prepares enough cells for 6 yeast transformations. <br><br />
<br />
<br><br />
<br />
1. Add the following to 50μL of yeast competent cells: <br><br />
<br />
240μL of Polyethylene Glycol - 3350 (PEG-3350) <br><br />
<br />
36μL of 1M Lithium Acetate <br><br />
<br />
32μL of dH2O <br><br />
<br />
<br />
<br />
2. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
3. Aliquot 59μL of the mixture into a 0.2mL microcentrifuge tube. <br><br />
<br />
4. Add 1uL (~100-200ng) of DNA. <br><br />
<br />
5. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
6. Incubate the mixture at 30C for 30 minutes. <br><br />
<br />
7. Heat shock the mixture at 42C for 20 minutes. <br><br />
<br />
8. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
9. Decant the supernatant. <br><br />
<br />
10. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
11. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
12. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
13. Plate 50-150μL of the mixture onto an appropriate Selective Dropout Media plate. <br><br />
<br />
14. Invert and incubate at 30C for 2 days. <br><br />
<br />
*The exact amount of DNA depends on the transformation efficiency of your competent cells. <br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnight Culturing </h3><br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Swipe 3 individually visible yeast colonies and add them to the culture tube media. <br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%.<br />
<br />
</p><br />
<br />
<br />
<h3> Culture Passaging </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Take 20-50μL from a previous overnight or passage culture and add it to the culture media.<br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%. <br><br />
<br />
Note: The exact amount of culture that you take from a previous culture is irrelevant as long as at least 1 living cell is passaged.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500uL of 40% glycerol and 500μL of Selective Dropout media or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Flow Cytometry"></a><br />
<br />
</html><br />
<br />
<h2> Flow Cytometry </h2><br />
<br />
<br />
<h3> Dilutions </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. From an overnight culture measure the optical density at 660nm by making 1:10 dilutions. <br><br />
<br />
2. Take enough culture to make a 1mL aliquot with OD 0.4. <br><br />
<br />
3. Spin down the aliquot in a 1.5mL centrifuge tube at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 800μL of the appropriate selective dropout media and 200μL of 20% glucose. <br><br />
<br />
6. Transfer the new culture to a 14mL culture tube. <br><br />
<br />
7. Incubate and shake at 30C and 250rpm for at least 6 hours (1.2-1.6 optical density).<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Preparations for Analysis using C6 Accuri Flow Cytometer </h3><br />
<br />
<br />
<p><br />
<br />
<br />
1. From the dilution previously made, measure the optical density, roughly 1.2-1.6. <br><br />
<br />
2. Make an aliquot of 500μL of the dilution culture in a 1.5mL centrifuge tube. <br><br />
<br />
3. Spin down the aliquot at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 500μL of PBS(F). <br><br />
<br />
6. Spin down the resuspension at 3000rpm for 3 minutes. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cell pellet in another 500uL of PBS(F).* <br><br />
<br />
9. Prepare the C6 Accuri Flow Cytometer by running a backflush cycle and a dH2O cycle. <br><br />
<br />
10. Load the sample onto the sip. <br><br />
<br />
11. Run the sample with 100,000 cell count. <br><br />
<br />
12. Repeat for all samples and make sure to change data cells otherwise the old data is erased. <br><br />
<br />
13. Once finished, run a cleaning cycle with Accuri approved cleaning solution, then run a dH2O cycle. <br><br />
<br />
<br />
<br />
*For special cases do not resuspend all samples, instead resuspend immediately before running the sample through the flow cytometer.<br />
<br />
<br />
</p><br />
<br />
<br />
<html><a name="Fluorescence Activated Cell Sorting"></a><br />
<br />
</html><br />
<br />
<h2> Fluorescence Activated Cell Sorting </h2><br />
<br />
<br />
<h3> Final Preparations </h3><br />
<br />
<br />
<br />
<p> <br />
<br />
Sample Prep:<br />
<br />
<br />
Spin down samples and negative control (5000 RPM, 1 min), keeping in mind the library size. Aspirate off supernatant. Resuspend in PBSF. Spin down cells. Aspirate off supernatant. Resuspend in PBSF. <br><br />
<br />
<br><br />
<br />
1. Open the “iGEM Template” file in the FACS Software and change name to current date and sort cycle<br><br />
<br />
2. Make sure the stream is stable (look for green light in bottom right corner). If not, run the Sort Calibration order. <br><br />
<br />
3. Run Negative Control from the PyE1 cells. <br><br />
<br />
a. Load cells onto carrier and into the machine. Press play button on screen. <br><br />
<br />
b. Set gate around lower left quadrant of cells to ensure single cell analysis using Forward Scatter Area and Side Scatter Area as your axes. Make sure oval gate covers around 80% of cell population. <br><br />
<br />
c. Set second gate on the first gated population by double-clicking on the gated population and using Forward Scatter Height and Forward Scatter Width as your axes. You will notice two distinct populations. Try to focus on the single cell portion of the plot. <br><br />
<br />
NOTE: If you see a large portion of the second gated population existing near the upper right edge of the first gate, you may need to enlarge the first gate to fit more of the population. <br><br />
<br />
d. Press record. Record 100,000 events and stop run. Move to Next Tube. <br><br />
<br />
4. Run first control (Gene clone) <br><br />
<br />
a. Follow step 3 to run the first control <br><br />
<br />
5. Run first library sample <br><br />
<br />
a. Follow steps 3b and 3c to set first two gates correctly. <br><br />
<br />
b. Set final gate for sort which includes top 1% of GFP producers from second gated population. <br><br />
<br />
c. Use final gate to set up the sort. <br><br />
<br />
d. Select sort conditions at the bottom of the screen. <br><br />
<br />
e. Insert and load collection tube. <br><br />
<br />
f. Record 100,000 events, and sort 10x the library size <br><br />
<br />
6. Run Bleach and diH2O through FACS to avoid cross-contamination. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Protein Expression"></a><br />
<br />
</html><br />
<br />
<h2> Protein Expression </h2><br />
<br />
<br />
<h3> Overnight Cultures </h3><br />
<br />
<p><br />
<br />
1. Add 25mL TB and 25uL Kan to a 250mL baffled flask <br><br />
<br />
2. Stab a glycerol stock with a p1000 pipette and swirl in the flask of media <br><br />
<br />
3. Put flask in 37C shaker overnight <br />
<br />
</p><br />
<br />
<h3> Protein Expression </h3><br />
<br />
<p> <br />
<br />
1. Add 500μL 1000x Kanamycin to 500mL TB in 2L baffled flask <br><br />
<br />
2. Transfer 10mL overnight culture to TB <br><br />
<br />
3. Shake at 37C (DO WE NEED THE RPM?) <br><br />
<br />
4. Remove flask from shaker when optical density is between 0.5 and 0.8 <br><br />
<br />
5. Allow flask to rest at room temp for 30 min <br><br />
<br />
6. Add 125μL 1M IPTG <br><br />
<br />
7. Shake flask at 18C overnight<br />
<br />
</p><br />
<br />
<h3> Protein Extraction and Purification </h3><br />
<br />
<p><br />
<br />
1. Transfer cell culture to centrifuge flask <br><br />
<br />
2. Centrifuge culture at 4000g for 10 min <br><br />
<br />
3. Discard supernatant <br><br />
<br />
4. Resuspend pellet in 25mL lysis buffer <br><br />
<br />
5. Add 250μL of 100x PMSF, 250μL of 100mg/mL lysozyme, and 250μL of 10mg/mL DNAse <br><br />
<br />
6. Sonicate sample with 0.25inch probe for 5 min at 70% amplitude with 20 sec on and off pulses <br><br />
<br />
7. Take 50μL total sample <br><br />
<br />
8. Transfer lysate to SS-34 centrifuge tube <br><br />
<br />
9. Centrifuge for 30 min at 18000g <br><br />
<br />
10. Take 50μL soluble sample<br />
<br />
</p><br />
<br />
<h3> Nickel Nitrotriacetic Acid Chromatography (Nickel-NTA Chromatography) </h3><br />
<br />
<p><br />
<br />
1. Add 5mL 50%(v/v) nickel resin in ethanol to 25mL column and allow to settle to ~2.5mL(CV) <br><br />
<br />
2. Rinse with 10CV H20 <br><br />
<br />
3. Equilibrate with 10CV lysis buffer <br><br />
<br />
4. Load sample onto column <br><br />
<br />
5. Wash column with 15CV lysis buffer <br><br />
<br />
6. Perform 2 additional wash steps with 15CV <br><br />
<br />
7. Elute sample in 10CV elution buffer and collect eluate <br><br />
<br />
8. Take 50μL pure sample <br />
<br />
</p><br />
<br />
<h3> Size Exclusion Chromatography (S.E.C.) </h3><br />
<p><br />
1. Concentrate sample to as high as possible without inducing protein aggregation<br><br />
2. Pre-equilibrate Superdex 75 column with 48 ml PBS <br><br />
3. Inject 500 ul sample onto column <br><br />
4. Run 36 ml PBS through column at 0.5 ml/min, collecting 1 ml fractions <br><br />
5. Verify presence of protein in fractions by measuring concentration on Nanodrop and running SDS-PAGE (15 kDa protein should elute at ~13 ml)<br><br />
6. Pool fractions containing protein <br />
</p><br />
<br />
<br />
<html><a name="Stability Analysis"></a><br />
<br />
</html><br />
<br />
<h2> Stability Analysis </h2><br />
<br />
<br />
<h3> Circular Dichroism: Wavelength Scan </h3><br />
<p><br />
1. Load 1mm cuvette with 400uL protein solution onto CD <br><br />
2. Take wavelength scan <br><br />
260nm-190nm <br><br />
sample every 1nm <br><br />
averaging time 3sec <br><br />
1 scan <br><br />
step scan <br><br />
25C <br><br />
3. Record wavelength which gives strongest signal (222nm)<br />
</p><br />
<br />
<h3> Circular Dichroism: Guanidine Melt </h3><br />
<p><br />
1. Load 1cm cuvette containing 1.996mL of 0.05mg/mL protein solution and stirrer onto CD <br><br />
2. Prepare 8mL of 0.05mg/mL protein in concentrated guanidine solution <br><br />
3. Set up Automixer with guanidine solution on one syringe and waste tube on other syringe <br><br />
4. Titrate up to 6 M guanidine, taking a CD measurement at 222 nm every 0.15 M interval <br><br />
5. Also measure the fluorescence at 280 nm to ensure the total protein concentration is not changing<br />
</p><br />
<br />
<br />
<br />
<html><br />
<br />
<a href="#linktotop">Back To Top</a><br />
</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2014-10-17T17:25:39Z<p>Kristanguyen: </p>
<hr />
<div>{{Template:Team:UW/CSS}}<br />
<br />
<br />
<h1> Protocols </h1><br />
<br />
<br />
<html><a name="Media, Plates, and Sol"></a><br />
<br />
</html><br />
<br />
<br />
<h2> Media, Plates, and Solutions </h2><br />
<br />
<br />
<h3> 40% and 20% Glucose </h3><br />
<br />
<br />
<p><br />
<br />
40g for 40% or 20g for 20% of Glucose <br><br />
<br />
Mix in 100mL dH2O <br><br />
<br />
Sterile filter into a 150mL bottle <br><br />
<br />
</p><br />
<br />
<br />
<h3> 20% Glycerol </h3><br />
<br />
<br />
<p><br />
<br />
20g Glycerol (Liquid) <br><br />
<br />
Mix in 100mL dH2O <br><br />
<br />
Sterile filter into a 150mL bottle <br><br />
<br />
</p><br />
<br />
<br />
<h3> Competent Cell Media Buffer (CCMB) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2 L container: <br><br />
<br />
100 g glycerol (liquid) <br><br />
<br />
10 mL x 1 M potassium acetate <br><br />
<br />
11.8 g CaCl2*H2O <br><br />
<br />
4 g MnCl2 <br><br />
<br />
2 g MgCl2 <br><br />
<br />
1 L of dH2O <br><br />
<br />
Sterile filter or autoclave (20min at 121 °C and 20psi) in a 1 L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Luria Broth (LB) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2L container: <br><br />
<br />
10g tryptone <br><br />
<br />
5g yeast extract <br><br />
<br />
10g NaCl <br> <br />
<br />
1L of dH2O <br><br />
<br />
Autoclave in two 500 ml bottle (20 min at 121C and 20psi) <br><br />
<br />
<br />
*If using antibiotics create a separate aliquot<br />
<br />
</p><br />
<br />
<br />
<h3> LB-Agar </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2L container: <br><br />
<br />
1000ml LB as above <br><br />
<br />
15g agar <br><br />
<br />
1L of dH2O <br><br />
<br />
Autoclave in two 500mL bottles (20 min at 121C at 20psi) <br> <br />
<br />
<br />
<br />
*If using antibiotics create a separate aliquot<br />
<br />
<br />
</p><br />
<br />
<br />
<h3> Super Optimal Broth (SOB) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2L container: <br><br />
<br />
20g BactoTryptone <br><br />
<br />
5g BactoYeast Extract <br><br />
<br />
10mL x 1M NaCl <br><br />
<br />
2.5mL x 1M KCl <br><br />
<br />
1L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121C and 20psi) in a 1L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Phosphate Buffered Saline (PBS) Solution </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2L container or 1L beaker: <br><br />
<br />
8g NaCl <br><br />
<br />
1.44g Na2HPO4 <br><br />
<br />
0.8g KCl <br><br />
<br />
0.24g KH2PO4 <br><br />
<br />
1L of dH2O <br><br />
<br />
Buffer to pH 7.4 <br><br />
<br />
Sterile filter or autoclave (20 min at 121C and 20psi) in a 1L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Terrific Broth (TB) </h3><br />
<br />
<br />
<p><br />
<br />
<br />
<br />
Mix the following in a 1L bottle: <br><br />
<br />
6g Tryptone <br><br />
<br />
12g Yeast Extract <br><br />
<br />
2mL Glycerol <br><br />
<br />
500mL of dH2O <br><br />
<br />
Autoclave (20min at 121C and 20psi) <br><br />
<br />
Cool and add 5mL of 100X Potassium Phosphate Salts (17mM KH2PO4 and 72mM K2HPO4)<br />
<br />
</p> <br />
<br />
<br />
<h3> Yeast Extract Peptone Dextrose (YPD) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following into 950mL of dH2O in a 1L bottle: <br><br />
<br />
20g Bacto Peptone <br><br />
<br />
10g Yeast Extract <br><br />
<br />
Autoclave (20min at 121C and 20psi) <br><br />
<br />
Add 50mL 40% Glucose <br><br />
<br />
Sterile filter into a 1L bottle <br><br />
<br />
<br />
<br />
For long-term liquid media storage, do not add 40% Glucose instead add the glucose directly into cell cultures. <br><br />
<br />
For YPD-plates add 24g Bacto Agar to the Bacto Peptone and Yeast Extract before autoclaving. <br />
<br />
</p><br />
<br />
<br />
<h3> Selective Dropout media, C-Uracil and C-Histidine (C-Ura and C-His) </h3><br />
<br />
<br />
<p><br />
<br />
Synthesized by the Yeast Resource Center at the University of Washington's <br />
<br />
Department of Genome Sciences and Department of Biochemistry.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<h3> Guanidinium Hydrogen Chloride </h3><br />
<br />
<br />
<p><br />
<br />
For maximum effectiveness, final concentration should be approximately 8.5M in PBS<br><br />
<br />
203g Guanidinium Hydrogen Chloride <br><br />
<br />
250mL PBS solution <br><br />
<br />
Add dilute HCl to 7.4pH <br><br />
<br />
<br />
*Alternatively add slightly less than 250mL of PBS in order to buffer the solution to the appropriate volume then add more dH2O as neccessary. <br />
<br />
</p><br />
<br />
<html><a name="Basic Cloning"></a><br />
<br />
</html><br />
<br />
<h2> Basic Cloning </h2><br />
<br />
<br />
<br />
<h3> Polymerase Chain Reaction </h3><br />
<br />
<br />
<p><br />
<br />
All PCRs were done using a standard 50μL reaction volume. <br><br />
<br />
PCRs were done using GoTaq Green Master Mix 2X purchased from PROMEGA Corporation. <br><br />
<br />
Protocols for the PROMEGA GoTaq Green Master Mix 2X: <br><br />
<br />
Mix the following in a 0.2mL microcentrifuge tube on ice: <br><br />
<br />
25μL GoTaq® Green Master Mix 2X <br><br />
<br />
1-5μL of 10μM forward primer <br><br />
<br />
1-5μL of 10μM reverse primer <br><br />
<br />
<250ng of DNA template <br><br />
<br />
Nuclease-free water to 50μl <br><br />
<br />
Conduct the reaction in a Thermocycler, adjusting anneal temperature and extension times accordingly. See your polymerase supplier protocol for more details on thermocycling.<br />
<br />
</p><br />
<br />
<h3> Error-prone Polymerase Chain Reaction </h3><br />
<br />
<br />
<p> <br />
<br />
<br />
<br />
Prepare 50μL reaction: <br><br />
<br />
5μL 10X Mutazyme II Rxn Buffer <br><br />
<br />
1μL 40mM dNTP mix (200μM each final) <br><br />
<br />
1μL 20μM forward primer <br><br />
<br />
1μL 20μM reverse primer <br><br />
<br />
1μL Mutazyme II DNA polymerase (2.5U/μL) <br><br />
<br />
0.01ng template <br><br />
<br />
QS 50μL diH2O <br><br />
<br />
<br><br />
<br />
Thermocycler: <br><br />
<br />
95C, 2min <br><br />
<br />
95C, 30sec <br><br />
<br />
XXC*, 30sec <br><br />
<br />
72C, Xmin** <br><br />
<br />
32 cycles <br><br />
<br />
72C, 10min <br><br />
<br />
4C, hold<br />
<br />
<br><br />
<br />
*Adjust annealing temperature according to Tm of primer. <br><br />
<br />
**Adjust extension time according to the length of amplified DNA. <br><br />
<br />
<br><br />
<br />
Note: Use 0.01ng (calculate by insert and not by total plasmid). <br><br />
<br />
Calculate amount of template to use. <br><br />
<br />
(bp for amplified region) / (bp in total plasmid) = % amplified region <br><br />
<br />
(conc of total plasmid) x (% amplified region as a decimal) = conc of amplified region <br><br />
<br />
<br><br />
<br />
Note: Will probably need to dilute. Never pipette less than 0.5μL. <br><br />
<br />
(0.01ng) / (conc of amplified region) = vol to add to PCR <br><br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Restriction Endonuclease Reaction (Digestion) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
All restriction enzyme reactions were done using a 50ul reaction volume. <br><br />
<br />
Restriction enzymes and buffers were purchased from New England Biolabs Incorporated. <br><br />
<br />
Protocols for various New England Biolab restriction enzyme reactions: <br><br />
<br />
Mix the following in a 0.2mL PCR tube: <br><br />
<br />
1μL of each Restriction Enzyme, add the RE last <br><br />
<br />
1μg of DNA <br><br />
<br />
5μL of the appropriate 10X New Englan Biolab Buffer <br><br />
<br />
Nuclease-free water to 50μL <br><br />
<br />
Incubate the reaction for 1hr <br><br />
<br />
Heat inactive the the reaction at the appropriate temperature <br><br />
<br />
<br />
Notes: Add the restriction enzyme(s) to the reaction last <br><br />
<br />
Thaw the restriction enzyme(s) on ice to improve shelf life <br><br />
<br />
<br />
<br />
</p> <br />
<br />
<br />
<h3> Ligation </h3><br />
<br />
<br />
<p><br />
<br />
T4 DNA Ligase and Buffer was purchased from New England BioLabs Corporation. <br><br />
<br />
1. Prepare the following in a 0.2mL microcentrifuge tube: <br><br />
<br />
50.0ng Vector DNA* <br><br />
<br />
37.5ng Vector DNA* <br><br />
<br />
2μL 10X T4 DNA Ligase Buffer <br><br />
<br />
1μL T4 DNA Ligase <br><br />
<br />
Add diH2O to 20μL <br><br />
<br />
2. Incubate the reaction at room temperature for 10-30 minutes or at 16C overnight. <br><br />
<br />
3. Heat inactivate at 65C for 10 minutes. <br><br />
<br />
4. Chill on ice before starting a transformation reaction. <br><br />
<br />
*The exact amount of DNA is dependent on the number of base pairs. In order to conduct a proper reaction consult the New England Biolab Ligation Calculator at:<br />
<br />
http://nebiocalculator.neb.com/#!/<br />
<br />
</p><br />
<br />
<br />
<html><a name="Escherichia coli Protocols"></a><br />
<br />
</html><br />
<br />
<br />
<h2> <i> Escherichia coli Protocols </i> (XL1-Blue and XL10-Gold) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Culturing </h3><br />
<br />
<br />
<br />
<p> <br />
<br />
<br />
Chemically Competent Cell Stocks – CCMB Transformation <br />
For BL21*, CJ236, etc. Cell Lines. <br><br />
<br />
NOTE: Make SOB-Mg media and autoclave the 250ml flasks with water in them the same day the cells are plated (step 1) <br><br />
<br><br />
<br />
1. Plate 10ul of cells onto an antibiotic free LB/Agar plate..* <br><br />
<br />
2. Incubate overnight at 37OC <br><br />
<br />
3. Inoculate lots of colonies into 50ml SOB-Mg media in a 250ml flask. <br><br />
4. Incubate at 37 OC, 200rpm, until OD at 550nm reaches 0.3, takes about 3 hours. I do my first check at 2 hours.<br><br />
<br />
5.Transfer the cell culture into a sterile 50ml Falcon tube and chill on ice for 10 minutes. <br><br />
<br />
6. Pellet the cells at 2500rpm for 15 minutes at 4 OC.<br><br />
<br />
7. Decant supernatant and invert tubes to remove excess culture medium. <br><br />
<br />
8. Resuspend the cells in 16ml CCMB by gentle vortexing or pipetting (I shake the falcon tube).<br />
(16ml CCMB per 50ml culture) <br><br />
<br />
9. Incubate on ice for 20 minutes. <br><br />
<br />
10. Centrifuge at 2500rpm for 10 minutes at 4 OC. <br><br />
<br />
11. Decant supernatant and invert tubes to remove excess liquid. <br><br />
<br />
12. Resuspend the cells in 4ml CCMB by gentle vortexing or pipetting.<br />
(4ml CCMB per culture) <br><br />
<br />
13. If making multiple 50ml cultures, combine all of the cells and mix before aliquoting. <br> <br />
<br />
14. In cold room: make 205ul aliquots, flash freeze in liquid nitrogen, and store at -80 OC.<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Chemically Competent Cell Transformations </h3><br />
<br />
<br />
<p> <br />
<br />
1. Thaw competent <i> E.coli </i> cells on ice (XL1-Blue or XL10-Gold)* <br><br />
<br />
2. Add 50μL of competent cells to sterile 15mL conical centrifuge tubes <br><br />
<br />
3. Add 1μL (~100-200ng)* of the mini-prep to each culture tube <br><br />
<br />
4. Equilibrate the cells on ice for 10 min <br><br />
<br />
5. Heat shock the cells at 42C for 30-45 seconds** <br><br />
<br />
6. Immediately place the cells back on ice for 3 min <br><br />
<br />
7. Add 250μL LB media without antibiotics and shake at 250 rpm and 37C for 30 min <br><br />
<br />
8. Spread 10μL and 290μL on an appropriate LB-antibiotic plate <br><br />
<br />
9. Invert the plate and incubate at 37C overnight<br />
<br />
<br />
*The exact amount of DNA to add depends on your cell's transformation efficiency. However, it is acceptable to add a larger amount to increase the number of transformed cells. <br><br />
<br />
** Do not heat shock for an extended duration as this may damage and/or kill your cells.<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnights </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottom tube, add 3-5mL of LB and an appropriate volume of antibiotic(s). <br><br />
<br />
2. Swipe several individual colonies, do not collect satellites or colony clumps, with a pipette tip. <br><br />
<br />
3. Swirl the colony tip in the tube, there should be no visible cell clumps. <br><br />
<br />
4. Incubate and shake the tube at 37C at 250rpm for 12-16 hours and no longer than 20 hours. <br><br />
<br />
</p><br />
<br />
<br />
<h3> DNA-Extraction and mini-preps </h3><br />
<br />
<br />
<p><br />
<br />
All DNA mini-preps were prepared using EPOCH mini-kits and following the supplied protocols.<br />
<br />
</p><br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500μL of 40% Glycerol and 500uL of LB (no antibiotics) or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Saccharomyces cerevisiae"></a><br />
<br />
</html><br />
<br />
<h2> <i> Saccharomyces cerevisiae </i> (PYE1 Yeast) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Culturing </h3><br />
<br />
<br />
<br />
<p><br />
<br />
This process take 4 days in lab with a 1 day wait for incubation. <br><br />
<br />
<br />
Day 1: <br><br />
<br />
1. Streak yeast cells onto a YPD plate.* <br><br />
<br />
2. Invert the plate and incubate at 30C for 2 days. <br><br />
<br />
<br />
Day 3: <br><br />
<br />
1. Add 50mL of YPD liquid media into a 250mL baffle flask. <br><br />
<br />
2. Swipe as many individual colonies as you can see into the YPD media.** <br><br />
<br />
3. Incubate and shake the culture at 30C at 250rpm overnight approximately 24 hours. <br><br />
<br />
<br />
Day 4: <br><br />
<br />
1. Take an optical density measurement. <br><br />
<br />
2. In three 250mL baffle flask add the portions of the overnight liquid culture. <br><br />
<br />
3. Dilute each culture to approximately 0.4 optical density with YPD. <br><br />
<br />
4. Incubate and shake the cultures at 30C at 250rpm until the optical density reaches 1.2-1.6. <br><br />
<br />
5. Collect each culture into separate 50ml flat-bottomed centrifuge tubes. <br><br />
<br />
6. Spin down the cells at 4000g for 5 minutes at 4C. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cells in 100mL total for all three culture of dH2O. <br><br />
<br />
9. Combine the suspensions into two 50mL flat-bottomed centrifuge tubes. <br><br />
<br />
10. Spin down the cells as above. <br><br />
<br />
11. Decant the supernatant. <br><br />
<br />
12. Resuspend each in 3mL of 100mM Lithium Acetate. <br><br />
<br />
13. Transfer both cultures into a single 15mL conical centrifuge tube. <br><br />
<br />
14. Spin down the cells at 3000rpm for 5 minutes. <br><br />
<br />
15. Resuspend the cells in 0.75mL of 100mM Lithium Acetate, total volume is roughly 2mL. <br><br />
<br />
16. Qualitatively bring up the volume to 3.5mL by adding 40% Glycerol. <br><br />
<br />
17. Aliquot the cells into 1.5mL centrifuge tubes or 1.7mL cryogenic vials.*** <br><br />
<br />
<br />
*Streak in such a way that there are individual colonies visible on the plate without clumps or satellite colonies. <br><br />
<br />
**Collect only individual visible colonies. Do not collect clumps or satellite colonies. <br><br />
<br />
***The volume of aliquots depends on the number to transformations you intend to do at a time. <br><br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Chemically Competent Transformations </h3><br />
<br />
<br />
<p><br />
<br />
This protocol assumes a 50μL aliquot of yeast competent cells were made. <br><br />
<br />
Furthermore, this protocol prepares enough cells for 6 yeast transformations. <br><br />
<br />
<br><br />
<br />
1. Add the following to 50μL of yeast competent cells: <br><br />
<br />
240μL of Polyethylene Glycol - 3350 (PEG-3350) <br><br />
<br />
36μL of 1M Lithium Acetate <br><br />
<br />
32μL of dH2O <br><br />
<br />
<br />
<br />
2. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
3. Aliquot 59μL of the mixture into a 0.2mL microcentrifuge tube. <br><br />
<br />
4. Add 1uL (~100-200ng) of DNA. <br><br />
<br />
5. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
6. Incubate the mixture at 30C for 30 minutes. <br><br />
<br />
7. Heat shock the mixture at 42C for 20 minutes. <br><br />
<br />
8. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
9. Decant the supernatant. <br><br />
<br />
10. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
11. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
12. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
13. Plate 50-150μL of the mixture onto an appropriate Selective Dropout Media plate. <br><br />
<br />
14. Invert and incubate at 30C for 2 days. <br><br />
<br />
*The exact amount of DNA depends on the transformation efficiency of your competent cells. <br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnight Culturing </h3><br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Swipe 3 individually visible yeast colonies and add them to the culture tube media. <br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%.<br />
<br />
</p><br />
<br />
<br />
<h3> Culture Passaging </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Take 20-50μL from a previous overnight or passage culture and add it to the culture media.<br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%. <br><br />
<br />
Note: The exact amount of culture that you take from a previous culture is irrelevant as long as at least 1 living cell is passaged.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500uL of 40% glycerol and 500μL of Selective Dropout media or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Flow Cytometry"></a><br />
<br />
</html><br />
<br />
<h2> Flow Cytometry </h2><br />
<br />
<br />
<h3> Dilutions </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. From an overnight culture measure the optical density at 660nm by making 1:10 dilutions. <br><br />
<br />
2. Take enough culture to make a 1mL aliquot with OD 0.4. <br><br />
<br />
3. Spin down the aliquot in a 1.5mL centrifuge tube at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 800μL of the appropriate selective dropout media and 200μL of 20% glucose. <br><br />
<br />
6. Transfer the new culture to a 14mL culture tube. <br><br />
<br />
7. Incubate and shake at 30C and 250rpm for at least 6 hours (1.2-1.6 optical density).<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Preparations for Analysis using C6 Accuri Flow Cytometer </h3><br />
<br />
<br />
<p><br />
<br />
<br />
1. From the dilution previously made, measure the optical density, roughly 1.2-1.6. <br><br />
<br />
2. Make an aliquot of 500μL of the dilution culture in a 1.5mL centrifuge tube. <br><br />
<br />
3. Spin down the aliquot at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 500μL of PBS(F). <br><br />
<br />
6. Spin down the resuspension at 3000rpm for 3 minutes. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cell pellet in another 500uL of PBS(F).* <br><br />
<br />
9. Prepare the C6 Accuri Flow Cytometer by running a backflush cycle and a dH2O cycle. <br><br />
<br />
10. Load the sample onto the sip. <br><br />
<br />
11. Run the sample with 100,000 cell count. <br><br />
<br />
12. Repeat for all samples and make sure to change data cells otherwise the old data is erased. <br><br />
<br />
13. Once finished, run a cleaning cycle with Accuri approved cleaning solution, then run a dH2O cycle. <br><br />
<br />
<br />
<br />
*For special cases do not resuspend all samples, instead resuspend immediately before running the sample through the flow cytometer.<br />
<br />
<br />
</p><br />
<br />
<br />
<html><a name="Fluorescence Activated Cell Sorting"></a><br />
<br />
</html><br />
<br />
<h2> Fluorescence Activated Cell Sorting </h2><br />
<br />
<br />
<h3> Final Preparations </h3><br />
<br />
<br />
<br />
<p> <br />
<br />
Sample Prep:<br />
<br />
<br />
Spin down samples and negative control (5000 RPM, 1 min), keeping in mind the library size. Aspirate off supernatant. Resuspend in PBSF. Spin down cells. Aspirate off supernatant. Resuspend in PBSF. <br><br />
<br />
<br><br />
<br />
1. Open the “iGEM Template” file in the FACS Software and change name to current date and sort cycle<br><br />
<br />
2. Make sure the stream is stable (look for green light in bottom right corner). If not, run the Sort Calibration order. <br><br />
<br />
3. Run Negative Control from the PyE1 cells. <br><br />
<br />
a. Load cells onto carrier and into the machine. Press play button on screen. <br><br />
<br />
b. Set gate around lower left quadrant of cells to ensure single cell analysis using Forward Scatter Area and Side Scatter Area as your axes. Make sure oval gate covers around 80% of cell population. <br><br />
<br />
c. Set second gate on the first gated population by double-clicking on the gated population and using Forward Scatter Height and Forward Scatter Width as your axes. You will notice two distinct populations. Try to focus on the single cell portion of the plot. <br><br />
<br />
NOTE: If you see a large portion of the second gated population existing near the upper right edge of the first gate, you may need to enlarge the first gate to fit more of the population. <br><br />
<br />
d. Press record. Record 100,000 events and stop run. Move to Next Tube. <br><br />
<br />
4. Run first control (Gene clone) <br><br />
<br />
a. Follow step 3 to run the first control <br><br />
<br />
5. Run first library sample <br><br />
<br />
a. Follow steps 3b and 3c to set first two gates correctly. <br><br />
<br />
b. Set final gate for sort which includes top 1% of GFP producers from second gated population. <br><br />
<br />
c. Use final gate to set up the sort. <br><br />
<br />
d. Select sort conditions at the bottom of the screen. <br><br />
<br />
e. Insert and load collection tube. <br><br />
<br />
f. Record 100,000 events, and sort 10x the library size <br><br />
<br />
6. Run Bleach and diH2O through FACS to avoid cross-contamination. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Protein Expression"></a><br />
<br />
</html><br />
<br />
<h2> Protein Expression </h2><br />
<br />
<br />
<h3> Overnight Cultures </h3><br />
<br />
<p><br />
<br />
1. Add 25mL TB and 25uL Kan to a 250mL baffled flask <br><br />
<br />
2. Stab a glycerol stock with a p1000 pipette and swirl in the flask of media <br><br />
<br />
3. Put flask in 37C shaker overnight <br />
<br />
</p><br />
<br />
<h3> Protein Expression </h3><br />
<br />
<p> <br />
<br />
1. Add 500μL 1000x Kanamycin to 500mL TB in 2L baffled flask <br><br />
<br />
2. Transfer 10mL overnight culture to TB <br><br />
<br />
3. Shake at 37C (DO WE NEED THE RPM?) <br><br />
<br />
4. Remove flask from shaker when optical density is between 0.5 and 0.8 <br><br />
<br />
5. Allow flask to rest at room temp for 30 min <br><br />
<br />
6. Add 125μL 1M IPTG <br><br />
<br />
7. Shake flask at 18C overnight<br />
<br />
</p><br />
<br />
<h3> Protein Extraction and Purification </h3><br />
<br />
<p><br />
<br />
1. Transfer cell culture to centrifuge flask <br><br />
<br />
2. Centrifuge culture at 4000g for 10 min <br><br />
<br />
3. Discard supernatant <br><br />
<br />
4. Resuspend pellet in 25mL lysis buffer <br><br />
<br />
5. Add 250μL of 100x PMSF, 250μL of 100mg/mL lysozyme, and 250μL of 10mg/mL DNAse <br><br />
<br />
6. Sonicate sample with 0.25inch probe for 5 min at 70% amplitude with 20 sec on and off pulses <br><br />
<br />
7. Take 50μL total sample <br><br />
<br />
8. Transfer lysate to SS-34 centrifuge tube <br><br />
<br />
9. Centrifuge for 30 min at 18000g <br><br />
<br />
10. Take 50μL soluble sample<br />
<br />
</p><br />
<br />
<h3> Nickel Nitrotriacetic Acid Chromatography (Nickel-NTA Chromatography) </h3><br />
<br />
<p><br />
<br />
1. Add 5mL 50%(v/v) nickel resin in ethanol to 25mL column and allow to settle to ~2.5mL(CV) <br><br />
<br />
2. Rinse with 10CV H20 <br><br />
<br />
3. Equilibrate with 10CV lysis buffer <br><br />
<br />
4. Load sample onto column <br><br />
<br />
5. Wash column with 15CV lysis buffer <br><br />
<br />
6. Perform 2 additional wash steps with 15CV <br><br />
<br />
7. Elute sample in 10CV elution buffer and collect eluate <br><br />
<br />
8. Take 50μL pure sample <br />
<br />
</p><br />
<br />
<h3> Size Exclusion Chromatography (S.E.C.) </h3><br />
<p><br />
1. Concentrate sample to as high as possible without inducing protein aggregation<br><br />
2. Pre-equilibrate Superdex 75 column with 48 ml PBS <br><br />
3. Inject 500 ul sample onto column <br><br />
4. Run 36 ml PBS through column at 0.5 ml/min, collecting 1 ml fractions <br><br />
5. Verify presence of protein in fractions by measuring concentration on Nanodrop and running SDS-PAGE (15 kDa protein should elute at ~13 ml)<br><br />
6. Pool fractions containing protein <br />
</p><br />
<br />
<br />
<html><a name="Stability Analysis"></a><br />
<br />
</html><br />
<br />
<h2> Stability Analysis </h2><br />
<br />
<br />
<h3> Circular Dichroism: Wavelength Scan </h3><br />
<p><br />
1. Load 1mm cuvette with 400uL protein solution onto CD <br><br />
2. Take wavelength scan <br><br />
260nm-190nm <br><br />
sample every 1nm <br><br />
averaging time 3sec <br><br />
1 scan <br><br />
step scan <br><br />
25C <br><br />
3. Record wavelength which gives strongest signal (222nm)<br />
</p><br />
<br />
<h3> Circular Dichroism: Guanidine Melt </h3><br />
<p><br />
1. Load 1cm cuvette containing 1.996mL of 0.05mg/mL protein solution and stirrer onto CD <br><br />
2. Prepare 8mL of 0.05mg/mL protein in concentrated guanidine solution <br><br />
3. Set up Automixer with guanidine solution on one syringe and waste tube on other syringe <br><br />
4. Titrate up to 6 M guanidine, taking a CD measurement at 222 nm every 0.15 M interval <br><br />
5. Also measure the fluorescence at 280 nm to ensure the total protein concentration is not changing<br />
</p><br />
<br />
<br />
<br />
<html><br />
<br />
<a href="#linktotop">Back To Top</a><br />
</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2014-10-17T04:28:04Z<p>Kristanguyen: </p>
<hr />
<div>{{Template:Team:UW/CSS}}<br />
<br />
<br />
<h1> Protocols </h1><br />
<br />
<br />
<html><a name="Media, Plates, and Sol"></a><br />
<br />
</html><br />
<br />
<br />
<h2> Media, Plates, and Solutions </h2><br />
<br />
<br />
<h3> 40% and 20% Glucose </h3><br />
<br />
<br />
<p><br />
<br />
40g for 40% or 20g for 20% of Glucose <br><br />
<br />
Mix in 100mL dH2O <br><br />
<br />
Sterile filter into a 150mL bottle <br><br />
<br />
</p><br />
<br />
<br />
<h3> 20% Glycerol </h3><br />
<br />
<br />
<p><br />
<br />
20g Glycerol (Liquid) <br><br />
<br />
Mix in 100mL dH2O <br><br />
<br />
Sterile filter into a 150mL bottle <br><br />
<br />
</p><br />
<br />
<br />
<h3> Competent Cell Media Buffer (CCMB) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2L container: <br><br />
<br />
100g Glycerol (liquid) <br><br />
<br />
10mL x 1M Potassium Acetate <br><br />
<br />
11.8g CaCl2*H2O <br><br />
<br />
4g MnCl2 <br><br />
<br />
2g MgCl2 <br><br />
<br />
1L of dH2O <br><br />
<br />
Sterile filter or autoclave (20min at 121 °C and 20psi) in a 1L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Luria Broth (LB) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2L container: <br><br />
<br />
10g tryptone <br><br />
<br />
5g yeast extract <br><br />
<br />
10g NaCl <br> <br />
<br />
1L of dH2O <br><br />
<br />
Autoclave in two 500 ml bottle (20 min at 121C and 20psi) <br><br />
<br />
<br />
*If using antibiotics create a separate aliquot<br />
<br />
</p><br />
<br />
<br />
<h3> LB-Agar </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2L container: <br><br />
<br />
1000ml LB as above <br><br />
<br />
15g agar <br><br />
<br />
1L of dH2O <br><br />
<br />
Autoclave in two 500mL bottles (20 min at 121C at 20psi) <br> <br />
<br />
<br />
<br />
*If using antibiotics create a separate aliquot<br />
<br />
<br />
</p><br />
<br />
<br />
<h3> Super Optimal Broth (SOB) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2L container: <br><br />
<br />
20g BactoTryptone <br><br />
<br />
5g BactoYeast Extract <br><br />
<br />
10mL x 1M NaCl <br><br />
<br />
2.5mL x 1M KCl <br><br />
<br />
1L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121C and 20psi) in a 1L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Phosphate Buffered Saline (PBS) Solution </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2L container or 1L beaker: <br><br />
<br />
8g NaCl <br><br />
<br />
1.44g Na2HPO4 <br><br />
<br />
0.8g KCl <br><br />
<br />
0.24g KH2PO4 <br><br />
<br />
1L of dH2O <br><br />
<br />
Buffer to pH 7.4 <br><br />
<br />
Sterile filter or autoclave (20 min at 121C and 20psi) in a 1L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Terrific Broth (TB) </h3><br />
<br />
<br />
<p><br />
<br />
<br />
<br />
Mix the following in a 1L bottle: <br><br />
<br />
6g Tryptone <br><br />
<br />
12g Yeast Extract <br><br />
<br />
2mL Glycerol <br><br />
<br />
500mL of dH2O <br><br />
<br />
Autoclave (20min at 121C and 20psi) <br><br />
<br />
Cool and add 5mL of 100X Potassium Phosphate Salts (17mM KH2PO4 and 72mM K2HPO4)<br />
<br />
</p> <br />
<br />
<br />
<h3> Yeast Extract Peptone Dextrose (YPD) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following into 950mL of dH2O in a 1L bottle: <br><br />
<br />
20g Bacto Peptone <br><br />
<br />
10g Yeast Extract <br><br />
<br />
Autoclave (20min at 121C and 20psi) <br><br />
<br />
Add 50mL 40% Glucose <br><br />
<br />
Sterile filter into a 1L bottle <br><br />
<br />
<br />
<br />
For long-term liquid media storage, do not add 40% Glucose instead add the glucose directly into cell cultures. <br><br />
<br />
For YPD-plates add 24g Bacto Agar to the Bacto Peptone and Yeast Extract before autoclaving. <br />
<br />
</p><br />
<br />
<br />
<h3> Selective Dropout media, C-Uracil and C-Histidine (C-Ura and C-His) </h3><br />
<br />
<br />
<p><br />
<br />
Synthesized by the Yeast Resource Center at the University of Washington's <br />
<br />
Department of Genome Sciences and Department of Biochemistry.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<h3> Guanidinium Hydrogen Chloride </h3><br />
<br />
<br />
<p><br />
<br />
For maximum effectiveness, final concentration should be approximately 8.5M in PBS<br><br />
<br />
203g Guanidinium Hydrogen Chloride <br><br />
<br />
250mL PBS solution <br><br />
<br />
Add dilute HCl to 7.4pH <br><br />
<br />
<br />
*Alternatively add slightly less than 250mL of PBS in order to buffer the solution to the appropriate volume then add more dH2O as neccessary. <br />
<br />
</p><br />
<br />
<html><a name="Basic Cloning"></a><br />
<br />
</html><br />
<br />
<h2> Basic Cloning </h2><br />
<br />
<br />
<br />
<h3> Polymerase Chain Reaction </h3><br />
<br />
<br />
<p><br />
<br />
All PCRs were done using a standard 50μL reaction volume. <br><br />
<br />
PCRs were done using GoTaq Green Master Mix 2X purchased from PROMEGA Corporation. <br><br />
<br />
Protocols for the PROMEGA GoTaq Green Master Mix 2X: <br><br />
<br />
Mix the following in a 0.2mL microcentrifuge tube on ice: <br><br />
<br />
25μL GoTaq® Green Master Mix 2X <br><br />
<br />
1-5μL of 10μM forward primer <br><br />
<br />
1-5μL of 10μM reverse primer <br><br />
<br />
<250ng of DNA template <br><br />
<br />
Nuclease-free water to 50μl <br><br />
<br />
Conduct the reaction in a Thermocycler, adjusting anneal temperature and extension times accordingly. See your polymerase supplier protocol for more details on thermocycling.<br />
<br />
</p><br />
<br />
<h3> Error-prone Polymerase Chain Reaction </h3><br />
<br />
<br />
<p> <br />
<br />
<br />
<br />
Prepare 50μL reaction: <br><br />
<br />
5μL 10X Mutazyme II Rxn Buffer <br><br />
<br />
1μL 40mM dNTP mix (200μM each final) <br><br />
<br />
1μL 20μM forward primer <br><br />
<br />
1μL 20μM reverse primer <br><br />
<br />
1μL Mutazyme II DNA polymerase (2.5U/μL) <br><br />
<br />
0.01ng template <br><br />
<br />
QS 50μL diH2O <br><br />
<br />
<br><br />
<br />
Thermocycler: <br><br />
<br />
95C, 2min <br><br />
<br />
95C, 30sec <br><br />
<br />
XXC*, 30sec <br><br />
<br />
72C, Xmin** <br><br />
<br />
32 cycles <br><br />
<br />
72C, 10min <br><br />
<br />
4C, hold<br />
<br />
<br><br />
<br />
*Adjust annealing temperature according to Tm of primer. <br><br />
<br />
**Adjust extension time according to the length of amplified DNA. <br><br />
<br />
<br><br />
<br />
Note: Use 0.01ng (calculate by insert and not by total plasmid). <br><br />
<br />
Calculate amount of template to use. <br><br />
<br />
(bp for amplified region) / (bp in total plasmid) = % amplified region <br><br />
<br />
(conc of total plasmid) x (% amplified region as a decimal) = conc of amplified region <br><br />
<br />
<br><br />
<br />
Note: Will probably need to dilute. Never pipette less than 0.5μL. <br><br />
<br />
(0.01ng) / (conc of amplified region) = vol to add to PCR <br><br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Restriction Endonuclease Reaction (Digestion) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
All restriction enzyme reactions were done using a 50ul reaction volume. <br><br />
<br />
Restriction enzymes and buffers were purchased from New England Biolabs Incorporated. <br><br />
<br />
Protocols for various New England Biolab restriction enzyme reactions: <br><br />
<br />
Mix the following in a 0.2mL PCR tube: <br><br />
<br />
1μL of each Restriction Enzyme, add the RE last <br><br />
<br />
1μg of DNA <br><br />
<br />
5μL of the appropriate 10X New Englan Biolab Buffer <br><br />
<br />
Nuclease-free water to 50μL <br><br />
<br />
Incubate the reaction for 1hr <br><br />
<br />
Heat inactive the the reaction at the appropriate temperature <br><br />
<br />
<br />
Notes: Add the restriction enzyme(s) to the reaction last <br><br />
<br />
Thaw the restriction enzyme(s) on ice to improve shelf life <br><br />
<br />
<br />
<br />
</p> <br />
<br />
<br />
<h3> Ligation </h3><br />
<br />
<br />
<p><br />
<br />
T4 DNA Ligase and Buffer was purchased from New England BioLabs Corporation. <br><br />
<br />
1. Prepare the following in a 0.2mL microcentrifuge tube: <br><br />
<br />
50.0ng Vector DNA* <br><br />
<br />
37.5ng Vector DNA* <br><br />
<br />
2μL 10X T4 DNA Ligase Buffer <br><br />
<br />
1μL T4 DNA Ligase <br><br />
<br />
Add diH2O to 20μL <br><br />
<br />
2. Incubate the reaction at room temperature for 10-30 minutes or at 16C overnight. <br><br />
<br />
3. Heat inactivate at 65C for 10 minutes. <br><br />
<br />
4. Chill on ice before starting a transformation reaction. <br><br />
<br />
*The exact amount of DNA is dependent on the number of base pairs. In order to conduct a proper reaction consult the New England Biolab Ligation Calculator at:<br />
<br />
http://nebiocalculator.neb.com/#!/<br />
<br />
</p><br />
<br />
<br />
<html><a name="Escherichia coli Protocols"></a><br />
<br />
</html><br />
<br />
<br />
<h2> <i> Escherichia coli Protocols </i> (XL1-Blue and XL10-Gold) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Culturing </h3><br />
<br />
<br />
<br />
<p> <br />
<br />
<br />
Chemically Competent Cell Stocks – CCMB Transformation <br />
For BL21*, CJ236, etc. Cell Lines. <br><br />
<br />
NOTE: Make SOB-Mg media and autoclave the 250ml flasks with water in them the same day the cells are plated (step 1) <br><br />
<br><br />
<br />
1. Plate 10ul of cells onto an antibiotic free LB/Agar plate..* <br><br />
<br />
2. Incubate overnight at 37OC <br><br />
<br />
3. Inoculate lots of colonies into 50ml SOB-Mg media in a 250ml flask. <br><br />
4. Incubate at 37 OC, 200rpm, until OD at 550nm reaches 0.3, takes about 3 hours. I do my first check at 2 hours.<br><br />
<br />
5.Transfer the cell culture into a sterile 50ml Falcon tube and chill on ice for 10 minutes. <br><br />
<br />
6. Pellet the cells at 2500rpm for 15 minutes at 4 OC.<br><br />
<br />
7. Decant supernatant and invert tubes to remove excess culture medium. <br><br />
<br />
8. Resuspend the cells in 16ml CCMB by gentle vortexing or pipetting (I shake the falcon tube).<br />
(16ml CCMB per 50ml culture) <br><br />
<br />
9. Incubate on ice for 20 minutes. <br><br />
<br />
10. Centrifuge at 2500rpm for 10 minutes at 4 OC. <br><br />
<br />
11. Decant supernatant and invert tubes to remove excess liquid. <br><br />
<br />
12. Resuspend the cells in 4ml CCMB by gentle vortexing or pipetting.<br />
(4ml CCMB per culture) <br><br />
<br />
13. If making multiple 50ml cultures, combine all of the cells and mix before aliquoting. <br> <br />
<br />
14. In cold room: make 205ul aliquots, flash freeze in liquid nitrogen, and store at -80 OC.<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Chemically Competent Cell Transformations </h3><br />
<br />
<br />
<p> <br />
<br />
1. Thaw competent <i> E.coli </i> cells on ice (XL1-Blue or XL10-Gold)* <br><br />
<br />
2. Add 50μL of competent cells to sterile 15mL conical centrifuge tubes <br><br />
<br />
3. Add 1μL (~100-200ng)* of the mini-prep to each culture tube <br><br />
<br />
4. Equilibrate the cells on ice for 10 min <br><br />
<br />
5. Heat shock the cells at 42C for 30-45 seconds** <br><br />
<br />
6. Immediately place the cells back on ice for 3 min <br><br />
<br />
7. Add 250μL LB media without antibiotics and shake at 250 rpm and 37C for 30 min <br><br />
<br />
8. Spread 10μL and 290μL on an appropriate LB-antibiotic plate <br><br />
<br />
9. Invert the plate and incubate at 37C overnight<br />
<br />
<br />
*The exact amount of DNA to add depends on your cell's transformation efficiency. However, it is acceptable to add a larger amount to increase the number of transformed cells. <br><br />
<br />
** Do not heat shock for an extended duration as this may damage and/or kill your cells.<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnights </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottom tube, add 3-5mL of LB and an appropriate volume of antibiotic(s). <br><br />
<br />
2. Swipe several individual colonies, do not collect satellites or colony clumps, with a pipette tip. <br><br />
<br />
3. Swirl the colony tip in the tube, there should be no visible cell clumps. <br><br />
<br />
4. Incubate and shake the tube at 37C at 250rpm for 12-16 hours and no longer than 20 hours. <br><br />
<br />
</p><br />
<br />
<br />
<h3> DNA-Extraction and mini-preps </h3><br />
<br />
<br />
<p><br />
<br />
All DNA mini-preps were prepared using EPOCH mini-kits and following the supplied protocols.<br />
<br />
</p><br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500μL of 40% Glycerol and 500uL of LB (no antibiotics) or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Saccharomyces cerevisiae"></a><br />
<br />
</html><br />
<br />
<h2> <i> Saccharomyces cerevisiae </i> (PYE1 Yeast) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Culturing </h3><br />
<br />
<br />
<br />
<p><br />
<br />
This process take 4 days in lab with a 1 day wait for incubation. <br><br />
<br />
<br />
Day 1: <br><br />
<br />
1. Streak yeast cells onto a YPD plate.* <br><br />
<br />
2. Invert the plate and incubate at 30C for 2 days. <br><br />
<br />
<br />
Day 3: <br><br />
<br />
1. Add 50mL of YPD liquid media into a 250mL baffle flask. <br><br />
<br />
2. Swipe as many individual colonies as you can see into the YPD media.** <br><br />
<br />
3. Incubate and shake the culture at 30C at 250rpm overnight approximately 24 hours. <br><br />
<br />
<br />
Day 4: <br><br />
<br />
1. Take an optical density measurement. <br><br />
<br />
2. In three 250mL baffle flask add the portions of the overnight liquid culture. <br><br />
<br />
3. Dilute each culture to approximately 0.4 optical density with YPD. <br><br />
<br />
4. Incubate and shake the cultures at 30C at 250rpm until the optical density reaches 1.2-1.6. <br><br />
<br />
5. Collect each culture into separate 50ml flat-bottomed centrifuge tubes. <br><br />
<br />
6. Spin down the cells at 4000g for 5 minutes at 4C. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cells in 100mL total for all three culture of dH2O. <br><br />
<br />
9. Combine the suspensions into two 50mL flat-bottomed centrifuge tubes. <br><br />
<br />
10. Spin down the cells as above. <br><br />
<br />
11. Decant the supernatant. <br><br />
<br />
12. Resuspend each in 3mL of 100mM Lithium Acetate. <br><br />
<br />
13. Transfer both cultures into a single 15mL conical centrifuge tube. <br><br />
<br />
14. Spin down the cells at 3000rpm for 5 minutes. <br><br />
<br />
15. Resuspend the cells in 0.75mL of 100mM Lithium Acetate, total volume is roughly 2mL. <br><br />
<br />
16. Qualitatively bring up the volume to 3.5mL by adding 40% Glycerol. <br><br />
<br />
17. Aliquot the cells into 1.5mL centrifuge tubes or 1.7mL cryogenic vials.*** <br><br />
<br />
<br />
*Streak in such a way that there are individual colonies visible on the plate without clumps or satellite colonies. <br><br />
<br />
**Collect only individual visible colonies. Do not collect clumps or satellite colonies. <br><br />
<br />
***The volume of aliquots depends on the number to transformations you intend to do at a time. <br><br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Chemically Competent Transformations </h3><br />
<br />
<br />
<p><br />
<br />
This protocol assumes a 50μL aliquot of yeast competent cells were made. <br><br />
<br />
Furthermore, this protocol prepares enough cells for 6 yeast transformations. <br><br />
<br />
<br><br />
<br />
1. Add the following to 50μL of yeast competent cells: <br><br />
<br />
240μL of Polyethylene Glycol - 3350 (PEG-3350) <br><br />
<br />
36μL of 1M Lithium Acetate <br><br />
<br />
32μL of dH2O <br><br />
<br />
<br />
<br />
2. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
3. Aliquot 59μL of the mixture into a 0.2mL microcentrifuge tube. <br><br />
<br />
4. Add 1uL (~100-200ng) of DNA. <br><br />
<br />
5. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
6. Incubate the mixture at 30C for 30 minutes. <br><br />
<br />
7. Heat shock the mixture at 42C for 20 minutes. <br><br />
<br />
8. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
9. Decant the supernatant. <br><br />
<br />
10. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
11. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
12. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
13. Plate 50-150μL of the mixture onto an appropriate Selective Dropout Media plate. <br><br />
<br />
14. Invert and incubate at 30C for 2 days. <br><br />
<br />
*The exact amount of DNA depends on the transformation efficiency of your competent cells. <br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnight Culturing </h3><br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Swipe 3 individually visible yeast colonies and add them to the culture tube media. <br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%.<br />
<br />
</p><br />
<br />
<br />
<h3> Culture Passaging </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Take 20-50μL from a previous overnight or passage culture and add it to the culture media.<br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%. <br><br />
<br />
Note: The exact amount of culture that you take from a previous culture is irrelevant as long as at least 1 living cell is passaged.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500uL of 40% glycerol and 500μL of Selective Dropout media or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Flow Cytometry"></a><br />
<br />
</html><br />
<br />
<h2> Flow Cytometry </h2><br />
<br />
<br />
<h3> Dilutions </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. From an overnight culture measure the optical density at 660nm by making 1:10 dilutions. <br><br />
<br />
2. Take enough culture to make a 1mL aliquot with OD 0.4. <br><br />
<br />
3. Spin down the aliquot in a 1.5mL centrifuge tube at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 800μL of the appropriate selective dropout media and 200μL of 20% glucose. <br><br />
<br />
6. Transfer the new culture to a 14mL culture tube. <br><br />
<br />
7. Incubate and shake at 30C and 250rpm for at least 6 hours (1.2-1.6 optical density).<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Preparations for Analysis using C6 Accuri Flow Cytometer </h3><br />
<br />
<br />
<p><br />
<br />
<br />
1. From the dilution previously made, measure the optical density, roughly 1.2-1.6. <br><br />
<br />
2. Make an aliquot of 500μL of the dilution culture in a 1.5mL centrifuge tube. <br><br />
<br />
3. Spin down the aliquot at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 500μL of PBS(F). <br><br />
<br />
6. Spin down the resuspension at 3000rpm for 3 minutes. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cell pellet in another 500uL of PBS(F).* <br><br />
<br />
9. Prepare the C6 Accuri Flow Cytometer by running a backflush cycle and a dH2O cycle. <br><br />
<br />
10. Load the sample onto the sip. <br><br />
<br />
11. Run the sample with 100,000 cell count. <br><br />
<br />
12. Repeat for all samples and make sure to change data cells otherwise the old data is erased. <br><br />
<br />
13. Once finished, run a cleaning cycle with Accuri approved cleaning solution, then run a dH2O cycle. <br><br />
<br />
<br />
<br />
*For special cases do not resuspend all samples, instead resuspend immediately before running the sample through the flow cytometer.<br />
<br />
<br />
</p><br />
<br />
<br />
<html><a name="Fluorescence Activated Cell Sorting"></a><br />
<br />
</html><br />
<br />
<h2> Fluorescence Activated Cell Sorting </h2><br />
<br />
<br />
<h3> Final Preparations </h3><br />
<br />
<br />
<br />
<p> <br />
<br />
Sample Prep:<br />
<br />
<br />
Spin down samples and negative control (5000 RPM, 1 min), keeping in mind the library size. Aspirate off supernatant. Resuspend in PBSF. Spin down cells. Aspirate off supernatant. Resuspend in PBSF. <br><br />
<br />
<br><br />
<br />
1. Open the “iGEM Template” file in the FACS Software and change name to current date and sort cycle<br><br />
<br />
2. Make sure the stream is stable (look for green light in bottom right corner). If not, run the Sort Calibration order. <br><br />
<br />
3. Run Negative Control from the PyE1 cells. <br><br />
<br />
a. Load cells onto carrier and into the machine. Press play button on screen. <br><br />
<br />
b. Set gate around lower left quadrant of cells to ensure single cell analysis using Forward Scatter Area and Side Scatter Area as your axes. Make sure oval gate covers around 80% of cell population. <br><br />
<br />
c. Set second gate on the first gated population by double-clicking on the gated population and using Forward Scatter Height and Forward Scatter Width as your axes. You will notice two distinct populations. Try to focus on the single cell portion of the plot. <br><br />
<br />
NOTE: If you see a large portion of the second gated population existing near the upper right edge of the first gate, you may need to enlarge the first gate to fit more of the population. <br><br />
<br />
d. Press record. Record 100,000 events and stop run. Move to Next Tube. <br><br />
<br />
4. Run first control (Gene clone) <br><br />
<br />
a. Follow step 3 to run the first control <br><br />
<br />
5. Run first library sample <br><br />
<br />
a. Follow steps 3b and 3c to set first two gates correctly. <br><br />
<br />
b. Set final gate for sort which includes top 1% of GFP producers from second gated population. <br><br />
<br />
c. Use final gate to set up the sort. <br><br />
<br />
d. Select sort conditions at the bottom of the screen. <br><br />
<br />
e. Insert and load collection tube. <br><br />
<br />
f. Record 100,000 events, and sort 10x the library size <br><br />
<br />
6. Run Bleach and diH2O through FACS to avoid cross-contamination. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Protein Expression"></a><br />
<br />
</html><br />
<br />
<h2> Protein Expression </h2><br />
<br />
<br />
<h3> Overnight Cultures </h3><br />
<br />
<p><br />
<br />
1. Add 25mL TB and 25uL Kan to a 250mL baffled flask <br><br />
<br />
2. Stab a glycerol stock with a p1000 pipette and swirl in the flask of media <br><br />
<br />
3. Put flask in 37C shaker overnight <br />
<br />
</p><br />
<br />
<h3> Protein Expression </h3><br />
<br />
<p> <br />
<br />
1. Add 500μL 1000x Kanamycin to 500mL TB in 2L baffled flask <br><br />
<br />
2. Transfer 10mL overnight culture to TB <br><br />
<br />
3. Shake at 37C (DO WE NEED THE RPM?) <br><br />
<br />
4. Remove flask from shaker when optical density is between 0.5 and 0.8 <br><br />
<br />
5. Allow flask to rest at room temp for 30 min <br><br />
<br />
6. Add 125μL 1M IPTG <br><br />
<br />
7. Shake flask at 18C overnight<br />
<br />
</p><br />
<br />
<h3> Protein Extraction and Purification </h3><br />
<br />
<p><br />
<br />
1. Transfer cell culture to centrifuge flask <br><br />
<br />
2. Centrifuge culture at 4000g for 10 min <br><br />
<br />
3. Discard supernatant <br><br />
<br />
4. Resuspend pellet in 25mL lysis buffer <br><br />
<br />
5. Add 250μL of 100x PMSF, 250μL of 100mg/mL lysozyme, and 250μL of 10mg/mL DNAse <br><br />
<br />
6. Sonicate sample with 0.25inch probe for 5 min at 70% amplitude with 20 sec on and off pulses <br><br />
<br />
7. Take 50μL total sample <br><br />
<br />
8. Transfer lysate to SS-34 centrifuge tube <br><br />
<br />
9. Centrifuge for 30 min at 18000g <br><br />
<br />
10. Take 50μL soluble sample<br />
<br />
</p><br />
<br />
<h3> Nickel Nitrotriacetic Acid Chromatography (Nickel-NTA Chromatography) </h3><br />
<br />
<p><br />
<br />
1. Add 5mL 50%(v/v) nickel resin in ethanol to 25mL column and allow to settle to ~2.5mL(CV) <br><br />
<br />
2. Rinse with 10CV H20 <br><br />
<br />
3. Equilibrate with 10CV lysis buffer <br><br />
<br />
4. Load sample onto column <br><br />
<br />
5. Wash column with 15CV lysis buffer <br><br />
<br />
6. Perform 2 additional wash steps with 15CV <br><br />
<br />
7. Elute sample in 10CV elution buffer and collect eluate <br><br />
<br />
8. Take 50μL pure sample <br />
<br />
</p><br />
<br />
<h3> Size Exclusion Chromatography (S.E.C.) </h3><br />
<p><br />
1. Concentrate sample to as high as possible without inducing protein aggregation<br><br />
2. Pre-equilibrate Superdex 75 column with 48 ml PBS <br><br />
3. Inject 500 ul sample onto column <br><br />
4. Run 36 ml PBS through column at 0.5 ml/min, collecting 1 ml fractions <br><br />
5. Verify presence of protein in fractions by measuring concentration on Nanodrop and running SDS-PAGE (15 kDa protein should elute at ~13 ml)<br><br />
6. Pool fractions containing protein <br />
</p><br />
<br />
<br />
<html><a name="Stability Analysis"></a><br />
<br />
</html><br />
<br />
<h2> Stability Analysis </h2><br />
<br />
<br />
<h3> Circular Dichroism: Wavelength Scan </h3><br />
<p><br />
1. Load 1mm cuvette with 400uL protein solution onto CD <br><br />
2. Take wavelength scan <br><br />
260nm-190nm <br><br />
sample every 1nm <br><br />
averaging time 3sec <br><br />
1 scan <br><br />
step scan <br><br />
25C <br><br />
3. Record wavelength which gives strongest signal (222nm)<br />
</p><br />
<br />
<h3> Circular Dichroism: Guanidine Melt </h3><br />
<p><br />
1. Load 1cm cuvette containing 1.996mL of 0.05mg/mL protein solution and stirrer onto CD <br><br />
2. Prepare 8mL of 0.05mg/mL protein in concentrated guanidine solution <br><br />
3. Set up Automixer with guanidine solution on one syringe and waste tube on other syringe <br><br />
4. Titrate up to 6 M guanidine, taking a CD measurement at 222 nm every 0.15 M interval <br><br />
5. Also measure the fluorescence at 280 nm to ensure the total protein concentration is not changing<br />
</p><br />
<br />
<br />
<br />
<html><br />
<br />
<a href="#linktotop">Back To Top</a><br />
</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/MethodsTeam:Washington/Methods2014-10-17T04:01:47Z<p>Kristanguyen: </p>
<hr />
<div>{{Template:Team:UW/CSS}}<br />
<br />
<html><br />
<br />
<body><br />
<br />
<br />
<h1> Method </h1><br />
<br />
<p align =left><br />
<br />
The essential process of our system involves cloning and manufacturing of a plasmid in <i> E. coli </i>. Once, the plasmids have been constructed and verified in <i> E. coli </i> they are transformed into <i>S. cerevisiae</i>. The plasmid constructs are then expressed. Following several days of growth the yeast cultures are passed through a Flow Cytometer and the fluorescence of each cell is measured. Higher fluorescence is associated with higher expression of the protein of interest which in-turn is indicative of higher protein stability. <br />
<br />
</p><br />
<br />
<br />
<h2> Cloning in <i> Escherichia coli </i> </h2><br />
<br />
<p align = left> <br />
<br />
There are five possible degron constructs corresponding to five different positions the degron can take in our construct. Four of the five vectors for our protein of interest DNA code contains contain a degron (Deg1-4) as well as an EcoRI and Nhel111 restriction enzyme cutsite between Gal4 and VP16. Furthermore, the vector also contains a region that encodes Ampicillin resistance as well as autotrophic region that encodes for uracil synthesis. The DNA that encodes our protein of interest, the insert, is amplified to include both cutsites through a polymerase chain reaction. In a subsequent step the amplified fragment is then digested and then ligated with the appropriate vector and transformed into chemically competent<i> E.coli </i> wither XL-1 Blue or XL10-Gold strains. Similarly, Deg0(no degron) vector has EcoRI along with a Hind111 cutsite. Using the different cutsites our DNA fragment is prepared using PCR and ligated into the Deg0 vector and then transformed. Once the transformation is complete, the cells are plated onto LB-agar plates supplimented with ampicillin in order to ensure that all <i> E.coli </i> colonies contain our recombinant plasmid. After being grown for a day, several colonies are swiped and added to an overnight culture. The overnight culture is grown overnight and their recombinant plasmid is harvested and sequenced (Sanger sequencing is used through Genewiz Inc.). If the plasmids are correct, we then proceed to create a glycerol stock of the cell culture as well as a miniprep stock of the plasmid in order to conduct further experimentation in yeast. <br><br />
<br />
</p> <br />
<br />
<br />
<h2> Preparation and Passaging of <i> Saccharomyces cerevisiae </i> </h2><br />
<p align = left> <br />
<br />
Once, plasmids of the five possible degron constructs have been cloned with our three test proteins, they are subsequently transformed in PyE1 a strain of <i> S. cerevisiae </i> with the ability to produce green fluorescent proteins. Following the transformation, the cells are plated onto plates with on a Selective Dropout (C-Uracil) media and incubated at 30<sup>o</sup>C for 2 days. The purpose of the dropout media is to ensure that only cells that contain our plasmid survive as the recombinant plasmid allows cells to produce uracil, an essential amino acid. Without the recombinant plasmid, the cell would be fatally deprived of uracil which has been "knocked out" of the plating media. After two days, three colonies are swiped from the plate and added to an overnight culture of 2-3mL Selective Dropout Media C-Uracil and 2% Glucose then incubated for another two days at 30<sup>o</sup>C. After another two days of incubation, a 20-50uL aliquot of each culture is "passaged" into another 3mL culture prepared in the same manner as before and incubated for the same duration and temperature as the previous culture. The passaging is done several times after each passage after the second passage, a glycerol stock is prepare from the culture and Flow Cytometry is run on the culture. <br><br />
<br />
</p><br />
<br />
<p align = left> <br />
<br />
The purpose of passaging is to gradually remove excess copies of the plasmid constructs. Excess copies, exceeding one per cell will lead to multiple fold increase in the expression of the degron protein construct. As a result of this, GFP expression will also be increased thus reducing the viability and accuracy of the Flow Cytometry measurements conducted on each cell culture. This problematice as GFP output will become related to the number of plasmids as well as the stability of the various degron constructs which will likely invalidate any results.<br />
<br />
</p> <br />
<br />
<h2> Construct Stability Analysis with Flow Cytometry </h2><br />
<br />
<p> <br />
<br />
After PYE1 cells have been passaged several times and have roughly one copy of our degron construct containing plasmid, the cells are now ready to be run through a flow cytometer. On the morning of the day that the instrument is to be used, the optical density of cultures is read and an aliquot is taken from each culture and diluted to similar optical densities. This is to ensure that all cultures are in the same growth phase and will express our protein of interest degron construct equally so there will be no discrepancies in expression levels that could skew our fluorescence measurements. Once diluted, the cultures are incubated for roughly six hours. Approximately 200 microliters of each culture is pelleted, and resuspened in buffer and run through the flow cytometer. <br><br />
<br><br />
The instrument reads forward and side scatter allowing us to see cell debris or contaminants which can then be gated (excluded) from the actual fluorescent readings. Each construct containing culture is analyzed and both mean fluorescence of a culture as well as fluorescent culture’s population data is collected. Both of which can be used to analyze the relative stabilities of each construct and protein of interest.<br />
<br />
</p> <br />
<br />
<br />
<br />
<h2> Mutagenesis through Error Prone Polymerase Chain Reactions (E-PCRs) </h2><br />
<br />
<p><br />
<br />
In order to validate our system as being capable of selecting more stable protein variants, we have to produce mutations in our protein of interest. Those mutations could potentially be beneficial and could carry a stabilizing affect on the protein of interest. Error-prone PCR utilizes DNA-polymerase's error-prone nature and further increases the likelyhood of mutations by manipulating the conditions in which, DNA-polymerase operates in, thereby causing the polymerase to create errors in DNA sequencing which in turn will create changes in the protein construct. Once, the DNA coding of our protein has been changed we can express the mutations and analyze those stabilizing or de-stabilizing affects they have on our protein. The possible mutations can then be analyzed using the degron system and more stable mutations can be easily seen then sequenced.<br />
<br />
</p><br />
<br />
<br />
<h2> Selecting Stable Variants through Fluorescence Activated Cell Sorting (FACS) </h2><br />
<br />
<p><br />
<br />
In order to validate our system as being capable of selecting more stable protein variants, we have to produce mutations in our protein of interest. Those mutations could potentially be beneficial and could carry a stabilizing affect on the protein of interest. Error-prone PCR utilizes DNA-polymerase's error-prone nature and further increases the likelyhood of mutations by manipulating the conditions in which, DNA-polymerase operates in, thereby causing the polymerase to create errors in DNA sequencing which in turn will create changes in the protein construct. Once, the DNA coding of our protein has been changed we can express the mutations and analyze those stabilizing or de-stabilizing affects they have on our protein. The possible mutations can then be analyzed using the degron system and more stable mutations can be easily seen through FACS (see below) then subsequently sequenced. As we sequence the various mutants, we are also looking for convergence in which the genetic sequence as well as the amino acid sequences converge on a single or a few mutations that lead to significantly higher fluorescence and therefore, higher stability.<br />
<br />
</p> <br />
<br />
</body><br />
<br />
</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2014-10-16T22:17:19Z<p>Kristanguyen: </p>
<hr />
<div>{{Template:Team:UW/CSS}}<br />
<br />
<br />
<h1> Protocols </h1><br />
<br />
<br />
<html><a name="Media, Plates, and Sol"></a><br />
<br />
</html><br />
<br />
<br />
<h2> Media, Plates, and Solutions </h2><br />
<br />
<br />
<h3> 40% and 20% Glucose </h3><br />
<br />
<br />
<p><br />
<br />
40g for 40% or 20g for 20% of Glucose <br><br />
<br />
Mix in 100mL dH2O <br><br />
<br />
Sterile filter into a 150mL bottle <br><br />
<br />
</p><br />
<br />
<br />
<h3> 20% Glycerol </h3><br />
<br />
<br />
<p><br />
<br />
20g Glycerol (Liquid) <br><br />
<br />
Mix in 100mL dH2O <br><br />
<br />
Sterile filter into a 150mL bottle <br><br />
<br />
</p><br />
<br />
<br />
<h3> Competent Cell Media Buffer (CCMB) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2L container: <br><br />
<br />
100g Glycerol (liquid) <br><br />
<br />
10mL x 1M Potassium Acetate <br><br />
<br />
11.8g CaCl2*H2O <br><br />
<br />
4g MnCl2 <br><br />
<br />
2g MgCl2 <br><br />
<br />
1L of dH2O <br><br />
<br />
Sterile filter or autoclave (20min at 121C and 20psi) in a 1L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Luria Broth (LB) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2L container: <br><br />
<br />
10g tryptone <br><br />
<br />
5g yeast extract <br><br />
<br />
10g NaCl <br> <br />
<br />
1L of dH2O <br><br />
<br />
Autoclave in two 500 ml bottle (20 min at 121C and 20psi) <br><br />
<br />
<br />
*If using antibiotics create a separate aliquot<br />
<br />
</p><br />
<br />
<br />
<h3> LB-Agar </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2L container: <br><br />
<br />
1000ml LB as above <br><br />
<br />
15g agar <br><br />
<br />
1L of dH2O <br><br />
<br />
Autoclave in two 500mL bottles (20 min at 121C at 20psi) <br> <br />
<br />
<br />
<br />
*If using antibiotics create a separate aliquot<br />
<br />
<br />
</p><br />
<br />
<br />
<h3> Super Optimal Broth (SOB) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2L container: <br><br />
<br />
20g BactoTryptone <br><br />
<br />
5g BactoYeast Extract <br><br />
<br />
10mL x 1M NaCl <br><br />
<br />
2.5mL x 1M KCl <br><br />
<br />
1L of dH2O <br><br />
<br />
Sterile filter or autoclave (20 min at 121C and 20psi) in a 1L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Phosphate Buffered Saline (PBS) Solution </h3><br />
<br />
<br />
<br />
<p><br />
<br />
Mix the following to a 2L container or 1L beaker: <br><br />
<br />
8g NaCl <br><br />
<br />
1.44g Na2HPO4 <br><br />
<br />
0.8g KCl <br><br />
<br />
0.24g KH2PO4 <br><br />
<br />
1L of dH2O <br><br />
<br />
Buffer to pH 7.4 <br><br />
<br />
Sterile filter or autoclave (20 min at 121C and 20psi) in a 1L bottle<br />
<br />
</p><br />
<br />
<br />
<h3> Terrific Broth (TB) </h3><br />
<br />
<br />
<p><br />
<br />
<br />
<br />
Mix the following in a 1L bottle: <br><br />
<br />
6g Tryptone <br><br />
<br />
12g Yeast Extract <br><br />
<br />
2mL Glycerol <br><br />
<br />
500mL of dH2O <br><br />
<br />
Autoclave (20min at 121C and 20psi) <br><br />
<br />
Cool and add 5mL of 100X Potassium Phosphate Salts (17mM KH2PO4 and 72mM K2HPO4)<br />
<br />
</p> <br />
<br />
<br />
<h3> Yeast Extract Peptone Dextrose (YPD) </h3><br />
<br />
<br />
<p><br />
<br />
Mix the following into 950mL of dH2O in a 1L bottle: <br><br />
<br />
20g Bacto Peptone <br><br />
<br />
10g Yeast Extract <br><br />
<br />
Autoclave (20min at 121C and 20psi) <br><br />
<br />
Add 50mL 40% Glucose <br><br />
<br />
Sterile filter into a 1L bottle <br><br />
<br />
<br />
<br />
For long-term liquid media storage, do not add 40% Glucose instead add the glucose directly into cell cultures. <br><br />
<br />
For YPD-plates add 24g Bacto Agar to the Bacto Peptone and Yeast Extract before autoclaving. <br />
<br />
</p><br />
<br />
<br />
<h3> Selective Dropout media, C-Uracil and C-Histidine (C-Ura and C-His) </h3><br />
<br />
<br />
<p><br />
<br />
Synthesized by the Yeast Resource Center at the University of Washington's <br />
<br />
Department of Genome Sciences and Department of Biochemistry.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<h3> Guanidinium Hydrogen Chloride </h3><br />
<br />
<br />
<p><br />
<br />
For maximum effectiveness, final concentration should be approximately 8.5M in PBS<br><br />
<br />
203g Guanidinium Hydrogen Chloride <br><br />
<br />
250mL PBS solution <br><br />
<br />
Add dilute HCl to 7.4pH <br><br />
<br />
<br />
*Alternatively add slightly less than 250mL of PBS in order to buffer the solution to the appropriate volume then add more dH2O as neccessary. <br />
<br />
</p><br />
<br />
<html><a name="Basic Cloning"></a><br />
<br />
</html><br />
<br />
<h2> Basic Cloning </h2><br />
<br />
<br />
<br />
<h3> Polymerase Chain Reaction </h3><br />
<br />
<br />
<p><br />
<br />
All PCRs were done using a standard 50μL reaction volume. <br><br />
<br />
PCRs were done using GoTaq Green Master Mix 2X purchased from PROMEGA Corporation. <br><br />
<br />
Protocols for the PROMEGA GoTaq Green Master Mix 2X: <br><br />
<br />
Mix the following in a 0.2mL microcentrifuge tube on ice: <br><br />
<br />
25μL GoTaq® Green Master Mix 2X <br><br />
<br />
1-5μL of 10μM forward primer <br><br />
<br />
1-5μL of 10μM reverse primer <br><br />
<br />
<250ng of DNA template <br><br />
<br />
Nuclease-free water to 50μl <br><br />
<br />
Conduct the reaction in a Thermocycler, adjusting anneal temperature and extension times accordingly. See your polymerase supplier protocol for more details on thermocycling.<br />
<br />
</p><br />
<br />
<h3> Error-prone Polymerase Chain Reaction </h3><br />
<br />
<br />
<p> <br />
<br />
<br />
<br />
Prepare 50μL reaction: <br><br />
<br />
5μL 10X Mutazyme II Rxn Buffer <br><br />
<br />
1μL 40mM dNTP mix (200μM each final) <br><br />
<br />
1μL 20μM forward primer <br><br />
<br />
1μL 20μM reverse primer <br><br />
<br />
1μL Mutazyme II DNA polymerase (2.5U/μL) <br><br />
<br />
0.01ng template <br><br />
<br />
QS 50μL diH2O <br><br />
<br />
<br><br />
<br />
Thermocycler: <br><br />
<br />
95C, 2min <br><br />
<br />
95C, 30sec <br><br />
<br />
XXC*, 30sec <br><br />
<br />
72C, Xmin** <br><br />
<br />
32 cycles <br><br />
<br />
72C, 10min <br><br />
<br />
4C, hold<br />
<br />
<br><br />
<br />
*Adjust annealing temperature according to Tm of primer. <br><br />
<br />
**Adjust extension time according to the length of amplified DNA. <br><br />
<br />
<br><br />
<br />
Note: Use 0.01ng (calculate by insert and not by total plasmid). <br><br />
<br />
Calculate amount of template to use. <br><br />
<br />
(bp for amplified region) / (bp in total plasmid) = % amplified region <br><br />
<br />
(conc of total plasmid) x (% amplified region as a decimal) = conc of amplified region <br><br />
<br />
<br><br />
<br />
Note: Will probably need to dilute. Never pipette less than 0.5μL. <br><br />
<br />
(0.01ng) / (conc of amplified region) = vol to add to PCR <br><br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Restriction Endonuclease Reaction (Digestion) </h3><br />
<br />
<br />
<br />
<p><br />
<br />
All restriction enzyme reactions were done using a 50ul reaction volume. <br><br />
<br />
Restriction enzymes and buffers were purchased from New England Biolabs Incorporated. <br><br />
<br />
Protocols for various New England Biolab restriction enzyme reactions: <br><br />
<br />
Mix the following in a 0.2mL PCR tube: <br><br />
<br />
1μL of each Restriction Enzyme, add the RE last <br><br />
<br />
1μg of DNA <br><br />
<br />
5μL of the appropriate 10X New Englan Biolab Buffer <br><br />
<br />
Nuclease-free water to 50μL <br><br />
<br />
Incubate the reaction for 1hr <br><br />
<br />
Heat inactive the the reaction at the appropriate temperature <br><br />
<br />
<br />
Notes: Add the restriction enzyme(s) to the reaction last <br><br />
<br />
Thaw the restriction enzyme(s) on ice to improve shelf life <br><br />
<br />
<br />
<br />
</p> <br />
<br />
<br />
<h3> Ligation </h3><br />
<br />
<br />
<p><br />
<br />
T4 DNA Ligase and Buffer was purchased from New England BioLabs Corporation. <br><br />
<br />
1. Prepare the following in a 0.2mL microcentrifuge tube: <br><br />
<br />
50.0ng Vector DNA* <br><br />
<br />
37.5ng Vector DNA* <br><br />
<br />
2μL 10X T4 DNA Ligase Buffer <br><br />
<br />
1μL T4 DNA Ligase <br><br />
<br />
Add diH2O to 20μL <br><br />
<br />
2. Incubate the reaction at room temperature for 10-30 minutes or at 16C overnight. <br><br />
<br />
3. Heat inactivate at 65C for 10 minutes. <br><br />
<br />
4. Chill on ice before starting a transformation reaction. <br><br />
<br />
*The exact amount of DNA is dependent on the number of base pairs. In order to conduct a proper reaction consult the New England Biolab Ligation Calculator at:<br />
<br />
http://nebiocalculator.neb.com/#!/<br />
<br />
</p><br />
<br />
<br />
<html><a name="Escherichia coli Protocols"></a><br />
<br />
</html><br />
<br />
<br />
<h2> <i> Escherichia coli Protocols </i> (XL1-Blue and XL10-Gold) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Culturing </h3><br />
<br />
<br />
<br />
<p> <br />
<br />
<br />
Chemically Competent Cell Stocks – CCMB Transformation <br />
For BL21*, CJ236, etc. Cell Lines. <br><br />
<br />
NOTE: Make SOB-Mg media and autoclave the 250ml flasks with water in them the same day the cells are plated (step 1) <br><br />
<br><br />
<br />
1. Plate 10ul of cells onto an antibiotic free LB/Agar plate..* <br><br />
<br />
2. Incubate overnight at 37OC <br><br />
<br />
3. Inoculate lots of colonies into 50ml SOB-Mg media in a 250ml flask. <br><br />
4. Incubate at 37 OC, 200rpm, until OD at 550nm reaches 0.3, takes about 3 hours. I do my first check at 2 hours.<br><br />
<br />
5.Transfer the cell culture into a sterile 50ml Falcon tube and chill on ice for 10 minutes. <br><br />
<br />
6. Pellet the cells at 2500rpm for 15 minutes at 4 OC.<br><br />
<br />
7. Decant supernatant and invert tubes to remove excess culture medium. <br><br />
<br />
8. Resuspend the cells in 16ml CCMB by gentle vortexing or pipetting (I shake the falcon tube).<br />
(16ml CCMB per 50ml culture) <br><br />
<br />
9. Incubate on ice for 20 minutes. <br><br />
<br />
10. Centrifuge at 2500rpm for 10 minutes at 4 OC. <br><br />
<br />
11. Decant supernatant and invert tubes to remove excess liquid. <br><br />
<br />
12. Resuspend the cells in 4ml CCMB by gentle vortexing or pipetting.<br />
(4ml CCMB per culture) <br><br />
<br />
13. If making multiple 50ml cultures, combine all of the cells and mix before aliquoting. <br> <br />
<br />
14. In cold room: make 205ul aliquots, flash freeze in liquid nitrogen, and store at -80 OC.<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Chemically Competent Cell Transformations </h3><br />
<br />
<br />
<p> <br />
<br />
1. Thaw competent <i> E.coli </i> cells on ice (XL1-Blue or XL10-Gold)* <br><br />
<br />
2. Add 50μL of competent cells to sterile 15mL conical centrifuge tubes <br><br />
<br />
3. Add 1μL (~100-200ng)* of the mini-prep to each culture tube <br><br />
<br />
4. Equilibrate the cells on ice for 10 min <br><br />
<br />
5. Heat shock the cells at 42C for 30-45 seconds** <br><br />
<br />
6. Immediately place the cells back on ice for 3 min <br><br />
<br />
7. Add 250μL LB media without antibiotics and shake at 250 rpm and 37C for 30 min <br><br />
<br />
8. Spread 10μL and 290μL on an appropriate LB-antibiotic plate <br><br />
<br />
9. Invert the plate and incubate at 37C overnight<br />
<br />
<br />
*The exact amount of DNA to add depends on your cell's transformation efficiency. However, it is acceptable to add a larger amount to increase the number of transformed cells. <br><br />
<br />
** Do not heat shock for an extended duration as this may damage and/or kill your cells.<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnights </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottom tube, add 3-5mL of LB and an appropriate volume of antibiotic(s). <br><br />
<br />
2. Swipe several individual colonies, do not collect satellites or colony clumps, with a pipette tip. <br><br />
<br />
3. Swirl the colony tip in the tube, there should be no visible cell clumps. <br><br />
<br />
4. Incubate and shake the tube at 37C at 250rpm for 12-16 hours and no longer than 20 hours. <br><br />
<br />
</p><br />
<br />
<br />
<h3> DNA-Extraction and mini-preps </h3><br />
<br />
<br />
<p><br />
<br />
All DNA mini-preps were prepared using EPOCH mini-kits and following the supplied protocols.<br />
<br />
</p><br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500μL of 40% Glycerol and 500uL of LB (no antibiotics) or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Saccharomyces cerevisiae"></a><br />
<br />
</html><br />
<br />
<h2> <i> Saccharomyces cerevisiae </i> (PYE1 Yeast) </h2><br />
<br />
<br />
<h3> Chemically Competent Cell Culturing </h3><br />
<br />
<br />
<br />
<p><br />
<br />
This process take 4 days in lab with a 1 day wait for incubation. <br><br />
<br />
<br />
Day 1: <br><br />
<br />
1. Streak yeast cells onto a YPD plate.* <br><br />
<br />
2. Invert the plate and incubate at 30C for 2 days. <br><br />
<br />
<br />
Day 3: <br><br />
<br />
1. Add 50mL of YPD liquid media into a 250mL baffle flask. <br><br />
<br />
2. Swipe as many individual colonies as you can see into the YPD media.** <br><br />
<br />
3. Incubate and shake the culture at 30C at 250rpm overnight approximately 24 hours. <br><br />
<br />
<br />
Day 4: <br><br />
<br />
1. Take an optical density measurement. <br><br />
<br />
2. In three 250mL baffle flask add the portions of the overnight liquid culture. <br><br />
<br />
3. Dilute each culture to approximately 0.4 optical density with YPD. <br><br />
<br />
4. Incubate and shake the cultures at 30C at 250rpm until the optical density reaches 1.2-1.6. <br><br />
<br />
5. Collect each culture into separate 50ml flat-bottomed centrifuge tubes. <br><br />
<br />
6. Spin down the cells at 4000g for 5 minutes at 4C. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cells in 100mL total for all three culture of dH2O. <br><br />
<br />
9. Combine the suspensions into two 50mL flat-bottomed centrifuge tubes. <br><br />
<br />
10. Spin down the cells as above. <br><br />
<br />
11. Decant the supernatant. <br><br />
<br />
12. Resuspend each in 3mL of 100mM Lithium Acetate. <br><br />
<br />
13. Transfer both cultures into a single 15mL conical centrifuge tube. <br><br />
<br />
14. Spin down the cells at 3000rpm for 5 minutes. <br><br />
<br />
15. Resuspend the cells in 0.75mL of 100mM Lithium Acetate, total volume is roughly 2mL. <br><br />
<br />
16. Qualitatively bring up the volume to 3.5mL by adding 40% Glycerol. <br><br />
<br />
17. Aliquot the cells into 1.5mL centrifuge tubes or 1.7mL cryogenic vials.*** <br><br />
<br />
<br />
*Streak in such a way that there are individual colonies visible on the plate without clumps or satellite colonies. <br><br />
<br />
**Collect only individual visible colonies. Do not collect clumps or satellite colonies. <br><br />
<br />
***The volume of aliquots depends on the number to transformations you intend to do at a time. <br><br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Chemically Competent Transformations </h3><br />
<br />
<br />
<p><br />
<br />
This protocol assumes a 50μL aliquot of yeast competent cells were made. <br><br />
<br />
Furthermore, this protocol prepares enough cells for 6 yeast transformations. <br><br />
<br />
<br><br />
<br />
1. Add the following to 50μL of yeast competent cells: <br><br />
<br />
240μL of Polyethylene Glycol - 3350 (PEG-3350) <br><br />
<br />
36μL of 1M Lithium Acetate <br><br />
<br />
32μL of dH2O <br><br />
<br />
<br />
<br />
2. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
3. Aliquot 59μL of the mixture into a 0.2mL microcentrifuge tube. <br><br />
<br />
4. Add 1uL (~100-200ng) of DNA. <br><br />
<br />
5. Mix the mixture by gentle pipetting or vortexing. <br><br />
<br />
6. Incubate the mixture at 30C for 30 minutes. <br><br />
<br />
7. Heat shock the mixture at 42C for 20 minutes. <br><br />
<br />
8. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
9. Decant the supernatant. <br><br />
<br />
10. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
11. Spin down the cells in a microcentrifuge for ~1 minute. <br><br />
<br />
12. Resuspend the cell pellets in 200μL of dH2O. <br><br />
<br />
13. Plate 50-150μL of the mixture onto an appropriate Selective Dropout Media plate. <br><br />
<br />
14. Invert and incubate at 30C for 2 days. <br><br />
<br />
*The exact amount of DNA depends on the transformation efficiency of your competent cells. <br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Overnight Culturing </h3><br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Swipe 3 individually visible yeast colonies and add them to the culture tube media. <br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%.<br />
<br />
</p><br />
<br />
<br />
<h3> Culture Passaging </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br><br />
<br />
2. Take 20-50μL from a previous overnight or passage culture and add it to the culture media.<br><br />
<br />
3. Incubate and shake at 37C at 250rpm for 2 days. <br><br />
<br />
<br />
Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%. <br><br />
<br />
Note: The exact amount of culture that you take from a previous culture is irrelevant as long as at least 1 living cell is passaged.<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3> Glycerol Stocks </h3><br />
<br />
<br />
<p><br />
<br />
1. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br><br />
<br />
2. Spin down the culture at 3000rpm for 3 minutes. <br><br />
<br />
3. Decant the supernatant. <br><br />
<br />
4. Resuspend the cells in 500uL of 40% glycerol and 500μL of Selective Dropout media or water. <br><br />
<br />
5. Transfer the resuspension to a cryogenic vial. <br><br />
<br />
6. Store the glycerol stock at -80C. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Flow Cytometry"></a><br />
<br />
</html><br />
<br />
<h2> Flow Cytometry </h2><br />
<br />
<br />
<h3> Dilutions </h3><br />
<br />
<br />
<br />
<p><br />
<br />
1. From an overnight culture measure the optical density at 660nm by making 1:10 dilutions. <br><br />
<br />
2. Take enough culture to make a 1mL aliquot with OD 0.4. <br><br />
<br />
3. Spin down the aliquot in a 1.5mL centrifuge tube at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 800μL of the appropriate selective dropout media and 200μL of 20% glucose. <br><br />
<br />
6. Transfer the new culture to a 14mL culture tube. <br><br />
<br />
7. Incubate and shake at 30C and 250rpm for at least 6 hours (1.2-1.6 optical density).<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h3> Preparations for Analysis using C6 Accuri Flow Cytometer </h3><br />
<br />
<br />
<p><br />
<br />
<br />
1. From the dilution previously made, measure the optical density, roughly 1.2-1.6. <br><br />
<br />
2. Make an aliquot of 500μL of the dilution culture in a 1.5mL centrifuge tube. <br><br />
<br />
3. Spin down the aliquot at 3000rpm for 3 minutes. <br><br />
<br />
4. Decant the supernatant. <br><br />
<br />
5. Resuspend the cell pellet in 500μL of PBS(F). <br><br />
<br />
6. Spin down the resuspension at 3000rpm for 3 minutes. <br><br />
<br />
7. Decant the supernatant. <br><br />
<br />
8. Resuspend the cell pellet in another 500uL of PBS(F).* <br><br />
<br />
9. Prepare the C6 Accuri Flow Cytometer by running a backflush cycle and a dH2O cycle. <br><br />
<br />
10. Load the sample onto the sip. <br><br />
<br />
11. Run the sample with 100,000 cell count. <br><br />
<br />
12. Repeat for all samples and make sure to change data cells otherwise the old data is erased. <br><br />
<br />
13. Once finished, run a cleaning cycle with Accuri approved cleaning solution, then run a dH2O cycle. <br><br />
<br />
<br />
<br />
*For special cases do not resuspend all samples, instead resuspend immediately before running the sample through the flow cytometer.<br />
<br />
<br />
</p><br />
<br />
<br />
<html><a name="Fluorescence Activated Cell Sorting"></a><br />
<br />
</html><br />
<br />
<h2> Fluorescence Activated Cell Sorting </h2><br />
<br />
<br />
<h3> Final Preparations </h3><br />
<br />
<br />
<br />
<p> <br />
<br />
Sample Prep:<br />
<br />
<br />
Spin down samples and negative control (5000 RPM, 1 min), keeping in mind the library size. Aspirate off supernatant. Resuspend in PBSF. Spin down cells. Aspirate off supernatant. Resuspend in PBSF. <br><br />
<br />
<br><br />
<br />
1. Open the “iGEM Template” file in the FACS Software and change name to current date and sort cycle<br><br />
<br />
2. Make sure the stream is stable (look for green light in bottom right corner). If not, run the Sort Calibration order. <br><br />
<br />
3. Run Negative Control from the PyE1 cells. <br><br />
<br />
a. Load cells onto carrier and into the machine. Press play button on screen. <br><br />
<br />
b. Set gate around lower left quadrant of cells to ensure single cell analysis using Forward Scatter Area and Side Scatter Area as your axes. Make sure oval gate covers around 80% of cell population. <br><br />
<br />
c. Set second gate on the first gated population by double-clicking on the gated population and using Forward Scatter Height and Forward Scatter Width as your axes. You will notice two distinct populations. Try to focus on the single cell portion of the plot. <br><br />
<br />
NOTE: If you see a large portion of the second gated population existing near the upper right edge of the first gate, you may need to enlarge the first gate to fit more of the population. <br><br />
<br />
d. Press record. Record 100,000 events and stop run. Move to Next Tube. <br><br />
<br />
4. Run first control (Gene clone) <br><br />
<br />
a. Follow step 3 to run the first control <br><br />
<br />
5. Run first library sample <br><br />
<br />
a. Follow steps 3b and 3c to set first two gates correctly. <br><br />
<br />
b. Set final gate for sort which includes top 1% of GFP producers from second gated population. <br><br />
<br />
c. Use final gate to set up the sort. <br><br />
<br />
d. Select sort conditions at the bottom of the screen. <br><br />
<br />
e. Insert and load collection tube. <br><br />
<br />
f. Record 100,000 events, and sort 10x the library size <br><br />
<br />
6. Run Bleach and diH2O through FACS to avoid cross-contamination. <br><br />
<br />
</p><br />
<br />
<br />
<html><a name="Protein Expression"></a><br />
<br />
</html><br />
<br />
<h2> Protein Expression </h2><br />
<br />
<br />
<h3> Overnight Cultures </h3><br />
<br />
<p><br />
<br />
1. Add 25mL TB and 25uL Kan to a 250mL baffled flask <br><br />
<br />
2. Stab a glycerol stock with a p1000 pipette and swirl in the flask of media <br><br />
<br />
3. Put flask in 37C shaker overnight <br />
<br />
</p><br />
<br />
<h3> Protein Expression </h3><br />
<br />
<p> <br />
<br />
1. Add 500μL 1000x Kanamycin to 500mL TB in 2L baffled flask <br><br />
<br />
2. Transfer 10mL overnight culture to TB <br><br />
<br />
3. Shake at 37C (DO WE NEED THE RPM?) <br><br />
<br />
4. Remove flask from shaker when optical density is between 0.5 and 0.8 <br><br />
<br />
5. Allow flask to rest at room temp for 30 min <br><br />
<br />
6. Add 125μL 1M IPTG <br><br />
<br />
7. Shake flask at 18C overnight<br />
<br />
</p><br />
<br />
<h3> Protein Extraction and Purification </h3><br />
<br />
<p><br />
<br />
1. Transfer cell culture to centrifuge flask <br><br />
<br />
2. Centrifuge culture at 4000g for 10 min <br><br />
<br />
3. Discard supernatant <br><br />
<br />
4. Resuspend pellet in 25mL lysis buffer <br><br />
<br />
5. Add 250μL of 100x PMSF, 250μL of 100mg/mL lysozyme, and 250μL of 10mg/mL DNAse <br><br />
<br />
6. Sonicate sample with 0.25inch probe for 5 min at 70% amplitude with 20 sec on and off pulses <br><br />
<br />
7. Take 50μL total sample <br><br />
<br />
8. Transfer lysate to SS-34 centrifuge tube <br><br />
<br />
9. Centrifuge for 30 min at 18000g <br><br />
<br />
10. Take 50μL soluble sample<br />
<br />
</p><br />
<br />
<h3> Nickel Nitrotriacetic Acid Chromatography (Nickel-NTA Chromatography) </h3><br />
<br />
<p><br />
<br />
1. Add 5mL 50%(v/v) nickel resin in ethanol to 25mL column and allow to settle to ~2.5mL(CV) <br><br />
<br />
2. Rinse with 10CV H20 <br><br />
<br />
3. Equilibrate with 10CV lysis buffer <br><br />
<br />
4. Load sample onto column <br><br />
<br />
5. Wash column with 15CV lysis buffer <br><br />
<br />
6. Perform 2 additional wash steps with 15CV <br><br />
<br />
7. Elute sample in 10CV elution buffer and collect eluate <br><br />
<br />
8. Take 50μL pure sample <br />
<br />
</p><br />
<br />
<h3> Size Exclusion Chromatography (S.E.C.) </h3><br />
<p><br />
1. Concentrate sample to as high as possible without inducing protein aggregation<br><br />
2. Pre-equilibrate Superdex 75 column with 48 ml PBS <br><br />
3. Inject 500 ul sample onto column <br><br />
4. Run 36 ml PBS through column at 0.5 ml/min, collecting 1 ml fractions <br><br />
5. Verify presence of protein in fractions by measuring concentration on Nanodrop and running SDS-PAGE (15 kDa protein should elute at ~13 ml)<br><br />
6. Pool fractions containing protein <br />
</p><br />
<br />
<br />
<html><a name="Stability Analysis"></a><br />
<br />
</html><br />
<br />
<h2> Stability Analysis </h2><br />
<br />
<br />
<h3> Circular Dichroism: Wavelength Scan </h3><br />
<p><br />
1. Load 1mm cuvette with 400uL protein solution onto CD <br><br />
2. Take wavelength scan <br><br />
260nm-190nm <br><br />
sample every 1nm <br><br />
averaging time 3sec <br><br />
1 scan <br><br />
step scan <br><br />
25C <br><br />
3. Record wavelength which gives strongest signal (222nm)<br />
</p><br />
<br />
<h3> Circular Dichroism: Guanidine Melt </h3><br />
<p><br />
1. Load 1cm cuvette containing 1.996mL of 0.05mg/mL protein solution and stirrer onto CD <br><br />
2. Prepare 8mL of 0.05mg/mL protein in concentrated guanidine solution <br><br />
3. Set up Automixer with guanidine solution on one syringe and waste tube on other syringe <br><br />
4. Titrate up to 6 M guanidine, taking a CD measurement at 222 nm every 0.15 M interval <br><br />
5. Also measure the fluorescence at 280 nm to ensure the total protein concentration is not changing<br />
</p><br />
<br />
<br />
<br />
<html><br />
<br />
<a href="#linktotop">Back To Top</a><br />
</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/Our_ProjectTeam:Washington/Our Project2014-10-16T21:40:11Z<p>Kristanguyen: </p>
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<h1> Our System </h1><br />
<br />
<h2> Components of the Degron Construct </h2><br />
<br />
<center> <img src="https://static.igem.org/mediawiki/2014/1/1d/UWPlasmid.png" alt="Degron Constructs" style="width:500px;height:406px"><br />
<br />
<br><br />
<br />
<sup> <b> Fig 1. </b> Potential Degron insert sites for our system. </sup> </center><br />
<br />
<br> <br><br />
<br />
<p><br />
<br />
Our experiment utilizes 5 different Degron constructs: <br><br />
-Deg0: This construct contains only the Gal4-VP16 transcriptional activator complex with the protein of interest in between the two (shortened as Gal4-Protein-VP16). <br><br />
-Deg1: This construct contains the Degron in front of our Gal4-Protein-VP16 complex. <br><br />
-Deg2: This construct contains the Degron in between Gal4 and the protein in our Gal4-Protein-VP15 complex. <br><br />
-Deg3: This construct contains the Degron in between the protein and VP16 in our Gal4-Protein-VP15 complex. <br><br />
-Deg4: This construct contains the Degron at the end of our Gal4-Protein-VP16 complex. <br><br />
<br />
</p><br />
<br />
<h3> Relative Stability Analyzed via Flow Cytometry </h3><br />
<br />
<p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/d/d9/Degron_construct.jpg" alt="Degron Constructs" style="width:750px;height:379px"><br />
<br />
<br><br />
<br />
<sup> <b> Fig 2. </b> Expected GFP output based on our Degron constructs </sup> </center> <br />
<br />
<br><br />
<br />
Flow cytometry is a high throughput method of analyzing cells for various optical outputs, namely fluorescence. A flow cytometer is an analytical instrument in which cells that have been suspended in a solution are passed through a narrow channel in which fluorescence of individual cells can be measured. <br><br />
<br><br />
By utilizing Flow Cytometry, we can measure the amount of GFP output within cells from each degron construct. Based on where the Degron is inserted, we expected a different level of fluorescence. As such, we expected to see the highest GFP production in our Deg0 construct, as it only contains the Gal4-Protein-VP16 complex with no Degron inserted, hence being the most stable. We expected that Deg2 and Deg3 would have a lower GFP production than Deg0 but higher than Deg1 and Deg4. This rationale was based on the fact that the Deg1 and Deg4 have the Degron exposed, making it more likely to be degraded by ubiquitination than in Deg2 and Deg3 which has the Degron buried inside the Gal4-Protein-VP16 complex.<br><br />
<br />
</p><br />
<br />
<br />
<br />
<h2> Test Protein </h2><br />
<br />
<p align = left> <br />
<br />
The test protein that must be chosen in testing a novel and new system must be a protein that has been well studied and rigorously examined through other existing and well accepted protein stability testing methods. <br />
Therefore,our team decided to use the protein known as BINDI. <br />
BINDI and two of its less stable variants, BbpD04 and BbpD04.3 were studied and examined in <i> A Computationally Designed Inhibitor of an Epstein-Barr Viral Bcl-2 Protein Induces Apoptosis in Infected Cells </i> by Procko et al. <br />
Many of the individuals who were cited in the paper including Dr. Procko as well as the lab that did the research was done in was a nearby lab at the University of Washington.<br />
Therefore, it was convenient for us to contact members of their lab and speak with them about the protein we intended on using as well as acquiring samples of the protein's DNA code.<br />
<br />
</p> <br />
<br />
<br />
<h2> PYE1 a strain of <i> Saccharomyces cerevisiae </i> </h2><br />
<p><br />
<br />
The lynchpin of our project is the usage of flow cytometry and fluorescence activated cell sorting for high throughput protein stability analysis. <br />
However, both analytical systems, flow cytometry and F.A.C.S. require fluorescence emission. <br />
Therefore we need a method of generating fluorescence within our cells in a way that also gives us insight into protein stability.<br />
Each degron construct contains a Gal4 promoter which can bind to an upstream activating site, Gal1, that induces downstream expression of something. <br />
It just so happens that PYE1, this something happens to be Green Fluorescent Proteins.<br />
Protein expressions in PYE1, will allow us to generate GFP relative to the amount of degron protein construct that exist within the cell. <br />
The more stable the degron protein construct is, the more likely it is that more GFP will be expressed.<br />
This relationship between stability and GFP forms the basis from which we will measure the relative protein stability of our degron constructs as well as the protein of interest degron construct.<br />
<br />
</p><br />
<br />
</body><br />
<br />
</html></div>Kristanguyenhttp://2014.igem.org/Team:Washington/Our_TeamTeam:Washington/Our Team2014-10-16T03:58:06Z<p>Kristanguyen: </p>
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<br />
<h1> UW iGEM 2014 Team </h1><br />
<br />
<h2> Undergraduate Team Members </h2><br />
<br />
<table><br />
<tr><br />
<td>Edward Chang, Biochemistry</td><br />
<td>Poster</td><br />
</tr><br />
<tr><br />
<td>Andrew Chau, Chemistry</td><br />
<td>Flow Cytometry and Team Page</td><br />
</tr><br />
<tr><br />
<td>Joshua Cho, Molecular, Cellular, & Developmental Biology</td><br />
<td>Fluorescence Activated Cell Sorting, Flow Cytometry, and Presentation</td><br />
</tr><br />
<tr><br />
<td>Chris Choe, Molecular, Cellular, & Developmental Biology</td><br />
<td>Expression and Stability Analysis</td><br />
</tr><br />
<tr><br />
<td>William Harvey, Biochemistry</td><br />
<td>Fluorescence Activated Cell Sorting and Presentation</td><br />
</tr><br />
<tr><br />
<td>Alex Kang, Biochemistry</td><br />
<td>Expression and Stability Analysis</td><br />
</tr><br />
<tr><br />
<td>Julia Lim, Microbiology</td><br />
<td>BioBricks, Flow Cytometry, and Team Page</td><br />
</tr><br />
<tr><br />
<td>Harman Malhi, Molecular, Cellular, & Developmental Biology</td><br />
<td>Expression and Stability Analysis</tr><br />
</tr><br />
<tr><br />
<td>Colton McDavid, Microbiology</td><br />
<td>Expression and Stability Analysis and Poster</td><br />
</tr><br />
<tr><br />
<td>Krista Nguyen, Biochemistry</td><br />
<td>Flow Cytometry, Poster, and Team Page</td><br />
</tr><br />
<tr><br />
<td>Anastasia Nicolov, Bioengineering and Violin Performance</td><br />
<td>Expression and Stability Analysis</td><br />
</tr><br />
<tr><br />
<td>Ahmed Qureshi, Biochemistry</td><br />
<td>BioBricks and Poster</td><br />
</tr><br />
<tr><br />
<td>Stephen Rettie, Microbiology</td><br />
<td>BioBricks, Fluorescence Activated Cell Sorting, and Presentation</td> <br />
</tr><br />
</table><br />
<br />
<br />
<h2> Advisors </h2><br />
<table><br />
<tr><br />
<td>Nick Bolten, Electrical Engineering</td><br />
</tr><br />
<tr><br />
<td>Cassie Bryan, Biochemistry</td><br />
</tr><br />
<tr><br />
<td>Arjun Khakhar, Bioengineering</td><br />
</tr><br />
<tr><br />
<td>Bob Lamm, Bioengineering</td><br />
</tr><br />
<tr><br />
<td>Erik Murphy, Molecular, Cellular, & Developmental Biology</td><br />
</tr><br />
<tr><br />
<td>Rashmi Ravichandran, Biochemistry</td><br />
</tr><br />
<tr><br />
<td>David Younger, Bioengineering</td><br />
</tr><br />
</table><br />
<br />
<h2> Faculty </h2><br />
<table><br />
<tr><br />
<td>David Baker, Biochemistry</td><br />
</tr><br />
<tr><br />
<td>Eric Klavins, Electrical Engineering</td><br />
</tr><br />
</table></div>Kristanguyenhttp://2014.igem.org/Team:Washington/Our_TeamTeam:Washington/Our Team2014-10-16T03:55:52Z<p>Kristanguyen: </p>
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<td>Edward Chang, Biochemistry</td><br />
<td>Poster</td><br />
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<tr><br />
<td>Andrew Chau, Chemistry</td><br />
<td>Flow Cytometry</td><br />
</tr><br />
<tr><br />
<td>Joshua Cho, Molecular, Cellular, & Developmental Biology</td><br />
<td>Fluorescence Activated Cell Sorting, Flow Cytometry, and Presentation</td><br />
</tr><br />
<tr><br />
<td>Chris Choe, Molecular, Cellular, & Developmental Biology</td><br />
<td>Expression and Stability Analysis</td><br />
</tr><br />
<tr><br />
<td>William Harvey, Biochemistry</td><br />
<td>Fluorescence Activated Cell Sorting and Presentation</td><br />
</tr><br />
<tr><br />
<td>Alex Kang, Biochemistry</td><br />
<td>Expression and Stability Analysis</td><br />
</tr><br />
<tr><br />
<td>Julia Lim, Microbiology</td><br />
<td>BioBricks, Flow Cytometry, and Team Page</td><br />
</tr><br />
<tr><br />
<td>Harman Malhi, Molecular, Cellular, & Developmental Biology</td><br />
<td>Expression and Stability Analysis</tr><br />
</tr><br />
<tr><br />
<td>Colton McDavid, Microbiology</td><br />
<td>Expression and Stability Analysis and Poster</td><br />
</tr><br />
<tr><br />
<td>Krista Nguyen, Biochemistry</td><br />
<td>Flow Cytometry, Poster, and Team Page</td><br />
</tr><br />
<tr><br />
<td>Anastasia Nicolov, Bioengineering and Violin Performance</td><br />
<td>Expression and Stability Analysis</td><br />
</tr><br />
<tr><br />
<td>Ahmed Qureshi, Biochemistry</td><br />
<td>BioBricks and Poster</td><br />
</tr><br />
<tr><br />
<td>Stephen Rettie, Microbiology</td><br />
<td>BioBricks, Fluorescence Activated Cell Sorting, and Presentation</td> <br />
</tr><br />
</table><br />
<br />
<br />
<h2> Advisors </h2><br />
<table><br />
<tr><br />
<td>Nick Bolten, Electrical Engineering</td><br />
</tr><br />
<tr><br />
<td>Cassie Bryan, Biochemistry</td><br />
</tr><br />
<tr><br />
<td>Arjun Khakhar, Bioengineering</td><br />
</tr><br />
<tr><br />
<td>Bob Lamm, Bioengineering</td><br />
</tr><br />
<tr><br />
<td>Erik Murphy, Molecular, Cellular, & Developmental Biology</td><br />
</tr><br />
<tr><br />
<td>Rashmi Ravichandran, Biochemistry</td><br />
</tr><br />
<tr><br />
<td>David Younger, Bioengineering</td><br />
</tr><br />
</table><br />
<br />
<h2> Faculty </h2><br />
<table><br />
<tr><br />
<td>David Baker, Biochemistry</td><br />
</tr><br />
<tr><br />
<td>Eric Klavins, Electrical Engineering</td><br />
</tr><br />
</table></div>Kristanguyenhttp://2014.igem.org/Team:Washington/Our_TeamTeam:Washington/Our Team2014-10-16T03:54:53Z<p>Kristanguyen: </p>
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<h1> UW iGEM 2014 Team </h1><br />
<br />
<h2> Undergraduate Team Members </h2><br />
<br />
<table><br />
<tr><br />
<td>Edward Chang, Biochemistry</td><br />
<td>Poster</td><br />
</tr><br />
<tr><br />
<td>Andrew Chau, Chemistry</td><br />
<td>Flow Cytometry</td><br />
</tr><br />
<tr><br />
<td>Joshua Cho, Molecular, Cellular, & Developmental Biology</td><br />
<td>Fluorescence Activated Cell Sorting, Flow Cytometry, and Presentation</td><br />
</tr><br />
<tr><br />
<td>Chris Choe, Molecular, Cellular, & Developmental Biology</td><br />
<td>Expression and Stability Analysis</td><br />
</tr><br />
<tr><br />
<td>William Harvey, Biochemistry</td><br />
<td>Fluorescence Activated Cell Sorting and Presentation</td><br />
</tr><br />
<tr><br />
<td>Alex Kang, Biochemistry</td><br />
<td>Expression and Stability Analysis</td><br />
</tr><br />
<tr><br />
<td>Julia Lim, Microbiology</td><br />
<td>BioBricks, Flow Cytometry, and Team Page</td><br />
</tr><br />
<tr><br />
<td>Harman Malhi, Molecular, Cellular, & Developmental Biology</td><br />
<td>Expression and Stability Analysis</tr><br />
</tr><br />
<tr><br />
<td>Colton McDavid, Microbiology</td><br />
<td>Expression and Stability Analysis and Poster</td><br />
</tr><br />
<tr><br />
<td>Krista Nguyen, Biochemistry</td><br />
<td>Flow Cytometry, Poster, and Team Page</td><br />
</tr><br />
<tr><br />
<td>Anastasia Nicolov, Bioengineering and Violin Performance</td><br />
<td>Expression and Stability Analysis</td><br />
</tr><br />
<tr><br />
<td>Ahmed Qureshi, Biochemistry</td><br />
<td>BioBricks and Poster</td><br />
</tr><br />
<tr><br />
<td>Stephen Rettie, Microbiology</td><br />
<td>BioBricks, Fluorescence Activated Cell Sorting, and Presentation</td> <br />
</tr><br />
</table><br />
<br />
<br />
<h2> Advisors </h2><br />
<table><br />
<tr><br />
<td>Nick Bolten, Electrical Engineering</td><br />
</tr><br />
<tr><br />
<td>Cassie Bryan, Biochemistry</td><br />
</tr><br />
<tr><br />
<td>Arjun Khakhar, Bioengineering</td><br />
</tr><br />
<tr><br />
<td>Bob Lamm, Bioengineering</td><br />
</tr><br />
<tr><br />
<td>Erik Murphy, Molecular Cellular Developmental Biology</td><br />
</tr><br />
<tr><br />
<td>Rashmi Ravichandran, Biochemistry</td><br />
</tr><br />
<tr><br />
<td>David Younger, Bioengineering</td><br />
</tr><br />
</table><br />
<br />
<h2> Faculty </h2><br />
<table><br />
<tr><br />
<td>David Baker, Biochemistry</td><br />
</tr><br />
<tr><br />
<td>Eric Klavins, Electrical Engineering</td><br />
</tr><br />
</table></div>Kristanguyenhttp://2014.igem.org/Team:Washington/Our_TeamTeam:Washington/Our Team2014-10-16T03:53:44Z<p>Kristanguyen: </p>
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<h1> UW iGEM 2014 Team </h1><br />
<br />
<h2> Undergraduate Team Members </h2><br />
<br />
<table><br />
<tr><br />
<td>Edward Chang, Biochemistry</td><br />
<td>Poster</td><br />
</tr><br />
<tr><br />
<td>Andrew Chau, Chemistry</td><br />
<td>Flow Cytometry</td><br />
</tr><br />
<tr><br />
<td>Joshua Cho, Molecular, Cellular, & Developmental Biology</td><br />
<td>Fluorescence Activated Cell Sorting, Flow Cytometry, and Presentation</td><br />
</tr><br />
<tr><br />
<td>Chris Choe, Molecular, Cellular, & Developmental Biology</td><br />
<td>Expression and Stability Analysis</td><br />
</tr><br />
<tr><br />
<td>William Harvey, Biochemistry</td><br />
<td>Fluorescence Activated Cell Sorting and Presentation</td><br />
</tr><br />
<tr><br />
<td>Alex Kang, Biochemistry</td><br />
<td>Expression and Stability Analysis</td><br />
</tr><br />
<tr><br />
<td>Julia Lim, Microbiology</td><br />
<td>BioBricks, Flow Cytometry, and Team Page</td><br />
</tr><br />
<tr><br />
<td>Harman Malhi, Molecular, Cellular, & Developmental Biology</td><br />
<td>Expression and Stability Analysis</tr><br />
</tr><br />
<tr><br />
<td>Colton McDavid, Microbiology</td><br />
<td>Expression and Stability Analysis and Poster</td><br />
</tr><br />
<tr><br />
<td>Krista Nguyen, Biochemistry</td><br />
<td>Flow Cytometry, Poster, and Team Page</td><br />
</tr><br />
<tr><br />
<td>Anastasia Nicolov, Bioengineering and Violin Performance</td><br />
<td>Expression and Stability Analysis</td><br />
</tr><br />
<tr><br />
<td>Ahmed Qureshi, Biochemistry</td><br />
<td>BioBricks and Poster</td><br />
</tr><br />
<tr><br />
<td>Stephen Rettie, Microbiology</td><br />
<td>BioBricks, Fluorescence Activated Cell Sorting and Presentation</td> <br />
</tr><br />
</table><br />
<br />
<br />
<h2> Advisors </h2><br />
<table><br />
<tr><br />
<td>Nick Bolten, Electrical Engineering</td><br />
</tr><br />
<tr><br />
<td>Cassie Bryan, Biochemistry</td><br />
</tr><br />
<tr><br />
<td>Arjun Khakhar, Bioengineering</td><br />
</tr><br />
<tr><br />
<td>Bob Lamm, Bioengineering</td><br />
</tr><br />
<tr><br />
<td>Erik Murphy, Molecular Cellular Developmental Biology</td><br />
</tr><br />
<tr><br />
<td>Rashmi Ravichandran, Biochemistry</td><br />
</tr><br />
<tr><br />
<td>David Younger, Bioengineering</td><br />
</tr><br />
</table><br />
<br />
<h2> Faculty </h2><br />
<table><br />
<tr><br />
<td>David Baker, Biochemistry</td><br />
</tr><br />
<tr><br />
<td>Eric Klavins, Electrical Engineering</td><br />
</tr><br />
</table></div>Kristanguyenhttp://2014.igem.org/Team:WashingtonTeam:Washington2014-10-16T03:53:26Z<p>Kristanguyen: </p>
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<tr><td colspan="3"> <h3> Overview</h3></td></tr><br />
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<p> Stabilizing proteins is an incredibly important and time consuming task in the field of protein engineering. Current methods require using intimate knowledge of the protein to hypothesize point mutations that could possibly improve stability. Extensive in vitro testing follows involving cloning the new construct into your model organism, expressing the construct, purifying your protein of interest, and producing a melting curve to verify if indeed your mutation did improve stability. In addition to being time intensive, this method is unreliably successful. <br><br><br />
<br />
Our team has developed a generalizable, high throughput method to select for the increased expression and stability of engineered proteins, making them more amenable to large-scale production in Escherichia coli and other downstream applications. Our method involves the insertion of proteins into a Gal4-VP16 transactivator that binds a promoter directly upstream of a GFP gene. The protein of interest is inserted into the middle of this complex using polypeptide linkers allowing for the subsequent selection of mutants associated with higher GFP output. We hypothesize that more stable proteins and their complexes will not be degraded by the cell’s natural machinery which will allow the Gal4-VP16 construct to produce higher levels of GFP. Less stable proteins will be degraded through natural mechanisms and will not produce GFP. The difference between these two populations can be evaluated using Flow Cytometry or Fluorescence Activated Cell Sorting. <br><br><br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/2/24/UWIgemsystem.png" alt="Overview of Project" style="width:800px;height:308px"></center><br />
<sup><b> Fig. 1. </b> This novel method of protein stabilization is potentially generalizable, as it utilizes cells' natural pathways for maintaining protein folding, and high throughput when coupled with Fluorescence-Activated Cell Sorting (FACS). </sup><br />
<br> <br><br />
<br />
Our method utilizes degrons to produce the desired range of GFP based off of the proteins relative stability while applying a destabilizing influence to the protein complex. The degron illuminates the differences between proteins of varying stabilities. This allows the system to operate with a higher clarity between stable and unstable protein variants. While the degron exaggerates differences between stabilities, it also can be used as a variable tool that can be adjusted to fit your protein of interest. By placing the degron in different positions along the protein complex, you can impose different destabilizing effects on the construct. <br><br><br />
<br />
The end goal of our project is to create a system that can be used in today’s protein engineering laboratories. By using Fluorescence Activated Cell Sorting of variants in a random mutagenesis library of our construct, we can sort out the highest GFP output variants which will correlate to the most stable variants. Through successive sorts the population will converge on a variant that improves stability of the protein complex. This revolutionary method is a generalizable alternative to current, labor intensive approaches for the selection of stable protein variants. Engineered proteins selected through this method could be produced in bacteria and aid in the development of thermostable, de novo protein therapeutics. </p><br />
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<h1> Background </h1><br />
<br />
<p align = left> Novel methods must be first tested for viability against other existing methods. Our project is no different. In order, to gauge the effectiveness and accuracy of our method we<br />
choose test proteins that are well studied and characterized. Therefore, BINDI and several of its mutant variants that have been well studied were chosen. The first step of our <br />
project was to replicate the results of the studies on BINDI and its variants by repeating the stability test experiments presented in "the paper." After verifying the results <br />
of "the paper", we proceeded to construct our degron protein constructs and expressed them in yeast cells containing an inducible mechanism for the expression of green fluorescence <br />
protein. Subsequently, as the fluorescent emission of each cell is measured as higher fluorescent corresponds to higher test protein stability. </p><br />
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<h1> Our System </h1><br />
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<h3> Components of the Degron Construct </h3><br />
<img src="https://static.igem.org/mediawiki/2014/d/d9/Degron_construct.jpg" alt="Degron Constructs" style="width:750px;height:379px"><br />
<p> Our experiment utilizes 5 different Degron constructs: <br><br />
-Deg0: This construct contains only the Gal4-VP16 transcriptional activator complex with the protein of interest in between the two (shortened as Gal4-Protein-VP16). <br><br />
-Deg1: This construct contains the Degron in front of our Gal4-Protein-VP16 complex. <br><br />
-Deg2: This construct contains the Degron in between Gal4 and the protein in our Gal4-Protein-VP15 complex. <br><br />
-Deg3: This construct contains the Degron in between the protein and VP16 in our Gal4-Protein-VP15 complex. <br><br />
-Deg4: This construct contains the Degron at the end of our Gal4-Protein-VP16 complex. <br><br />
</p><br />
<br />
<h3> Test Protein </h3><br />
<br />
<p align = left> <br />
<br />
The test protein that must be chosen in testing a novel and new system must be a protein that has been well studied and rigorously examined through other existing and well accepted protein stability testing methods. <br />
Therefore,our team decided to use the protein known as BINDI. <br />
BINDI and two of its less stable variants, BbpD04 and BbpD04.3 were studied and examined in "CITE THE BINDI PAPER."<br />
Many of the individuals who were cited in the paper as well as the lab that did the research was done in was a nearby lab at the University of Washington.<br />
Therefore, it was convient for us to contact members of the Baker Lab and speak with them about the protein we intended on using as well as accquiring samples of the protein's DNA code.<br />
<br />
</p> <br />
<h3> PYE1 a strain of <i> Saccharomyces cerevisiae </i> </h3><br />
<p><br />
The lynchpin of our project is the usage of flow cytometry and fluorescence activated cell sorting for high throughput protein stability analysis. <br />
However, both analytical systems, flow cytometry and F.A.C.S. require fluorescence emmission. <br />
Therefore we need a method of generating fluorescence within our cells in a way that also gives us insight into protein stability.<br />
Each degron construct contains a Gal4 promoter which can bind to an upstream activating site, Gal1, that induces downstream expression of something. <br />
It just so happens that PYE1, this something happens to be Green Fluorescent Proteins.<br />
Protein expressions in PYE1, will allow us to generate GFP relative to the amount of degron protein construct that exist within the cell. <br />
The more stable the degron protein construct is, the more likely it is that more GFP will be expressed.<br />
This relationship between stability and GFP forms the basis from which we will measure the relative protein stability of our degron constructs as well as the protein of interest degron construct.<br />
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<h1> Method </h1><br />
<br />
<p align =left> The essential process of our system involves cloning and manufacturing of a plasmid in <i> E. coli </i>. Once, the plasmids have been constructed and verified in <i> E. coli </i> <br />
they are transformed into <i>S. cerevisiae</i>. The plasmid constructs are then expressed. Following several days of growth the yeast cultures are passed through a Flow Cytometer<br />
and the fluorescence of each cell is measured. Higher fluorescence is associated with higher expression of the protein of interest which in-turn is indicative of higher protein <br />
stability. </p><br />
<br />
<br />
<h3> Cloning in <i> Escherichia coli </i> </h3><br />
<br />
<p align = left> <br />
There are five possible degron constructs corresponding to five different positions the degron can take in our construct.<br />
Four of the five vectors for our protein of interest DNA code contains contain a degron (Deg1-4) as well as an EcoRI and Nhel111 restriction enzyme cutsite between Gal4 and VP16.<br />
Furthermore, the vector also contains a region that encodes Ampicillin resistance as well as autotrophic region that encodes for uracil synthesis. <br />
The DNA that encodes our protein of interest, the insert, is amplified to include both cutsites through a polymerase chain reaction. <br />
In a subsequent step the amplified fragment is then digested and then ligated with the appropriate vector and transformed into chemically competent<i> E.coli </i> wither XL-1 Blue or XL10-Gold strains. <br />
Similarly, Deg0(no degron) vector has EcoRI along with a Hind111 cutsite. Using the different cutsites our DNA fragment is prepared using PCR and ligated into the Deg0 vector and then transformed.<br />
Once the transformation is complete, the cells are plated onto LB-agar plates supplimented with ampicillin in order to ensure that all <i> E.coli </i> colonies contain our recombinant plasmid.<br />
After being grown for a day, several colonies are swiped and added to an overnight culture. The overnight culture is grown overnight and their recombinant plasmid is harvested and sequenced (Sanger sequencing is used through Genewiz Inc.).<br />
If the plasmids are correct, we then proceed to create a glycerol stock of the cell culture as well as a miniprep stock of the plasmid in order to conduct further experimentation in yeast. <br><br />
</p> <br />
<br />
<h3> Preparation and Passaging of <i> Saccharomyces cerevisiae </i> </h3><br />
<p align = left> <br />
Once, plasmids of the five possible degron constructs have been cloned with our three test proteins, they are subsequently transformed in PyE1 a strain of <i> S. cerevisiae </i> with the ability to produce green fluorescent proteins.<br />
Following the transformation, the cells are plated onto plates with on a Selective Dropout (C-Uracil) media and incubated at 30<sup>o</sup>C for 2 days. <br />
The purpose of the dropout media is to ensure that only cells that contain our plasmid survive as the recombinant plasmid allows cells to produce uracil, an essential amino acid. <br />
Without the recombinant plasmid, the cell would be fatally deprived of uracil which has been "knocked out" of the plating media. <br />
After two days, three colonies are swiped from the plate and added to an overnight culture of 2-3mL Selective Dropout Media C-Uracil and 2% Glucose then incubated for another two days at 30<sup>o</sup>C.<br />
After another two days of incubation, a 20-50uL aliquot of each culture is "passaged" into another 3mL culture prepared in the same manner as before and incubated for the same duration and temperature as the previous culture. <br />
The passaging is done several times after each passage after the second passage, a glycerol stock is prepare from the culture and Flow Cytometry is run on the culture. <br><br />
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<p align = left> <br />
The purpose of passaging is to gradually remove excess copies of the plasmid constructs. Excess copies, exceeding one per cell will lead to multiple fold increase in the<br />
expression of the degron protein construct. As a result of this, GFP expression will also be increased thus reducing the viability and accuracy of the Flow Cytometry <br />
measurements conducted on each cell culture. This problematice as GFP output will become related to the number of plasmids as well as the stability of the various degron constructs which will likely invalidate any results.<br />
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<h3> Relative Stability Analyzed via Flow Cytometry </h3><br />
<p> Flow cytometry is a high throughput method of analyzing cells for various optical outputs, namely fluorescnce. A flow cytometer is an analytical instrument in which cells that have been suspended in a solution are passed through a narrow channel in which fluorescence of invidual cells can be measured. <br><br />
<br><br />
By utilizing Flow Cytometry, we can measure the amount of GFP output within cells from each degron construct. Based on where the Degron is inserted, we expected a different level of fluorescence. As such, we expected to see the highest GFP production in our Deg0 construct, as it only contains the Gal4-Protein-VP16 complex with no Degron inserted, hence being the most stable. We expected that Deg2 and Deg3 would have a lower GFP production than Deg0 but higher than Deg1 and Deg4. This rationale was based on the fact that the Deg1 and Deg4 have the Degron exposed, making it more likely to be degraded by ubiquitination than in Deg2 and Deg3 which has the Degron buried inside the Gal4-Protein-VP16 complex.<br> </p><br />
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<h3> Mutagenesis through Error Prone Polymerase Chain Reactions (E-PCRs) </h3><br />
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<p><br />
In order to validate our system as being capable of selecting more stable protein variants, we have to produce mutations in our protein of interest. <br />
Those mutations could potentially be beneficial and could carry a stabilizing affect on the protein of interest. <br />
Error-prone PCR utilizes DNA-polymerase's error-prone nature and further increases the likelyhood of mutations by manipulating the conditions in which, <br />
DNA-polymerase operates in, thereby causing the polymerase to create errors in DNA sequencing which in turn will create changes in the protein construct.<br />
Once, the DNA coding of our protein has been changed we can express the mutations and analyze those stabilizing or de-stabilizing affects they have on our protein.<br />
The possible mutations can then be analyzed using the degron system and more stable mutations can be easily seen then sequenced. <br />
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<br />
<h3> Selecting Stable Variants through Fluorescence Activated Cell Sorting (F.A.C.S.) </h3><br />
<br />
<p><br />
In order to validate our system as being capable of selecting more stable protein variants, we have to produce mutations in our protein of interest. <br />
Those mutations could potentially be beneficial and could carry a stabilizing affect on the protein of interest. <br />
Error-prone PCR utilizes DNA-polymerase's error-prone nature and further increases the likelyhood of mutations by manipulating the conditions in which, <br />
DNA-polymerase operates in, thereby causing the polymerase to create errors in DNA sequencing which in turn will create changes in the protein construct.<br />
Once, the DNA coding of our protein has been changed we can express the mutations and analyze those stabilizing or de-stabilizing affects they have on our protein.<br />
The possible mutations can then be analyzed using the degron system and more stable mutations can be easily seen through FACS (see below) then subsequently sequenced. <br />
As we sequence the various mutants, we are also looking for convergence in which the genetic sequence as well as the amino acid sequences converge on a single or a few mutations that lead to significantly higher fluorescence and therefore, higher stability.<br />
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</html></div>Kristanguyen