Team:UGA-Georgia/Notebook
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<a href="https://2014.igem.org/Team:UGA-Georgia"><p style="font-family: Basic L"><font size="2">HOME</font></p> </a> </td> | <a href="https://2014.igem.org/Team:UGA-Georgia"><p style="font-family: Basic L"><font size="2">HOME</font></p> </a> </td> | ||
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+ | <a href=“https://2014.igem.org/Team:UGA-Georgia/Overview">Overview</a> | ||
<a href="https://2014.igem.org/Team:UGA-Georgia/Geraniol">Geraniol</a> | <a href="https://2014.igem.org/Team:UGA-Georgia/Geraniol">Geraniol</a> | ||
<a href="https://2014.igem.org/Team:UGA-Georgia/Modeling">Modeling</a> | <a href="https://2014.igem.org/Team:UGA-Georgia/Modeling">Modeling</a> | ||
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+ | <a href="https://igem.org/Team.cgi?id=1383">Official Team Profile</a> | ||
<a href="https://2014.igem.org/Team:UGA-Georgia/Team">Members</a> | <a href="https://2014.igem.org/Team:UGA-Georgia/Team">Members</a> | ||
<a href="https://2014.igem.org/Team:UGA-Georgia/Attributions">Attributions</a> | <a href="https://2014.igem.org/Team:UGA-Georgia/Attributions">Attributions</a> | ||
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<li> Creation of general salts solution. </li> | <li> Creation of general salts solution. </li> | ||
<li> Creation of glycylglycine buffer. </li> | <li> Creation of glycylglycine buffer. </li> | ||
+ | <ul> | ||
+ | <li> All of these solution protocols can be found on our <a href="https://2014.igem.org/Team:UGA-Georgia/Protocols">protocol</a> page. </li> | ||
+ | </ul> | ||
<br> | <br> | ||
<br> | <br> | ||
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<h5> Week 1</h5> | <h5> Week 1</h5> | ||
<li> Determined the optical density (OD) of the previously revived cultures. </li> | <li> Determined the optical density (OD) of the previously revived cultures. </li> | ||
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+ | |||
<br> | <br> | ||
<h5> Week 2 </h5> | <h5> Week 2 </h5> | ||
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<li> Picked colonies from the previous day of plating. </li> | <li> Picked colonies from the previous day of plating. </li> | ||
<li> Plasmid extraction, digestion, gel electrophoresis, and verification. </li> | <li> Plasmid extraction, digestion, gel electrophoresis, and verification. </li> | ||
+ | <li>[E. Coli Lab] Extracted pMEV4 from bacteria.</li> | ||
+ | <li>[E.Coli Lab] PRC for mCherry with 11 mutated RBS sites and 1 native RBS.</li> | ||
+ | <li>[E. Coli Lab] Vector pMEV4 was digested with Spe1, Pst1 and buffer 2. Gel Extraction, Ligation and Heat Shock Transformation were also performed.</li> | ||
+ | <li>[E. Coli Lab] Colonies were successfully formed from 1,3,4,8, and 11; PCR was redone for 2,5,6,7,10, and 12.</li> | ||
+ | <li>[E. Coli Lab] Created new LB plates with amplicillin and LB Broth.</li> | ||
+ | <li>[E. Coli Lab] Prepared competent cells for transformation</li> | ||
+ | <li>[E. Coli Lab] Obtained PCR product samples 2,5,6,7,10 and 12, and redid vector digestion, gel extraction, ligation, and heat shock transformation.</li> | ||
+ | <li>[E. Coli Lab] Sample 5,6,7 and 10 were unsuccessfully transformed. Redid vector digestion, gel extraction, ligation, and heat shock transformation.</li> | ||
+ | |||
+ | |||
+ | |||
<br> | <br> | ||
<h5> Week 3</h5> | <h5> Week 3</h5> | ||
<li> Revival of pAW50-mCherry frozen stock. </li> | <li> Revival of pAW50-mCherry frozen stock. </li> | ||
<li> Revival of S0001 (WT) frozen stock. </li> | <li> Revival of S0001 (WT) frozen stock. </li> | ||
+ | <li>[E. Coli Lab] Inoculation, plasmid extraction, digestion and screening were performed on 1, 3,4,5,8,9,10,11 and 12. </li> | ||
+ | <li>[E. Coli Lab] Screening <a href= "https://static.igem.org/mediawiki/2014/7/7f/Screening1-12.png">Part I</a>,<a href="https://2014.igem.org/File:Part2screening.png"> Part II</a> for 1,3,4,5,8,9,10,11 and 12. Verification was done by using KpnI and NcoI ( if positive:2750 and 2414; if negative: 2414 and 1986; control: pMEV4). </li> | ||
+ | <li>[E. Coli Lab] Cloning for 2,6, and 7 in progress</li> | ||
<br> | <br> | ||
<h5> Week 4 </h5> | <h5> Week 4 </h5> | ||
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<li> Made glycerol stocks of original transformants. </li> | <li> Made glycerol stocks of original transformants. </li> | ||
<li> Fluorescent microscopy of transformants/pAW50-mCherry/S0001. </li> | <li> Fluorescent microscopy of transformants/pAW50-mCherry/S0001. </li> | ||
+ | <li> [E. Coli Lab] Plasmid extraction was done for 1,3,4,5,8,9,10,11 and 12. Also made 2 permanent stocks for each of them.</li> | ||
+ | <li> [E. Coli Lab] Plasmid extraction, inoculation, and screening were performed on 2, 6 and 7. Also made 2 permanent stocks for each of them</li> | ||
<br> | <br> | ||
<br> | <br> | ||
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<li> Subcultured the parent strains of WT and native RBS (12) into triplicates. The triplicates were wrapped in foil to prevent any photobleaching. </li> | <li> Subcultured the parent strains of WT and native RBS (12) into triplicates. The triplicates were wrapped in foil to prevent any photobleaching. </li> | ||
<li> The pAW50-mcherry came up and then all samples were taken to a plate reader to perform fluorescence tests. </li> | <li> The pAW50-mcherry came up and then all samples were taken to a plate reader to perform fluorescence tests. </li> | ||
+ | <li> [E. Coli Lab] the primers for the negative and positive control group were received; ran PCR on the negative and control groups in order to create a large amount of the DNA.</li> | ||
+ | <li> [E. Coli Lab] PCR amplification for mCherry gene with the negative and positive control.</li> | ||
+ | <li> [E. Coli Lab] Solid media was made.</li> | ||
+ | <li> [E. Coli Lab] <a href="https://static.igem.org/mediawiki/2014/a/a4/13-15PCRgel.png">PCR</a> was performed on 13, 14 and 15; vector digestion was also performed with Spe1, Pst1 and buffer2. </li> | ||
+ | <li> [E. Coli Lab] Gel electrophoresis was done to confirm <a href="https://static.igem.org/mediawiki/2014/4/4b/13-15Diggel.png">digestion </a>of 13-15. </li> | ||
<br> | <br> | ||
<h5> Week 5 </h5> | <h5> Week 5 </h5> | ||
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</ul> | </ul> | ||
<li> Track selection. </li> | <li> Track selection. </li> | ||
+ | |||
<br> | <br> | ||
<br> | <br> | ||
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<li> Since this pre-study gave us consistent, positive values for fluorescence, we were able to now refine the oxygen exposure protocol and apply it to characterization of our parts. </li> | <li> Since this pre-study gave us consistent, positive values for fluorescence, we were able to now refine the oxygen exposure protocol and apply it to characterization of our parts. </li> | ||
<ul> | <ul> | ||
- | <li> This detailed protocol can be found in our protocol section </li> | + | <li> This detailed protocol can be found in our <a href="https://2014.igem.org/Team:UGA-Georgia/Protocols">protocol</a> section. </li> |
</ul> | </ul> | ||
+ | <li> Revival of frozen stocks of cells containing plasmids with our theoretical "perfect" RBS (14), cells containing our theoretical "negative" RBS (15), and each of the 3 colonies of 12. Also, revival of WT with no plasmid were revived from a room temperature stock (negative control). </li> | ||
+ | <li> [E. Coli Lab] PCR Verification of 12C1, 12C2 and 12C3 was done with F12 as forward primer and R as reverse primer.</li> | ||
+ | <br> | ||
+ | <br> | ||
+ | <p><h4><font size="5">October</font></h4></p> | ||
+ | <br> | ||
+ | <h5> Week 1</h5> | ||
+ | <li> The revived parent strains of 14, 15, WT, and 12 C-1,2,3 were inoculated into varying volumes, and in triplicates, to test for a linear relationship of mCherry production to volume of culture which would be determined after the samples were taken to the plate reader. </li> | ||
+ | <ul> | ||
+ | <li> The triplicates were grown in 5mL, 25mL, and one single sample each of 12 C-1, 14, and 15 were grown into 100mL as a reference. </li> | ||
+ | </ul> | ||
+ | <li> [E. Coli Lab] Cloning for BioBrick (Genes: BBa_K1383000{Native RBS; F12 with F12 and R primers}, BBa_K1383001{Theoretical Best RBS; F14 with F14 and R primers}, and BBa_K1383002{Theoretical Worst RBS; F15 with F15 and R primers}; Vector: pSB1C3); PCR <a href="https://static.igem.org/mediawiki/2014/0/08/Biobrickpcr.png">result </a> was observed by using gel electrophoresis and UV light. </li> | ||
+ | <li> [E. Coli Lab] Plasmid extraction, purification, and <a href="https://static.igem.org/mediawiki/2014/a/a4/Biobrickscreening10-03-2014.JPG"> screening </a> were performed on F12A, F12B, F12C, F14A, F14B, F14C, F15A, F15B, and F15C. </li> | ||
+ | <br> | ||
+ | <h5> Week 2</h5> | ||
+ | <li> The oxygen exposure <a href="https://2014.igem.org/Team:UGA-Georgia/Protocols">protocol</a> was performed on the triplicates and the results of the experiment can be found on our <a href="https://2014.igem.org/Team:UGA-Georgia/RBS">RBS Library</a> page. </li> | ||
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Latest revision as of 02:30, 18 October 2014
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Notebook | |||||
FebruaryWeek 1Week 3Week 4MarchWeek 1Week 3
AprilWeek 1Week 2Week 3MayWeek 4JuneWeek 1Week 2Week 3Week 4JulyWeek 1Week 2Week 3Week 5
AugustWeek 2
Week 3Week 4Week 5SeptemberWeek 2
Week 3
Week 5
OctoberWeek 1
Week 2 |