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| <!-- Header --> | | <!-- Header --> |
− | <header id="header">
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| <div class="inner"> | | <div class="inner"> |
| <a href="https://2016.igem.org/Team:Warwick" class="logo" > | | <a href="https://2016.igem.org/Team:Warwick" class="logo" > |
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| <a href="https://2016.igem.org/Team:Warwick/Description">Project</a> | | <a href="https://2016.igem.org/Team:Warwick/Description">Project</a> |
| <div class="dropdown-content"> | | <div class="dropdown-content"> |
− | <a href="https://2016.igem.org/Team:Warwick/Project">Overview</a> | + | <a href="https://2016.igem.org/Team:Warwick/Description">Description</a> |
| <a href="https://2016.igem.org/Team:Warwick/Design">Design</a> | | <a href="https://2016.igem.org/Team:Warwick/Design">Design</a> |
| <a href="https://2016.igem.org/Team:Warwick/Parts">Parts</a> | | <a href="https://2016.igem.org/Team:Warwick/Parts">Parts</a> |
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| <a href="https://2016.igem.org/Team:Warwick/Human_Practices">Human Practices</a> | | <a href="https://2016.igem.org/Team:Warwick/Human_Practices">Human Practices</a> |
| <div class="dropdown-content"> | | <div class="dropdown-content"> |
| + | <a href="https://2016.igem.org/Team:Warwick/Human_Practices">Summary</a> |
| + | <a href="https://2016.igem.org/Team:Warwick/HP/Silver">Silver</a> |
| + | <a href="https://2016.igem.org/Team:Warwick/HP/Gold">Gold</a> |
| <a href="https://2016.igem.org/Team:Warwick/Integrated_Practices">Integrated Practices</a> | | <a href="https://2016.igem.org/Team:Warwick/Integrated_Practices">Integrated Practices</a> |
| + | <a href="https://2016.igem.org/Team:Warwick/Education">Education</a> |
| </div> | | </div> |
− | </div>
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− | <div class ="dropdown">
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− | <a href="https://2016.igem.org/Team:Warwick/Awards">Awards</a>
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− | <div class="dropdown-content">
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− | <a href="https://2016.igem.org/Team:Warwick/HP/Silver">Silver</a>
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− | <a href="https://2016.igem.org/Team:Warwick/HP/Gold">Gold</a>
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− | </div>
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| </div> | | </div> |
| </nav> | | </nav> |
| <a href="#navPanel" class="navPanelToggle"><span class="fa fa-bars"></span></a> | | <a href="#navPanel" class="navPanelToggle"><span class="fa fa-bars"></span></a> |
| </div> | | </div> |
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| <section id="banner"> | | <section id="banner"> |
| <h1>Lab Book</h1> | | <h1>Lab Book</h1> |
− | <p>Lab Book, sadly, not containing Labradors.</p>
| + | <p>Week by Week Summary</p> |
| </section> | | </section> |
| + | |
| + | <section id="three" class="wrapper style1 special"> |
| + | <div class="inner"> |
| + | <header class="align-center"> |
| + | <h2>Lab Book</h2> |
| + | </header> |
| + | <div class="flex flex-2"> |
| + | <article class="nopicture"> |
| + | <h3>Week 1 (04/07 – 08/07)</h3> |
| + | <p>Overview of molecular biology, CRISPR, cloning/plasmids, RNA design<br /> |
| + | Primers for Gibson assembly of GFP-dCAS9 in pSB1C3 designed and ordered<br /> |
| + | 1st batch of competent cells (DH5α) prepared<br /> |
| + | Transformed interlab plasmids into DH5alpha competent cells |
| + | </p> |
| + | <h3>Week 2 (11/07 – 15/07)</h3> |
| + | <p>GFP and dCas9 was amplified by PCR and extracted |
| + | Prepared more competent cells (Top10)<br /> |
| + | Issues with low PCR yield addressed by testing with gradient PCR<br /> |
| + | Gibson assembled and transformed first construct (GFP-dCas9) into Top10 E.coli |
| + | </p> |
| + | <h3>Week 3 (18/07 – 22/07)</h3> |
| + | <p>Interlab samples were transformed into our electrocompetent Top10 cells<br /> |
| + | Colony PCR of first construct failed twice but restriction digest was successful |
| + | Colonies that showed highest uptake of constructed plasmid were sent for sequencing and stabilates prepared.<br /> |
| + | Sequencing data showed one colony had no mutations present in the required construct<br /> |
| + | Made up additional antibiotic plates (ampicillin, tetracycline, kanamycin) |
| + | Various biobrick plasmids obtained and transformed into Top10 cells. |
| + | </p> |
| + | <h3>Week 4 (25/07 – 29/07)</h3> |
| + | <p>More primers arrived so we could begin constructing components that need to go into our second plasmid.<br /> |
| + | Plasmids amplified by PCR with Golden gate or Gibson primers to form backbones. T7 RNAP was also amplified. It took a few attempts for the amplification to succeed with both Gibson and Goldengate.<br /> |
| + | Interlab study continued |
| + | </p> |
| + | <h3>Week 5 (01/08 – 05/08)</h3> |
| + | <p>GFP-dCas9 assembled into pSB4A5, transformed into Top10 cells and grown up.<br /> |
| + | Colony PCR of eight of the colonies was completed – showed negative results for all colonies<br /> |
| + | Colony PCR of sixteen new colonies was completed - continued to show negative results<br /> |
| + | Different fusion protein sequences amplified – all successful (no Ms2-sigma or any of the T7's at this stage) |
| + | </p> |
| + | <h3>Week 6 (08/08 – 12/08)</h3> |
| + | <p>Protein fusion sequences ligated into pSB3T5 backbone, transformed into Top10 cells and grown up.<br /> |
| + | RpoZ competent cells produced<br /> |
| + | Digest set up for GFP-dCas9 in pSB1C3<br /> |
| + | </p> |
| + | <h3>Week 7 (15/08 – 19/08)</h3> |
| + | <p>olony PCR of plasmid 2 constructs undertaken – partially successful<br /> |
| + | Glycerol stocks of successful colonies produced and sent for sequencing |
| + | </p> |
| + | <h3>Week 8 (22/08 – 26/08)</h3> |
| + | <p>There was at least one colony that came back successfully from sequencing for each of the following; Com-omega, Ms2-Omega, Ms2-sigma54, Pp7-omega and Pp7-sigma54<br /> |
| + | The five plasmid 2 inserts above were digested successfully and extracted<br /> |
| + | Amplified pSB3T5 backbone |
| + | </p> |
| + | <h3>Week 9 (29/08 – 02/09)</h3> |
| + | <p>First extraction of the five plasmid 2 inserts failed so the process was repeated from the original glycerol stocks – successful<br /> |
| + | Attempted to amplify sgRNA – Partially successful<br /> |
| + | The five plasmid 2 inserts were digested (EcoRI/SpeI)<br /> |
| + | pSB3T5 and pSB1C3 digested (EcoRI/SpeI) and <br /> |
| + | Ligation reaction set up to insert the five plasmid 2 constructs into pSB1C3 backbone<br /> |
| + | PCR of sgRNA cassette – successful |
| + | </p> |
| + | <h3>Week 10 (05/09 – 09/09)</h3> |
| + | <p>Successfully cotransformed Top10 cells with pSB1C3/pSB3T5, pSB1C3/pSB4A5, pSB3T5/pSB4A5 and pSB1C3/pSB3T5/pSB4A5 also successfully transformed with sgRNA in pSB1C3<br /> |
| + | PheA terminator successfully added to the 3' end of the GFP in the GFP-dCas9 construct in the pSB1C3 backbone. |
| + | </p> |
| + | <h3>Week 11 (12/09 – 16/09)</h3> |
| + | <p>GFP-PheA-dCas9 digested with (EcoRI/SpeI) and successfully ligated into pSB4A5 backbone<br /> |
| + | PCR of sgRNA and pSB1C3 extracted and Gibson assembled |
| + | </p> |
| + | <h3>Week 12 (19/09 – 23/09)</h3> |
| + | <p>Amplified and assembled PAM staggered sequence into GFP-PheA-dCas9 in pSB1C3<br /> |
| + | Cloned PAM-GFP-PheA-dCas9 into pSB4A5 |
| + | </p> |
| + | <h3>Week 13 (26/09 - 30/09)</h3> |
| + | <p>Assembled different promoters for insertion into the PAM-GFP-PheA-dCas9 construct<br /> |
| + | Made multiple attempts to insert promoters |
| + | </p> |
| + | <h3>Week 14 (03/10 - 07/10)</h3> |
| + | <p>Insertion of promoters partially successful<br /> |
| + | Attempts made to insert fusion proteins (e.g. MS2-sigma) into pSB1C3 |
| + | </p> |
| + | <h3>Week 15 (10/10 - 14/10)</h3> |
| + | <p>Created a library of sgRNA targeting regions by annealing complementary primers<br /> |
| + | Golden gate assembled library into sgRNA construct in pSB1C3<br /> |
| + | Transformed cells with PP7-omega in pSB3T5, and PAM-omegaProm-GFP-PheA-dCas9 in pSB4A5 in preparation for insertion of sgRNA in pSB1C3.<br /> |
| + | Transformed prepared cells with library. |
| + | </p> |
| + | <h3>Week 16 (17/10 - 21/10)</h3> |
| + | <p>Cloned versions of our fusion proteins into PSB1C3<br/>Tested a full construct version of our device with PP7 omega, PAM-omegaProm-GFP-PheA-dCas9, library-sgRNA in order to determine whether fluorescence difference was significant between constructs including sgRNAs targeting the PAMs and constructs that include sgRNAs with no target site. |
| + | </p> |
| + | </article> |
| + | </div> |
| + | </div> |
| + | </section> |
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| <div class="flex"> | | <div class="flex"> |
| <div class="copyright"> | | <div class="copyright"> |
− | © Warwick iGem 2016. | + | © Warwick iGEM 2016. |
| <ul class="icons"> | | <ul class="icons"> |
| <li><a href="https://www.facebook.com/WarwickIGEM" class="logo" ><img src="https://static.igem.org/mediawiki/2016/6/6f/T--Warwick--Facebook.png" alt="Facebook" class="logo"> | | <li><a href="https://www.facebook.com/WarwickIGEM" class="logo" ><img src="https://static.igem.org/mediawiki/2016/6/6f/T--Warwick--Facebook.png" alt="Facebook" class="logo"> |
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| </a></li> | | </a></li> |
| </ul> | | </ul> |
− | <a href="https://2016.igem.org/Team:Warwick" class="logo" > | + | <p>We thankfully acknowledge generous funding support from our sponsors below.</p> |
| + | <a href="https://2016.igem.org/Team:Warwick/Attributions" class="logo" > |
| <img src="https://static.igem.org/mediawiki/2016/f/f6/T--Warwick--banner.png" alt="IGEM WARWICK" class="bannertop"> | | <img src="https://static.igem.org/mediawiki/2016/f/f6/T--Warwick--banner.png" alt="IGEM WARWICK" class="bannertop"> |
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− | <a href="https://2016.igem.org/Team:Warwick">Home</a>
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− | <a href="https://2016.igem.org/Team:Warwick/Team">Team</a>
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− | <a href="https://2016.igem.org/Team:Warwick/Project">Project</a>
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− | <a href="https://2016.igem.org/Team:Warwick/Design">Design</a>
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− | <a href="https://2016.igem.org/Team:Warwick/Parts">Parts</a>
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− | <a href="https://2016.igem.org/Team:Warwick/Safety">Safety</a>
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− | <a href="https://2016.igem.org/Team:Warwick/Attributions">Attributions</a>
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− | <a href="https://2016.igem.org/Team:Warwick/Human_Practices">Human Practices</a>
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− | <a href="https://2016.igem.org/Team:Warwick/Awards">Awards</a>
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− | </div>
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| </div> | | </div> |
| </div> | | </div> |