Line 522: | Line 522: | ||
Eco gsp::crtZY /pSIM5-tet + gspC::dhfr<br> | Eco gsp::crtZY /pSIM5-tet + gspC::dhfr<br> | ||
Plate on tmp<br> | Plate on tmp<br> | ||
− | + | ||
− | + | ||
</p> | </p> | ||
</div> | </div> | ||
Line 578: | Line 577: | ||
</div> | </div> | ||
<div class="col-sm-4"> | <div class="col-sm-4"> | ||
− | <h3></h3> | + | <h3>Week: 11/9 - 17/9</h3> |
<p> | <p> | ||
− | - <br> | + | - Purification of CsADH2946 on IMAC<br> |
− | + | </p> | |
− | - <br> | + | |
+ | <h3>Lambda red integration:</h3> | ||
+ | |||
+ | <p> | ||
+ | - Screening of crtEB clones, run screening PCR, gel analysis<br> | ||
</p> | </p> | ||
Line 591: | Line 594: | ||
</div> | </div> | ||
<div class="col-sm-4"> | <div class="col-sm-4"> | ||
− | <h3></h3> | + | <h3>Week: 18/9 - 24/9</h3> |
<p> | <p> | ||
− | - <br> | + | - SDS-PAGE of CsADH2946 IMAC fractions.<br> |
− | - <br> | + | - Changed buffer and concentrated pooled enzyme fractions.<br> |
− | - <br> | + | - Activity measurements of CsADH2946.<br> |
+ | - Created BioBricks of UGTCs2 by inserting it into the linearised iGEM plasmid psB1C3-J04500 using Gibson Assembly.<br> | ||
+ | </p> | ||
+ | |||
+ | <h3>Lambda red integration:</h3> | ||
+ | |||
+ | <p> | ||
+ | - Screening of crtEB clones<br> | ||
+ | Wrong band size! Redo Lambda Red.<br> | ||
+ | |||
+ | </p> | ||
+ | </div> | ||
+ | <div class="col-sm-1"> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="item"> | ||
+ | <div class="container-fluid"> | ||
+ | <div class="row entry"> | ||
+ | <div class="col-sm-1"> | ||
+ | </div> | ||
+ | <div class="col-sm-4"> | ||
+ | <h3>Week: 25/9 - 1/10</h3> | ||
+ | |||
+ | <p> | ||
+ | - Colony PCR of UGTCs2, gel electrophoresis and send for sequencing.<br> | ||
+ | - Transformation into BL21(DE3)* for expression<br> | ||
+ | - Ordered more CaCCD2 construct from IDT.<br> | ||
+ | - Sat overnight culture with UGTCs2 and performed expression testing with different concentrations of IPTG.<br> | ||
+ | - Harvested cells and performed IMAC purification on ÄKTA. Ran fractions on SDS-gel. No significant bands could be seen.<br> | ||
+ | </p> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/a/a9/T--Uppsala--notebook_gel2.png" style="width:50%;margin:auto;"></img> | ||
+ | |||
+ | |||
+ | </div> | ||
+ | <div class="col-sm-2"> | ||
+ | </div> | ||
+ | <div class="col-sm-4"> | ||
+ | <h3>Week: 2/10 - 8/10</h3> | ||
+ | |||
+ | <p> | ||
+ | - Inserted construct of CaCCD2 into plasmid with Gibson Assembly.<br> | ||
+ | - Colony PCR of CaCCD2, gel electrophoresis and send for sequencing. <br> | ||
+ | - Expression testing of UGTCs2 and CaCCD2 with different concentrations of IPTG. SDS-PAGE on the crude lysates.<br> | ||
+ | </p> | ||
+ | |||
+ | <h3>Lambda red integration:</h3> | ||
+ | |||
+ | <p> | ||
+ | - Start cultures for Lambda Red<br> | ||
+ | Eco IS150::crtI bgl::cat-sacB /pSIM5-tet<br> | ||
+ | - Lambda Red<br> | ||
+ | Eco IS150::crtI bgl::cat-sacB /pSIM5-tet<br> | ||
+ | + crtEB<br> | ||
+ | - Plate Lambda Red, incubate in RT over weekend<br> | ||
+ | Eco IS150::crtI bgl::cat-sacB /pSIM5-tet<br> | ||
+ | crtEB plate on sucrose agar (4 plates)<br> | ||
+ | </p> | ||
+ | </div> | ||
+ | <div class="col-sm-1"> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="item"> | ||
+ | <div class="container-fluid"> | ||
+ | <div class="row entry"> | ||
+ | <div class="col-sm-1"> | ||
+ | </div> | ||
+ | <div class="col-sm-4"> | ||
+ | <h3>Week: 9/10 - 15/10</h3> | ||
+ | |||
+ | <p> | ||
+ | - Set up pulling simulations for CsADH2946<br> | ||
+ | - Combining CaCCD2 and CsADH2946 into one BioBrick using 3A assembly.<br> | ||
+ | - Sent for sequencing.<br> | ||
+ | </p> | ||
+ | |||
+ | |||
+ | </div> | ||
+ | <div class="col-sm-2"> | ||
+ | </div> | ||
+ | <div class="col-sm-4"> | ||
+ | <h3>Lambda red integration:</h3> | ||
+ | |||
+ | <p> | ||
+ | - Restreak crtEB clones - There are a few colonies that look reddish | ||
+ | 16 clones on 4 LA agar plates<br> | ||
+ | - Restreak crtEB transformants - red colonies. | ||
+ | They grow really bad, and look quite sick. | ||
+ | Transduce crtYZ into crtEB strain with P1 | ||
+ | Clone 1-1 and 2-1-1.<br> | ||
+ | - Restreak 6 clones of the crtYZ + crtEB transductants on tmp agar | ||
+ | Start overnight cultures of restreaked crtEB clones in 30°C | ||
+ | 4 clones started<br> | ||
+ | - Freeze overnight cultures of restreaked crtEB clones in 30°C<br> | ||
+ | 4 clones frozen<br> | ||
+ | Restreak crtZY transductants on tmp agar<br> | ||
+ | Clone 1-1 and 2-1-1<br> | ||
+ | Restreak crtZY transductants a second time on LB agar<br> | ||
+ | Clone 1-1 and 2-1-1<br> | ||
+ | - Move plates to fridge or RT<br> | ||
+ | Crt strains from 30°C<br> | ||
+ | |||
+ | </p> | ||
+ | </div> | ||
+ | <div class="col-sm-1"> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="item"> | ||
+ | <div class="container-fluid"> | ||
+ | <div class="row entry"> | ||
+ | <div class="col-sm-1"> | ||
+ | </div> | ||
+ | <div class="col-sm-4"> | ||
+ | <h3>Week: 16/10 - 22/10</h3> | ||
+ | |||
+ | <p> | ||
+ | - Prep of UGTCs2, CsADH2946 and CaCCD2 for BioBrick submission.<br> | ||
+ | - Assembled J04500-CaCCD2 and J04500-CsADH2946 on pSB1K3 plasmid using 3A assembly into zeaxanthin producing E. coli strain.<br> | ||
+ | - Transformed zeaxanthin strain with the assembled plasmid and another batch with plasmid containing only CaCCD2.<br> | ||
+ | - Extracted zeaxanthin from the zeaxanthin producing strain<br> | ||
+ | </p> | ||
+ | |||
+ | <h3>Lambda red integration:</h3> | ||
+ | |||
+ | <p> | ||
+ | - Take photos of crtEBIZY strains<br> | ||
+ | Start overnight cultures<br> | ||
+ | CrtEBIZY strains (LB, 30°C)<br> | ||
+ | - Freeze overnight cultures<br> | ||
+ | CrtEBIZY strains (LB, 30°C)<br> | ||
+ | </p> | ||
+ | |||
+ | |||
+ | </div> | ||
+ | <div class="col-sm-2"> | ||
+ | </div> | ||
+ | <div class="col-sm-4"> | ||
+ | <h3>Week: 23/10 - 29/10</h3> | ||
+ | |||
+ | <p> | ||
+ | - Large scale expression of zeaxanthin strain containing CaCCD2+CsADH2946.<br> | ||
+ | - Assembled J04500-CaCCD2-J04500-CsADH2946 and J04500-UGTCs2 on pSB1A3 using 3A assembly into our zeaxanthin producing E. coli strain.<br> | ||
+ | - TLC comparing cultures of different zeaxanthin strain transformations (Zeaxanthin-strain only, zeaxanthin-strain with CaCCD2, zeaxanthin-strain with CaCCD2+CsADH2946 and zeaxanthin-strain with CaCCD2+CsADH2946+UGTCs2).<br> | ||
</p> | </p> | ||
</div> | </div> | ||
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<a class="left carousel-control" href="#dairy" data-slide="prev" style="background-image:none;width:50px;margin:30%2%;"> | <a class="left carousel-control" href="#dairy" data-slide="prev" style="background-image:none;width:50px;margin:30%2%;"> | ||
− | + | <img src="https://static.igem.org/mediawiki/2017/a/a9/Uppsala_notebook_arrow_right.svg" class="img-responsive pointy"></img> | |
</a> | </a> | ||
Revision as of 16:11, 31 October 2017