Team:Caltech/week7
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- | <tr><td colspan= | + | <tr> <td bgColor=#FFFFFF colspan = 3 height = 60px> <font size = +5> <center>Notebook </center> </td> </tr> |
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<b><a href='week11'>Week 11</a></b><br><br> | <b><a href='week11'>Week 11</a></b><br><br> | ||
<b><a href='week12'>Week 12</a></b><br><br> | <b><a href='week12'>Week 12</a></b><br><br> | ||
+ | <b><a href='week13'>Week 13</a></b><br><br> | ||
+ | <b><a href='week14'>Week 14</a></b><br><br> | ||
+ | <b><a href='week15'>Week 15</a></b><br><br> | ||
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- | <ul> | + | <b>Export Systems</b> |
+ | <ul><li>Ran colony PCR on pTG002/3/6-transformed JM109 cultures grown up over the weekend. Bad results for all of them</li> | ||
+ | <li>Created and ordered new primers that will hopefully avoid secondary-structure-formation when the exonuclease attacks the 5' end during Gibson assembly.</li> | ||
+ | </ul> | ||
+ | <b>lamBCDA & fsrABC Reception Systems</b> | ||
+ | <ul><li>Single-transformations run on each of the constructs used in the double-transformations: | ||
+ | <ol><li>pMB001</li><li>pMB002</li><li>pMB003</li><li>pMB004</li><li>pMB005</li><li>pMB006</li></ol></li> | ||
+ | </ul> | ||
+ | <b>agrBCDA Reception System and Combinatorial Promoters</b> | ||
+ | <ul><li>Colonies were picked from pAA008 transformations incubated over the weekend, as well as the pAA009 & pAA010 transformations, which had been set up last Thursday.</li> | ||
+ | <li>Set up liquid cultures of the colonies to grow overnight in preparation for miniprep and sequencing tomorrow</li> | ||
</ul> | </ul> | ||
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- | <ul> | + | <b>Export Systems</b> |
+ | <ul><li>Ran Gibson assembly products on a gel. Results inconclusive</li> | ||
+ | <li>Started 3 mL liquid cultures of bacteria (with pTG004 & pTG005) with varying levels of aTc induction: | ||
+ | <ul><li>0 nM, 50 nM, 150 nM, 250 nM, 350 nM, & 450 nM</li></ul></li> | ||
+ | </ul> | ||
+ | <b>lamBCDA & fsrABC Reception Systems</b> | ||
+ | <ul><li>Colony PCR was run on several of the colonies that emerged from the single-transformations last night</li> | ||
+ | <li>Gel was run, but wrong primers were used. New primers for sequencing/colPCR were ordered, and colPCR will be redone tomorrow.</li> | ||
+ | </ul> | ||
+ | <b>agrBCDA Reception System and Combinatorial Promoters</b> | ||
+ | <ul><li>Miniprepped liquid cultures grown last night and shipped samples for sequencing.</li> | ||
</ul> | </ul> | ||
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- | <ul> | + | <b>Export Systems</b> |
+ | <ul><li>Ran new PCR reactions with newly designed primers to extract backbones of pTG002/3/6.</li> | ||
+ | <li>DpnI-digested (4 hrs) and PCR-purified the PCR products.</li> | ||
+ | <li>Products were run on a gel to confirm their creation.</li> | ||
+ | <li>Gibson assembled the backbones with their geneblock inserts</li> | ||
+ | <li>Gibson assemblies were transformed into JM109, which were plated & incubated overnight.</li> | ||
+ | </ul> | ||
+ | Additionally we began experimentation on the pTG004 (lam system) & pTG005 (fsr system) constructs we had successfully constructed in Week 5, using the liquid cultures induced with aTc yesterday. | ||
+ | <ul><li>Western blots were run on both supernatant and cell lysate samples (6 concentrations of aTc, so 12 Western lanes for each system) from the 2 liquid cultures grown up last night. 3xFLAG-antibodies appear to mark proteins in the fsr supernatant and cell lysate lanes.</li></ul> | ||
+ | <b>agrBCDA Reception System and Combinatorial Promoters</b> | ||
+ | <ul><li>Analyzed sequencing results: | ||
+ | <ul><li>pAA008 successfully constructed</li> | ||
+ | <li>Minipreps supposedly containing pAA009 & pAA010 instead contain pKS001 backbone template</li> | ||
+ | </ul></li> | ||
+ | <li>We redid PCR of the pAA009/010 backbone</li> | ||
+ | <li>DpnI digestion for 2 hours, followed by PCR purification. The purified product was then run on a gel.</li> | ||
</ul> | </ul> | ||
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- | <ul> | + | <b>Export Systems</b> |
+ | <ul><li>5 colonies from each of the overnight transformations were picked (15 total), and we ran colony PCR (52°C annealing step) on them using the newly ordered sequencing primers.</li> | ||
+ | <li>colony PCR products were run on a gel, showing that none of them contained the correct construct.</li> | ||
+ | <li>10 new colonies were picked and "colony-PCR-ed" (this time with a 47° annealing step). However, running the product on a gel showed these didn't work either.</li> | ||
+ | </ul> | ||
+ | <b>lamBCDA & fsrABC Reception Systems</b> | ||
+ | <ul><li>colony PCR was redone on the pMB001-006 transformations using the newly ordered primers. Still no binding.</li> | ||
+ | </ul> | ||
+ | <b>agrBCDA Reception System and Combinatorial Promoters</b> | ||
+ | <br>agrBCDA system: | ||
+ | <ul><li>We used a gel extraction kit to extract the pAA009/010 backbone from the gel run yesterday.</li> | ||
+ | </ul> | ||
+ | Combinatorial Promoters: | ||
+ | <ul><li>PCRed the plasmid backbone and combinatorial promoter parts of pAA002.</li> | ||
+ | <li>DpnI digested the PCR products</li> | ||
+ | <li>Ran a gel on the products to confirm sequences were of the correct length</li> | ||
+ | <li>PCR-purified the DpnI-digested products</li> | ||
</ul> | </ul> | ||
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- | <ul> | + | <b>Export Systems</b> |
+ | <ul><li>To debug our colony PCRs from yesterday, we set up 8 colony-PCR reactions on a annealing temperature gradient, using E. coli containing pKS001 (the positive control in our earlier colPCRs) as the template. | ||
+ | <ul><li>Annealing temperature was allowed to range from 47°C to 54°C</li> | ||
+ | <li>Optimal annealing temperature for our new sequencing primers was determined to be around 48.6°C, with no binding beyond 52°C.</li> | ||
+ | </ul></li> | ||
+ | <li>To create a negative control for our Western blots, we obtained a pTet_GFP plasmid that had been assembled via Golden Braid assembly. This assembly product was transformed into JM109 and plated on a chloramphenicol plate. | ||
+ | <ul><li>We will eventually place a 3xFLAG behind the GFP in this plasmid to be used in a Western.</li></ul></li> | ||
+ | <li>In preparation for the LCMS run to detect the lam system's signalling peptide, we are going to remove the 3xFLAG from pTG004 (as it would be in any actual signal-reception system involving lam we would design): | ||
+ | <ul><li>3xFLAG was PCRed out (linearizing the pTG004 plasmid) using 2 new primers we had designed with phosphorylated 5' ends. 50°C annealing step was used.</li> | ||
+ | <li>PCR-purified PCR product.</li> | ||
+ | <li>Around-the-horn ligation (to recircularize pTG004-without-FLAG) was run using Quick Ligation kit</li> | ||
+ | <li>Ligation product was transformed into JM109, plated on carbenicillin, and incubated.</li> | ||
+ | <li>Colony PCR (49°C annealing step) was then run on the 3 colonies that grew up.</li> | ||
+ | <li>A gel was run on the colony PCR products, revealing ____</li> | ||
+ | </ul></li> | ||
+ | </ul> | ||
+ | <b>lamBCDA & fsrABC Reception Systems</b> | ||
+ | <ul><li>Still no success with the colony PCR.</li> | ||
+ | </ul> | ||
+ | <b>agrBCDA Reception System and Combinatorial Promoters</b> | ||
+ | <ul><li>Did a Gibson assembly of pAA002 (containing a combinatorial promoter), pAA009, and pAA010 (containing agr reception system), using PCRed/gel-extracted (respectively) products from yesterday.</li> | ||
</ul> | </ul> | ||
</td> | </td> |
Latest revision as of 00:35, 15 September 2014
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