Team:UC Davis/Notebook/Week 9


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Week 9

We decided to investigate the possible difference in GFP expression of three different strains with respect to arabinose and aTc induction. To begin this process, we began transformations of BBa_K750000 in MG1655Z1 and DH10B. We also transformed J23101+GFP BBa_I20260 into DH10B and MG1655Z1. We also wanted to make DH10B and BW22826 chemically competent for future use. In addition, we grew up a culture containing BBa_R0040+BBa_E0240 to investigate the behavior of pTet in these three different strains. Only MG1655Z1 has a specific insertion of constitutive TetR production.

Only a few of the transformations from yesterday were successful. We performed a DNA extraction on BBa_R0040+BBa_E0240 and eventually transformed MG1655Z1 with the resultant miniprep. We began growing up BW22826 and DH10B for the eventual making of chemically competent cells.

This morning we had a meeting with our advisors and gave a preliminary presentation of our project, which will be refined for the jamboree. We got a lot of good advice and constructive criticism on what we need to work on. We also discussed which strain we want to use to test our final constructs in. The transformations from yesterday were successful. However, our cultures of BW22826 and DH10B still needed to be grown up today. Taking another look at our Golden Gate assemblies, we decided to try decreasing the reaction volume in order to not use up the small amount of synthesized oligos we received last week before we get our new primers and ran a reaction to assemble our pBAD+Riboswitch+GFP (BBa_K1212010) testing construct.

The transformations of the pBAD+Riboswitch+GFP (BBa_K1212010) appeared to work, but after discussion, we’re worried that they may in fact be false positives with only pSB1C3 template DNA in the colonies. We originally ran the PCR reaction with BBa_K750000 to amplify the pSB1C3 and make it Golden Gate compatible. After PCR purification, the template DNA (BBa_K750000) may have been taken up by the cells. To check for the possibility of this false positive, we ran a PCR reaction with universal primers for some of our Golden Gate assembled colonies and a PCR control reaction involving BBa_K750000. The two band sizes of the resultant PCR will be compared. We also made BW22826 cells competent for transformation. We realized we were running low on Golden Gate compatible PCR product consisting of pSB1C3 and ran some additional PCR reactions and plan to gel purify those tomorrow. We continued Golden Gate assembly reactions with our other colonies in femtomole ratios and made transformations out of each of them.

The transformations involving our Golden Gate assembled constructs appeared to work, but we ran several colony PCR reactions for the possibility of positives. We ran the reaction for the colony PCR screen on a 2% agarose gel. Three out of six of the colonies showed the band sizes near our reference. We decided to run the same PCR products in a 0.5% gel at 100 volts to see if there was a more discernible difference. There were very few bands on the gel, which may have been due to an issue of aliquoting the sample into the poorly formed gel. We realized that we were running low on kanamycin and chloramphenicol plates, so we decided to make additional ones. After looking at the gel of the Golden Gate compatible backbone of pSB1C3, we realized that we should do additional reactions to ensure a high yield of product.

We tried another colony PCR run to see if we could see a discernible difference in the band sizes. The bands were about the same in size. We also amplified more pSB1C3 for Golden Gate assembly. We finally got around to running the Tecan with the different strains containing pTet+GFP (BBa_R0040+BBa_E0040) or pBAD+GFP (K206000+BBa_E0040).

We did a gel extraction and purification of the pSB1C3 PCR product and hope to measure the DNA concentration on Monday to use it for Golden Gate assembly. We also continued rehearsing and updating our presentation for the regional jamboree.