Team:UC Davis/Notebook/Week 12


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Week 12

The transformations involving the SOE reactions were unsuccessful. We plated them leftover transformants in hopes of finding colonies. We did a miniprep of pTet+TBS 1+GFP, pBAD+RS 2+TAL 8, and pBAD+RS 1+TAL 8. We hope to sequence the pTet+TBS 1+GFP and pBAD+RS 2+TAL 8 tomorrow. We also looked at some of the Tecan data for our pBAD+RS 1+GFP and pBAD+RS 2+GFP constructs. The fold change in GFP output was quite low but this can be explained due to the low copy number in each cell. Colonies were visible on the cotransformation plates, which were then grown up. They will be made into glycerol stocks tomorrow. No colonies were present for the CPEC assemblies (R1T8, R2T8, and pTetTBS1GFP) in either backbone (pSB1C3 or pSB3K3), or for the second attempt at Standard Assembly of R2T8 + pTetTBS2GFP. SOE amplification was repeated for pSB1c3, RS1, pSB3K3, pTetTBS1RBS, GFP, and pTet+TBS2+RBS, as well as for R1T8, R2T8, and pTet+TBS2+RBS+GFP in order to assemble the full construct. Assembly will be reattempted tomorrow after running a gel to check for the correct bands.

We sent out a number of constructs for sequencing. The concentrations of pBAD+RS 1+GFP and pBAD+RS 2+GFP from the gel extraction were too low for ligation. So another restriction digest was carried out, along with gel extraction, and ligation into pSB1C3. We also took a look at the Tecan data involving pBAD+RS 1+GFP and pBAD+RS 2+GFP. According to the data, the fluorescence per cell was quite low in both instances of maximal induction. In other words, pBAD+RS 1+GFP and pBAD+RS 2+GFP at 2 mM Theophylline and 1% arabinose did not show high amounts of fluorescence. We believe this may be due to the low copy number of the plasmid and the high degradation rate of GFP. We will run another Tecan run with higher induction levels of theophylline with a higher copy backbone such as pSB1C3.

We checked all of our sequencing we sent in yesterday. The sequence of pTet+TBS 1+GFP looks good and is not missing the operator like its TBS2 counterpart; however, it does still have one mutation in the second pTet operator that we need to fix; Site Directed Mutagenesis(SDM) is now underway. Sequencing for pTet+TBS2+GFP also looks good, containing pTet plus an additional second operator. The TAL repressor sequences are still being a little difficult to get good sequencing of, but the sequences for our pTet+TBS2+GFP+pBAD+RS2+TAL8 and pTet+TBS2+GPF+pBAD+RS1+TAL1 full constructs look promising. Unfortunately, the pTet in these constructs is still missing the first operator, so they still need to be worked on. We used primers to amplify out our parts starting from the TBS2 and will use Standard Assembly to try to put a correct pTet sequence into our constructs. We also tried to use CPEC assembly for our full constructs and to move them into pSB1C3.

We started testing our constructs today. We started a Tecan run of pBAD+RS 1+TAL 8//pTet+TBS 2+GFP, pBAD+RS 2+TAL 8//pTet+TBS 2+GFP, and pBAD+RS 2+TAL 1//pTet+TBS 2+GFP cotransformations, with pTet+TBS2+GFP in pSB1C3 and the pBAD+RS+TAL in pSB3K3. We used the strain MG1655Z1 which expresses both of the repressors we need for our system, AraC and TetR. We started with a wide range of concentrations for our inducers, arabinose, theophylline and aTc. We plan to use the data from this run to see if our constructs are working as expected and will refine our range of concentrations for our later runs. Unfortunately, there were no colonies present on our plating of our SDM for pTet+TBS 1+GFP, so it was replated in hopes of getting additional transformants tomorrow. More SDM PCR was also run as a back up. We gave our latest version of our presentation to our advisors but there is still a lot of work to be done on it. Assembly of our full constructs continues to be a work in progress.

The Tecan run of pBAD+RS 1+TAL8//pTet+TBS 2+GFP, pBAD+RS 2+TAL 8//pTet+TBS 2+GFP, and pBAD+RS 2+TAL 1//pTet+TBS 2+GFP showed some interesting and some unexpected results, so we did two more runs to see if the results can be duplicated. pTet+TBS 1+GFP SDM transformants were present on the replated plates, and some were picked for colony PCR. Our second SDM for pTet+TBS1+GFP, was digested with DpnI and run on a gel, but no bands were present. Since there were bands for the colony PCR of our previous SDM transformation, we are going to culture and send them for sequencing tomorrow, and save our second SDM for after the sequencing comes back. Lots of standard assembly is being done in order to get all of our constructs into pSB1C3 and to fix the missing pTet operator in our pTet+TBS2+GFP constructs for the parts submission deadline that is fast approaching.

We set up our potentially mutated pTet+TBS 1+GFP constructs out for sequencing on Monday. We are also sequencing our pBAD+RS 1+GFP and pBAD+RS 2+GFP that were in the pSB1C3 backbone. The Tecan run from yesterday’s results showed that the three cotransformations exhibited the same output as before. We set up another Tecan run testing our pBAD+RS 1+GFP, pBAD+RS 2+GFP, and pTet+TBS 2+GFP constructs. Hopefully these constructs will perform as expected. We also have continued doing ligations, transformations, and other procedures involved in our standard assembly of our remaining constructs.

The Tecan run for pBAD+RS1+GFP, pBAD+RS2+GFP and pTet+TBS2+GFP did not all show the results we were expecting. There appeared to be issues with the growth of our cultures, so we set up another run with only varying theophylline levels to check if it is affecting the culture growth in any way. Assembly is also still on going, but the results are mostly still pending.