Team:UC Davis/Notebook/Week 10


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September 30-October 2815+

Week 10

We received more feedback on our presentation and found new ways to improve upon what we already have. We set up additional Golden Gate assembly reactions involving the two halves of our entire construct. The assembled constructs were then transformed into MG1655Z1. The Tecan data was also analyzed with the help of David’s scripts. We’ve decided to use MG1655Z1 for any transformations involving our construct. By growing the cells in a minimal media, we will avoid the prospect of arabinose degradation if the cells are saturated with glucose. We realized that we were running out of Golden Gate ready GFP for additional assembly reactions. Therefore, we ran additional PCR reactions for future gel extractions.

All of the transformations involving our Golden Gate products had colonies, and we are going to screen them through colony PCR. According to the gel, we still saw leftover template DNA, which indicates that all of the colonies may still have pSB1C3 or some other contaminant. So far we have been screening 4 colonies from each of our plates without getting any correct ones yet. We are going to continue screening more of our colonies, as well as look into ways to make our Golden Gate assembly reactions more efficient with less background. Our primers to amplify our synthesized parts have finally arrived, so we shouldn’t need to worry about running out of them anymore.

We ran additional colony PCR reactions and could not find any evidence of successful assembly within the colonies. Many of the transformants appear to contain pSB1C3 only. In an effort to remove pSB1C3 template, we did a DpnI digestion. We also amplified the Golden Gate product of pSB1C3 further as another step to avoid false positives and give us more product to work with. After mulling over the difficulties of what may have gone wrong with assembling our construct, we realized that the addition of Bovine Serum Albumin (BSA) would be advantageous in optimizing BsaI activity. In addition to adding BSA to each Golden Gate assembly reaction, we used different concentrations of ligase to see if we could optimize the output of the reaction. We used 5 uL of the Golden Gate reactions for transformations in hopes of getting colonies. We ran a gel with all of the colony PCR that we did yesterday in hopes of still finding an assembled reaction. Unfortunately, none of our colonies appear to have the correctly assembled plasmid.

After checking the plates, we realized that there were no colonies with the exception of the positive control. We decided to plate the rest of the media containing possible transformants. We also ran a gel of the Golden Gate products and realized that it appeared pSB1C3 was not binding to the sticky ends of our inserts. However, the promoter and GFP were assembling together. It also appeared that the higher concentration ligase was less effective in assembly, since the bands were generally fainter. In an effort to troubleshoot our Golden Gate assembly reaction, we decided to not do the one pot reaction and instead, do a separate digestion and ligation step. We tried using a different template to amplify pSB1C3 to get rid of an aberrant band that was appearing. Unfortunately, the primers got mixed up and the reaction most likely failed. We will try to redo it tomorrow. Since our backbone seems to be the problematic aspect of our assembly, we tried using pSB3K3 instead to see if proper assembly would occur.

We ran a gel of our Golden Gate assembly consisting of pSB3K3 and our other components. There were faint bands consisting of our entire construct and also some fragments. We decided to do a transformation anyway. The addition of liquid media containing transformants to some of the plates from yesterday worked. We ran a colony PCR screen in an attempt to deduce if any of them had the correct constructs. The construct consisting of an arabinose promoter, a riboswitch and GFP seemed to be properly assembled. We also amplified out more pSB1C3 from BBa_K750000 for Golden Gate assembly. The separate BsaI digest from yesterday was ligated together and then used for a transformation. In case our Golden Gate assembly does not work in time for our upcoming deadline, we ordered additional primers for another type of assembly method, Splice Overlap Extension (SOE).

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).