Team:UC Davis/Notebook/Week 3


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September 30-October 2815+

Week 3

Our transformation from last Friday yielded very few colonies, so we transferred the part BBa_K598001 and BBa_B0034 from last Wednesday into cultures to check for the correct constructs through PCR later. We also ran the PCR product from the Golden Gate Assembly practice in a gel and realized that the construct had not properly annealed. Two of the three lanes contained bands that were not the proper size and multiple bands were present from the individual parts. For most of the day, we designed primers for our Golden Gate Assembly and obtained sequences for our riboswitches of interest.

We began the day with a DNA miniprep of the colonies from BBa_K598001 and BBa_B0034. The DNA concentrations from the miniprep were too low for PCR, so we had to redo the DNA extraction from our original colonies. After redoing the DNA extractions, we measured the DNA concentrations and aliquoted the corrected amount of DNA for PCR. We then began the PCR reaction with the other appropriate reagents. In our spare time, we continued designing primers for our final construct involving Golden Gate Assembly and figuring out the most efficient way assemble our construct.

After the cycles of PCR amplified BBa_K598001 and BBa_B0034 finished, we ran them in a gel to see if the correct band sizes were present. The bands were present at the right size, so the transformation for BBa_K598001 and BBa_B0034 worked! Neither band was a contaminant. We then began determining the volumes necessary for a restriction digest of our inserts of interest and left the digests to incubate overnight. We wanted to eventually ligate the BBa_K598001 to the promoter BBa_J23101 through standard assembly. In our spare time, we continued looking at our primer list and making sure that they had the correct design for Golden Gate Assembly.

After incubating our digests of BBa_K598001 and BBa_J23101 overnight, we performed a gel extraction. When performing the extraction, we made sure to have the proper protection against the harmful UV light. The extracted gel was purified to obtain our desired products, the riboswitch reporter part and a backbone with a promoter. We then measured the concentration of our purified products and performed a ligation of the vector and the insert to complete our first standard assembly. We then performed four transformations involving a positive control and combinations of our vector and/or insert. In addition we hydrated BBa_K537003 and BBa_K537004 and then transformed both parts. Each construct contains a riboswitch relevant to our library. However, the parts remain unsequenced and need to be verified for use.

We looked at our ligation transformation of the insert part BBa_K598001 and the vector BBa_J23101 with its backbone and saw no colonies. There were colonies on the plate treated with only the insert and no colonies with E. coli treated with only the vector. The evidence of colonies with the insert only suggests that the restriction digest was unsuccessful. We decided to compare and contrast the sizes of the restriction enzyme digest and the DNA miniprep from BBa_K598001 on a gel to see if any digestion occurred. After viewing the gel, there was presence of a 3 kb plasmid in the DNA miniprep, but there were two 1 kb bands in the lane containing the restriction enzyme digest product, even though only one band should have been remaining after the gel extraction. We decided to verify BBa_K598001 through sequencing. We are awaiting verification of our primer list by our advisers. The transformations of BBa_K537004 and BBa_K537003 were successful. The colonies were inoculated for further propagation in LB media.