4/22-4/26 3-Way Gibson Assembly

 At the start of the week we had prepared 3 plates from freezebacs. One being D. Rad, one being PRAD 1 in LB Amp, and the other being PHRAM in LB Kan. We started to make sure that these were constantly growing in case we needed to do any plasmid or genomic extractions to get new samples for the Gibson Assembly project. We had also inoculated D. Metalli into R2B and let it incubate for 24 hours to observe any biofilm formation. Surprisingly by the next day there appeared to be some biofilm on the bottom of the flask, while at this point it was still pretty weak and there was little biofilm growth, any growth was a surprise since that was not what I had hypothesized. Under gram stain the cells were consistent to growth in TGY Broth in which they were not uniform and had a stringy structure, however we did not see any biofilm so we had let it sit in the incubator for another 24 hours to see how much more would grow. 

Slight Biofilm Growth of D. Metalli in R2B

The next day we did PCR on our 3 insert fragments just to see if we could get this linear assembly done. The 3 fragments were the D. Rad Left Flank, The Kan Resistance, and the D. Rad Right Flank. This would make a linear 3-Way fragment that we could use to eventually assemble with the PRAD 1 Vector. The size of the 3-way fragment was 2,527 base pairs, since it is just the 3 insert fragment sizes added up. We also viewed the D. Metalli growth to see if there were any changes as the biofilm growth was even more present than the day before. 
D. Metalli With More Biofilm Growth in R2B

On 4/25 we ran a gel to see if there was any presence of the 3-way fragment, again looking for a fragment at around 2500 base pairs. 
3-Way Gibson Gel 
In the first lane is the 1KB ladder, in the second lane was the PCR’d 3-Way Gibson, in the 3rd lane was the 100bp ladder, and the 4th lane was the genomic extract which was meant to be used as a sort of positive control. Oddly enough, nothing really showed in our 4th lane however, we did get a band at the predicted size of 2500bp in lane 2. This had meant that we had gotten our 3-Way Gibson Assembly between the 3 insert fragments, which is not something that we have gotten before. With this we could attempt to get even more 3-Way Assemblies if we PCR the product again. Furthermore we could attempt to form the whole plasmid under Gibson Assembly with this 3-Way Assembly and the PRAD 1 Vector.





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