We reached out to Team Northwestern to establish a collaboration that would allow us to test our homemade Gibson Assembly recipe on Northwestern’s construct. We received the backbone BBa_J32015, the PelB insert, and the associated PCR primers to test out our method and assemble the PelB-saCas9-His construct. The expected workflow involved designing Gibson assembly primers specific to the vector and insert, assembling the construct, transforming the assembled construct into DH5α cells, and verifying the assembly success via colony PCR and sequencing.
Once testing proved successful, Northwestern would have received the DNA polymerase and DNA ligase lysate to assemble their PelB-saCas9-His construct using optimal ratios that we established. The success of the assembly would be verified via colony PCR and sequencing.
Unfortunately, we faced unforeseen delays in the verification of our construct and in successful assembly of our test devices. Although we were in communication throughout the project, we were not able to pursue this plan completely.