Team:Caltech/Project/Data

comX export

The export domains team made many attempts at isolating a properly assembled construct (export domain--ligand comX--linker--mNeonGreen) in vector pKS001. However, our colony PCRs were never able to verify a properly assembled construct. Instead, gels with lanes containing colony PCRs of colonies that grew on carbenicillin plates were run and ended up showing bands at roughly 100 base pairs (see Figure 1 below).

These bands at ~100bp suggest that the vector does not contain any insert, since a resealed vector containing no insert should contain around 109bp, assuming that the ends of the linearized backbone do not overlap when recircularizing. In fact, on examination of the biobrick prefix and suffix, which were used as the "overlap regions" for gibson assembly, it was found that there was enough sequence homology between the two to suggest significant binding of the prefix to the suffix. In particular, the sequence of similarity (14 base pairs in length [include that sequence]) had a very high estimated melting temperature of 46.2°C, owing to its rich GC-content. Since the melting temperature is so close to the 50°C used as our incubation temperature for Gibson assembly, it can be concluded that a significant number of backbone ends were "chewed back" by exonucleases in the Gibson reaction and then annealed to each other, recircularizing the backbone without the desired insert.