This part codes the sequence for our QSP peptide string. By placing our peptide string under the OmpC promoter biobrick (BBa_R0082), it will be induced under conditions of high osmolarity, and therefore be responsive to environments of high glucose concentrations. The peptide string is marked with the HlyA secretion tag biobrick (BBa_K554002), as this will allow the peptide string to be secreted from the cell when the tag is recognized by the HlyB/D membrane proteins.
This part presents the code for the proline iminopeptidase from the organism Bacillus Coagulans. This enzyme is an exoprotease that is able to cleave a proline from the N terminal of a polypeptide chain. While this protease would be ineffective in cleaving our peptide string into tripeptides, its design was intended to provide baseline kinetics in comparison for a future construct expressing this enzyme on the membrane of the cell as verification that the unobtainable protease, proline endopeptidase, would function as well. It is our hope that we have made it easier for future teams to work with this protease by contributing it to the registry.
Melittin Optimus BBa_K1810003
Improvement on Melittin BioBrick: BBa_K1382000 Last year the Stony Brook iGEM team designed the Melittin biobrick (BBa_K1382000). The sequence for melittin, an antimicrobial peptide naturally found in bee venom that is capable of lysing cells by puncturing their membranes, was obtained directly from Apis mellifera. This resulted in difficulty in expressing melittin in E. coli as the sequence was not codon optimized. After confirming that optimization would increase the yield of melittin by the fluorescence assay obtained from the construction of the biobrick described below, the optimized sequence of melittin has been added as an improvement in the registry.
BBa_K1810002 Melittin Optimus/Mcherry
With the suspicion that incompatible codons between Apis mellifera and E. coli were responsible for the low yield of melittin reported by the 2014 Stony Brook iGEM team, two constructs were designed, both the regular and optimized sequence of melittin tagged with the mCherry fluorescent protein, that allowed the optimization to be quantified. Using a plate reader to measure fluorescence, a near 100 fold increase in production was seen in the codon optimized version of melittin.