Team:Austin UTexas/Results/Parts
Part Plasmids
Before the plasmids could be assembled, the contributing parts must be isolated, affixed with restriction sites and customized overhangs, and inserted into a backbone so the parts could be stabilized and stored in a part plasmid. In a Golden Gate Assembly cloning reaction, the DNA sequence for the plasmid part is inserted into a plasmid containing a gene for green fluorescent protein production (GFP), on either side of which, are sites where the BsmBI enzyme cuts. The GFP gene is replaced by the plasmid part, so colonies that do not fluoresce green under UV light are sustaining the part plasmid. Over the course of this project we have made a total of 48 parts. For coding sequences and promoter/RBS parts, not all of the varieties of the part will be used in the assemblies. Rather, they are meant to provide a resource of alternative parts to a researcher constructing their own plasmid from the kit. For barcodes and 1-5 bridges, each must be unique to the origin in their plasmid because they are meant to identify which origin the plasmid contains. Having many varieties of antibiotic resistance and origin of replication strengthens the kit, as it allows researchers to rapidly test more origins at a time, in bacteria that may be naturally resistant to some antibiotics.
