Difference between revisions of "Team:William and Mary/Parts"

We utilized a suite of 6 protein degradation tags (pdts) originally designed by [Collins et al 2014]. Each tag confers a distinct level of protein degradation; tags are lettered (A-F) in order of protease affinity, from strongest (highest level of degradation) to weakest. These parts have been made BioBrick compatible, codon-optimized for E. coli, and placed in between UNS sites, so that teams may easily insert them after their gene of choice using Gibson Assembly. They are also flanked by BsaI cut sites, for ease of use with Golden Gate Assembly. This allows future teams to efficiently adapt the speed-control system to their own parts and circuits, regardless of their preferred assembly method.

Our next collection of parts consist of a protein degradation tagged mScarlet reporter under the control of the strong constitutive promoter J23100. These parts, in combination with inducible mf-Lon protease constructs (listed below), allowed us to characterize the degradation properties of each protein degradation tag on a plasmid-based system. We successfully demonstrated distinct levels of protein degradation by each of the 6 pdt’s; see our full characterization here [HYPERLINK TO RESULTS PAGE]. We also included a tagless control construct (J23100 mScarlet with no pdt) as a comparison. In order to demonstrate that our protein degradation tags operated similarly regardless of the tagged protein, we also built and characterized analogous constructs with an sfGFP reporter; these are listed below in the “All Parts” table. mScarlet and sfGFP reporters have been codon-optomized for E. coli and feature a double stop codon for ….(enhanced efficiency or something idk).

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