Difference between revisions of "Part:BBa K2333405"

Revision as of 20:01, 16 June 2017

Cloning ready protein degradation tag E (medium-weak) with double terminator

This part is designed to easily facilitate appending the pdt#3 tag to the end of an arbitrary protein using Gibson assembly, without requiring multiple cloning steps. UNS pdt#3 DT contains a tail that can be degrade Mesoplasma florum’s Lon protease Link mf-Lon here, which is orthogonal to E. Coli’s own degradation machinery. As this part contains both a double stop codon and the B0015 double terminator, it can be added before the stop codons of an arbitrary protein, preventing a multistep assembly to incorporate double stop codons and a double terminator.

UNS pdt#3d DT is on the William and Mary iGEM standard backbone, which contains 40bp Universal Nucleotide Sequences (UNS) on the inside of the prefix/suffix as a standardized flanking region to facilitate oligo-based cloning methods like PCR and gibson assembly. See Torella, J. P., Boehm, C. R., Lienert, F., Chen, J. H., Way, J. C., & Silver, P. A. (2013).

This part belongs to a series comprising 6 parts with pdt tags of different strengths BBa_ K2333401-K2333406. Of this series, the pdt in this part has the second lowest degradation rate. See characterization from William and Mary 2017, and also Collins et al. 2014 "Tunable Protein Degradation in Bacteria" for background informaiton.

This design significantly increases the accessibility of the mf- Lon tags, which can be easily added to the end of any arbitrary protein on either William and Mary's UNS backbone system or a standard Biobrick vector. Using a reverse primer with a pdt overhang to the end of a given protein, any team can easily create and amplify their own linear fragment with parts BBa_ K2333401-K2333406 to append a pdt tag onto a protein in a given circuit without changing other underlying architecture.

Sequence and Features