Difference between revisions of "Part:BBa K2333401"

Revision as of 16:53, 28 October 2017

Cloning ready protein degradation tag A (strong) with double terminator

This part is designed to facilitate quick, easy and reproducible cloning of protein degradation tag (pdt) A, onto an arbitrary gene, regardless of cloning method. William and Mary iGEM 2017 used pdts as a method to control gene expression speed. Utilizing this part along with results and mathematical modeling from William and Mary should enable the tuning of gene expression speed for any arbitrary protein in a circuit, without having to perform a multistep re-cloning process. See 2017.igem.org/Team:William_and_Mary/Results for more details.

Usage and Biology

Protein degradation tag A is the strongest of the 6 protein degradation tags that William and Mary 2017 characterized, and is associated with the E. Coli orthogonal protease mf-Lon (Bba_K2333011). While any mf-Lon generating part can be used alongside this tag to increase degradation rate/speed of a given protein of interest, the majority of William and Mary 2017's characterization was done using BBa_K2333434, which is a LacI regulated (IPTG inducible) mf-Lon. In cases where LacI cannot be used, the leakier Arabinose inducible mf-Lon Bba_K2333435 can be used instead. (Note, it is recommended that these parts be used on a low copy backbone such as Bba_psb3k3)

This part contains pdt A, a double stop codon and Bba_B0015 (double terminator) in the William and Mary iGEM Universal Nucleotide Sequences (UNS) format. This enables easy cloning with Gibson Assembly, as UNS primers are designed for easy PCRs and high yield Gibson Assembly. See Torella, et. al (2013). On the interior of each UNS are BsaI cut sites, which enables Golden Gate Assembly as an alternative to Gibson Assembly. For groups that want to use restriction enzyme cloning, or a different Golden Gate enzyme/overhang sequence, we recommend that they PCR using the primers below, and add on up to 30 basepairs of overhang.

Since this part contains both a double stop codon and a double terminator, to tag an arbitrary protein all that is required is to append this part without UNS2 to the end of your protein of choice. (Note, that the double stop codons of your protein should be removed, as this will prevent translation of the tag.)

Primers

UNS pdt#3 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 highest 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