Composite

Part:BBa_K2333401

Designed by: Ethan M Jones   Group: iGEM17_William_and_Mary   (2017-06-14)
Revision as of 18:11, 28 October 2017 by Ethan801 (Talk | contribs)


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 [http://2017.igem.org/Team:William_and_Mary/Results William and Mary's 2017 project] for more details

This part is one of a series of easy cloning pdt parts. Series range is from BBa_K2333401 to BBa_K2333406


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 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

The primers below should be useful for cloning purposes. They each are short enough that 20+ basepairs of overhang can be added on, have annealing temperatures in Q5 greater than 60C, and have no significant homo-dimers, hairpins or hetero-dimers. UNS2 F and UNS3 R can be used for sequencing, or amplification to move parts to a new plasmid backbone. Since all of the protein degradation tags have the same first 33 base pairs, the Protein Degradation Primer can be used for any of the pdts in this part series. While these parts should be useful for any group using Gibson Cloning (either in or not in the W&M UNS backbone), they can also be used to add any arbitrary restriction site as well. Using the pdt F and B0015 R primers with restriction site overhangs added on should work robustly, as W&M 2017's used variants of this method to clone most of their tagged reporters. See [http://2017.igem.org/Team:William_and_Mary/Parts here] for a complete list.

Protein Degradation Primer F(standard): GCTGCTAACAAAAACGAAGAAAACAC

UNS2 Primer F: GCTGGGAGTTCGTAGACG

UNS3 Primer R: CGACCTTGATGTTTCCAGTG

B0015 end R: tataaacgcagaaaggccca

Double stop + B0015 beginning F: TAATAAccaggcatcaaataaaacg


Characterization

W&M 2017 characterized this tag's degradation rate and speed change effects as part of their iGEM project. The graphs below show this data along with the data from the other tags in this series Graph 1 Graph 2


Associated Parts

Promoter RBS Coding Region Terminator Device
BBa_K801020 BBa_K1084104 BBa_K1172501 BBa_K731721 BBa_K863005
BBa_K1216007 BBa_K1017202 BBa_K525515 BBa_K404108 BBa_K404163


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 41
    Illegal BsaI.rc site found at 263

References

  1. To Do


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