Coding

Part:BBa_K3755004

Designed by: Qi Xin   Group: iGEM21_ShanghaiTech_China   (2021-09-28)


cfa-N

Codon optimized N-terminal spontaneous splicing−ligation peptide sequnece for 3*mfp5 CDS assembly, connected downstream of the 2*mfp5 CDS.



Usage and Biology

Split inteins(SIs) are autocatalytic peptides that catalyze the spontaneous splicing−ligation reactions between two SI-fused target proteins, assembling the two target proteins covalently in trans[1]. A recently engineered Cfa SI was employed due to its rapid protein splicing rate in denaturing conditions (e.g., 8 M urea), which we used to efficiently extract and solubilize the target Mfp5 proteins[2].

The Cfa-n is the N-terminal Cfa SI which is genetically fused to 2*Mfp5 CDS when Cfa-c is fused to 1*Mfp5 CDS. The two fusion proteins in whole cell lysate mixtures were then mixed to yield 3 repeats of Mfp5 protein, which can not be expressed by simple 3*Mfp5 CDS[3].

We learned form Eugene Kim's literatur that repeating the sequence of Mfp5 can lead to higher adhesion[3]. So, we built the expression system of 3 repeats of Mfp5 protein(Mfp5-3) to improve our Mfp5-1(part:BBa_K3755011) and Mfp5-2(part:BBa_K3755012). The expression system consists of two composite part part:BBa_K3755029 and part:BBa_K3755030, and this part is the basic component of part:BBa_K3755030.

Experiment and Results

We characterized this part in a larger composite part part:BBa_K3755030, please go to that page for more details.

References

[1]Carvajal-Vallejos P, Pallissé R, Mootz HD, Schmidt SR. Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources. J Biol Chem. 2012 Aug 17;287(34):28686-96. doi: 10.1074/jbc.M112.372680. Epub 2012 Jun 28. PMID: 22753413; PMCID: PMC3436554.

[2]Stevens AJ, Brown ZZ, Shah NH, Sekar G, Cowburn D, Muir TW. Design of a Split Intein with Exceptional Protein Splicing Activity. J Am Chem Soc. 2016 Feb 24;138(7):2162-5. doi: 10.1021/jacs.5b13528. Epub 2016 Feb 8. PMID: 26854538; PMCID: PMC4894280.

[3]Kim E, Dai B, Qiao JB, Li W, Fortner JD, Zhang F. Microbially Synthesized Repeats of Mussel Foot Protein Display Enhanced Underwater Adhesion. ACS Appl Mater Interfaces. 2018 Dec 12;10(49):43003-43012. doi: 10.1021/acsami.8b14890. Epub 2018 Nov 27. PMID: 30480422. 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
    COMPATIBLE WITH RFC[1000]
[edit]
Categories
//awards/basic_part
//awards/part_collection
Parameters
None