Coding

Part:BBa_K1462460:Design

Designed by: zhanru Chen   Group: iGEM14_SCUT   (2014-10-11)

GBD-lig


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]


Design Notes

This is the GBD ligand of the GBD protein. The GBD ligand, with the fusion of the GBD domain , we can use it as a binding unit to built up the scaffold protein that contains the funtion we want. We hope that we can use it to combine the PRK and funtion as the part of the carbon capture.

Source

This is the ligand which can we use it to make fusion protein with GBD protein.

At the date of 7th, July, we did the overlap PCR to get this gene with the help of our designed primer.
Upstream primer 1 : GTTTCTTCGA ATTCGCGGCC GCTTCTAGAT TGGTTGGTGC TTTGATGCAC GTCATGCAGA
Upstream primer 2 : TTTGATGCAC GTCATGCAGA AGAGGTCTAG GGCTATTCAC TCTTCTGACG AGGGTGAGGA
Downstream primer 1：GTTTCTTCCT GCAGCGGCCG CTACTAGTAT CTTCATCTTC ATCACCAGCT TGGTCCTCAC
Downstream primer 2: ATCACCAGCT TGGTCCTCAC CCTCGTCAGA AGAGTGAATA GCCCTAGACC TCTTCTGCAT

We added the RFC 23 into the GBD ligand.First we use upstream/downstream primer 2 to get the intermediate. After purification of PCR, we carried on the next PCR with the help of upstream/downstream primer 1.

References

[1] John E Dueber, Gabriel C Wu, G Reza Malmirchegini, et al.Synthetic protein scaffolds provide modular control over metabolic flux.Nat Biotechnol. 2009 Aug;27(8):753-9.
[2] Schultz, J. et al. Specific interactions between the syntrophin PDZ domain and voltage-gated sodium channels. Nat Struct Biol 5, 19-24 (1998).
[3] Kim, A.S., Kakalis, L.T., Abdul-Manan, N., Liu, G.A. & Rosen, M.K. Autoinhibition and activation mechanisms of the Wiskott-Aldrich syndrome protein. Nature 404, 151-158 (2000).
[4] Wu, X. et al. Structural basis for the specific interaction of lysine-containing proline-rich peptides with the N-terminal SH3 domain of c-Crk. Structure 3, 215-226 (1995).
[5] Harris, B.Z., Hillier, B.J. & Lim, W.A. Energetic determinants of internal motif recognition by PDZ domains. Biochemistry 40, 5921-5930 (2001).
[6] Dueber, J.E., Yeh, B.J., Chak, K. & Lim, W.A. Reprogramming control of an allosteric signaling switch through modular recombination. Science 301, 1904-1908 (2003).
[7] Nguyen, J.T., Turck, C.W., Cohen, F.E., Zuckermann, R.N. & Lim, W.A. Exploiting the basis of proline recognition by SH3 and WW domains: design of N-substituted inhibitors. Science 282, 2088-2092 (1998).