Composite

Part:BBa_K1371015:Design

Designed by: Qifeng Tan   Group: iGEM14_SCUT-China   (2014-10-16)

pT7+birA+accA+pccB+2TM-pT7+sfp+2TM


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2946
    Illegal BglII site found at 3362
    Illegal BamHI site found at 1489
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1880
    Illegal NgoMIV site found at 1895
    Illegal NgoMIV site found at 3610
    Illegal NgoMIV site found at 3687
    Illegal AgeI site found at 2252
    Illegal AgeI site found at 2384
    Illegal AgeI site found at 2579
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1712
    Illegal BsaI.rc site found at 2426
    Illegal BsaI.rc site found at 3826
    Illegal SapI.rc site found at 2039


Design Notes

As the three enzymes are suitable to produce the substrate for all of our polyketide synthases(PKS), we decided to co-express them in one plasmid—the low copy plasmid pSB4A5 and make it the chassis for our expression of PKSs.

we use T7 promoter and terminator to stay the same with our plasmid pSB1C3 carrying the genes encoding DEBS1 PKS so that they will be co-expressed under the same condition. RBS is placed between every two genes in order to ensure the transcription of every gene. Since the sfp gene knocked into the prp operon was frame shifted, we worried about whether it works as expected. Therefore，we constructed two chassis, of which one carries sfp(chassis 1) while another(chassis 2) doesn’t, and we will verify the activity of the sfp by transforming them into the E.coli BL21(DE3) separately with pSB1C3 carrying DEBS1 at the same time and see if there will be any difference between these two strain of E.coli BL21(DE3) under the same condition.


Source

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References

[1] Pfeifer B A, Admiraal S J, Gramajo H, et al. Biosynthesis of complex polyketides in a metabolically engineered strain of E. coli ［J］. Science,2001,291:1790
[2] M. Madyagol, H. Al-Alami, Z. Levarski, H. Drahovská, J. Tur˜na, S. Stuchlik, Gene replacement techniques for Escherichia coli genome modification, Folia Microbiol. 56 (2011) 253–263.
[3] A.R. Horswill, J.C. Escalante-Semerena, The prpE gene of Salmonella typhimurium LT2 encodes propionyl-CoA synthetase, Microbiology 145 (1999) 1381–1388
[4] Pfeifer, B.A., Khosla, C., 2001. Biosynthesis of polyketides in heterologous hosts. Microbiol. Mol. Biol. Rev. 65, 106.