Difference between revisions of "Part:BBa K1598008:Design"

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Revision as of 23:23, 24 September 2015

J23101 promoter + RBS + Human FDXR + RBS + Human FDX1 + RBS + Human CYP11A1 + rrnb double terminator


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 3275
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 565
    Illegal XhoI site found at 1457
    Illegal XhoI site found at 3710
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1653
    Illegal AgeI site found at 1090
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This biobrick expresses the CYP11A1 gene that codes for Cytochrome P450scc (P450scc), which converts cholesterol into pregnenlone, as well as the genes for its 2 essential electron transport partners, FDX1 and FDXR, which code for Ferredoxin and Ferredoxin Reductase. The biobrick consists of the iGEM prefix followed by the J23101 constitutive promoter, RBS, FDXR gene, RBS, FDX1 GENE, RBS, CYP11A1 gene, rrnb double terminator, and the iGEM suffix, ligated onto a PSB1C3 backbone. The design was influenced by previous studies that successfully produced Pregnenolone using E Coli, and was made under the guidance of Dr. Fiona Truscott, who specializes in Cytochrome P450 enzymes [1].

The biobrick was broken into 5 parts for quick synthesis and assembled using Golden Gate assembly. Hence, each part was made compatible for golden gate assembly, and had overhangs for the Type II restriction endonuclease, SapI. Thus an almost 4000 base pairs large insert was created, which codes for 3 different protein/enzymes. This is the first time that all the 3 genes (CYP11A1, FDX1, AND FDXR) used in a P450scc steroidogenic system are human.



"Preg_3.png" 
 "Preg_4.png"

Source

http://www.ncbi.nlm.nih.gov/nuccore/153218645?report=fasta

http://www.ncbi.nlm.nih.gov/nuccore/16877631?report=fasta

http://www.ncbi.nlm.nih.gov/nuccore/39645178?report=fasta

https://parts.igem.org/Part:BBa_J34801

https://parts.igem.org/Part:BBa_J23101

http://file.scirp.org/Html/38129.html

References

[1] Desislava S. Makeeva Functional reconstruction of bovine P450scc steroidogenic system in Escherichia coli in American Journal of Molecular Biology, 2013, 3, 173-182