Composite

Part:BBa_K2570009:Design

Designed by: Luying He   Group: iGEM18_FJNU-China   (2018-09-30)
Revision as of 02:05, 16 October 2018 by Clivia (Talk | contribs)


Degrading enzyme complex：araC＋atoD＋atoA＋adhE2


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2085
    Illegal BamHI site found at 1144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 3478
    Illegal AgeI site found at 979
    Illegal AgeI site found at 2731
    Illegal AgeI site found at 3133
    Illegal AgeI site found at 3570
    Illegal AgeI site found at 4504
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961


Design Notes

Mutation of restriction sites EcoRⅠand PstⅠ, codon optimization

We obtained atoD and atoA from E.coli Bw25113 by PCR. AdhE2 was synthesized from a gene synthesis company and was codon optimized of the sequence. Also we did mutation of restriction sites EcoRⅠand PstⅠto meet the requirements of RFC10. We chose pRB1s as the backbone and successfully construct pRB1s-atoD-atoA-adhE2 by gibson assembly. Then we transformed it into the odorless E. coli tnaA-/- and verified its function.

Fig.1 Schematic map of the recombinant plasmid pRB1s-atoD-atoA-adhE2.

Source

atoD and atoA from E.coli, adhE2 from Clostridium acetobutylicum ATCC 824.

https://www.ncbi.nlm.nih.gov/nuccore/U00096.3?from=2323447&to=2324109

https://www.ncbi.nlm.nih.gov/nuccore/U00096.3?from=2323447&to=2324109

https://www.ncbi.nlm.nih.gov/gene/1116040


References

[1] Andreas Natsch*. What Makes Us Smell: The Biochemistry of Body Odour and the Design of New Deodorant Ingredients. KGF-SCS Industrial Investigator Award 2014

[2] Saini M, Chen M H, Chiang C J, et al. Potential production platform of n-butanol in Escherichia coli[J]. Metabolic Engineering, 2015, 27:76.

[3] Anu Jose Mattam and Syed Shams Yazdan. Engineering E. coli strain for conversion of short chain fatty acids to bioalcohols. Biotechnol Biofuels. 2013; 6: 128.