Generator

Part:BBa_K1403006:Design

Designed by: Antonio Villarreal Larrauri   Group: iGEM14_aris_Bettencourt   (2014-10-06)
Revision as of 00:37, 19 October 2014 by Tovila (Talk | contribs) (→‎References)


Jasmonic acid carboxyl methyltransferase (JMT) expression cassette


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 222
    Illegal BamHI site found at 793
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 71
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The purpose of this plasmid is to produce methyl jasmonate in E.coli. We used JMT CDS from NCBI (AY008434.1). An IDT gBlock was synthesized with the codon optimized CDS downstream of constitutive promoter BBa_J23108 and a sRBS. Jasmonic acid carboxyl methyltransferase catalyzes the conversion of jasmonic acid to methyl jasmonate, which is one component of jasmine scent. The gBlock and pSB1C3 vector were amplified by PCR, digested and ligated. The finished plasmid is in standard biobricks format.

Silently removed an illegal PstI site from the codon optimized sequence. Added a C-terminus 6-His tag.

BioBrick transcriptional terminators were not added because the vector already has a terminator for E. coli downstream the BioBrick suffix. Promoter BBa_J23108 from the Anderson library of constitutive promoters has a relative activity of 0.51.

Source

CDS of Arabidopsis thaliana JMT (NCBI: AY008434.1), codon optimized for expression in E. coli..

References

Anderson Promoter Collection: https://parts.igem.org/Promoters/Catalog/Anderson

Salis lab RBS calculator: https://salis.psu.edu/software/forward_withConstraints 

H.M Salis, Methods in Enzymology 2011
H.M. Salis, E.A. Mirsky, C.A. Voigt, Nat. Biotech., 2009

Seo, H., Song, J. & Cheong, J., 2001. Jasmonic acid carboxyl methyltransferase: a key enzyme for jasmonate-regulated plant responses. Proceedings of the National Academy of Sciences. Available at: http://www.pnas.org/content/98/8/4788.short [Accessed June 19, 2014].