Part:BBa_K1403009:Design
Benzoic acid/salicylic acid carboxyl methyltransferase 1 (BSMT1) expression cassette
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 71
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
The purpose of this part is to biosynthesize methyl salicylate in E.coli.
We used BSMT1 CDS from part BBa_J45004 [http://www.ncbi.nlm.nih.gov/nuccore/AY233465.1 (NCBI:AY233465.1)] and inserted the constitutive promoter BBa_J23108 and a sRBS upstream of the CDS. The sequence was synthesized as a gBlock by IDT with the codon optimized CDS along with a C-terminus 6-His tag downstream of constitutive promoter BBa_J23108 and a sRBS. Benzoic acid/salicylic acid carboxyl methyltransferase 1 converts farnesyl-diphosphate to methyl salicylate or methyl benzoate, which are components of mint and flower scents respectively. The gBlock and linearized pSB1C3 vector were amplified by PCR, digested, and ligated. The finished plasmid is in standard BioBrick format.
BioBrick transcriptional terminators were not added because the vector already has a terminator for E. coli downstream of the BioBrick suffix. Promoter BBa_J23108from the Anderson library of constitutive promoters has a relative activity of 0.51.
Source
CDS of Petunia x hybrida BSMT1 (NCBI:AY233465.1), codon optimized for expression in E. coli.
References
Negre, F., Kish, C. & Boatright, J., 2003. Regulation of methylbenzoate emission after pollination in snapdragon and petunia flowers. The Plant Cell …, 15(December), pp.2992–3006. Available at: http://www.plantcell.org/content/15/12/2992.short [Accessed August 27, 2014].
Salis lab RBS calculator
H.M Salis, Methods in Enzymology 2011
H.M. Salis, E.A. Mirsky, C.A. Voigt, Nat. Biotech., 2009