Part:BBa_K1403012:Design
Alcohol acetyltransferase I (ATF1) expression cassette
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 163
Illegal BamHI site found at 1490 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 590
Illegal SapI.rc site found at 1598
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 a constitutive promoter BBa_J23108 and a sRBS upstream of the CDS. An IDT gBlock was synthesized 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 the BioBrick suffix. Constitutive promoter BBa_J23108 has a relative activity of 0.51.
Source
BBa_J45014 CDS of ATF1 from Saccharomyces cerevisiae.
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