Difference between revisions of "Part:BBa K1403006:Design"
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===Design Notes=== | ===Design Notes=== | ||
The purpose of this part is to biosynthesize methyl jasmonate with <i>E. coli</i>. | The purpose of this part is to biosynthesize methyl jasmonate with <i>E. coli</i>. | ||
| − | + | The sequence was synthesized as a gBlock by IDT with the codon optimized CDS downstream of the constitutive promoter <partinfo>BBa_J23108</partinfo> 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 linearized pSB1C3 vector were amplified by PCR, digested and ligated. | + | The gBlock and linearized pSB1C3 vector were amplified by PCR, digested, and ligated. |
The finished plasmid is in standard BioBrick format. | The finished plasmid is in standard BioBrick format. | ||
| Line 17: | Line 17: | ||
===Source=== | ===Source=== | ||
| − | CDS of <i>Arabidopsis thaliana</i> JMT | + | CDS of <i>Arabidopsis thaliana</i> JMT from [http://www.ncbi.nlm.nih.gov/nuccore/AY008434.1 GenBank (AY008434.1)], codon optimized for expression in <i>E. coli</i>. |
===References=== | ===References=== | ||
| − | + | [https://parts.igem.org/Promoters/Catalog/Anderson Anderson Promoter Collection] | |
| − | + | [https://salis.psu.edu/software/forward_withConstraints Salis lab RBS calculator]<br> | |
H.M Salis, Methods in Enzymology 2011<br> | H.M Salis, Methods in Enzymology 2011<br> | ||
H.M. Salis, E.A. Mirsky, C.A. Voigt, Nat. Biotech., 2009 | 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]. | 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]. | ||
Latest revision as of 07:07, 31 October 2014
Jasmonic acid carboxyl methyltransferase (JMT) 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 222
Illegal BamHI site found at 793 - 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 jasmonate with E. coli. The sequence was synthesized as a gBlock by IDT with the codon optimized CDS downstream of the 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 linearized pSB1C3 vector were amplified by PCR, digested, and ligated. The finished plasmid is in standard BioBrick 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.
Source
CDS of Arabidopsis thaliana JMT from [http://www.ncbi.nlm.nih.gov/nuccore/AY008434.1 GenBank (AY008434.1)], codon optimized for expression in E. coli.
References
Salis lab RBS calculator
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].