Difference between revisions of "Part:BBa K1403009:Design"

 
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<partinfo>BBa_K1403009 short</partinfo>
 
<partinfo>BBa_K1403009 short</partinfo>
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===Design Notes===
 
===Design Notes===
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RBS geneator
+
The purpose of this part is to biosynthesize methyl salicylate in <i>E.coli</i>. 
  
 +
We used BSMT1 CDS from part BBa_J45004 (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. Promoter BBa_J23108 from the Anderson library of constitutive promoters has a relative activity of 0.51.
  
 
===Source===
 
===Source===
−
 
+
CDS of <i>Petunia x hybrida</i> BSMT1 (NCBI:AY233465.1), codon optimized for expression in <i>E. coli</i>.
−
Salis Lab RBS generator
+
  
 
===References===
 
===References===
 +
Anderson Promoter Collection: https://parts.igem.org/Promoters/Catalog/Anderson
 +
 +
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].
 +
 +
iGEM 2006 MIT: https://parts.igem.org/Part:BBa_J45004
 +
 +
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

Revision as of 01:59, 19 October 2014

Benzoic acid/salicylic acid carboxyl methyltransferase 1 (BSMT1) 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
    COMPATIBLE WITH RFC[21]
  • 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 part is to biosynthesize methyl salicylate in E.coli.

We used BSMT1 CDS from part BBa_J45004 (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. Promoter BBa_J23108 from 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

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

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].

iGEM 2006 MIT: https://parts.igem.org/Part:BBa_J45004

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