Difference between revisions of "Part:BBa K1403012:Design"

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===Design Notes===
 
===Design Notes===
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The purpose of this part is to biosynthesize methyl salicylate in <i>E.coli</i>.   
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The purpose of this part is to biosynthesize isoamyl acetate in <i>E.coli</i>.   
  
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We used BSMT1 CDS from part <partinfo>BBa_J45004</partinfo> ([http://www.ncbi.nlm.nih.gov/nuccore/AY233465.1 NCBI:AY233465.1]) and inserted a constitutive promoter <partinfo>BBa_J23108</partinfo> 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 <partinfo>BBa_J23108</partinfo> 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.
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We used the ATF1 generator <partinfo>BBa_J45199</partinfo> and inserted a constitutive promoter <partinfo>BBa_J23108</partinfo>  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.
  
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BioBrick transcriptional terminators were not added because the vector already has a terminator for E. coli downstream the BioBrick suffix. Constitutive promoter <partinfo>BBa_J23108</partinfo> has a relative activity of 0.51.
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Constitutive promoter <partinfo>BBa_J23108</partinfo> has a relative activity of 0.51.
  
 
===Source===
 
===Source===

Revision as of 02:31, 19 October 2014

Alcohol acetyltransferase I (ATF1) 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 163
    Illegal BamHI site found at 1490
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE 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 isoamyl acetate in E.coli.

We used the ATF1 generator BBa_J45199 and inserted a constitutive promoter BBa_J23108 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.

Constitutive promoter BBa_J23108 has a relative activity of 0.51.

Source

BBa_J45014 CDS of ATF1 from Saccharomyces cerevisiae.

References

Anderson Promoter Collection

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

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