Complete caffeine synthesis pathway under pTET constitutive promoter
The constitutive promoter we used was from the 2012 Distribution Kit, part BBa_J23118. The caffeine biosynthesis genes we assembled were from the 2013 Distribution Kit and were submitted by the 2012 TU Munich team: parts BBa_K801070, BBa_K801071, BBa_K801072.
We combined the three caffeine synthesis genes in the Registry by standard assembly into a pSB1C3 vector. This vector had a constitutive promoter, BBa_J23118, and a bacterial consensus sequence cloned into it previously, so that the completed construct would allow constitutive expression of the entire caffeine biosynthesis pathway when the substrate xanthosine was present.
We have yet to test for expression, and we may find that codon optimization is needed.
RBS (Elowitz 1999) -- defines RBS efficiency
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix. No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info for this part: Brian Chow
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
Double terminator consisting of BBa_B0010 and BBa_B0012
xanthosine methyltransferase CaXMT1-strep
http://www.ncbi.nlm.nih.gov/nuccore/AB048793 synthesized by IDT, yeast codon optimized
xanthosine methyltransferase CaXMT1-strep from ''Coffea arabica'' followed by a strep-tag for purification and/or detection via western blot.
7-methylxanthine N-methyltransferase CaMXMT1-strep
Synthesized by IDT, Yeast codon optimized
7-methylxanthine N-methyltransferase CaMXMT1 from ''Coffea arabica'' followed by a strep-tag for purification and/or detection via western blot.
3,7-dimethylxanthine N-methyltransferase CaDXMT1-strep
http://www.ncbi.nlm.nih.gov/nuccore/AB084125 synthesized by IDT, yeast codon optimized
3,7-dimethylxanthine N-methyltransferase CaDXMT1 from ''Coffea arabica'' followed by a strep-tag for purification and/or detection via western blot.
Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 <genbank>V01146</genbank>.
Transcription terminator for the E.coli RNA polymerase.
Suggested by Sri Kosuri and Drew Endy as a high efficiency terminator. The 5' end cutoff was placed immediately after the TAA stop codon and the 3' end cutoff was placed just prior to the RBS of Gene 1.4 (before AAGGAG). Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator.
Transcriptional terminator consisting of a 64 bp stem-loop.
Conversion of the iGEM parts registry to SBOL2.1