Part:BBa_K734000
Caffeine demethylation pathway
Synthetic operon consisting of methylxanthine N-demethylases: NdmA,NdmB, NdmC, and associated reductase NdmD from Psuedomonas putida CBB5. Also includes gst9 from Janthinobacterium sp. Marseille which is required for NdmC functionality. Constitutive expression is driven by promoter BBa_J23100.
Enables Escherichia coli cells to degrade caffeine (1,3,7-Methylxanthine) and related methylxanthines to xanthine.
See 2012 UT Austin wiki: [http://2012.igem.org/Team:Austin_Texas/Caffeinated_coli Caffeinated coli]
2015 Austin UTexas iGEM Team Improvements
The 2015 Austin UTexas iGEM team worked to improve this operon. In this operon, the NdmB, NdmC, NdmD, and gst9 genes are all under the control of the same RBS sequence BBa_B0034 with the surrounding sequence kept the same, meaning the same 25 bp are repeated four times throughout the sequence. This repeated region introduces a higher possibility of repeat-mediated deletion of entire genes in the operon when not strongly selected for. The 2015 Austin UTexas iGEM team replaced three of these four RBS sequences with different RBS sequences of similar strengths so as to reduce this risk. In addition, two restriction sites, a PstI cut site in the NdmA gene and an EcoRI cut site in the NdmB gene, were removed in order to make the synthetic operon BioBrick compatible. We submitted this part to the iGEM Registry as BBa_K1627006.
The use of this pathway to measure caffeine (as seen in the 2012 Austin Texas iGEM team's Caffeinated coli project) was also expanded on by making plasmids containing all the subsets of the NdmA, NdmB, and NdmC genes in order to measure mixtures of methylxanthines in organic beverages such as coffee or tea. For further information, see the 2015 Austin UTexas wiki: [http://2015.igem.org/Team:Austin_UTexas/Project/Caffeine Austin UTexas 2015.]
Usage and Biology
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1229
Illegal XbaI site found at 1126
Illegal XbaI site found at 2229
Illegal XbaI site found at 3109
Illegal PstI site found at 548 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1229
Illegal NheI site found at 8
Illegal NheI site found at 31
Illegal NheI site found at 1730
Illegal NheI site found at 1925
Illegal PstI site found at 548 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1229
Illegal BglII site found at 2687
Illegal BglII site found at 3181
Illegal BglII site found at 3652
Illegal XhoI site found at 4793 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1229
Illegal XbaI site found at 1126
Illegal XbaI site found at 2229
Illegal XbaI site found at 3109
Illegal PstI site found at 548 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1229
Illegal XbaI site found at 1126
Illegal XbaI site found at 2229
Illegal XbaI site found at 3109
Illegal PstI site found at 548
Illegal NgoMIV site found at 5491
Illegal NgoMIV site found at 5604
Illegal AgeI site found at 2224
Illegal AgeI site found at 3320 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 984
Illegal BsaI.rc site found at 2221
| None |