Quandt EM, Hammerling MJ, et al. (2013) Decaffeination and measurement of caffeine content by addicted Escherichia coli with a refactored N-demethylation operon from Pseudomonas putida CBB5. ACS Synth Biol. DOI: 10.1021/sb4000146.
As part of an undergraduate project for the International Genetically Engineered Machine (iGEM) competition, this research group from the University of Texas at Austin imported a refactored N-demethylation operon that provides a caffeine degradation pathway from Pseudomonas putida CBB5 to E. coli, an organism that is more amenable to genetic engineering and industrial applications. The E. coli strain was a guaB knockout that required xanthine, a precursor of guanine, to grow. Xanthine is produced by N-demethylation of caffeine; thus, these cells required caffeine to survive.
IDT gBlocks® Gene Fragments were used in Gibson Isothermal AssemblyTM reactions to generate some of the plasmid constructs within this work. In addition to characterizing the sequence elements regulating the caffeine degradation pathway, the researchers were able to use the growth yield of the “addicted” E. coli strain to precisely measure caffeine concentrations, for example, in sodas and energy drinks. The synthetic N-demethylation operon has applications for cheaply producing pharmaceuticals or their precursor molecules, and for detoxifying waste that could subsequently be recycled into animal feed and biofuels.