The adaptive and direct evolution cycle are intended to mimic natural selection by inducing mutagenesis on NdmB to produce new variants every generation in hopes of obtaining a variant that is highly favorable. Improvement is necessary so that the best cultures of NdmB are chosen for the highest enzymatic efficiency for the production of 7-methylxanthine and paraxanthine when combined with NdmA and NdmD. Enzymatic efficiency is determined through the kcat/km ratio, which allows a way to test how effective an enzyme is to a particular substrate. The higher the kcat/km ratio, the higher the rate of catalytic activity, and thus the more efficient the enzyme-substrate system is.

The proof of concept will be conducted by creating a construct via Gibson Assembly with NdmABD, GFP, and the pSB1C3 backbone. This construct will undergo a baseline assay to observe the natural conversion of caffeine to our desired products using serially diluted amounts of caffeine. These will then be compared to the mutated constructs that underwent adaptive and direct evolution via GFP marker to observe if mutagenesis produced more theobromine, then later converting theobromine to paraxanthine and 7-methylxanthine. The best growers will be sequenced and further analyzed via caffeine cultures to identify the most favorable mutant, thus establishing our new and improved part.

Sequences would be compared via high-throughput screening and utilizing genome analysis tools such as NCBI BLAST to compare the mutated constructs to the wild-type constructs. Our intended NdmB construct advances the production of both paraxanthine and 7-methylxanthine as it is crucial in the conversion of theobromine to 7-methylxanthine as well as the demethylation of caffeine into paraxanthine.

NCBI BLAST will be used to compare protein sequences, as shown below. Protein alignment allows us to identify percent identity as well as E-values to better quantify the relationship between our mutated NdmB construct and the wild-type construct.

Through our adaptive and directed evolution cycle, we have the ability to obtain a mutated NdmB construct that is more efficient in its enzymatic abilities to produce more theobromine and paraxanthine, which further enhances the production of 7-methylxanthine while maintaining a high percent identity.

UT Austin’s part BBa_K734000 has the original operon of NdmABCD. With Cornell’s focus on improving the enzymatic efficiency of NdmB in that operon, the overall part can be improved. Through the epPCR and ADE cycle designed by Team Cornell, a new NdmB part, named αNdmB-6x his, can be created with the mutation that shows the best increase in catalytic efficiency. After sequencing with our sponsor Plasmidsaurus and comparing sequence differences with NCBI Blast, αNdmB-6x his can be identified and later documented with the updated Kcat/ Km value.

While time ran out before wiki freeze, continued work on ENERGEM can allow for an improvement that will not only let Team Cornell to create a more efficient, single strand method for developing useful methylxanthines for the pharmaceutical industry, but allow for the discovery of a mutation that overall helped the caffeine demethylation pathway for future iGEM teams to use in any future project that requires breaking down coffee or caffeine into other chemicals. The new basic parts created by Team Cornell will allow future teams to work with the enzymes separately rather than the full operon and gain increased flexibility and control within their projects involving the caffeine demethylation pathway. As ENERGEM continues to be developed, αNdmB-6x his will allow for a general improved NdmABCD operon and increase in 7-MX production.