Part:BBa_K4580001:Design

Single Nucleotide Polymorphism (SNP)

To maintain compatibility with the iGEM Parts Registry, a SNP was done at base 80 and maintains the same amino acid sequence.

Enzyme Isolation

To allow for easy enzyme isolation, a 6x-histidine tag was added to the NdmB. This design choice allowed Team Cornell to use Ni-NTA Affinity Chromatography to isolate our enzymes of interest for further encapsulation into our reactor.

When conjugated to GFP (such as in BBa_K4580004), the his tag prevented the potential for loss of function between NdmB and GFP rather than if the two proteins were fused together. This his-tag, in turn, acts as a linker.

Source

NdmA, NdmB, and, NdmD were amplified from the bacteria Pseudomonas putida CBB5 genomic DNA.

References

Summers, R. M., Louie, T. M., Yu, C. L., Gakhar, L., Louie, K. C., & Subramanian, M. (2012). Novel, highly specific N-demethylases enable bacteria to live on caffeine and related purine alkaloids. Journal of bacteriology, 194(8), 2041–2049. https://doi.org/10.1128/JB.06637-11

UT Austin. (2012). BBA_K734000. NdmABCD operon. https://parts.igem.org/Part:BBa_K734000