Part:BBa_K4580004: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. Because the GFP is conjugated to NdmB-6x his, this design choice allows for a direct correlation between a qualitative GFP readout and NdmB expression as NdmB was Team Cornell's protein of interest for 2023.

Source

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

GFP is sourced from the jellyfish Aequorea victor 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