Coding

Part:BBa_K4580002

Designed by: Michael Constant   Group: iGEM23_Cornell   (2023-10-09)


NdmD- 6x his

This gene encodes for the reductase in the caffeine demethylation pathway within the NdmABCD operon [1]. Attached to it is a 6x-histidine tag, this part is utilized for easy enzyme isolation via affinity chromatography and/ or specific mutation without the other genes within the original operon.

Serves as a co-enzyme to allows for Escherichia coli cells to efficiency utilize NdmA's and NdmB's enzymes within caffeine demethylation [2].


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 48
    Illegal BglII site found at 519
    Illegal XhoI site found at 1660
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 187
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

While caffeine is a simple molecule and relatively cheap to purchase, its derivatives hold a medical application that is not easily seen. Studies performed by Dr. Ryan Summers and their team have utilized a multi-strand method of biosynthetically creating 7-methxylanthine, one of caffeine's derivatives [2]. The usage for this compound comes from its medical application within ophthalmology. Clinical trials have revealed that 7-methxylanthine (or 7-MX for short) has a correlation on moderating severe forms of myopia (nearsightedness) on patients where surgeries, such as LASIK, cannot be performed. By producing this compound biologically, the traditional and expensive method of chemical synthesis can be evaded by utilizing the type III caffeine demethylation pathway shown below [3].

Figure 1: Type III Caffeine Demethylation Pathway

NdmD specifically is gene that encodes for a co-enzyme that serves as a reductase. It passes a H+ proton from NAD(P)H to help drive the reaction caused by NdmA and NdmB (BBa_K4580000 and BBa_K4580001 respectively).

After designing BBa_K4580002, a gel confirmation was done to check for the proper size of the part.

Figure 2: Gel Confirmation of BBa_K4580002

References

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

[2] 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

[3] Caffeine Demethylation Pathway Type III. MetaCyc caffeine degradation III (bacteria, via Demethylation). (n.d.). http://vm-trypanocyc.toulouse.inra.fr/META/NEW-IMAGE?type=PATHWAY&object=PWY-6538&detail-level=4

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