Part:BBa_K4580004
NdmB-6x his-GFP
This is a composite part of GFP and Team Cornell's BBa_K4580001 BioBrick which encodes for NdmB-6x his. The conjugation of GFP and NdmB-6x his allows for confirmation that the NdmB enzyme is being expressed qualitatively via GFP readout before more conclusive experiments such as western blots.
Allows for Escherichia coli cells to degrade caffeine into paraxanthine and theobromine into 7-Methxylxanthine (7-MX) while allowing for a direct correlation between NdmB expression and GFP fluorescence.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 580
Illegal NheI site found at 775 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1730
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].
NdmB-6x his-GFP specifically is the second N-demethylase within the pathway conjugated to a fluorescence protein. It's enzymatic purpose is to produce paraxanthine ( 1,7-dimethylxanthine) converted from caffeine (1,3,7-trimethylxanthine) and 7-MX from theobromine (3,7-dimethylxanthine). To complete this, a co-enzyme found from NdmD passes a H+ proton from NAD(P)H to help drive the reaction.
After designing BBa_K4580004, a gel confirmation was done along with BBa_K4580000 to check for noticeable size difference which was successful.
Functionality
Catalytic Efficiency
| Substrate | Product | Km (uM) | kcat (min^-1) | kcat/Km (min^-1 / uM) |
|---|---|---|---|---|
| 1. Caffeine | Theobromine | 37 | 190 | 5.1 |
| 2. Paraxanthine | 7-methylxanthine | 53 | 130 | 2.5 |
After conjugating NdmB and GFP together, a concern of loss of function of the proteins came up. To qualitatively test for this, BBa_K4580004 was placed within Team Cornell's full plasmid for the 2023 project ENERGEM and observed a bright green fluorescence from GFP under UV light.
Theoretically, with conjugated GFP having a working and bright readout, conjugated NdmB would have the same conclusion and therefore keep the similar functionality to NdmB-6x his (BBa_K4580002). To ensure the flourscence was coming from the full plasmid pENGM42 (and in turn BBa_K4580004), the liquid cultures were miniprepped and gel electrophoresis was performed again as shown below. As GFP is only around 700 bp and there was no band around the 500 or 1000 bp mark, it was confirmed the source of light came from NdmB-6x his-GFP that was expressed from Team Cornell's pENGM42.
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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