Part:BBa_K5034217
PolyP <->Pi, Poly P -> NADP
Contents
Basic Description
This composite part includes the PPK2 gene from Pseudomonas aeruginosa and the NADK gene from Mycobacterium tuberculosis H37Rv, both we performed codon optimization on and are expressed in the PYYDT plasmid together. The PPK2 enzyme facilitates the reversible conversion between inorganic polyphosphate (PolyP) and inorganic phosphate (Pi), while the NADK enzyme converts PolyP to NADP. Importing them separately was successful, thus we intend to proceed with continued optimisation by their combination. This part consists of two enzymes, one is a reversible enzyme that converts Pi and Polyp, and the other is an enzyme that converts Polyp to NADK. The tandem connection of the two enzymes actually promoted the synthesis of NADK, and by maintaining some polyp reserves, it was able to improve the efficiency of electrical production and improve the phosphorus accumulation capacity of Shewanella.
Construct features(only coding sequence included in)
Promoter: Constitutive promoter for continuous expression. We use tac promoter in our experiment. RBS: Strong ribosome binding site for efficient translation. We use BBa-B0034 which shows the strongest translation in our experiment. PPK2 Coding Sequence: Encodes the polyphosphate kinase 2 enzyme. NADK Coding Sequence: Encodes the NAD kinase enzyme. Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use T7Te terminator in our experiment.
Origin (Organism)
PPK2 Gene: Pseudomonas aeruginosa PAO1 strain. NADK Gene: Mycobacterium tuberculosis H37Rv strain.
Experimental Characterization and results
Students from dry lab group using mathematical modelling to introduce the two enzymes and found an enhancement in the polyphosphate and electroproduction capabilities of Shewanella. Using half-cell reaction(electrochemistry) to measure the electricity production ability and conducting molybdate assays to determine Pi concentration, we also found a notable enhancement in the polyphosphate, ATP and electroproduction capabilities. The results of the half-cell experiments indicated an elevated electron transfer activity, with currents of 137.4 ± 16.34 µA/cm² for the SPPK2 strain, 134.56 ± 17.01 µA/cm² for the SNADK strain, and 164.2 ± 17.64 µA/cm² for the SPPK2-NADK strain.
Figure 1: statistical data on phosphorus accumulation capacity of Shewanella with the introduction of PPK2-NADK
Figure 2: statistical data on electricity production capacity of Shewanella with the introduction of PPK2-NADK
Figure 3: statistical data on ATP level of Shewanella with the introduction of PPK2-NADK
Potential Applications
In bioelectrochemical Systems, utilizing PolyP and NADP in microbial fuel cells for further improved electron transfer and energy production.
References
1.Mori S, Yamasaki M, Maruyama Y, Momma K, Kawai S, Hashimoto W, Mikami B, Murata K. Crystallographic studies of Mycobacterium tuberculosis polyphosphate/ATP-NAD kinase complexed with NAD. J Biosci Bioeng. 2004;98(5):391-3. 2. Zhang, H., Ishige, K., & Kornberg, A. (2002). A polyphosphate kinase (PPK2) widely conserved in bacteria. Proceedings of the National Academy of Sciences, 99(26), 16678-16683. 3. Neville N, Roberge N, Jia Z. Polyphosphate Kinase 2 (PPK2) Enzymes: Structure, Function, and Roles in Bacterial Physiology and Virulence. Int J Mol Sci. 2022 Jan 8;23(2):670. Sequence and Features
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