Part:BBa_K5034217

PolyP <->Pi, Poly P -> NADP

This composite part includes the PPK2 gene from Pseudomonas aeruginosa and the NADK gene from Mycobacterium tuberculosis H37Rv. We performed codon optimization on both and expressed in the pBBR1MCS-terminator plasmid together. The PPK2 enzyme facilitates the reversible conversion between inorganic polyphosphate (PolyP) and inorganic phosphate (Pi), and the NADK enzyme converts PolyP to NADP. Importing them separately was successful, thus we intend to proceed with continued optimisation by their combination.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 S.oneidensis.

Figure 1: Basic structure of PPK2-NADK

Sequence and Features

Promoter: We use lac promoter in our experiment. There isn't lacI downstream，so it's constitutive promoter for continuous expression.

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 a double terminator rrnBT1-T7TE(BBa_B0015) in our experiment.

The translational unit is composed of the components above. In this composite part, promotor and terminator is not included.Because the backbone has promotor sequence and terminator sequence.

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Origin

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 simulate the introduction of the two enzymes and found an enhancement in the polyphosphate and electroproduction capabilities of S.oneidensis.(Fig.2)

Figure 2: Experimental modelling proves that importing PPK2 and NADK simultaneously is better than importing PPK2 or NADK separately

Then, the students in the wet lab constructed a component that linked two enzymes in series, and the results of colony PCR confirmed the success of our construction. （Fig.3-4）

Figure 3: Plasmid profile of PBBR1mcs-Terminator-PPK2-NADK

Since the PPK2 gene is approximately 1.1 kb and the NADK gene is about 1.0 kb, the PPK2-NADK construct should be approximately 2.1 kb. The colony PCR results show a band at about 2.1 kb, confirming that we successfully introduce the plasmid containing PPK2-NADK into S.oneidensis.(Fig4)

Figure 4: Colony PCR to prove that PPK2-NADK plasmid is introduced to S.oneidensis

After successful construction, we transferred it into S.oneidensis and conducted measurements of its electricity production and phosphorus accumulation effects. We found that after transferring into the S.oneidensis, both the electricity production and phosphorus accumulation efficiency were significantly improved compared to the wild type.(Fig.5)

Figure 5: Electricity production capacity and phosphorus accumulation capacity of S.oneidensis with the introduction of PPK2-NADK

The phosphorus accumulation effect was measured in M9 cultural medium, and the electricity generation effect was measured in LB medium, because M9 medium is the medium used in practical applications and can be better combined with practical applications.

Subsequently, we also investigated the reasons for the improvement in electricity generation and phosphorus accumulate efficiency. We found that the levels of ATP and NADH/NAD⁺ inside the cell were significantly increased(Fig.6), indicating that the metabolic level of S.oneidensis increased, leading to an increase in electricity production and phosphorus accumulation levels.

Figure 6: Levels of ATP and NADH/NAD⁺ of S.oneidensis with the introduction of PPK2-NADK

To explore the possible applications of this component, we conducted a full cell experiment using Shewanella bacteria.Firstly, we use cyclic voltammetry (CV) and linear sweep voltammetry (LSV) techniques for testing. The CV curve shows higher redox activity in the SPPK2-NADK strain.(Fig.7)

Figure 7: Cyclic voltammetry show higher redox activity in the SPPK2-NADK strain

The LSV curve indicates lower internal resistance in the MFC cells of the SPPK2-NADK strain.(Fig.8)

Figure 8: LSV curve indicates lower internal resistance in the MFC cells of the SPPK2-NADK strain

Next, we measure the relative output power. The power density results show that the SPPK2-NADK strain has a maximum output power of 243.77 ± 25.2 mW/m², which is 2.32 times higher than the WT strain’s output power density (105.06 ± 11.72 mW/m²) (Fig. 9).

Figure 9: Output power of the PPK2-NADK strain

Chassis and genetic context

We express this gene on Shawanella oneidensis MR-1

Potential Applications

In bioelectrochemical systems, we can utilize PolyP and NADP in microbial fuel cells for further improved electron transfer and energy production.

In fact, based on the results, we make a hardware to demostrate its application.We can use it to collect Pi in the soil and produce electricity to be used by human.(Fig.10)

Figure 10: Hardware about its application

There are a list of other probably applications:

Managing phosphate levels in contaminated environments;

Enhancing phosphate metabolism in engineered microbial systems;

Optimizing phosphate utilization in industrial microbial processes.

Enhancing the performance of bioelectrochemical systems for electricity generation in providing a renewable and sustainable source of electricity, reducing reliance on fossil fuels and contributing to cleaner 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.