Difference between revisions of "Part:BBa K5034205"

Revision as of 10:07, 29 September 2024

PolyP <->Pi

Basic Description

This basic part encodes the PPK2 gene which is initially from Pseudomonas aeruginosa and we performed codon optimization on, is expressed in the PYYDT plasmid. The PPK2 enzyme facilitates the reversible conversion between inorganic polyphosphate (PolyP) and inorganic phosphate (Pi), playing a crucial role in phosphate metabolism. It distinguished from PPK1 by the following: synthesis of poly P from GTP or ATP, a preference for Mn2+ over Mg2+, and a stimulation by Poly P. The forward reaction, a poly P-driven nucleoside diphosphate kinase synthesis of GTP from GDP, is 75-fold greater than the reverse reaction, Poly P synthesis from GTP.

In a sentence,It can reversibly convert Poly p and Pi. For the first time, we expressed this element in a strain of Shewanella and conducted codon optimization based on Shewanella.

Figure 1: Basic function of PPK2

Sequence and Features

Chassis and Genetic Context

Successfully expressed in Escherichia coli DH5α and BL21(DE3) strains.

Construct features(only coding sequence included in basic part)

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 relatively strongest translation in our experiment. PPK2 Coding Sequence: Encodes the polyphosphate kinase 2 enzyme. Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use T7Te terminator in our experiment.

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Figure 2: PCR of target genes before plasmids construction (The extra small fragment in the picture is primer dimer)

Origin (Organism)

Gene Source: Pseudomonas aeruginosa PAO1 strain.

Experimental Characterization and results

In our team’s previous research we found that the behavior of the modified Shewanella did not reach our expectation and the electron microscopic observation also showed an abnormal morphology of the bacterium, we postulated that too much PPK1 may lead to an abnormal charge distribution in the bacterium thus result in a decrease in the bacterium's activity and a reduction in its capacity for electricity production, so we planed to improve the situation by introducing different polyphosphate hydrolases which influence the phosphorus metabolism of Shewanella. Electricity production: Using half-cell reaction(electrochemistry) to measure the electricity production ability. Capacity to polymerize phosphorus: Conducting molybdate assays to determine Pi concentration. The expression of hydrolase PPK2 showed relatively high phosphorus accumulation and electricity generation ability. Also, the ATP level is considerably enhanced.

Figure 3: statistical data on electricity production capacity of Shewanella with the introduction of different hydrolases

Figure 4: statistical data on the phosphorus accumulation capacity of Shewanella with PPK2

Figure 5: ATP level in Shewanella with the introduction of different hydrolases

Potential 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.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. 2.Neville, N., Roberge, N., & Jia, Z. (2022). Polyphosphate Kinase 2 (PPK2) enzymes: Structure, function, and roles in bacterial physiology and virulence. International Journal of Molecular Sciences, 23(2), 670.  3.Itoh, H., & Shiba, T. (2004). Polyphosphate synthetic activity of polyphosphate:AMP phosphotransferase in Acinetobacter johnsonii 210A. Journal of Bacteriology, 186(15), 5178-5181.