Part:BBa_K5034227

Pi <-> PolyP

Basic Description

This plasmid is the expression vector of PPK2 gene(BBa_K5034205).

This basic part encodes the PPK2 gene which is sourced from Pseudomonas aeruginosa and we performed codon optimization on, is expressed in the pBBR1MCS-terminator 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 PolyP-driven nucleoside diphosphate kinase synthesis of GTP from GDP, is 75-fold greater than the reverse reaction, PolyP synthesis from GTP.

Figure 1: Basic function of PPK2

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 4981
  Illegal suffix found in sequence at 1
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 4981
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 2834
  Illegal NotI site found at 4987
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 4981
  Illegal BglII site found at 3580
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 4981
  Illegal suffix found in sequence at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 4981
  Illegal XbaI site found at 4996
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NgoMIV site found at 562
  Illegal NgoMIV site found at 4244
  Illegal NgoMIV site found at 4527
  Illegal AgeI site found at 402
  
• 1000
  COMPATIBLE WITH RFC[1000]

Construct features

• 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.

• Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use a double terminator rrnBT1-T7TE(BBa_B0015) in our experiment.

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

Figure 3: Basic construction of PPK2 plasmid

Figure 4: Construction of PPK2 plasmid

Figure 5: Bacterial PCR indicating that different plasmids can replicate in S. oneidensis

Origin (Organism)

PPK2 Gene Source: Pseudomonas aeruginosa PAO1 strain.

Plasmid Backbone: pBBR1MCS plasmid, a standard vector used for gene expression in synthetic biology. The plasmid backbone(BBa_K5034201) of this part is a modified version of pBBR1MCS, with a double terminator(BBa_B0015) on it.

Chassis and Genetic Context

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

Experimental Characterization and results

In our team’s previous research we found that the behavior of the modified S. oneidensis 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 S. oneidensis.

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.

Details of all experiments can be found at the Experiments section on the Wiki.

Figure 6: Statistical data on electricity production capacity of S. oneidensis with the introduction of different hydrolases

Figure 7: Statistical data on the phosphorus accumulation capacity of S. oneidensis with PPK2

Figure 8: ATP level in S. oneidensis 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.