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

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.

Experimental Characterization and results

We first performed Colony PCR on the target gene in S. oneidensis(fig5), and the length of PPK2 was 1074 base pairs, which was consistent with the results obtained from the experiments.

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

Then, we determined the electroproduction capacity of S. oneidensis after introduction of the SPPN1 enzyme (e.g. fig6)

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

We were surprised to find that, similar to after the introduction of NADK, S. oneidensis had a qualitative leap in its ability to produce electricity

Similarly, we determined the ability of S. oneidensis to aggregate phosphorus after its introduction of PPK2(fig7). We also found that it has a superior ability to aggregate phosphorus compared to WT.

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

At the same time, we can also see from the data measuring ATP levels in S. oneidensis(fig8) that PPK2 increased the metabolic strength of S. oneidensis, which is consistent with the results brought about by the introduction of NADK into S. oneidensis.

Figure 8: ATP level in S. oneidensis with the introduction of different hydrolases

All of the above results suggest that PPK2 will be the key enzyme that will be beneficial in enhancing both of these abilities in S. oneidensis, and we have entered it into the next optimisation of the igem cycle.

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

Chassis and Genetic Context

Chassis：Shewanella oneidensis MR-1.

The gene can be expressed and function properly in S. oneidensis.

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.