Part:BBa_K5034222

Pi <-> Poly P

Basic Description

This composite part includes the PPK1 gene which is initially from Citrobacter freundii and we performed codon optimization on, is expressed in the pBBR1MCS-terminator plasmid with the BBa-B0032 RBS, which is a medium RBS compared to others. This composite part is designed to facilitate the reversible conversion between inorganic polyphosphate (PolyP) and inorganic phosphate (Pi). The PPK1 enzyme is known for its ability to synthesize PolyP from ATP and Pi and to degrade PolyP back to Pi, with a preference for the synthetic reaction, making it a versatile tool for managing phosphate metabolism in engineered systems.

Figure 1: Basic function of PPK1

Construct 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]

• Plasmid Backbone: pBBR1MCS-terminator plasmid

• Promoter: Constitutive promoter for continuous expression. We use Lac promoter in our experiment.

• RBS: Ribosome binding site for efficient translation. We use BBa-B0034 here.

• PPK1 Coding Sequence: Encodes the polyphosphate kinase 1 enzyme.

• Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use rrnB T1 terminator and T7Te terminator in our experiment.

Figure 2: Basic construction of PPK1 with BBa-B0034 RBS plasmid

Figure 3: Construction of PPK1 with BBa-B0034 RBS plasmid

We transformed the plasmids into wild-type S. oneidensis., expressed it, and performed colony PCR. The results showed that PPK1 was successfully introduced into S. oneidensis. for replication.

Figure 4: Colony PCR indicating plasmid replication in S. oneidensis.

DNA agarose gel electrophoresis results showed that we obtained the plasmid with BBa-B0034 RBS, which is approximately 2.1 kb in size.

Figure 5: Agarose gel electrophoresis indicating we got the target gene with the corresponding RBS

We performed protein extraction for SDS-PAGE. SDS-PAGE results showed that protein expression of the plasmid with BBa-B0034 RBS is the maximum, corresponding to the strength of RBS.

Figure 6: SDS-PAGE results showing that the BBa-B0034 one’s protein expression is the maximum, corresponding to the strength of RBS

Origin (Organism)

The PPK1 gene was sourced from Citrobacter freundii. The pBBR1MCS-terminator plasmid backbone is a standard vector used for gene expression in synthetic biology applications.

Experimental Characterization and results

Alteration of protein expression intensity can regulate the metabolic networks, so we focused on RBS with varying translation strengths to facilitate the regulation of PPK1 concentration in S. oneidensis. to develop the best ability to produce electricity and polymerize phosphorus.

We conducted Pi content detection to determine Pi concentration and half-cell experiment to measure the electricity production ability, we found SPK1 with RBS BBa-B0034 has the greatest capacity to polymerize phosphorus but a worst electroproduction capability.

Figure 7: Electricity production capacity of S. oneidensis. after the introduction of PPK1 with different RBS

Figure 8: Phosphorus accumulation capacity of S. oneidensis. after the introduction of PPK1 with different RBS

We then analyzed factors contributing to the reduced electricity generation capability of SPK1.

Scanning electron microscope and transmission electron microscope results showed that the strains with high phosphorus content had abnormal morphology, indicating that allowing bacteria to be in a high phosphorus content state in advance is not conducive to the rapid transfer of electrons and is not conducive to the ability to produce electricity.

Figure 9: Scanning electron microscope of the S. oneidensis. morphology of WT and modified S. oneidensis. strains with BBa-B0034 RBS(strong RBS)

Figure 10: Transmission electron microscopy of the S. oneidensis. morphology of WT and modified S. oneidensis. strains with BBa-B0034 RBS

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

Chassis and genetic

Chassis：Shewanella onediensis MR-1

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

Potential applications

The PPK1 gene (polyphosphate kinase 1) has potential applications in:

Industrial Microbial Engineering: Enhances the production of biofuels, amino acids, or antibiotics by boosting polyphosphate synthesis in microorganisms.

Environmental Bioremediation: Assists in the accumulation of heavy metals or radioactive substances for pollution control.

References

Wang X , Wang X , Hui K , et al. Highly Effective Polyphosphate Synthesis, Phosphate Removal and Concentration Using Engineered Environmental Bacteria Based on a Simple Solo Medium-copy Plasmid Strategy[J]. Environmental Science & Technology, 2017:acs.est.7b04532.