Part:BBa_K5034220

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 PYYDT plasmid with the BBa-B0031 RBS, which is a weaker one 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 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 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: Ribosome binding site for efficient translation. BBa-B0031 here. PPK1 Coding Sequence: Encodes the polyphosphate kinase 1 enzyme. Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use T7Te terminator in our experiment.

Figure 2: Basic construction of PPK1 with B0031-RBS plasmid

Figure 3: Construction of PPK1 with B0031 RBS plasmid

Figure 4: Colony PCR indicating plasmid replication in Shewanella

Figure 5: Agarose gel electrophoresis indicating the target gene was successfully introduced into Shewanella

Figure 6: SDS-PAGE results showing that the BBa-B0031 one’s protein expression is the minimum, corresponding to the strength of RBS.

Origin (Organism)

The PPK1 gene was sourced from Citrobacter freundii. The PYYDT 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 Shewanella thus developing the best ability to produce electricity and polymerize phosphorus. Conducting molybdate assays to determine Pi concentration and half-cell reaction(electrochemistry) to measure the electricity production ability, we found SPK1(with RBS BBa-B0031) has the worst capacity to polymerize phosphorus but a greatest electroproduction capability.

Figure 7: statistical data on electricity production capacity of Shewanella with the introduction of PPK1 with different RBS

Figure 8: statistical data on phosphorus accumulation capacity of Shewanella with the introduction of PPK1 with different RBS

References

1.Itoh, H., & Shiba, T. (2004). Polyphosphate synthetic activity of polyphosphate:AMP phosphotransferase in Acinetobacter johnsonii 210A. Journal of Bacteriology, 186(15), 5178-5181.