Part:BBa_K5034222

Pi <-> Poly P

Basic Description

This basic part encodes the PPK1 gene which is initially from Citrobacter freundii and we performed codon optimization on, is expressed in the PYYDT plasmid. This basic 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

Construct features

Promoter: Constitutive promoter for continuous expression. We use tac promoter in our experiment. 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 B0034-RBS plasmid

Figure 3: Construction of PPK1 with B0034 RBS plasmid

Figure 4: Colony PCR indicating plasmid replication in Shewanell

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

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 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. Using molybdate assays to determine Pi concentration and half-cell reaction(electrochemistry) to measure the electricity production ability, we found SPK3(with RBS BBa-B0034) has the greatest capacity to polymerize phosphorus but a worst 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. Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 11
  Illegal PstI site found at 3787
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 11
  Illegal PstI site found at 3787
  Illegal NotI site found at 2828
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 11
  Illegal BglII site found at 3574
  Illegal XhoI site found at 4985
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 11
  Illegal PstI site found at 3787
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 11
  Illegal PstI site found at 3787
  Illegal NgoMIV site found at 556
  Illegal NgoMIV site found at 4238
  Illegal NgoMIV site found at 4521
  Illegal AgeI site found at 396
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 3150
  Illegal SapI.rc site found at 4087
  Illegal SapI.rc site found at 4297