Part:BBa_K5034231

Poly P -> Poly P(smaller) or Pi

Basic Description

This basic part encodes the PPN1 gene which is initially from Saccharomyces cerevisiae and we performed codon optimization on, is expressed in the pBBR1MCS-terminatorplasmid. This basic part is designed to facilitate the conversion of long-chain inorganic polyphosphate (PolyP) into shorter fragments without completely degrading it to inorganic phosphate (Pi). The PPN1 enzyme exhibits both exopolyphosphatase and endopolyphosphatase activities, depending on the presence of specific metal ions. Inactivation of the PPN1 gene encoding another protein, which exhibited exopolyPase activity in the yeast (CRN and CNX strains), resulted in almost total elimination of the nuclear exopolyPase activities in both growth phases.

Figure 1: Basic function of PPN1

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.
• PPN1 Coding Sequence: Encodes the polyphosphatase enzyme.
• RBS: BBa_B0034
• Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use T7Te terminator in our experiment.

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

Figure 3: Basic construction of PPN1 plasmid

Figure 4: Construction of PPN1 plasmid

Origin (Organism)

The PPN1 gene was sourced from Saccharomyces cerevisiae. The pBBR1MCS-terminatorplasmid backbone is a standard vector used for gene expression in synthetic biology applications.

Experimental Characterization and results

We first performed Colony PCR on the target gene in S. oneidensis, and the length of PPN1 was 2025 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 found that the power production efficiency of S. oneidensis did not improve significantly after SPPN1 introduction, and even decreased a bit, so we did not choose it for our subsequent power production optimisation experiments.

Afterwards, we also measured the phosphorus aggregation capacity(fig7), and frustratingly, the programme still did not perform well.

Figure 7: Statistical data on the phosphorus accumulation capacity of S. oneidensis with PPN1

Subsequent experiments(fig8) also showed that the PPN1 did not result in an increase in the metabolic strength of S. oneidensis either, suggesting that the enzyme did not have any of the effects that we wanted it to have.

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

Overall, the PPN1 enzyme did not perform well in our scenario and did not have the ability to enhance the electroproduction and phosphorus-polymerisation capacity of S. oneidensis.

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

Chassis and genetic

Chassis：Shewanella oneidensis MR-1.

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

Potential application

This plasmid contains PPN1 enzyme, as an important enzyme class for hydrolysis of polyP, which can be used as a key plasmid for regulating phosphorus metabolism in other microorganisms.

References

1.Lichko, L. P., Kulakovskaya, T. V., & Kulaev, I. S. (2006). Inorganic polyphosphate and exopolyphosphatase in the nuclei of Saccharomyces cerevisiae: dependence on the growth phase and inactivation of the PPX1 and PPN1 genes. Biochemistry (Moscow), 71(11), 1171-1175.