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

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

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

In our team’s previous research we found that the behavior of the modified S. oneidensis did not reach our expectation and the electron microscopic observation also showed an abnormal morphology of the bacterium, we postulated that too much PPK1 may lead to an abnormal charge distribution in the bacterium thus result in a decrease in the bacterium's activity and a reduction in its capacity for electricity production, so we planed to improve the situation by introducing different polyphosphate hydrolases which influence the phosphorus metabolism of S. oneidensis.

• Electricity production: Using half-cell reaction(electrochemistry) to measure the electricity production ability.
• Capacity to polymerize phosphorus: Conducting molybdate assays to determine Pi concentration.
• Conducting molybdate assays to determine Pi concentration and found PPN1 a bad capacity to polymerize phosphorus.

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

Figure 4: Statistical data on electricity production capacity of S. oneidensis with the introduction of different hydrolases

Figure 5: Statistical data on the phosphorus accumulation capacity of S. oneidensis with PPN1

Figure 6: ATP level in S. oneidensis with the introduction of different hydrolases

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.