Part:BBa_K5034209

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 PYYDT plasmid. 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.In a sentence, this enzyme can convert Poly P to Poly P with smaller fragments, but not to Pi completely. For the first time, we expressed this element in a strain of S. oneidensis and conducted codon optimization based on S. oneidensis. We tested the effects of the introduction of this element on electricity production and phosphorus metabolism.

Figure 1: Basic function of PPN1

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Construct feature

Figure 2: Basic construction of PPN1 plasmid

• Promoter: Constitutive promoter for continuous expression. We use tac promoter in our experiment.
• RBS: BBa_B0034
• PPN1 Coding Sequence: Encodes the polyphosphatase enzyme.
• Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use T7Te terminator in our experiment.

Figure 3: PCR of target genes PCR before plasmids construction (The extra small fragment in the picture is primer dimer)

The length of the PPN1 gene is about 2025 base pairs, which is consistent with the results on the DNA electropherogram, proving that we have obtained the PPN1 gene.

Origin (Organism)

The PPN1 gene was sourced from Saccharomyces cerevisiae.

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：S. oneidensis onediensis MR-1.

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

Potential applications

PPN1, as an enzyme that hydrolyses PolyP, is an important player in the regulation of intracellular PolyP metabolism.

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.