Difference between revisions of "Part:BBa K5034231"

Revision as of 08:57, 1 October 2024

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.

Figure 1: Basic function of PPN1

Construct features

Promoter: Constitutive promoter for continuous expression. We use tac promoter in our experiment. PPN1 Coding Sequence: Encodes the polyphosphatase enzyme. 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 Shewanella

Origin (Organism)

The PPN1 gene was sourced from Saccharomyces cerevisiae. The PYYDT plasmid 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 Shewanella 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 Shewanella. 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.

Figure 6: statistical data on electricity production capacity of Shewanella with the introduction of different hydrolases

Figure 7: statistical data on the phosphorus accumulation capacity of Shewanella with PPN1

Figure 8: ATP level in Shewanella with the introduction of different hydrolases

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.

Sequence and Features