Difference between revisions of "Part:BBa K5034229"

Revision as of 06:16, 29 September 2024

Poly P -> Pi Basic Description: This basic part encodes the PPX gene which is initially from Escherichia coli and we performed codon optimization on, is expressed in the PYYDT plasmid. This basic part is designed to facilitate the complete conversion of inorganic polyphosphate (PolyP) to inorganic phosphate (Pi). The PPX enzyme, also known as exopolyphosphatase, is crucial for degrading PolyP into Pi, which is essential for various cellular processes. Inactivation of PPX1 had no effect on the Poly P level in nuclei in the stationary phase, Poly P level in the nuclei increased 1.5- and 2-fold in the exponential phase in the parent strain and PPX1 mutant, respectively.

Figure 1: Basic function of PPX

Construct features : Promoter: Constitutive promoter for continuous expression. We use tac promoter in our experiment. PPX Coding Sequence: Encodes the exopolyphosphatase 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 PPX plasmid

Figure 4: Construction of PPX plasmid

Figure 5: Bacterial PCR indicating that different plasmids can replicate in Shewanella

Origin (Organism): The PPX gene was sourced from Yeast. 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 PPX 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 PPX

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