Difference between revisions of "Part:BBa K5034229"

Revision as of 05:13, 1 October 2024

Poly P -> Pi

Basic Description

This plasmid is the expression vector of PPX gene(BBa_K5034210).

The basic part(BBa_K5034210) encodes the PPX gene which is sourced from E. coli and we performed codon optimization on. The basic part(BBa_K5034210) 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 PPX had no effect on the PolyP level in nuclei in the stationary phase, PolyP level in the nuclei increased 1.5- and 2-fold in the exponential phase in the parent strain and PPX mutant, respectively.

Figure 1: Basic function of PPX

Sequence and Features

Construct features

Promoter: Constitutive promoter for continuous expression. We use Lac promoter in our experiment.

RBS: Ribosome binding site for efficient translation. In this part, we use BBa_B0034 which has highest strength.

PPX Coding Sequence: Encodes the exopolyphosphatase enzyme.

Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use a double terminator rrnBT1-T7TE(BBa_B0015) in our experiment.

Figure 3: Basic construction of PPX plasmid

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

Figure 4: Construction of PPX plasmid

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

Origin (Organism)

The PPX gene was sourced from S. cerevisiae.

The pBBR1MCS plasmid backbone is a standard vector used for gene expression in synthetic biology applications. The plasmid backbone(BBa_K5034201) of this part is a modified version of pBBR1MCS, with a double terminator(BBa_B0015) on it.

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 resulting in a decrease in the bacterium's activity and a reduction in its capacity of electricity production, so we planned to improve the situation by introducing different polyphosphate hydrolases which influence the phosphorus metabolism of S. oneidensis, including PPX, PPK2, NADK, PAP, PPN1.

Electricity production: Using half-cell reaction(electrochemistry) to measure the electricity production ability.

Capacity to polymerize phosphorus: Conducting molybdate assays to determine Pi concentration. We conducted molybdate assays to determine Pi concentration and found that PPX has a bad capacity to polymerize phosphorus.

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

Figure 6: Statistical data on electricity production capacity of S. oneidensis with the introduction of different hydrolases

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

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

Chasis and genetic context

This part can be normally expressed and function properly in S. oneidensis.

Potential applications

PPX can hydrolyze inorganic polyphosphate (PolyP) to inorganic phosphate (Pi), which can be a crucial part in phosphate 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.