Part:BBa_K5034210

PolyP ---> Pi

Basic Description

This basic part encodes the PPX gene which is initially from E. coli and we performed codon optimization on. 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 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.

In a sentence, it can reversibly convert PolyP to Pi thoroughly. 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 PPX

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 features

In basic parts, only coding sequence is included.

Plasmid backbone: pBBR1MCS-terminator. The Lac promoter and the double terminator are on the plasmid backbone.

Promoter: We use Lac promoter in our experiment. Since there is no LacI protein on plasmid backbone, the gene expression is constitutive. Since the plasmid backbone does not encode the regulatory gene lacI for the repressor protein, the Lac promoter can be used as a constitutive promoter. This allows the subsequent genes to be constantly expressed.

RBS: Strong ribosome binding site for efficient translation. We use BBa-B0034 which shows the relatively strongest translation in our experiment.

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.

Origin (Organism)

The PPX gene was sourced from S. cerevisiae.

Experimental Characterization and results

The results of our amplification of this enzyme are shown in the figure.(Fig.3)

Figure 2: PCR of target genes before plasmids construction (The extra small fragment in the picture is primer dimer)

In our team’s previous research, we found that the introduction of PolyP synthase in Shewanella decrease the current significantly. So we planned to improve the situation by introducing different polyphosphate hydrolases which influence the phosphate metabolism of S. oneidensis, and this part(PPX) was one of the PolyP hydrolases.

However, there is no significant improvement on electricity producing capacity after the introduction of PPX gene.(Fig.3) In addition, the phosphorus accumulation capacity also decreased compared with WT.(Fig.4)

Figure 3: Statistical data on electricity production capacity of S. oneidensis with the introduction of different hydrolases

Figure 4: Statistical data on the phosphorus accumulation capacity of S. oneidensis with PPX

We postulated that PolyP hydrolases could promote the conversion of PolyP to phosphorus-containing small molecules, such as ATP or NADPH, which could have an impact on electricity generation and phosphorus accumulation. So, the concentration of ATP in the bacteria was measured. The results showed that PPX had no significant impact on ATP concentration of engineered bacteria.(Fig.5)

Figure 5: ATP level in S. oneidensis with the introduction of different hydrolases

In the end, PPK2(BBa_K5034205) and NADK(BBa_K5034206) was selected to further improve electricity producing capacity and phosphorus accumulation capacity.

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.

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.