Part:BBa_K5034216

PolyP <->Pi

Basic Description

This basic part encodes the PPK1 gene which is initially from Citrobacter freundii and we performed codon optimization on, is expressed in the pBBR1MCS-terminator plasmid with the BBa-B0034 RBS, which is a stronger RBS compared to others. This basic part is designed to facilitate the reversible conversion between inorganic polyphosphate (PolyP) and inorganic phosphate (Pi). The PPK1 enzyme is known for its ability to synthesize PolyP from ATP and Pi and to degrade PolyP back to Pi, with a preference for the synthetic reaction, making it a versatile tool for managing phosphate metabolism in engineered systems.

In a sentence, this part is activated by an efficient RBS. It can reversibly convert PolyP and Pi. This reversible process favors the generation of PolyP. For the first time, we expressed this element in a strain of S. oneidensis. and conducted codon optimization based on S. oneidensis..

Figure 1: Basic function of PPK1

Construct 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]

• Promoter: Constitutive promoter for continuous expression. We use Lac promoter in our experiment. Since our sequence does not encode the regulatory gene lacI for the repressor protein, the promoter we introduced is a constitutive promoter. This allows the subsequent genes to be continuously expressed.

• RBS: Ribosome binding site for efficient translation. We use BBa-B0034 here.

• PPK1 Coding Sequence: Encodes the polyphosphate kinase 1 enzyme.

• Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use rrnB T1 terminator and T7Te terminator in our experiment.

The basic structure of the part is shown as follows:

Figure 2: Basic construction of PPK1 plasmid with BBa-B0034 RBS

Figure 3: Construction of PPK1 with BBa-B0034 RBS plasmid

We transformed the plasmids into wild-type S. oneidensis., expressed it, and performed colony PCR. The results showed that PPK1 was successfully introduced into Shewanella for replication.

Figure 4: Colony PCR indicating plasmid replication in S. oneidensis.

DNA agarose gel electrophoresis results showed that we obtained the plasmid with BBa-B0034 RBS, which is approximately 2.1 kb in size.

Figure 5: Agarose gel electrophoresis indicating we got the target gene with the corresponding RBS

We performed protein extraction for SDS-PAGE. SDS-PAGE results showed that protein expression of the plasmid with BBa-B0034 RBS is the maximum, corresponding to the strength of RBS.

Figure 6: SDS-PAGE results showing that the BBa-B0034 one’s protein expression is the maximum, corresponding to the strength of RBS

Origin (Organism)

The PPK1 gene was sourced from Citrobacter freundii.

Experimental Characterization and results

Alteration of protein expression intensity can regulate the metabolic networks, so we focused on RBS with varying translation strengths to facilitate the regulation of PPK1 concentration in S. oneidensis. to develop the best ability to produce electricity and polymerize phosphorus.

We conducted Pi content detection to determine Pi concentration and half-cell experiment to measure the electricity production ability, we found SPK1 with RBS BBa-B0034 has the greatest capacity to polymerize phosphorus but a worst electroproduction capability.

Figure 7: Electricity production capacity of S. oneidensis. with the introduction of PPK1 with different RBS

Figure 8: Phosphorus accumulation capacity of S. oneidensis. with the introduction of PPK1 with different RBS

We then analyzed factors contributing to the reduced electricity generation capability of SPK1.

Scanning electron microscope and transmission electron microscope results showed that the strains with high phosphorus content had abnormal morphology, indicating that allowing bacteria to be in a high phosphorus content state in advance is not conducive to the rapid transfer of electrons and is not conducive to the ability to produce electricity.

Figure 9: Scanning electron microscope of the S. oneidensis. morphology of WT and modified S. oneidensis. strains with BBa-B0034 RBS(strong RBS)

Figure 10: Transmission electron microscopy of the S. oneidensis. morphology of WT and modified S. oneidensis. strains with BBa-B0034 RBS

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

Chassis and genetic

Chassis：Shewanella onediensis MR-1

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

Potential applications

The PPK1 gene (polyphosphate kinase 1) has potential applications in:

Industrial Microbial Engineering: Enhances the production of biofuels, amino acids, or antibiotics by boosting polyphosphate synthesis in microorganisms.

Environmental Bioremediation: Assists in the accumulation of heavy metals or radioactive substances for pollution control.

References

1.Itoh, H., & Shiba, T. (2004). Polyphosphate synthetic activity of polyphosphate:AMP phosphotransferase in Acinetobacter johnsonii 210A. Journal of Bacteriology, 186(15), 5178-5181.