Part:BBa_K5034213

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. 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, 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 Shewanella 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.

RBS: Ribosome binding site for efficient translation. In our experiments, we used BBa-B0031, BBa-B0032 and BBa-B0034, with BBa-B0031 having the lowest strength and BBa-B0034 having the highest strength.

PPK1 Coding Sequence: Encodes the polyphosphate kinase 1 enzyme.

Terminator: Efficient transcription terminator to ensure proper mRNA processing. We used rrnB T1 terminator and T7Te terminator in our experiments.

In basic parts, only coding sequence is included in our sequence. In this part, it is PPK1 Coding Sequence.

Figure 2: Basic construction of PPK1 plasmid

Origin (Organism)

The PPK1 gene was sourced from Citrobacter freundii. The pBBR1MCS-terminator plasmid backbone is a standard vector used for gene expression in synthetic biology applications.

Experimental Characterization and results

Trying to enhance S. oneidensis.’s capacity to produce electricity and polymerize phosphorus, we first introduced PPK1 into it. As the strength of the RBS decreases, the electrochemical activity of S. oneidensis. significantly increases, as detected by the Pi content detection method, while its polyphosphate capacity decreases, as observed through half-cell experiment.

Figure 3: Phosphorus accumulation capacity in S. oneidensis. after the introduction of PPK1

Figure 4: Electricity production capacity of S. oneidensis. after the introduction of PPK1

We then analyzed factors contributing to the reduced electricity generation capability of SPK1 Reflection electron microscopy 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 5: Scanning electron microscope of the S. oneidensis. morphology of WT and modified S. oneidensis. strains with BBa-B0034 RBS

Figure 6: 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.