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 Shewanella and conducted codon optimization based on Shewanella.

Figure 1: Basic function of PPK1

Construct features

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]

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

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

We transform the plasmids into wild-type Shewanella, express it, and perform colony PCR. The results show that PPK1 is successfully introduced into Shewanella for replication.

Figure 3: Colony PCR indicating plasmid replication in Shewanell

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

Figure 4: Agarose gel electrophoresis indicating we got the target gene with the corresponding RBS

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

Figure 5: 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 Shewanella thus developing the best ability to produce electricity and polymerize phosphorus. We conduct Pi content detection to determine Pi concentration and half-cell experiment to measure the electricity production ability, we found SPK3 with RBS BBa-B0034 has the greatest capacity to polymerize phosphorus but a worst electroproduction capability.

Figure 6: Electricity production capacity of Shewanella with the introduction of PPK1 with different RBS

Figure 7: Phosphorus accumulation capacity of Shewanella with the introduction of PPK1 with different RBS

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.