Part:BBa_K5034214

PolyP <->Pi

Basic Description

This composite part includes the PPK1 gene which is initially from Citrobacter freundii and we performed codon optimization on. It is expressed in the pBBR1MCS-terminator plasmid(BBa-K5034201) with the BBa-B0031 RBS, which is a weaker RBS compared compared to others. This composite 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 a weaker 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

Sequenece 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.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-B0031 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.

In this composite part, only coding sequence and RBS is included.But the translational unit is composed of components above.

The basic structure of the part is shown as follows:

Figure 2: Basic construction of PPK1 plasmid with BBa-B0031 RBS

Figure 3: Construction of PPK1 with BBa-B0031 RBS plasmid

DNA agarose gel electrophoresis results showed that we obtained the target gene with BBa-B0031 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 transformed the plasmids into wild-type S. oneidensis., expressed it, and performed colony PCR. The results showed that PPK1 was successfully introduced into S. oneidensis. for replication.

Figure 5: Colony PCR indicating plasmid replication in S. oneidensis.

We performed protein extraction for SDS-PAGE. The SDS-PAGE results showed that protein expression of the plasmid with BBa-B0031 RBS is the minimum, corresponding to the strength of RBS.

Figure 6: SDS-PAGE results showing that the BBa-B0031 one's protein expression is the minimum, 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 that SPK3 with RBS BBa-B0031 has the lowest capacity for phosphorus polymerization but the highest electroproduction capability compared to other RBS.

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

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

This is the end of our one cycle, and through this successful cycle, we have achieved an improvement in phosphorus accumulation ability while maintaining almost unchanged electricity production capacity of S.oneidensis.

Experimental manipulation：

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

Capacity to absorb phosphorus:Conducting molybdate assays to determine Pi concentration.

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

Chassis and genetic

Chassis：Shewanella onediensis MR-1

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

Potential applications

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

It can enhance the production of some phosphorus-related substances production by boosting polyphosphate synthesis in microorganisms and create greater economic value

It can manage phosphate levels in contaminated environments.

It can enhance the performance of bioelectrochemical systems for electricity generation in providing a renewable and sustainable source of electricity, reducing reliance on fossil fuels and contributing to cleaner energy production.

References

Wang X , Wang X , Hui K , et al. Highly Effective Polyphosphate Synthesis, Phosphate Removal and Concentration Using Engineered Environmental Bacteria Based on a Simple Solo Medium-copy Plasmid Strategy[J]. Environmental Science & Technology, 2017:acs.est.7b04532.