Part:BBa_K5034215
PolyP <->Pi
Contents
Basic Description
This composite part includes 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-B0032 RBS, which is a medium RBS 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 initiated by a medium promoter. 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
- Plasmid backbone: pBBR1MCS-terminator. The Lac promoter and the double terminator are on the plasmid backbone.<a href="https://parts.igem.org/Part:BBa_K5034201">BBa_K5034201</a>
- Promoter: Constitutive promoter for continuous expression. We used 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 used BBa-B0032 here.
- 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 experiment.
The basic structure of the part is shown as follows:
Figure 2: Basic construction of PPK1 plasmid with BBa-B0032 RBS
Figure 3: Construction of PPK1 with BBa-B0032 RBS plasmid
We transform the plasmids into wild-type S. oneidensis., express it, and perform colony PCR. The results show that PPK1 is successfully introduced into S. oneidensis. 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-B0032 RBS, which is approximately 2.1 kb in size.
Figure 5: Agarose gel electrophoresis indicating we got the target gene with corresponding RBS
We performed protein extraction for SDS-PAGE. SDS-PAGE results showed that protein expression of the plasmid with BBa-B0032 RBS is the medium, corresponding to the strength of RBS.
Figure 6: SDS-PAGE results showing that the BBa-B0032 one's protein expression is the medium, 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 SPK2 with RBS BBa-B0032 has the medium capacity to polymerize phosphorus and a medium electroproduction capability.
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
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
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.
| None |