Part:BBa_K5034220
Pi <-> Poly P
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 PYYDT plasmid with the BBa-B0031 RBS, which is a weaker one 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 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.
Figure 1: Basic function of PPK1 Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 11
Illegal PstI site found at 3787 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 11
Illegal PstI site found at 3787
Illegal NotI site found at 2828 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 11
Illegal BglII site found at 3574
Illegal XhoI site found at 4985 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 11
Illegal PstI site found at 3787 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 11
Illegal PstI site found at 3787
Illegal NgoMIV site found at 556
Illegal NgoMIV site found at 4238
Illegal NgoMIV site found at 4521
Illegal AgeI site found at 396 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 3150
Illegal SapI.rc site found at 4087
Illegal SapI.rc site found at 4297
Construct features
Promoter: Constitutive promoter for continuous expression. We use tac promoter in our experiment. RBS: Ribosome binding site for efficient translation. BBa-B0031 here. PPK1 Coding Sequence: Encodes the polyphosphate kinase 1 enzyme. Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use T7Te terminator in our experiment.
Figure 2: Basic construction of PPK1 with B0031-RBS plasmid
Figure 3: Construction of PPK1 with B0031 RBS plasmid
Figure 4: Colony PCR indicating plasmid replication in Shewanella
Figure 5: Agarose gel electrophoresis indicating the target gene was successfully introduced into Shewanella
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. The PYYDT plasmid backbone is a standard vector used for gene expression in synthetic biology applications.
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. Conducting molybdate assays to determine Pi concentration and half-cell reaction(electrochemistry) to measure the electricity production ability, we found SPK1(with RBS BBa-B0031) has the worst capacity to polymerize phosphorus but a greatest electroproduction capability.
Figure 7: statistical data on electricity production capacity of Shewanella with the introduction of PPK1 with different RBS
Figure 8: statistical data on 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.
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