Part:BBa_K5034221
Pi <-> Poly P
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 PYYDT plasmid with the BBa-B0032 RBS, which is a medium 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
Construct features
Promoter: Constitutive promoter for continuous expression. We use tac promoter in our experiment. RBS: Ribosome binding site for efficient translation. BBa-B0032 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 B0032-RBS plasmid
Figure 3: Construction of PPK1 with B0032 RBS plasmid
Figure 4: Colony PCR indicating plasmid replication in Shewanell
Figure 5: Agarose gel electrophoresis indicating the target gene was successfully introduced into Shewanella
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. 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 SPK2(with RBS BBa-B0032) has the medium capacity to polymerize phosphorus and a medium 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.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 4981
Illegal suffix found in sequence at 1 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 4981
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NotI site found at 9
Illegal NotI site found at 2834
Illegal NotI site found at 4987 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 4981
Illegal BglII site found at 3580 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 4981
Illegal suffix found in sequence at 2 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 4981
Illegal XbaI site found at 4996
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NgoMIV site found at 562
Illegal NgoMIV site found at 4244
Illegal NgoMIV site found at 4527
Illegal AgeI site found at 402 - 1000COMPATIBLE WITH RFC[1000]
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