Difference between revisions of "Part:BBa K5034215"

 
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This part is initiated by a medium promoter.It can reversibly convert Poly p and Pi. This reversible process favors the generation of Poly P.For the first time, we expressed this element in a strain of Shewanella and conducted codon optimization based on Shewanella.
 
This part is initiated by a medium promoter.It can reversibly convert Poly p and Pi. This reversible process favors the generation of Poly P.For the first time, we expressed this element in a strain of Shewanella and conducted codon optimization based on Shewanella.
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<b>Basic Description:</b>
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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.
  
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Figure 1: Basic function of PPK1
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<b>Construct features :</b>
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Promoter: Constitutive promoter for continuous expression. We use tac promoter in our experiment.
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RBS: Ribosome binding site for efficient translation. BBa-B0032 here.
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PPK1 Coding Sequence: Encodes the polyphosphate kinase 1 enzyme.
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Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use T7Te terminator in our experiment.
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Figure 2: Colony PCR indicating plasmid replication in Shewanell
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Figure 3: Agarose gel electrophoresis indicating the target gene was successfully introduced into Shewanella
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Figure 4: SDS-PAGE results showing that the BBa-B0032 one’s protein expression is the medium, corresponding to the strength of RBS
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<b>Origin (Organism):</b>
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The PPK1 gene was sourced from Citrobacter freundii.
 +
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<b>Experimental Characterization and results:</b>
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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.
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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.
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Figure 5: statistical data on electricity production capacity of Shewanella with the introduction of PPK1 with different RBS
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Figure 6: statistical data on phosphorus accumulation capacity of Shewanella with the introduction of PPK1 with different RBS
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<b>References:</b>
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1.Itoh, H., & Shiba, T. (2004). Polyphosphate synthetic activity of polyphosphate:AMP phosphotransferase in Acinetobacter johnsonii 210A. Journal of Bacteriology, 186(15), 5178-5181.
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Revision as of 05:57, 29 September 2024


PolyP <->Pi

This part is initiated by a medium promoter.It can reversibly convert Poly p and Pi. This reversible process favors the generation of Poly P.For the first time, we expressed this element in a strain of Shewanella and conducted codon optimization based on Shewanella. 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: Colony PCR indicating plasmid replication in Shewanell





Figure 3: Agarose gel electrophoresis indicating the target gene was successfully introduced into Shewanella





Figure 4: 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 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 5: statistical data on electricity production capacity of Shewanella with the introduction of PPK1 with different RBS




Figure 6: 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


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]