Difference between revisions of "Part:BBa K5034213"
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===Basic Description===
 
===Basic Description===
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This basic part encodes the PPK1 gene which is initially from Citrobacter freundii and we performed codon optimization on, is expressed in the PYYDT plasmid. This basic 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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This basic part encodes the PPK1 gene which is initially from *Citrobacter freundii* and we performed codon optimization on, is expressed in the pBBR1MCS-terminator plasmid. This basic 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.
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In a sentence, 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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In a sentence, it can reversibly convert PolyP 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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Figure 1: Basic function of PPK1
 
Figure 1: Basic function of PPK1
  
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===Construct features(only coding sequence included in basic part)===
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===Construct features===
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Promoter: Constitutive promoter for continuous expression. We use tac promoter in our experiment.
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Promoter: Constitutive promoter for continuous expression. We use Lac promoter in our experiment.
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RBS: Ribosome binding site for efficient translation. In our experiments, we used BBa-B0031, BBa-B0032 and BBa-B0034.
 
PPK1 Coding Sequence: Encodes the polyphosphate kinase 1 enzyme.
 
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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Terminator: Efficient transcription terminator to ensure proper mRNA processing. We used rrnB T1 terminator and T7Te terminator in our experiments.
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In basic parts, only coding sequence is included in our sequence. In this part, it is PPK1 Coding Sequence.
 
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         <img src="https://static.igem.wiki/teams/5034/engineering/fig3.png" style="width: 500px; height: auto;">
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===Origin (Organism)===
 
===Origin (Organism)===
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The PPK1 gene was sourced from Citrobacter freundii. The PYYDT plasmid backbone is a standard vector used for gene expression in synthetic biology applications.
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The PPK1 gene was sourced from *Citrobacter freundii*. The pBBR1MCS-terminator plasmid backbone is a standard vector used for gene expression in synthetic biology applications.
  
 
===Experimental Characterization and results===
 
===Experimental Characterization and results===
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Trying to enhance Shewanella’s capacity to produce electricity and polymerize phosphorus, we first introduce PPK1 into it.  
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Trying to enhance *Shewanella*’s capacity to produce electricity and polymerize phosphorus, we first introduced PPK1 into it.  
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Conducting molybdate assays, the level of phosphorus uptake was measured in the Shewanella following the introduction of the plasmid. It was observed that the Shewanella exhibited the most pronounced capacity to polymerize phosphorus following the introduction of PPK1.
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The level of phosphorus uptake was measured in the *Shewanella* after the introduction of the plasmid, using Pi content detection(https://2024.igem.wiki/nanjing-china/experiments) method. It was observed that the Shewanella exhibited the most pronounced capacity to polymerize phosphorus following the introduction of PPK1.
 
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Revision as of 13:52, 30 September 2024


PolyP <->Pi


Basic Description

This basic part encodes the PPK1 gene which is initially from *Citrobacter freundii* and we performed codon optimization on, is expressed in the pBBR1MCS-terminator plasmid. This basic 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, it can reversibly convert PolyP 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*.

Figure 1: Basic function of PPK1

Construct features

Promoter: Constitutive promoter for continuous expression. We use Lac promoter in our experiment. RBS: Ribosome binding site for efficient translation. In our experiments, we used BBa-B0031, BBa-B0032 and BBa-B0034. 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 experiments. In basic parts, only coding sequence is included in our sequence. In this part, it is PPK1 Coding Sequence.

Figure 2: Basic construction of PPK1 plasmid

Origin (Organism)

The PPK1 gene was sourced from *Citrobacter freundii*. The pBBR1MCS-terminator plasmid backbone is a standard vector used for gene expression in synthetic biology applications.

Experimental Characterization and results

Trying to enhance *Shewanella*’s capacity to produce electricity and polymerize phosphorus, we first introduced PPK1 into it. The level of phosphorus uptake was measured in the *Shewanella* after the introduction of the plasmid, using Pi content detection(https://2024.igem.wiki/nanjing-china/experiments) method. It was observed that the Shewanella exhibited the most pronounced capacity to polymerize phosphorus following the introduction of PPK1.

Figure 3: phosphorus accumulation capacity in Shewanella with the introduction of PPK1(SPK1 in the diagram)
Figure 4: electricity production capacity of Shewanella with the introduction of PPK1(SPK1 in the diagram)

SEM and TEM electron microscopy showed that the strains with high phosphorus content had abnormal morphology, indicating that allowing bacteria to be in a high phosphorus content state in advance is not conducive to the rapid transfer of electrons and is not conducive to the ability to produce electricity.

Figure 5: Scanning electron microscope of the Shewanella morphology of WT and modified Shewanella strains with B0034-RBS
Figure 6: Transmission electron microscopy of the Shewanella morphology of WT and modified Shewanella strains with B0034-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]