Difference between revisions of "Part:BBa K5034209"
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<partinfo>BBa_K5034209 short</partinfo>
 
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===Basic Description===
 
===Basic Description===
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This basic part encodes the PPN1 gene which is initially from Saccharomyces cerevisiae and we performed codon optimization on, is expressed in the PYYDT plasmid. This basic part is designed to facilitate the conversion of long-chain inorganic polyphosphate (PolyP) into shorter fragments without completely degrading it to inorganic phosphate (Pi). The PPN1 enzyme exhibits both exopolyphosphatase and endopolyphosphatase activities, depending on the presence of specific metal ions. Inactivation of the PPN1 gene encoding another protein, which exhibited exopolyPase activity in the yeast (CRN and CNX strains), resulted in almost total elimination of the nuclear exopolyPase activities in both growth phases.In a sentence, this enzyme can convert Poly P to Poly P with smaller fragments, but not to Pi completely. For the first time, we expressed this element in a strain of Shewanella and conducted codon optimization based on Shewanella.We tested the effects of the introduction of this element on electricity production and phosphorus metabolism.
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This basic part encodes the <i>PPN1</i> gene which is initially from Saccharomyces cerevisiae and we performed codon optimization on, is expressed in the PYYDT plasmid. This basic part is designed to facilitate the conversion of long-chain inorganic polyphosphate (PolyP) into shorter fragments without completely degrading it to inorganic phosphate (Pi). The PPN1 enzyme exhibits both exopolyphosphatase and endopolyphosphatase activities, depending on the presence of specific metal ions. Inactivation of the <i>PPN1</i> gene encoding another protein, which exhibited exopolyPase activity in the yeast (CRN and CNX strains), resulted in almost total elimination of the nuclear exopolyPase activities in both growth phases.In a sentence, this enzyme can convert Poly P to Poly P with smaller fragments, but not to Pi completely. For the first time, we expressed this element in a strain of <i>S. oneidensis</i> and conducted codon optimization based on <i>S. oneidensis</i>. We tested the effects of the introduction of this element on electricity production and phosphorus metabolism.
  
 
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     <img src="https://static.igem.wiki/teams/5034/engineering/ppn1-function.png" style="width: 300px; height: auto;">
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     <div style="text-align: center;">
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        <img src="https://static.igem.wiki/teams/5034/engineering/ppn1-function.png" style="width: 500px; height: auto;">
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        <div style="text-align: center;"><p>Figure 1: Basic function of PPN1</p></div>
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    </div>
 
</body>
 
</body>
 
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Figure 1: Basic function of PPN1
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===Sequence and Features===
  
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===Construct features(only coding sequence included in basic part)===
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<partinfo>BBa_K5034209 SequenceAndFeatures</partinfo>
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Promoter: Constitutive promoter for continuous expression. We use tac promoter in our experiment.
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PPN1 Coding Sequence: Encodes the polyphosphatase enzyme.
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===Construct feature===
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Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use T7Te terminator in our experiment.
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     <img src="https://static.igem.wiki/teams/5034/engineering/fig17.png" style="width: 300px; height: auto;">
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     <div style="text-align: center;">
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        <img src="https://static.igem.wiki/teams/5034/engineering/sppn1.png" style="width: 500px; height: auto;">
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        <div style="text-align: center;"><p>Figure 2: Basic construction of PPN1 plasmid</p></div>
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    </div>
 
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Figure 2: PCR of target genes PCR before plasmids construction (The extra small fragment in the picture is primer dimer)
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* Promoter: Constitutive promoter for continuous expression. We use tac promoter in our experiment.
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* RBS: <html><body><a href="https://parts.igem.org/Part:BBa_B0034">BBa_B0034</a></body></html>
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* <i>PPN1</i> Coding Sequence: Encodes the polyphosphatase 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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<html>
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<body>
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    <div style="text-align: center;">
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        <img src="https://static.igem.wiki/teams/5034/engineering/fig17.png" style="width: 500px; height: auto;">
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        <div style="text-align: center;"><p>Figure 3: PCR of target genes PCR before plasmids construction</p></div>
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    </div>
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</body>
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</html>
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The length of the <i>PPN1</i> gene is about 2025 base pairs, which is consistent with the results on the DNA electropherogram, proving that we have obtained the <i>PPN1</i> gene.
  
 
===Origin (Organism)===
 
===Origin (Organism)===
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The PPN1 gene was sourced from Saccharomyces cerevisiae.
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The <i>PPN1</i> gene was sourced from <i>Saccharomyces cerevisiae</i>.
 +
 
  
 
===Experimental Characterization and results===
 
===Experimental Characterization and results===
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In our team’s previous research we found that the behavior of the modified Shewanella did not reach our expectation and the electron microscopic observation also showed an abnormal morphology of the bacterium, we postulated that too much PPK1 may lead to an abnormal charge distribution in the bacterium thus result in a decrease in the bacterium's activity and a reduction in its capacity for electricity production, so we planed to improve the situation by introducing different polyphosphate hydrolases which influence the phosphorus metabolism of Shewanella.
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We first determined the electroproduction capacity of <i>S. oneidensis</i> after introduction of the SPPN1 enzyme (e.g. fig4)
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Electricity production: Using half-cell reaction(electrochemistry) to measure the electricity production ability.
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Capacity to polymerize phosphorus: Conducting molybdate assays to determine Pi concentration.
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Conducting molybdate assays to determine Pi concentration and found PPN1 a bad capacity to polymerize phosphorus.
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     <img src="https://static.igem.wiki/teams/5034/engineering/current-with-different-hydrolases.png" style="width: 300px; height: auto;">
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     <div style="text-align: center;">
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        <img src="https://static.igem.wiki/teams/5034/engineering/current-with-different-hydrolases.png" style="width: 500px; height: auto;">
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        <div style="text-align: center;"><p>Figure 4: Statistical data on electricity production capacity of <i>S. oneidensis</i> with the introduction of different hydrolases</p></div>
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    </div>
 
</body>
 
</body>
 
</html>
 
</html>
  
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Figure 3: statistical data on electricity production capacity of Shewanella with the introduction of different hydrolases
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We found that the power production efficiency of <i>S. oneidensis</i> did not improve significantly after SPPN1 introduction, and even decreased a bit, so we did not choose it for our subsequent power production optimisation experiments.
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Afterwards, we also measured the phosphorus aggregation capacity(fig5), and frustratingly, the programme still did not perform well.
  
 
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<body>
 
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     <img src="https://static.igem.wiki/teams/5034/engineering/ppn1-pi.png" style="width: 300px; height: auto;">
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     <div style="text-align: center;">
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        <img src="https://static.igem.wiki/teams/5034/engineering/ppn1-pi.png" style="width: 500px; height: auto;">
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        <div style="text-align: center;"><p>Figure 5: Statistical data on the phosphorus accumulation capacity of <i>S. oneidensis</i> with PPN1</p></div>
 +
    </div>
 
</body>
 
</body>
 
</html>
 
</html>
  
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Figure 4: statistical data on the phosphorus accumulation capacity of Shewanella with PPN1
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Subsequent experiments(fig6) also showed that the PPN1 did not result in an increase in the metabolic strength of <i>S. oneidensis</i> either, suggesting that the enzyme did not have any of the effects that we wanted it to have.
  
 
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<body>
 
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     <img src="https://static.igem.wiki/teams/5034/engineering/atp-level-with-different-hydrolyases.png" style="width: 300px; height: auto;">
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     <div style="text-align: center;">
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        <img src="https://static.igem.wiki/teams/5034/engineering/atp-level-with-different-hydrolyases.png" style="width: 50%; height: auto;">
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        <div style="text-align: center;"><p>Figure 6: ATP level in <i>S. oneidensis</i> with the introduction of different hydrolases</p></div>
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    </div>
 
</body>
 
</body>
 
</html>
 
</html>
  
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Figure 5: ATP level in Shewanella with the introduction of different hydrolases
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Overall, the PPN1 enzyme did not perform well in our scenario and did not have the ability to enhance the electroproduction and phosphorus-polymerisation capacity of <i>S. oneidensis</i>.
  
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===References===
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Details of all experiments can be found at the <html><body><a href="https://2024.igem.wiki/nanjing-china/experiments">Experiments  section on the Wiki.</a></body></html>
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1.Lichko, L. P., Kulakovskaya, T. V., & Kulaev, I. S. (2006). Inorganic polyphosphate and exopolyphosphatase in the nuclei of Saccharomyces cerevisiae: dependence on the growth phase and inactivation of the PPX1 and PPN1 genes. Biochemistry (Moscow), 71(11), 1171-1175.  
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===Usage and Biology===
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===Chassis and genetic===
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<span class='h3bb'>Sequence and Features</span>
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Chassis：<i>Shewanella oneidensis</i> MR-1.
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<partinfo>BBa_K5034209 SequenceAndFeatures</partinfo>
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The gene can be expressed and function properly in <i>S. oneidensis</i>.
  
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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===Potential applications===
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<partinfo>BBa_K5034209 parameters</partinfo>
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PPN1, as an enzyme that hydrolyses PolyP, is an important player in the regulation of intracellular PolyP metabolism.
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<!-- -->
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===References===
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1.Lichko, L. P., Kulakovskaya, T. V., & Kulaev, I. S. (2006). Inorganic polyphosphate and exopolyphosphatase in the nuclei of Saccharomyces cerevisiae: dependence on the growth phase and inactivation of the <i>PPX1</i> and <i>PPN1</i> genes. Biochemistry (Moscow), 71(11), 1171-1175.

Latest revision as of 13:15, 1 October 2024

Poly P -> Poly P(smaller) or Pi


Basic Description

This basic part encodes the PPN1 gene which is initially from Saccharomyces cerevisiae and we performed codon optimization on, is expressed in the PYYDT plasmid. This basic part is designed to facilitate the conversion of long-chain inorganic polyphosphate (PolyP) into shorter fragments without completely degrading it to inorganic phosphate (Pi). The PPN1 enzyme exhibits both exopolyphosphatase and endopolyphosphatase activities, depending on the presence of specific metal ions. Inactivation of the PPN1 gene encoding another protein, which exhibited exopolyPase activity in the yeast (CRN and CNX strains), resulted in almost total elimination of the nuclear exopolyPase activities in both growth phases.In a sentence, this enzyme can convert Poly P to Poly P with smaller fragments, but not to Pi completely. For the first time, we expressed this element in a strain of S. oneidensis and conducted codon optimization based on S. oneidensis. We tested the effects of the introduction of this element on electricity production and phosphorus metabolism.

Figure 1: Basic function of PPN1

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]

Construct feature

Figure 2: Basic construction of PPN1 plasmid

  • Promoter: Constitutive promoter for continuous expression. We use tac promoter in our experiment.
  • RBS: BBa_B0034
  • PPN1 Coding Sequence: Encodes the polyphosphatase enzyme.
  • Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use T7Te terminator in our experiment.

Figure 3: PCR of target genes PCR before plasmids construction

The length of the PPN1 gene is about 2025 base pairs, which is consistent with the results on the DNA electropherogram, proving that we have obtained the PPN1 gene.

Origin (Organism)

The PPN1 gene was sourced from Saccharomyces cerevisiae.


Experimental Characterization and results

We first determined the electroproduction capacity of S. oneidensis after introduction of the SPPN1 enzyme (e.g. fig4)

Figure 4: Statistical data on electricity production capacity of S. oneidensis with the introduction of different hydrolases

We found that the power production efficiency of S. oneidensis did not improve significantly after SPPN1 introduction, and even decreased a bit, so we did not choose it for our subsequent power production optimisation experiments.

Afterwards, we also measured the phosphorus aggregation capacity(fig5), and frustratingly, the programme still did not perform well.

Figure 5: Statistical data on the phosphorus accumulation capacity of S. oneidensis with PPN1

Subsequent experiments(fig6) also showed that the PPN1 did not result in an increase in the metabolic strength of S. oneidensis either, suggesting that the enzyme did not have any of the effects that we wanted it to have.

Figure 6: ATP level in S. oneidensis with the introduction of different hydrolases

Overall, the PPN1 enzyme did not perform well in our scenario and did not have the ability to enhance the electroproduction and phosphorus-polymerisation capacity of S. oneidensis.

Details of all experiments can be found at the Experiments section on the Wiki.

Chassis and genetic

Chassis：Shewanella oneidensis MR-1.

The gene can be expressed and function properly in S. oneidensis.


Potential applications

PPN1, as an enzyme that hydrolyses PolyP, is an important player in the regulation of intracellular PolyP metabolism.


References

1.Lichko, L. P., Kulakovskaya, T. V., & Kulaev, I. S. (2006). Inorganic polyphosphate and exopolyphosphatase in the nuclei of Saccharomyces cerevisiae: dependence on the growth phase and inactivation of the PPX1 and PPN1 genes. Biochemistry (Moscow), 71(11), 1171-1175.