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 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.
+
This basic part encodes the <i>PPK1</i> gene which is initially from <i>Citrobacter freundii</i> 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 <i>PPK1</i> 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 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*.
+
In a sentence, it can reversibly convert PolyP and Pi. This reversible process favors the generation of PolyP. 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>.
 
<html>
 
<html>
 
<body>
 
<body>
 
     <div style="text-align: center;">
 
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         <img src="https://static.igem.wiki/teams/5034/engineering/mechanism-of-ppk1.png" style="width: 500px; height: auto;">
 
         <img src="https://static.igem.wiki/teams/5034/engineering/mechanism-of-ppk1.png" style="width: 500px; height: auto;">
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<p>Figure 1: Basic function of <i>PPK1</i></p>
 
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     </div>
 
</body>
 
</body>
 
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Figure 1: Basic function of PPK1
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===Construct features===
 
===Construct features===
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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.
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<partinfo>BBa_K5034213 SequenceAndFeatures</partinfo>
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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 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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* Promoter: Constitutive promoter for continuous expression. We use Lac promoter in our experiment.Since our sequence does not encode the regulatory gene ''lacI'' for the repressor protein, the promoter we introduced is a constitutive promoter. This allows the subsequent genes to be continuously expressed.
 +
 
 +
* RBS: Ribosome binding site for efficient translation. In our experiments, we used BBa-B0031, BBa-B0032 and BBa-B0034, with BBa-B0031 having the lowest strength and BBa-B0034 having the highest strength.
 +
 
 +
* <i>PPK1</i> Coding Sequence: Encodes the polyphosphate kinase 1 enzyme.
 +
 
 +
* Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use BBa-B0015 in our experiments,which is rrnB T1 terminator and T7Te terminator.
 +
 
 +
In basic parts, only coding sequence is included in our sequence. In this part, it is PPK1 Coding Sequence.But the translational unit is composed of components above.
 
<html>
 
<html>
 
<body>
 
<body>
 
     <div style="text-align: center;">
 
     <div style="text-align: center;">
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         <img src="https://static.igem.wiki/teams/5034/engineering/fig8.png" style="width: 500px; height: auto;">
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         <img src="https://static.igem.wiki/teams/5034/results/new/basic-structure-of-ppk1.png" style="width: 500px; height: auto;">
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<p>Figure 2: Basic construction of PPK1 plasmid</p>
 
     </div>
 
     </div>
 
</body>
 
</body>
 
</html>
 
</html>
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Figure 2: Basic construction of PPK1 plasmid
 
  
 
===Origin (Organism)===
 
===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.
+
The <i>PPK1</i> gene was sourced from <i>Citrobacter freundii</i>. 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===
−
Trying to enhance *Shewanella*’s capacity to produce electricity and polymerize phosphorus, we first introduced PPK1 into it.  
+
Trying to enhance <i>S. oneidensis.</i>'s capacity to produce electricity and polymerize phosphorus, we first introduced <i>PPK1</i> into it.  
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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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 +
As the strength of the RBS decreases, the electrochemical activity of <i>S. oneidensis.</i> significantly increases, as detected by the Pi content detection method, while its polyphosphate capacity decreases, as observed through half-cell experiment.
 
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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/fig4.png" style="width: 500px; height: auto;">
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         <img src="https://static.igem.wiki/teams/5034/engineering/fig11.png" style="width: 500px; height: auto;">
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<p>Figure 3: Phosphorus accumulation capacity in <i>S. oneidensis.</i> after the introduction of <i>PPK1</i></p>
 
     </div>
 
     </div>
 
</body>
 
</body>
 
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Figure 3:  phosphorus accumulation capacity in Shewanella with the introduction of PPK1(SPK1 in the diagram)
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         <img src="https://static.igem.wiki/teams/5034/engineering/fig5.png" style="width: 500px; height: auto;">
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         <img src="https://static.igem.wiki/teams/5034/engineering/fig12.png" style="width: 500px; height: auto;">
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<p>Figure 4: Electricity production capacity of <i>S. oneidensis.</i> after the introduction of <i>PPK1</i></p>
 
     </div>
 
     </div>
 
</body>
 
</body>
 
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Figure 4: electricity production capacity of Shewanella with the introduction of PPK1(SPK1 in the diagram)
 
  
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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.
+
We then analyzed factors contributing to the reduced electricity generation capability of SPK1
 +
 
 +
Scanning electron microscope and transmission electron microscope results 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.
 
<html>
 
<html>
 
<body>
 
<body>
 
     <div style="text-align: center;">
 
     <div style="text-align: center;">
 
         <img src="https://static.igem.wiki/teams/5034/engineering/fig6.png" style="width: 500px; height: auto;">
 
         <img src="https://static.igem.wiki/teams/5034/engineering/fig6.png" style="width: 500px; height: auto;">
 +
<p>Figure 5: Scanning electron microscope of the <i>S. oneidensis.</i> morphology of WT and modified <i>S. oneidensis.</i> strains with BBa-B0034 RBS(strong RBS)</p>
 
     </div>
 
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Figure 5: Scanning electron microscope of the Shewanella morphology of WT and modified Shewanella strains with B0034-RBS
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     <div style="text-align: center;">
 
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         <img src="https://static.igem.wiki/teams/5034/engineering/fig7.png" style="width: 500px; height: auto;">
 
         <img src="https://static.igem.wiki/teams/5034/engineering/fig7.png" style="width: 500px; height: auto;">
 +
<p>Figure 6: Transmission electron microscopy of the <i>S. oneidensis.</i> morphology of WT and modified <i>S. oneidensis.</i> strains with BBa-B0034 RBS</p>
 
     </div>
 
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Figure 6: Transmission electron microscopy of the Shewanella morphology of WT and modified Shewanella strains with B0034-RBS
 
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===References===
 
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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.
 
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===Usage and Biology===
 
  
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K5034213 SequenceAndFeatures</partinfo>
 
  
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Details of all experiments can be found in the <html><body><a href="https://2024.igem.wiki/nanjing-china/experiments">Experiments section on the Wiki.</a></body></html>
  
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<!-- Uncomment this to enable Functional Parameter display
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===Chassis and genetic===
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===Functional Parameters===
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Chassis：<i>Shewanella onediensis</i> MR-1
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<partinfo>BBa_K5034213 parameters</partinfo>
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<!-- -->
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The gene can be expressed and function properly in <i>S. oneidensis.</i>.
 +
 
 +
===Potential applications===
 +
The <i>PPK1</i> gene (polyphosphate kinase 1) has potential applications in:
 +
 
 +
Industrial Microbial Engineering: Enhances the production of biofuels, amino acids, or antibiotics by boosting polyphosphate synthesis in microorganisms.
 +
 
 +
Environmental Bioremediation: Assists in the accumulation of heavy metals or radioactive substances for pollution control.
 +
 
 +
===References===
 +
<i>Wang X , Wang X , Hui K , et al. Highly Effective Polyphosphate Synthesis, Phosphate Removal and Concentration Using Engineered Environmental Bacteria Based on a Simple Solo Medium-copy Plasmid Strategy[J]. Environmental Science & Technology, 2017:acs.est.7b04532.</i>

Latest revision as of 08:10, 2 October 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 PolyP. For the first time, we expressed this element in a strain of S. oneidensis. and conducted codon optimization based on S. oneidensis..

Figure 1: Basic function of PPK1


Construct 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]


  • Promoter: Constitutive promoter for continuous expression. We use Lac promoter in our experiment.Since our sequence does not encode the regulatory gene lacI for the repressor protein, the promoter we introduced is a constitutive promoter. This allows the subsequent genes to be continuously expressed.
  • RBS: Ribosome binding site for efficient translation. In our experiments, we used BBa-B0031, BBa-B0032 and BBa-B0034, with BBa-B0031 having the lowest strength and BBa-B0034 having the highest strength.
  • PPK1 Coding Sequence: Encodes the polyphosphate kinase 1 enzyme.
  • Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use BBa-B0015 in our experiments,which is rrnB T1 terminator and T7Te terminator.

In basic parts, only coding sequence is included in our sequence. In this part, it is PPK1 Coding Sequence.But the translational unit is composed of components above.

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 S. oneidensis.'s capacity to produce electricity and polymerize phosphorus, we first introduced PPK1 into it.

As the strength of the RBS decreases, the electrochemical activity of S. oneidensis. significantly increases, as detected by the Pi content detection method, while its polyphosphate capacity decreases, as observed through half-cell experiment.

Figure 3: Phosphorus accumulation capacity in S. oneidensis. after the introduction of PPK1

Figure 4: Electricity production capacity of S. oneidensis. after the introduction of PPK1

We then analyzed factors contributing to the reduced electricity generation capability of SPK1

Scanning electron microscope and transmission electron microscope results 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 S. oneidensis. morphology of WT and modified S. oneidensis. strains with BBa-B0034 RBS(strong RBS)

Figure 6: Transmission electron microscopy of the S. oneidensis. morphology of WT and modified S. oneidensis. strains with BBa-B0034 RBS


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

Chassis and genetic

Chassis：Shewanella onediensis MR-1

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

Potential applications

The PPK1 gene (polyphosphate kinase 1) has potential applications in:

Industrial Microbial Engineering: Enhances the production of biofuels, amino acids, or antibiotics by boosting polyphosphate synthesis in microorganisms.

Environmental Bioremediation: Assists in the accumulation of heavy metals or radioactive substances for pollution control.

References

Wang X , Wang X , Hui K , et al. Highly Effective Polyphosphate Synthesis, Phosphate Removal and Concentration Using Engineered Environmental Bacteria Based on a Simple Solo Medium-copy Plasmid Strategy[J]. Environmental Science & Technology, 2017:acs.est.7b04532.