Difference between revisions of "Part:BBa K5034222"
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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.
+
This composite part includes 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 with the BBa-B0032 RBS, which is a medium RBS 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 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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<html>
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<body>
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    <div style="text-align: center;">
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Figure 1: Basic function of PPK1
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        <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 PPK1</p>
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    </div>
 +
</body>
 +
</html>
  
 
===Construct features===
 
===Construct features===
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Promoter: Constitutive promoter for continuous expression. We use tac promoter in our experiment.
 
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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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<partinfo>BBa_K5034222 SequenceAndFeatures</partinfo>
  
  
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Figure 2: Basic construction of PPK1 with B0034-RBS plasmid
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* Plasmid Backbone: pBBR1MCS-terminator plasmid
  
 +
* Promoter: Constitutive promoter for continuous expression. We use Lac promoter in our experiment.
  
 +
* RBS: Ribosome binding site for efficient translation. We use BBa-B0034 here.
  
 +
* PPK1 Coding Sequence: Encodes the polyphosphate kinase 1 enzyme.
  
 +
* Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use rrnB T1 terminator and T7Te terminator in our experiment.
  
 +
<html>
 +
<body>
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    <div style="text-align: center;">
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        <img src="https://static.igem.wiki/teams/5034/results/new/basic-structure-of-spk1.png" style="width: 500px; height: auto;">
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<p>Figure 2: Basic construction of <i>PPK1</i> with BBa-B0034 RBS plasmid</p>
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    </div>
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</body>
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</html>
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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/pbbr1mcs-terminator-34-ppk1.png" style="width: 500px; height: auto;">
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<p>Figure 3: Construction of <i>PPK1</i> with BBa-B0034 RBS plasmid</p>
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    </div>
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</body>
 +
</html>
  
  
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Figure 3: Construction of PPK1 with B0034 RBS plasmid
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We transformed the plasmids into wild-type <i>S. oneidensis.</i>, expressed it, and performed colony PCR. The results showed that <i>PPK1</i> was successfully introduced into <i>S. oneidensis.</i> for replication.
 +
<html>
 +
<body>
 +
    <div style="text-align: center;">
 +
        <img src="https://static.igem.wiki/teams/5034/engineering/fig9.png" style="width: 500px; height: auto;">
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<p>Figure 4: Colony PCR indicating plasmid replication in <i>S. oneidensis.</i></p>
 +
    </div>
 +
</body>
 +
</html>
  
  
 +
DNA agarose gel electrophoresis results showed that we obtained the plasmid with BBa-B0034 RBS, which is approximately 2.1 kb in size.
 +
<html>
 +
<body>
 +
    <div style="text-align: center;">
 +
        <img src="https://static.igem.wiki/teams/5034/engineering/gel-ppk1.png" style="width: 500px; height: auto;">
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<p>Figure 5: Agarose gel electrophoresis indicating we got the target gene with the corresponding RBS</p>
 +
    </div>
 +
</body>
 +
</html>
  
  
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We performed protein extraction for SDS-PAGE. SDS-PAGE results showed that protein expression of the plasmid with BBa-B0034 RBS is the maximum, corresponding to the strength of RBS.
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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/fig10.png" style="width: 500px; height: auto;">
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<p>Figure 6: SDS-PAGE results showing that the BBa-B0034 one’s protein expression is the maximum, corresponding to the strength of RBS</p>
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Figure 4: Colony PCR indicating plasmid replication in Shewanell
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    </div>
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</body>
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</html>
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Figure 5: Agarose gel electrophoresis indicating the target gene was successfully introduced into Shewanella
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Figure 6: SDS-PAGE results showing that the BBa-B0034 one’s protein expression is the maximum, corresponding to the strength of RBS
+
  
 
===Origin (Organism)===
 
===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.
+
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===
−
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.
+
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 <i>S. oneidensis.</i> to develop the best ability to produce electricity and polymerize phosphorus.
−
Using molybdate assays to determine Pi concentration and half-cell reaction(electrochemistry) to measure the electricity production ability, we found SPK3(with RBS BBa-B0034) has the greatest capacity to polymerize phosphorus but a worst electroproduction capability.
+
  
 +
We conducted Pi content detection to determine Pi concentration and half-cell experiment to measure the electricity production ability, we found SPK1 with RBS BBa-B0034 has the greatest capacity to polymerize phosphorus but a worst electroproduction capability.
 +
<html>
 +
<body>
 +
    <div style="text-align: center;">
 +
        <img src="https://static.igem.wiki/teams/5034/engineering/fig12.png" style="width: 500px; height: auto;">
 +
<p>Figure 7: Electricity production capacity of <i>S. oneidensis.</i> after the introduction of PPK1 with different RBS</p>
 +
    </div>
 +
</body>
 +
</html>
 +
<html>
 +
<body>
 +
    <div style="text-align: center;">
 +
        <img src="https://static.igem.wiki/teams/5034/engineering/fig11.png" style="width: 500px; height: auto;">
 +
<p>Figure 8: Phosphorus accumulation capacity of <i>S. oneidensis.</i> after the introduction of PPK1 with different RBS</p>
 +
    </div>
 +
</body>
 +
</html>
  
 +
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>
 +
<body>
 +
    <div style="text-align: center;">
 +
        <img src="https://static.igem.wiki/teams/5034/engineering/fig6.png" style="width: 500px; height: auto;">
 +
<p>Figure 9: 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>
 +
</body>
 +
</html>
  
 +
<html>
 +
<body>
 +
    <div style="text-align: center;">
 +
        <img src="https://static.igem.wiki/teams/5034/engineering/fig7.png" style="width: 500px; height: auto;">
 +
<p>Figure 10: 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>
 +
</body>
 +
</html>
  
  
 +
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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Figure 7: statistical data on electricity production capacity of Shewanella with the introduction of PPK1 with different RBS
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===Chassis and genetic===
 +
Chassis：<i>Shewanella onediensis</i> MR-1
  
 +
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.
−
 
+
−
 
+
−
Figure 8: statistical data on phosphorus accumulation capacity of Shewanella with the introduction of PPK1 with different RBS
+
  
 
===References===
 
===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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<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>
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K5034222 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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<partinfo>BBa_K5034222 parameters</partinfo>
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Latest revision as of 08:21, 2 October 2024


Pi <-> Poly P

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 pBBR1MCS-terminator plasmid with the BBa-B0032 RBS, which is a medium RBS 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 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.

Figure 1: Basic function of PPK1

Construct features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 4981
    Illegal suffix found in sequence at 1
  • 12
    INCOMPATIBLE 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
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 4981
    Illegal BglII site found at 3580
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 4981
    Illegal suffix found in sequence at 2
  • 25
    INCOMPATIBLE 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
  • 1000
    COMPATIBLE WITH RFC[1000]


  • Plasmid Backbone: pBBR1MCS-terminator plasmid
  • Promoter: Constitutive promoter for continuous expression. We use Lac promoter in our experiment.
  • RBS: Ribosome binding site for efficient translation. We use BBa-B0034 here.
  • PPK1 Coding Sequence: Encodes the polyphosphate kinase 1 enzyme.
  • Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use rrnB T1 terminator and T7Te terminator in our experiment.

Figure 2: Basic construction of PPK1 with BBa-B0034 RBS plasmid

Figure 3: Construction of PPK1 with BBa-B0034 RBS plasmid


We transformed the plasmids into wild-type S. oneidensis., expressed it, and performed colony PCR. The results showed that PPK1 was successfully introduced into S. oneidensis. for replication.

Figure 4: Colony PCR indicating plasmid replication in S. oneidensis.


DNA agarose gel electrophoresis results showed that we obtained the plasmid with BBa-B0034 RBS, which is approximately 2.1 kb in size.

Figure 5: Agarose gel electrophoresis indicating we got the target gene with the corresponding RBS


We performed protein extraction for SDS-PAGE. SDS-PAGE results showed that protein expression of the plasmid with BBa-B0034 RBS is the maximum, corresponding to the strength of RBS.

Figure 6: SDS-PAGE results showing that the BBa-B0034 one’s protein expression is the maximum, corresponding to the strength of RBS

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

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 S. oneidensis. to develop the best ability to produce electricity and polymerize phosphorus.

We conducted Pi content detection to determine Pi concentration and half-cell experiment to measure the electricity production ability, we found SPK1 with RBS BBa-B0034 has the greatest capacity to polymerize phosphorus but a worst electroproduction capability.

Figure 7: Electricity production capacity of S. oneidensis. after the introduction of PPK1 with different RBS

Figure 8: Phosphorus accumulation capacity of S. oneidensis. after the introduction of PPK1 with different RBS

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

Figure 10: 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.