Difference between revisions of "Part:BBa K5034215"
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<partinfo>BBa_K5034215 short</partinfo>
 
<partinfo>BBa_K5034215 short</partinfo>
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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.
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===Basic Description===
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<b>Basic Description:</b>
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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.In a sentence, this part is initiated by a medium promoter. 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>.
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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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<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/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>
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</body>
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</html>
  
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===Construct features===
  
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<partinfo>BBa_K5034215 SequenceAndFeatures</partinfo>
  
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Figure 1: Basic function of PPK1
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* Plasmid backbone: pBBR1MCS-terminator. The Lac promoter and the double terminator are on the plasmid backbone.<html><body><a href="https://parts.igem.org/Part:BBa_K5034201">BBa_K5034201</a></body></html>
  
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<b>Construct features :</b>
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* Promoter: Constitutive promoter for continuous expression. We used 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.
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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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* RBS: Ribosome binding site for efficient translation. We used 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 used rrnB T1 terminator and T7Te terminator in our experiment.
  
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The basic structure of the part is shown as follows:
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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/results/new/basic-structure-of-spk2.png" style="width: 500px; height: auto;">
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<p>Figure 2: Basic construction of <i>PPK1</i> plasmid with BBa-B0032 RBS</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-32-ppk1.png" style="width: 500px; height: auto;">
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<p>Figure 3: Construction of <i>PPK1</i> with BBa-B0032 RBS plasmid</p>
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    </div>
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</body>
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</html>
  
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We transform the plasmids into wild-type <i>S. oneidensis.</i>, express it, and perform colony PCR. The results show that <i>PPK1</i> is successfully introduced into <i>S. oneidensis.</i> for replication.
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<html>
 +
<body>
 +
    <div style="text-align: center;">
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        <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>
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    </div>
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</body>
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</html>
  
  
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DNA agarose gel electrophoresis results showed that we obtained the plasmid with BBa-B0032 RBS, which is approximately 2.1 kb in size.
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<html>
 +
<body>
 +
    <div style="text-align: center;">
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        <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 corresponding RBS</p>
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    </div>
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</body>
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</html>
  
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Figure 2: Colony PCR indicating plasmid replication in Shewanell
 
  
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We performed protein extraction for SDS-PAGE. SDS-PAGE results showed that protein expression of the plasmid with BBa-B0032 RBS is the medium, corresponding to the strength of RBS.
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<html>
 +
<body>
 +
    <div style="text-align: center;">
 +
        <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-B0032 one's protein expression is the medium, corresponding to the strength of RBS</p>
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    </div>
 +
</body>
 +
</html>
  
 +
===Origin (Organism)===
 +
The <i>PPK1</i> gene was sourced from <i>Citrobacter freundii</i>.
  
 +
===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 <i>S. oneidensis.</i> 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 SPK2 with RBS BBa-B0032 has the medium capacity to polymerize phosphorus and a medium electroproduction capability.
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<html>
 +
<body>
 +
    <div style="text-align: center;">
 +
        <img src="https://static.igem.wiki/teams/5034/engineering/fig12.png" style="width: 500px; height: auto;">
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<p>Figure 7: Electricity production capacity of <i>S. oneidensis.</i> after the introduction of <i>PPK1</i> with different RBS</p>
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    </div>
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</body>
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</html>
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<html>
 +
<body>
 +
    <div style="text-align: center;">
 +
        <img src="https://static.igem.wiki/teams/5034/engineering/fig11.png" style="width: 500px; height: auto;">
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<p>Figure 8: Phosphorus accumulation capacity of <i>S. oneidensis.</i> after the introduction of <i>PPK1</i> with different RBS</p>
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    </div>
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</body>
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</html>
  
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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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===Chassis and genetic===
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Chassis：<i>Shewanella onediensis</i> MR-1
  
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The gene can be expressed and function properly in <i>S. oneidensis.</i>.
  
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Figure 3: Agarose gel electrophoresis indicating the target gene was successfully introduced into Shewanella
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===Potential applications===
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The <i>PPK1</i> gene (polyphosphate kinase 1) has potential applications in:
  
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Industrial Microbial Engineering: Enhances the production of biofuels, amino acids, or antibiotics by boosting polyphosphate synthesis in microorganisms.
  
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Environmental Bioremediation: Assists in the accumulation of heavy metals or radioactive substances for pollution control.
  
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===References===
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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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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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−
 
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<b>Experimental Characterization and results:</b>
+
−
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.
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K5034215 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_K5034215 parameters</partinfo>
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Latest revision as of 12:03, 2 October 2024


PolyP <->Pi

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.In a sentence, this part is initiated by a medium promoter. 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]
  • Plasmid backbone: pBBR1MCS-terminator. The Lac promoter and the double terminator are on the plasmid backbone.BBa_K5034201
  • Promoter: Constitutive promoter for continuous expression. We used 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. We used BBa-B0032 here.
  • 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 experiment.

The basic structure of the part is shown as follows:

Figure 2: Basic construction of PPK1 plasmid with BBa-B0032 RBS

Figure 3: Construction of PPK1 with BBa-B0032 RBS plasmid

We transform the plasmids into wild-type S. oneidensis., express it, and perform colony PCR. The results show that PPK1 is 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-B0032 RBS, which is approximately 2.1 kb in size.

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


We performed protein extraction for SDS-PAGE. SDS-PAGE results showed that protein expression of the plasmid with BBa-B0032 RBS is the medium, corresponding to the strength of RBS.

Figure 6: 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 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 SPK2 with RBS BBa-B0032 has the medium capacity to polymerize phosphorus and a medium 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

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.