Difference between revisions of "Part:BBa K5034214"
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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 PPK1</p>
 
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Figure 1: Basic function of PPK1
 
  
 
===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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* Promoter: Constitutive promoter for continuous expression. We use Lac promoter in our experiment.
  
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RBS: Ribosome binding site for efficient translation. We use BBa-B0031 here.
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* RBS: Ribosome binding site for efficient translation. We use BBa-B0031 here.
  
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PPK1 Coding Sequence: Encodes the polyphosphate kinase 1 enzyme.
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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 rrnB T1 terminator and T7Te terminator in our experiment.
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* Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use rrnB T1 terminator and T7Te terminator in our experiment.
  
 
The basic structure of the part is shown as follows:
 
The basic structure of the part is shown as follows:
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         <img src="https://static.igem.wiki/teams/5034/results/new/basic-structure-of-spk3.png" style="width: 500px; height: auto;">
 
         <img src="https://static.igem.wiki/teams/5034/results/new/basic-structure-of-spk3.png" style="width: 500px; height: auto;">
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<p>Figure 2: Basic construction of <i>PPK1</i> plasmid with BBa-B0031 RBS</p>
 
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Figure 2: Basic construction of <i>PPK1</i> plasmid with BBa-B0031 RBS
 
  
  
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         <img src="https://static.igem.wiki/teams/5034/engineering/fig9.png" style="width: 500px; height: auto;">
 
         <img src="https://static.igem.wiki/teams/5034/engineering/fig9.png" style="width: 500px; height: auto;">
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<p>Figure 3: Colony PCR indicating plasmid replication in <i>S. oneidensis.</i></p>
 
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Figure 3: Colony PCR indicating plasmid replication in <i>S. oneidensis.</i>
 
  
  
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         <img src="https://static.igem.wiki/teams/5034/engineering/gel-ppk1.png" style="width: 500px; height: auto;">
 
         <img src="https://static.igem.wiki/teams/5034/engineering/gel-ppk1.png" style="width: 500px; height: auto;">
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<p>Figure 4: Agarose gel electrophoresis indicating we got the target gene with the corresponding RBS</p>
 
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Figure 4: Agarose gel electrophoresis indicating we got the target gene with the corresponding RBS
 
  
  
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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 5: SDS-PAGE results showing that the BBa-B0031 one's protein expression is the minimum, corresponding to the strength of RBS</p>
 
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Figure 5: SDS-PAGE results showing that the BBa-B0031 one's protein expression is the minimum, corresponding to the strength of RBS
 
  
  
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<p>Figure 6: Electricity production capacity of <i>S. oneidensis.</i> after the introduction of <i>PPK1</i> with different RBS</p>
 
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Figure 6: Electricity production capacity of <i>S. oneidensis.</i> after the introduction of <i>PPK1</i> with different RBS
 
 
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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 7: Phosphorus accumulation capacity of <i>S. oneidensis.</i> after the introduction of <i>PPK1</i> with different RBS</p>
 
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Figure 7: Phosphorus accumulation capacity of <i>S. oneidensis.</i> after the introduction of <i>PPK1</i> with different RBS
 
  
  

Revision as of 09:57, 1 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-B0031 RBS, which is a weaker 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 activated by a weaker RBS. 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.
  • RBS: Ribosome binding site for efficient translation. We use BBa-B0031 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.

The basic structure of the part is shown as follows:

Figure 2: Basic construction of PPK1 plasmid with BBa-B0031 RBS


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 3: Colony PCR indicating plasmid replication in S. oneidensis.


DNA agarose gel electrophoresis results showed that we obtained the plasmid with BBa-B0031 RBS, which is approximately 2.1 kb in size.

Figure 4: Agarose gel electrophoresis indicating we got the target gene with the corresponding RBS


We performed protein extraction for SDS-PAGE. The SDS-PAGE results showed that protein expression of the plasmid with BBa-B0031 RBS is the minimum, corresponding to the strength of RBS.

Figure 5: SDS-PAGE results showing that the BBa-B0031 one's protein expression is the minimum, 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 that SPK3 with RBS BBa-B0031 has the lowest capacity for phosphorus polymerization but the highest electroproduction capability compared to other RBS.

Figure 6: Electricity production capacity of S. oneidensis. after the introduction of PPK1 with different RBS

Figure 7: 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

1.Itoh, H., & Shiba, T. (2004). Polyphosphate synthetic activity of polyphosphate:AMP phosphotransferase in Acinetobacter johnsonii 210A. Journal of Bacteriology, 186(15), 5178-5181.