Difference between revisions of "Part:BBa K5034216"
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<partinfo>BBa_K5034216 short</partinfo>
 
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===Basic Description===
 
===Basic Description===
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This basic part encodes the PPK1 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-B0034 RBS, which is a stronger RBS compared to others. 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, this part is activated by an efficient 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 Shewanella and conducted codon optimization based on Shewanella.
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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 with the BBa-B0034 RBS, which is a stronger RBS compared to others. 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, this part is activated by an efficient 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 <i>S. oneidensis.</i> and conducted codon optimization based on <i>S. oneidensis.</i>.
 
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===Construct features===
 
===Construct features===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K5034216 SequenceAndFeatures</partinfo>
 
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Promoter: Constitutive promoter for continuous expression. We use Lac promoter in our experiment.
 
Promoter: Constitutive promoter for continuous expression. We use Lac promoter in our experiment.
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Figure 2: Basic construction of PPK1 plasmid with BBa-B0034 RBS
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Figure 2: Basic construction of <i>PPK1</i> plasmid with BBa-B0034 RBS
  
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We transform the plasmids into wild-type Shewanella, express it, and perform colony PCR. The results show that PPK1 is successfully introduced into <i>Shewanella</i> for replication.
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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>Shewanella</i> for replication.
 
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Figure 3: Colony PCR indicating plasmid replication in Shewanell
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Figure 3: Colony PCR indicating plasmid replication in <i>S. oneidensis.</i>
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DNA agarose gel electrophoresis results showed that we obtained the plasmid with BBa-B0034 RBS, which is approximately 2.1 kb in size.
 
DNA agarose gel electrophoresis results showed that we obtained the plasmid with BBa-B0034 RBS, which is approximately 2.1 kb in size.
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Figure 4: Agarose gel electrophoresis indicating we got the target gene with the corresponding RBS
 
Figure 4: Agarose gel electrophoresis indicating we got the target gene with the corresponding RBS
  
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We performed protein extraction for SDS-PAGE. The 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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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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===Origin (Organism)===
 
===Origin (Organism)===
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The PPK1 gene was sourced from Citrobacter freundii.  
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The <i>PPK1</i> gene was sourced from <i>Citrobacter freundii</i>.  
  
 
===Experimental Characterization and results===
 
===Experimental Characterization and results===
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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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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.
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We conduct Pi content detection to determine Pi concentration and half-cell experiment 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.
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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.
 
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Figure 6: Electricity production capacity of Shewanella with the introduction of PPK1 with different RBS
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Figure 6: Electricity production capacity of <i>S. oneidensis.</i> with the introduction of <i>PPK1</i> with different RBS
 
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Figure 7: Phosphorus accumulation capacity of Shewanella with the introduction of PPK1 with different RBS
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Figure 7: Phosphorus accumulation capacity of <i>S. oneidensis.</i> with the introduction of <i>PPK1</i> with different RBS
  
 
===References===
 
===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.
 
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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===Functional Parameters===
 
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<partinfo>BBa_K5034216 parameters</partinfo>
 
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Revision as of 01:44, 1 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 with the BBa-B0034 RBS, which is a stronger RBS compared to others. 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, this part is activated by an efficient 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-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.

The basic structure of the part is shown as follows:

Figure 2: Basic construction of PPK1 plasmid with BBa-B0034 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 Shewanella 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-B0034 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. SDS-PAGE results showed that protein expression of the plasmid with BBa-B0034 RBS is the maximum, corresponding to the strength of RBS.

Figure 5: 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.

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

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.