Difference between revisions of "Part:BBa K5034212"
 
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<partinfo>BBa_K5034212 short</partinfo>
 
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It can reversibly convert Poly p and AMP to ADP. For the first time, we expressed this element in a strain of Shewanella and conducted codon optimization based on Shewanella.We tested the effects of the introduction of this element on electricity production and phosphorus metabolism
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It can reversibly convert PolyP and AMP to ADP. 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>. We tested the effects of the introduction of this element on electricity production and phosphorus metabolism.
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<b>Basic Description:</b>
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This basic part encodes the PAP gene which is initially from Acinetobacter johnsonii and we performed codon optimization on, is expressed in the PYYDT plasmid. This basic part is designed to facilitate the reversible conversion of inorganic polyphosphate (PolyP) and adenosine monophosphate (AMP) to adenosine diphosphate (ADP). The PAP enzyme plays a crucial role in phosphate and energy metabolism. PAP and PPK2 have strong
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poly(P) utilization activity, and the activity of PAP is 20-fold higher than that in PPK1, even though the Km values for utilization of all three enzymes are almost the same. This implies that PAP and PPK2 work mainly for poly(P) utilization in the cell. However, PAP has the same level of poly(P) synthetic activity (Km and Vmax) as PPK1, and this suggests that PAP also works for poly(P) synthesis at a significant level.
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===Basic Description===
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This basic part encodes the <i>PAP</i> gene which is initially from Acinetobacter johnsonii and we performed codon optimization on, is expressed in the pBBR1MCS-terminator plasmid. This basic part is designed to facilitate the reversible conversion of inorganic polyphosphate (PolyP) and adenosine monophosphate (AMP) to adenosine diphosphate (ADP). The PAP enzyme plays a crucial role in phosphate and energy metabolism. PAP and PPK2 have strong PolyP utilization activity, and the activity of PAP is 20-fold higher than that in PPK1, even though the Km values for utilization of all three enzymes are almost the same. This implies that PAP and PPK2 work mainly for PolyP utilization in the cell. However, PAP has the same level of PolyP synthetic activity (Km and Vmax) as PPK1, and this suggests that PAP also works for PolyP synthesis at a significant level.
  
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Figure 1: Basic function of PAP
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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-pap.png" style="width: 50%; height: auto;">
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        <div style="text-align: center;"><p>Figure 1: Basic function of PAP</p></div>
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    </div>
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</body>
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</html>
  
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<b>Construct features(only coding sequence included in basic part) :</b>
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===Sequence and Features===
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Promoter: Constitutive promoter for continuous expression. We use tac promoter in our experiment.
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PAP Coding Sequence: Encodes the polyphosphate:AMP phosphotransferase 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_K5034212 SequenceAndFeatures</partinfo>
  
  
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===Construct features===
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* Promoter: We use Lac promoter in our experiment. Since there is no LacI protein on plasmid backbone, the gene expression is constitutive. Since the plasmid backbone does not encode the regulatory gene lacI for the repressor protein, the Lac promoter can be used as a constitutive promoter. This allows the subsequent genes to be constantly expressed.
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* RBS: <html><body><a href="https://parts.igem.org/Part:BBa_B0034">BBa_B0034</a></body></html>
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* <i>PAP</i> Coding Sequence: Encodes the polyphosphate:AMP phosphotransferase enzyme.
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* Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use a double terminator rrnBT1-T7TE(BBa_B0015) in our experiment.
  
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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/fig17.png" style="width: 50%; height: auto;">
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        <div style="text-align: center;"><p>Figure 2: PCR of target genes PCR before plasmids construction (The extra small fragment in the picture is primer dimer)</p></div>
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    </div>
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</body>
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</html>
  
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The length of the <i>PAP</i> gene is about 1428 base pairs, which is consistent with the results on the DNA electropherogram, proving that we have obtained the <i>PAP</i> gene.
  
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===Origin (Organism)===
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The <i>PAP</i> gene was sourced from ''Acinetobacter johnsonii''.
  
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Figure 2: PCR of target genes PCR before plasmids construction (The extra small fragment in the picture is primer dimer)
 
  
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===Experimental Characterization and results===
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We first determined the electroproduction capacity of <i>S. oneidensis</i> after introduction of the PAP enzyme.(Fig.3)
  
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<b>Origin (Organism):</b>
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<html>
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The PAP gene was sourced from Acinetobacter johnsonii.  
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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/current-with-different-hydrolases.png" style="width: 50%; height: auto;">
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        <div style="text-align: center;"><p>Figure 3: Statistical data on electricity production capacity of <i>S. oneidensis</i> with the introduction of different hydrolases</p></div>
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    </div>
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</body>
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</html>
  
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<b>Experimental Characterization and results:</b>
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We found that the efficiency of bacterial inhibition of electricity production did not significantly increase after SPAP introduction, and even showed a significant decrease, so it is not a good synthetic biology component to enhance the electricity production capacity of <i>S. oneidensis</i>.
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In our team’s previous research we found that the behavior of the modified Shewanella did not reach our expectation and the electron microscopic observation also showed an abnormal morphology of the bacterium, we postulated that too much PPK1 may lead to an abnormal charge distribution in the bacterium thus result in a decrease in the bacterium's activity and a reduction in its capacity for electricity production, so we planed to improve the situation by introducing different polyphosphate hydrolases which influence the phosphorus metabolism of Shewanella.
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Electricity production: Using half-cell reaction(electrochemistry) to measure the electricity production ability.
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Capacity to polymerize phosphorus: Conducting molybdate assays to determine Pi concentration.
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Conducting molybdate assays to determine Pi concentration and found that PAP do not have a good capacity to polymerize phosphorus (but better than PPN1 and PPX).
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Afterwards, we also measured the phosphorus aggregation capacity of <i>S. oneidensis</i>(Fig.4) and found no significant difference from the wild type, indicating that its introduction did not promote phosphorus uptake in <i>S. oneidensis</i> either.
  
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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/pap-pi.png" style="width: 50%; height: auto;">
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        <div style="text-align: center;"><p>Figure 4: Statistical data on the phosphorus accumulation capacity of <i>S. oneidensis</i> with PAP</p></div>
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    </div>
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</body>
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</html>
  
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Tests to measure ATP levels in <i>S. oneidensis</i>(Fig.5) also showed that its introduction did not result in an increase in the metabolic strength of <i>S. oneidensis</i> either.
  
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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/atp-level-with-different-hydrolyases.png" style="width: 50%; height: auto;">
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        <div style="text-align: center;"><p>Figure 5: ATP level in <i>S. oneidensis</i> with the introduction of different hydrolases</p></div>
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    </div>
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</body>
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</html>
  
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In summary, the option of importing PAP is not a good one.
  
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Details of all experiments can be found at 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 3: statistical data on electricity production capacity of Shewanella with the introduction of different hydrolases
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===Chassis and genetic context===
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Chassis：<i>Shewanella oneidensis</i> MR-1.
  
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Figure 4: statistical data on the phosphorus accumulation capacity of Shewanella with PAP
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The gene can be expressed and function properly in <i>S. oneidensis</i>.
  
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===Potential applications===
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PAP, as an enzyme class for the synthesis of ADP, plays an important role in the synthesis of intracellular high-energy phosphate compounds and is a key enzyme in the regulation of the intensity of intracellular metabolism.
  
  
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===References===
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Figure 5: ATP level in Shewanella with the introduction of different hydrolases
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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.
 
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_K5034212 SequenceAndFeatures</partinfo>
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===Functional Parameters===
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<partinfo>BBa_K5034212 parameters</partinfo>
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Latest revision as of 07:48, 2 October 2024

PolyP + AMP --> ADP

It can reversibly convert PolyP and AMP to ADP. For the first time, we expressed this element in a strain of S. oneidensis and conducted codon optimization based on S. oneidensis. We tested the effects of the introduction of this element on electricity production and phosphorus metabolism.


Basic Description

This basic part encodes the PAP gene which is initially from Acinetobacter johnsonii and we performed codon optimization on, is expressed in the pBBR1MCS-terminator plasmid. This basic part is designed to facilitate the reversible conversion of inorganic polyphosphate (PolyP) and adenosine monophosphate (AMP) to adenosine diphosphate (ADP). The PAP enzyme plays a crucial role in phosphate and energy metabolism. PAP and PPK2 have strong PolyP utilization activity, and the activity of PAP is 20-fold higher than that in PPK1, even though the Km values for utilization of all three enzymes are almost the same. This implies that PAP and PPK2 work mainly for PolyP utilization in the cell. However, PAP has the same level of PolyP synthetic activity (Km and Vmax) as PPK1, and this suggests that PAP also works for PolyP synthesis at a significant level.

Figure 1: Basic function of PAP

Sequence and 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]


Construct features

  • Promoter: We use Lac promoter in our experiment. Since there is no LacI protein on plasmid backbone, the gene expression is constitutive. Since the plasmid backbone does not encode the regulatory gene lacI for the repressor protein, the Lac promoter can be used as a constitutive promoter. This allows the subsequent genes to be constantly expressed.
  • RBS: BBa_B0034
  • PAP Coding Sequence: Encodes the polyphosphate:AMP phosphotransferase enzyme.
  • Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use a double terminator rrnBT1-T7TE(BBa_B0015) in our experiment.

Figure 2: PCR of target genes PCR before plasmids construction (The extra small fragment in the picture is primer dimer)

The length of the PAP gene is about 1428 base pairs, which is consistent with the results on the DNA electropherogram, proving that we have obtained the PAP gene.

Origin (Organism)

The PAP gene was sourced from Acinetobacter johnsonii.


Experimental Characterization and results

We first determined the electroproduction capacity of S. oneidensis after introduction of the PAP enzyme.(Fig.3)

Figure 3: Statistical data on electricity production capacity of S. oneidensis with the introduction of different hydrolases

We found that the efficiency of bacterial inhibition of electricity production did not significantly increase after SPAP introduction, and even showed a significant decrease, so it is not a good synthetic biology component to enhance the electricity production capacity of S. oneidensis.

Afterwards, we also measured the phosphorus aggregation capacity of S. oneidensis(Fig.4) and found no significant difference from the wild type, indicating that its introduction did not promote phosphorus uptake in S. oneidensis either.

Figure 4: Statistical data on the phosphorus accumulation capacity of S. oneidensis with PAP

Tests to measure ATP levels in S. oneidensis(Fig.5) also showed that its introduction did not result in an increase in the metabolic strength of S. oneidensis either.

Figure 5: ATP level in S. oneidensis with the introduction of different hydrolases

In summary, the option of importing PAP is not a good one.

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

Chassis and genetic context

Chassis：Shewanella oneidensis MR-1.

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

Potential applications

PAP, as an enzyme class for the synthesis of ADP, plays an important role in the synthesis of intracellular high-energy phosphate compounds and is a key enzyme in the regulation of the intensity of intracellular metabolism.


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.