Difference between revisions of "Part:BBa K5034219"
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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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         <img src="https://static.igem.wiki/teams/5034/engineering/mechanism-of-pap.png" style="width: 60%; height: auto;">
 
         <div style="text-align: center;"><p>Figure 1: Basic function of PAP</p></div>
 
         <div style="text-align: center;"><p>Figure 1: Basic function of PAP</p></div>
 
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<partinfo>BBa_K5034219 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5034219 SequenceAndFeatures</partinfo>
  
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Promoter: Constitutive promoter for continuous expression. We use tac promoter in our experiment.
+
===Construct features===
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PAP Coding Sequence: Encodes the polyphosphate:AMP phosphotransferase enzyme.
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Plasmid backbone: pBBR1MCS-terminator. The Lac promoter and the double terminator are on the plasmid backbone.
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Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use T7Te terminator in our experiment.
+
 
 +
* 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: <html><body><a href="https://parts.igem.org/Part:BBa_B0034">BBa_B0034</a></body></html>
 +
* <i>PAP</i> 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.
  
 
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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: 300px; height: auto;">
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<img src="https://static.igem.wiki/teams/5034/results/new/basic-structure-of-pap.png" style="width: 60%; height: auto;">
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<p>Figure 2: Basic construction of PAP plasmid</p>
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</div>
 
</html>
 
</html>
  
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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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The plasmid map of this part is shown in the figure below.(Fig.4)
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<div style="text-align: center;">
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    <img src="https://static.igem.wiki/teams/5034/engineering/pap-construct.jpg" style="width: 300px; height: auto;">
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<img src="https://static.igem.wiki/teams/5034/engineering/pbbr1mcs-terminator-pap.png" style="width: 60%; height: auto;">
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</body>
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<p>Figure 3: Construction of PAP plasmid</p>
 +
</div>
 
</html>
 
</html>
  
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Figure 3: Basic construction of PAP plasmid
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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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<div style="text-align: center;">
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    <img src="https://static.igem.wiki/teams/5034/engineering/pap-plasmid.png" style="width: 300px; height: auto;">
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<img src="https://static.igem.wiki/teams/5034/engineering/fig17.png" style="width: 60%; height: auto;">
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</body>
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<p>Figure 4: PCR of target genes PCR before plasmids construction (The extra small fragment in the picture is primer dimer)</p>
 +
</div>
 
</html>
 
</html>
  
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Figure 4: Construction of PAP plasmid
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We conducted colony PCR assays to verify that the plasmids can replicate in ''S. oneidensis''. The results showed that all plasmids can replicate normally.(Fig.5)
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    <img src="https://static.igem.wiki/teams/5034/engineering/hydrolyase-colony-pcr.png" style="width: 300px; height: auto;">
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<img src="https://static.igem.wiki/teams/5034/engineering/colony-pcr.png" style="width: 60%; height: auto;">
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<p>Figure 5: Colony PCR indicating that different plasmids can replicate in <i>S. oneidensis</i></p>
 +
</div>
 
</html>
 
</html>
  
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Figure 5: Colony PCR indicating that different plasmids can replicate in Shewanella
 
  
 
===Origin (Organism)===
 
===Origin (Organism)===
−
The PAP gene was sourced from Acinetobacter johnsonii. The PYYDT plasmid backbone is a standard vector used for gene expression in synthetic biology applications.
+
The PAP gene was sourced from ''Acinetobacter johnsonii''.  
 +
 
 +
The pBBR1MCS plasmid backbone is a standard vector used for gene expression in synthetic biology applications. The plasmid backbone(BBa_K5034201) of this part is a modified version of pBBR1MCS, with a double terminator(BBa_B0015) on it.
  
 
===Experimental Characterization and results===
 
===Experimental Characterization and results===
−
Using molybdate assays to determine Pi concentration and found that PAP do not have a good capacity to polymerise phosphorus (but better than PPN1 and PPX).
+
We first determined the electroproduction capacity of <i>S. oneidensis</i> after introduction of the PAP enzyme.(Fig.6)
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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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<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: 300px; height: auto;">
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<img src="https://static.igem.wiki/teams/5034/engineering/current-with-different-hydrolases.png" style="width: 60%; height: auto;">
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</body>
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<p>Figure 6: Statistical data on electricity production capacity of <i>S. oneidensis</i> with the introduction of different hydrolases</p>
 +
</div>
 
</html>
 
</html>
  
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Figure 6: statistical data on electricity production capacity of Shewanella 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 <i>S. oneidensis</i>
 +
 
 +
Afterwards, we also measured the phosphorus aggregation capacity of <i>S. oneidensis</i>(Fig.7) and found no significant difference from the wild type, indicating that its introduction did not promote phosphorus uptake in <i>S. oneidensis</i> either.
  
 
<html>
 
<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: 300px; height: auto;">
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<img src="https://static.igem.wiki/teams/5034/engineering/pap-pi.png" style="width: 60%; height: auto;">
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</body>
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<p>Figure 7: Statistical data on the phosphorus accumulation capacity of <i>S. oneidensis</i> with PAP</p>
 +
</div>
 
</html>
 
</html>
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Tests to measure ATP levels in <i>S. oneidensis</i>(Fig.8) 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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Figure 7: statistical data on the phosphorus accumulation capacity of Shewanella with PAP
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<html>
 
<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: 300px; height: auto;">
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<img src="https://static.igem.wiki/teams/5034/engineering/atp-level-with-different-hydrolyases.png" style="width: 60%; height: auto;">
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</body>
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<p>Figure 8: ATP level in <i>S. oneidensis</i> with the introduction of different hydrolases</p>
 +
</div>
 
</html>
 
</html>
 +
In summary, the option of importing PAP is not a good one.
  
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Figure 8: ATP level in Shewanella with the introduction of different hydrolases
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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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===Usage and Biology===
+
===Chassis and genetic context===
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The PAP gene from Acinetobacter johnsonii is cloned into the PYYDT plasmid. This composite 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.
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Chassis：''Shewanella onediensis'' MR-1
  
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The gene can be expressed and function properly in <i>S. oneidensis</i>.
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<span class='h3bb'>Sequence and Features</span>
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-->
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+
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===Chassis and genetic===
+
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Chassis：Shewanella onediensis MR-1
+
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The gene can be expressed and function properly in Shewanella.
+
  
 
===Potential applications===
 
===Potential applications===
−
PAP (polyphosphate:AMP phosphotransferase) has several 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.
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* Phosphate Metabolism: Key role in energy and phosphate metabolism studies.
+
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* Bioenergy Production: Enhances microbial electricity generation.
+
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* Industrial Biotechnology: Optimizes bioprocesses involving polyphosphate.
+
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* Microbial Metabolic Engineering: Improves microbial performance in synthetic biology.
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* Environmental Remediation: Aids in phosphate degradation to combat eutrophication.
+
  
 
===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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===Functional Parameters===
 
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<partinfo>BBa_K5034219 parameters</partinfo>
 
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Revision as of 07:41, 2 October 2024


Poly P + AMP-> ADP

Basic Description

This plasmid is the expression vector of PAP gene(BBa_K5034212).

The basic part(BBa_K5034212) encodes the PAP gene which is initially from Acinetobacter johnsonii and we performed codon optimization on, is expressed in the pBBR1MCS-terminator plasmid. This 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
    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]

Construct features

Plasmid backbone: pBBR1MCS-terminator. The Lac promoter and the double terminator are on the plasmid backbone.

  • 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: Basic construction of PAP plasmid

The plasmid map of this part is shown in the figure below.(Fig.4)

Figure 3: Construction of PAP plasmid

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.

Figure 4: PCR of target genes PCR before plasmids construction (The extra small fragment in the picture is primer dimer)

We conducted colony PCR assays to verify that the plasmids can replicate in S. oneidensis. The results showed that all plasmids can replicate normally.(Fig.5)

Figure 5: Colony PCR indicating that different plasmids can replicate in S. oneidensis


Origin (Organism)

The PAP gene was sourced from Acinetobacter johnsonii.

The pBBR1MCS plasmid backbone is a standard vector used for gene expression in synthetic biology applications. The plasmid backbone(BBa_K5034201) of this part is a modified version of pBBR1MCS, with a double terminator(BBa_B0015) on it.

Experimental Characterization and results

We first determined the electroproduction capacity of S. oneidensis after introduction of the PAP enzyme.(Fig.6)

Figure 6: 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.7) and found no significant difference from the wild type, indicating that its introduction did not promote phosphorus uptake in S. oneidensis either.

Figure 7: Statistical data on the phosphorus accumulation capacity of S. oneidensis with PAP

Tests to measure ATP levels in S. oneidensis(Fig.8) also showed that its introduction did not result in an increase in the metabolic strength of S. oneidensis either.

Figure 8: 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 onediensis 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.