Difference between revisions of "Part:BBa K5034219"
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Figure 1: Basic function of PAP
 
Figure 1: Basic function of PAP
  
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===Usage and Biology===
 
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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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<span class='h3bb'>Sequence and Features</span>
 
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===Sequence and Features===
 
===Sequence and Features===
 
<partinfo>BBa_K5034219 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5034219 SequenceAndFeatures</partinfo>
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===Origin (Organism)===
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The PAP gene was sourced from Acinetobacter johnsonii. The PYYDT plasmid backbone is a standard vector used for gene expression in synthetic biology applications.
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===Construct features===
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Promoter: Constitutive promoter for continuous expression. We use tac promoter in our experiment.
 
Promoter: Constitutive promoter for continuous expression. We use tac promoter in our experiment.
 
PAP Coding Sequence: Encodes the polyphosphate:AMP phosphotransferase enzyme.
 
PAP Coding Sequence: Encodes the polyphosphate:AMP phosphotransferase enzyme.
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Figure 5: Colony PCR indicating that different plasmids can replicate in Shewanella
 
Figure 5: Colony PCR indicating that different plasmids can replicate in Shewanella
 +
 +
===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.
 +
 
===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).
 
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).
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     <img src="https://static.igem.wiki/teams/5034/engineering/current-with-different-hydrolases.png<html>" 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: 300px; height: auto;">
 
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Figure 8: ATP level in Shewanella with the introduction of different hydrolases
 
Figure 8: ATP level in Shewanella with the introduction of different hydrolases
 +
 +
===Usage and Biology===
 +
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.
 +
 +
<!--
 +
<span class='h3bb'>Sequence and Features</span>
 +
-->
 +
 +
===Chassis and genetic===
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Chassis：Shewanella onediensis MR-1
 +
The gene can be expressed and function properly in Shewanella.
 +
 +
===Potential applications===
 +
PAP (polyphosphate:AMP phosphotransferase) has several potential applications:
 +
* Phosphate Metabolism: Key role in energy and phosphate metabolism studies.
 +
* Bioenergy Production: Enhances microbial electricity generation.
 +
* Industrial Biotechnology: Optimizes bioprocesses involving polyphosphate.
 +
* Microbial Metabolic Engineering: Improves microbial performance in synthetic biology.
 +
* 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.

Revision as of 14:31, 29 September 2024


Poly P + AMP-> ADP

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 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 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.

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]

Promoter: Constitutive promoter for continuous expression. We use tac promoter in our experiment. PAP Coding Sequence: Encodes the polyphosphate:AMP phosphotransferase enzyme. Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use T7Te terminator in our experiment.

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

Figure 3: Basic construction of PAP plasmid

Figure 4: Construction of PAP plasmid

Figure 5: Colony PCR indicating that different plasmids can replicate in Shewanella

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.

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). 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. Electricity production: Using half-cell reaction(electrochemistry) to measure the electricity production ability. Capacity to polymerize phosphorus: Conducting molybdate assays to determine Pi concentration. 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).

Figure 6: statistical data on electricity production capacity of Shewanella with the introduction of different hydrolases

Figure 7: statistical data on the phosphorus accumulation capacity of Shewanella with PAP

Figure 8: ATP level in Shewanella with the introduction of different hydrolases

Usage and Biology

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.


Chassis and genetic

Chassis：Shewanella onediensis MR-1 The gene can be expressed and function properly in Shewanella.

Potential applications

PAP (polyphosphate:AMP phosphotransferase) has several potential applications:

  • Phosphate Metabolism: Key role in energy and phosphate metabolism studies.
  • Bioenergy Production: Enhances microbial electricity generation.
  • Industrial Biotechnology: Optimizes bioprocesses involving polyphosphate.
  • Microbial Metabolic Engineering: Improves microbial performance in synthetic biology.
  • Environmental Remediation: Aids in phosphate degradation to combat eutrophication.

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.