Difference between revisions of "Part:BBa K5034227"
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===Basic Description===
 
===Basic Description===
 
This basic part encodes the PPK2 gene which is initially from Pseudomonas aeruginosa and we performed codon optimization on, is expressed in the PYYDT plasmid. The PPK2 enzyme facilitates the reversible conversion between inorganic polyphosphate (PolyP) and inorganic phosphate (Pi), playing a crucial role in phosphate metabolism. It distinguished from PPK1 by the following: synthesis of poly P from GTP or ATP, a preference for Mn2+ over Mg2+, and a stimulation by Poly P. The reverse reaction, a poly P-driven nucleoside diphosphate kinase synthesis of GTP from GDP, is 75-fold greater than the forward reaction, Poly P synthesis from GTP.
 
This basic part encodes the PPK2 gene which is initially from Pseudomonas aeruginosa and we performed codon optimization on, is expressed in the PYYDT plasmid. The PPK2 enzyme facilitates the reversible conversion between inorganic polyphosphate (PolyP) and inorganic phosphate (Pi), playing a crucial role in phosphate metabolism. It distinguished from PPK1 by the following: synthesis of poly P from GTP or ATP, a preference for Mn2+ over Mg2+, and a stimulation by Poly P. The reverse reaction, a poly P-driven nucleoside diphosphate kinase synthesis of GTP from GDP, is 75-fold greater than the forward reaction, Poly P synthesis from GTP.
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<img style="width:60%;height:auto;" src="https://static.igem.wiki/teams/5034/engineering/mechanism-of-ppk2.png">
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<p>
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Figure 1: Basic function of PPK2
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</p>
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</html>
  
 
===Sequence and Features===
 
===Sequence and Features===
 
<partinfo>BBa_K5034227 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5034227 SequenceAndFeatures</partinfo>
  
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Figure 1: Basic function of PPK2
 
  
 
===Chassis and Genetic Context===
 
===Chassis and Genetic Context===
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===Construct features===
 
===Construct features===
 
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.
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RBS: Strong ribosome binding site for efficient translation. We use BBa-B0034 which shows the strongest translation in our experiment.
 
RBS: Strong ribosome binding site for efficient translation. We use BBa-B0034 which shows the strongest translation in our experiment.
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PPK2 Coding Sequence: Encodes the polyphosphate kinase 2 enzyme.
 
PPK2 Coding Sequence: Encodes the polyphosphate kinase 2 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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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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<img style="width:60%;height:auto;" src="https://static.igem.wiki/teams/5034/engineering/fig17.png">
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<p>
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Figure 2: PCR of target genes before plasmids construction (The extra small fragment in the picture is primer dimer)
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</p>
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</html>
  
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Figure 2: PCR of target genes before plasmids construction (The extra small fragment in the picture is primer dimer)
 
  
  
  
 
Figure 3: Basic construction of PPK2 plasmid
 
Figure 3: Basic construction of PPK2 plasmid
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Figure 4: Construction of PPK2 plasmid
 
Figure 4: Construction of PPK2 plasmid
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===Experimental Characterization and results===
 
===Experimental Characterization and results===
 
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.
 
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.
 
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.
 
Capacity to polymerize phosphorus: Conducting molybdate assays to determine Pi concentration.
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The expression of hydrolase PPK2 showed relatively high phosphorus accumulation and electricity generation ability. Also, the ATP level is considerably enhanced.
 
The expression of hydrolase PPK2 showed relatively high phosphorus accumulation and electricity generation ability. Also, the ATP level is considerably enhanced.
  
  
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<html>
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<img style="width:60%;height:auto;" src="https://static.igem.wiki/teams/5034/current.png">
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<p>
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Figure 6: Statistical data on electricity production capacity of Shewanella with the introduction of different hydrolases
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</p>
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</html>
  
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<img style="width:60%;height:auto;" src="https://static.igem.wiki/teams/5034/engineering/pi-of-ppk2.png">
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Figure 6: statistical data on electricity production capacity of Shewanella with the introduction of different hydrolases
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Figure 7: statistical data on the phosphorus accumulation capacity of Shewanella with PPK2  
 
Figure 7: statistical data on the phosphorus accumulation capacity of Shewanella with PPK2  
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</p>
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<img style="width:60%;height:auto;" src="https://static.igem.wiki/teams/5034/atp.png">
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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
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</p>
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</html>
  
 
===Potential Applications===
 
===Potential Applications===

Revision as of 11:53, 30 September 2024


Pi <-> PolyP

Basic Description

This basic part encodes the PPK2 gene which is initially from Pseudomonas aeruginosa and we performed codon optimization on, is expressed in the PYYDT plasmid. The PPK2 enzyme facilitates the reversible conversion between inorganic polyphosphate (PolyP) and inorganic phosphate (Pi), playing a crucial role in phosphate metabolism. It distinguished from PPK1 by the following: synthesis of poly P from GTP or ATP, a preference for Mn2+ over Mg2+, and a stimulation by Poly P. The reverse reaction, a poly P-driven nucleoside diphosphate kinase synthesis of GTP from GDP, is 75-fold greater than the forward reaction, Poly P synthesis from GTP.

Figure 1: Basic function of PPK2

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]


Chassis and Genetic Context

Successfully expressed in Escherichia coli DH5α and BL21(DE3) strains.

Construct features

Promoter: Constitutive promoter for continuous expression. We use tac promoter in our experiment.

RBS: Strong ribosome binding site for efficient translation. We use BBa-B0034 which shows the strongest translation in our experiment.

PPK2 Coding Sequence: Encodes the polyphosphate kinase 2 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 before plasmids construction (The extra small fragment in the picture is primer dimer)



Figure 3: Basic construction of PPK2 plasmid



Figure 4: Construction of PPK2 plasmid



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

Origin (Organism)

PPK2 Gene Source: Pseudomonas aeruginosa PAO1 strain.

Plasmid Backbone: pBBR1MCS plasmid, a standard vector used for gene expression in synthetic biology. 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

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.

The expression of hydrolase PPK2 showed relatively high phosphorus accumulation and electricity generation ability. Also, the ATP level is considerably enhanced.


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 PPK2

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

Potential Applications

Managing phosphate levels in contaminated environments;

Enhancing phosphate metabolism in engineered microbial systems;

Optimizing phosphate utilization in industrial microbial processes.

Enhancing the performance of bioelectrochemical systems for electricity generation in providing a renewable and sustainable source of electricity, reducing reliance on fossil fuels and contributing to cleaner energy production.

References

1.Zhang, H., Ishige, K., & Kornberg, A. (2002). A polyphosphate kinase (PPK2) widely conserved in bacteria. Proceedings of the National Academy of Sciences, 99(26), 16678-16683. 2. Neville, N., Roberge, N., & Jia, Z. (2022). Polyphosphate Kinase 2 (PPK2) enzymes: Structure, function, and roles in bacterial physiology and virulence. International Journal of Molecular Sciences, 23(2), 670.  3. Itoh, H., & Shiba, T. (2004). Polyphosphate synthetic activity of polyphosphate:AMP phosphotransferase in Acinetobacter johnsonii 210A. Journal of Bacteriology, 186(15), 5178-5181.