Difference between revisions of "Part:BBa K5034217"
Line 6: Line 6:
 
===Basic Description===
 
===Basic Description===
 
This composite part includes the <i>PPK2</i> gene from <i>Pseudomonas aeruginosa</i> and the <i>NADK</i> gene from <i>Mycobacterium tuberculosis</i> H37Rv. we performed codon optimization on both and expressed in the <html><a href="https://parts.igem.org/Part:BBa_K5034201">pBBR1MCS-terminator</a></html> plasmid together. The PPK2 enzyme facilitates the reversible conversion between inorganic polyphosphate (PolyP) and inorganic phosphate (Pi), while the NADK enzyme converts PolyP to NADP. Importing them separately was successful, thus we intend to proceed with continued optimisation by their combination.
 
This composite part includes the <i>PPK2</i> gene from <i>Pseudomonas aeruginosa</i> and the <i>NADK</i> gene from <i>Mycobacterium tuberculosis</i> H37Rv. we performed codon optimization on both and expressed in the <html><a href="https://parts.igem.org/Part:BBa_K5034201">pBBR1MCS-terminator</a></html> plasmid together. The PPK2 enzyme facilitates the reversible conversion between inorganic polyphosphate (PolyP) and inorganic phosphate (Pi), while the NADK enzyme converts PolyP to NADP. Importing them separately was successful, thus we intend to proceed with continued optimisation by their combination.
−
This part consists of two enzymes, one is a reversible enzyme that converts Pi and PolyP, and the other is an enzyme that converts PolyP to NADK. The tandem connection of the two enzymes actually promoted the synthesis of NADK, and by maintaining some polyp reserves, it was able to improve the efficiency of electrical production and improve the phosphorus accumulation capacity of <i>S.oneidensis</i>.
+
This part consists of two enzymes, one is a reversible enzyme that converts Pi and PolyP, and the other is an enzyme that converts PolyP to NADK. The tandem connection of the two enzymes actually promoted the synthesis of NADK, and by maintaining some PolyP reserves, it was able to improve the efficiency of electrical production and improve the phosphorus accumulation capacity of <i>S.oneidensis</i>.
  
  
 
===Construct features===
 
===Construct features===
 
<html>
 
<html>
−
<p>Promoter: Constitutive promoter for continuous expression. We use tac promoter in our experiment.</p>
+
<p>Promoter: We use lac promoter in our experiment. There isn't lacI downstream，so it's constitutive promoter for continuous expression.</p>
−
<p>RBS: Strong ribosome binding site for efficient translation. We use BBa-B0034 which shows the strongest translation in our experiment.</p>
+
<p>RBS: Strong ribosome binding site for efficient translation. We use <a href="https://parts.igem.org/Part:BBa_B0034">BBa-B0034</a> which shows the strongest translation in our experiment.</p>
 
<p><i>PPK2</i> Coding Sequence: Encodes the polyphosphate kinase 2 enzyme.</p>
 
<p><i>PPK2</i> Coding Sequence: Encodes the polyphosphate kinase 2 enzyme.</p>
 
<p><i>NADK</i> Coding Sequence: Encodes the NAD kinase enzyme.</p>
 
<p><i>NADK</i> Coding Sequence: Encodes the NAD kinase enzyme.</p>
 
<p>Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use a double terminator rrnBT1-T7TE(BBa_B0015) in our experiment.
 
<p>Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use a double terminator rrnBT1-T7TE(BBa_B0015) in our experiment.
 
</p>
 
</p>
−
<p>The translational unit is composed of the components above. In this composite part, promotor and terminator is not included.Because the bachbone has promotor sequence and terminator sequence.</p>
+
<p>The translational unit is composed of the components above. In this composite part, promotor and terminator is not included.Because the backbone has promotor sequence and terminator sequence.</p>
 
</html>
 
</html>
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K5034217 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5034217 SequenceAndFeatures</partinfo>
  
−
===Origin (Organism)===
+
===Origin===
 
<i>PPK2</i> Gene: <i>Pseudomonas aeruginosa</i> PAO1 strain.
 
<i>PPK2</i> Gene: <i>Pseudomonas aeruginosa</i> PAO1 strain.
 
<i>NADK</i> Gene: <i>Mycobacterium tuberculosis</i> H37Rv strain.
 
<i>NADK</i> Gene: <i>Mycobacterium tuberculosis</i> H37Rv strain.
  
 
===Experimental Characterization and results===
 
===Experimental Characterization and results===
−
Students from dry lab group using mathematical modelling to simulate the introduction of the two enzymes and found an enhancement in the polyphosphate and electroproduction capabilities of <i>S.oneidensis</i>.
+
Students from dry lab group using mathematical modelling to simulate the introduction of the two enzymes and found an enhancement in the polyphosphate and electroproduction capabilities of <i>S.oneidensis</i>.(Fig.1)
 
<html>
 
<html>
 
<div align="center">
 
<div align="center">
Line 36: Line 36:
 
</div>
 
</div>
 
</html>
 
</html>
−
Then, the students in the wet experiment constructed a component that linked two enzymes in series, and the results of colony PCR confirmed the success of our construction. （Fig.2）
+
Then, the students in the wet lab constructed a component that linked two enzymes in series, and the results of colony PCR confirmed the success of our construction. （Fig.2）
 
<html>
 
<html>
 
<div align="center">
 
<div align="center">
 
<img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/results/figure16.png">
 
<img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/results/figure16.png">
 
<p>
 
<p>
−
Figure 2: Clony PCR to prove that PPK2-NADK plasmid is introduced to <i>S.oneidensis</i>
+
Figure 2: Colony PCR to prove that PPK2-NADK plasmid is introduced to <i>S.oneidensis</i>
 
</p>
 
</p>
 
</div>
 
</div>
 
</html>
 
</html>
−
After successful construction, we transferred it into Shewanella and conducted measurements of its electricity production and phosphorus accumulation effects. We found that after transferring into the Shewanella bacterium, both the electricity production and phosphorus accumulation efficiency were significantly improved compared to the wild type.
+
After successful construction, we transferred it into <i>S.oneidensis</i> and conducted measurements of its electricity production and phosphorus accumulation effects. We found that after transferring into the <i>S.oneidensis</i>, both the electricity production and phosphorus accumulation efficiency were significantly improved compared to the wild type.
 
<html>
 
<html>
 
<div align="center">
 
<div align="center">
 
<img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/engineering/fig21.png">
 
<img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/engineering/fig21.png">
 
<p>
 
<p>
−
Figure 3: statistical data on electricity production capacity and phosphorus accumulation capacity of Shewanella with the introduction of PPK2-NADK
+
Figure 3: Electricity production capacity and phosphorus accumulation capacity of <i>S.oneidensis</i> with the introduction of <i>PPK2-NADK</i>
 
</p>
 
</p>
 
</div>
 
</div>
 
</html>
 
</html>
−
The phosphorus aggregation effect was measured in M9 cultural medium, and the electricity generation effect was measured in LB medium, because M9 medium is the medium used in practical applications and can be better combined with practical applications.
+
The phosphorus accumulation effect was measured in M9 cultural medium, and the electricity generation effect was measured in LB medium, because M9 medium is the medium used in practical applications and can be better combined with practical applications.
  
−
Subsequently, we also investigated the reasons for the improvement in electricity generation and phosphorus aggregation efficiency. We found that the levels of ATP and NADH inside the cell were significantly increased(Fig.4), indicating that the metabolic level of Shewanella increased, leading to an increase in electricity production and phosphorus accumulation levels.
+
Subsequently, we also investigated the reasons for the improvement in electricity generation and phosphorus accumulate efficiency. We found that the levels of ATP and NADH/NAD<sup>+</sup> inside the cell were significantly increased(Fig.4), indicating that the metabolic level of <i>S.oneidensis</i> increased, leading to an increase in electricity production and phosphorus accumulation levels.
  
 
<html>
 
<html>
Line 62: Line 62:
 
<img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/results/figure18.png">
 
<img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/results/figure18.png">
 
<p>
 
<p>
−
Figure 4: levels of ATP and NADH/NAD<sup>+</sup> of S.oneidensis with the introduction of PPK2-NADK
+
Figure 4: Levels of ATP and NADH/NAD<sup>+</sup> of S.oneidensis with the introduction of PPK2-NADK
 
</p>
 
</p>
 
</div>
 
</div>
Line 71: Line 71:
 
<img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/results/figure19.png">
 
<img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/results/figure19.png">
 
<p>
 
<p>
−
Figure 5: cyclic voltammetry show higher redox activity in the SPPK2-NADK strain
+
Figure 5: Cyclic voltammetry show higher redox activity in the SPPK2-NADK strain
 
</p>
 
</p>
 
</div>
 
</div>
Line 89: Line 89:
 
<img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/results/figure21.png">
 
<img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/results/figure21.png">
 
<p>
 
<p>
−
Figure 7:  output power of the PPK2-NADK strain
+
Figure 7:  Output power of the PPK2-NADK strain
 
</p>
 
</p>
 
</div>
 
</div>
Line 98: Line 98:
  
 
===Potential Applications===
 
===Potential Applications===
−
In bioelectrochemical Systems, we can utilize PolyP and NADP in microbial fuel cells for further improved electron transfer and energy production.
+
In bioelectrochemical systems, we can utilize PolyP and NADP in microbial fuel cells for further improved electron transfer and energy production.
  
−
In fact, based on the outcome, we make a hardware to demostrate its application.We can use it to collect Pi in the soil and produce electricity to be used by human.
+
In fact, based on the results, we make a hardware to demostrate its application.We can use it to collect Pi in the soil and produce electricity to be used by human.
 
<html>
 
<html>
 
<div align="center">
 
<div align="center">
Line 109: Line 109:
 
</div>
 
</div>
 
</html>
 
</html>
−
There are a list of probably application:
+
There are a list of otherprobably applications:
  
 
Managing phosphate levels in contaminated environments;  
 
Managing phosphate levels in contaminated environments;  

Revision as of 19:18, 1 October 2024

PolyP <->Pi, Poly P -> NADP


Basic Description

This composite part includes the PPK2 gene from Pseudomonas aeruginosa and the NADK gene from Mycobacterium tuberculosis H37Rv. we performed codon optimization on both and expressed in the pBBR1MCS-terminator plasmid together. The PPK2 enzyme facilitates the reversible conversion between inorganic polyphosphate (PolyP) and inorganic phosphate (Pi), while the NADK enzyme converts PolyP to NADP. Importing them separately was successful, thus we intend to proceed with continued optimisation by their combination. This part consists of two enzymes, one is a reversible enzyme that converts Pi and PolyP, and the other is an enzyme that converts PolyP to NADK. The tandem connection of the two enzymes actually promoted the synthesis of NADK, and by maintaining some PolyP reserves, it was able to improve the efficiency of electrical production and improve the phosphorus accumulation capacity of S.oneidensis.


Construct features

Promoter: We use lac promoter in our experiment. There isn't lacI downstream，so it's constitutive promoter for continuous expression.

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.

NADK Coding Sequence: Encodes the NAD kinase enzyme.

Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use a double terminator rrnBT1-T7TE(BBa_B0015) in our experiment.

The translational unit is composed of the components above. In this composite part, promotor and terminator is not included.Because the backbone has promotor sequence and terminator sequence.

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]

Origin

PPK2 Gene: Pseudomonas aeruginosa PAO1 strain. NADK Gene: Mycobacterium tuberculosis H37Rv strain.

Experimental Characterization and results

Students from dry lab group using mathematical modelling to simulate the introduction of the two enzymes and found an enhancement in the polyphosphate and electroproduction capabilities of S.oneidensis.(Fig.1)

Figure 1: Experimental modelling proves that importing PPK2 and NADK simultaneously is better than importing PPK2 or NADK separately

Then, the students in the wet lab constructed a component that linked two enzymes in series, and the results of colony PCR confirmed the success of our construction. （Fig.2）

Figure 2: Colony PCR to prove that PPK2-NADK plasmid is introduced to S.oneidensis

After successful construction, we transferred it into S.oneidensis and conducted measurements of its electricity production and phosphorus accumulation effects. We found that after transferring into the S.oneidensis, both the electricity production and phosphorus accumulation efficiency were significantly improved compared to the wild type.

Figure 3: Electricity production capacity and phosphorus accumulation capacity of S.oneidensis with the introduction of PPK2-NADK

The phosphorus accumulation effect was measured in M9 cultural medium, and the electricity generation effect was measured in LB medium, because M9 medium is the medium used in practical applications and can be better combined with practical applications.

Subsequently, we also investigated the reasons for the improvement in electricity generation and phosphorus accumulate efficiency. We found that the levels of ATP and NADH/NAD+ inside the cell were significantly increased(Fig.4), indicating that the metabolic level of S.oneidensis increased, leading to an increase in electricity production and phosphorus accumulation levels.

Figure 4: Levels of ATP and NADH/NAD+ of S.oneidensis with the introduction of PPK2-NADK

To explore the possible applications of this component, we conducted a full cell experiment using Shewanella bacteria.Firstly, we use cyclic voltammetry (CV) and linear sweep voltammetry (LSV) techniques for testing. The CV curve shows higher redox activity in the SPPK2-NADK strain.(Fig.5)

Figure 5: Cyclic voltammetry show higher redox activity in the SPPK2-NADK strain

the LSV curve indicates lower internal resistance in the MFC cells of the SPPK2-NADK strain.(Fig.6)

Figure 6: LSV curve indicates lower internal resistance in the MFC cells of the SPPK2-NADK strain

Next, we measure the relative output power. The power density results show that the SPPK2-NADK strain has a maximum output power of 243.77 ± 25.2 mW/m², which is 2.32 times higher than the WT strain’s output power density (105.06 ± 11.72 mW/m²) (Fig. 7).

Figure 7: Output power of the PPK2-NADK strain

Chassis and genetic context

We express this gene on Shawanella oneidensis MR-1

Potential Applications

In bioelectrochemical systems, we can utilize PolyP and NADP in microbial fuel cells for further improved electron transfer and energy production.

In fact, based on the results, we make a hardware to demostrate its application.We can use it to collect Pi in the soil and produce electricity to be used by human.

Figure 7: Hardware about its application

There are a list of otherprobably 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.Mori S, Yamasaki M, Maruyama Y, Momma K, Kawai S, Hashimoto W, Mikami B, Murata K. Crystallographic studies of Mycobacterium tuberculosis polyphosphate/ATP-NAD kinase complexed with NAD. J Biosci Bioeng. 2004;98(5):391-3.

2. 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.

3. Neville N, Roberge N, Jia Z. Polyphosphate Kinase 2 (PPK2) Enzymes: Structure, Function, and Roles in Bacterial Physiology and Virulence. Int J Mol Sci. 2022 Jan 8;23(2):670.