Difference between revisions of "Part:BBa K5034217"
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===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), | + | 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), and the NADK enzyme converts PolyP to NADP.(Fig.1-2) Importing them separately was successful, thus we intended to proceed with continued optimisation by their combination.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>. |
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+ | <html> | ||
+ | <div align="center"> | ||
+ | <img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/engineering/mechanism-of-nadk.png"> | ||
+ | <p> | ||
+ | Figure 1: Mechanism of <i>NADK</i> | ||
+ | </p> | ||
+ | </div> | ||
+ | </html> | ||
+ | <html> | ||
+ | <div align="center"> | ||
+ | <img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/engineering/mechanism-of-ppk2.png"> | ||
+ | <p> | ||
+ | Figure 2: Mechanism of <i>PPK2</i> | ||
+ | </p> | ||
+ | </div> | ||
+ | </html> | ||
===Sequence and Features=== | ===Sequence and Features=== | ||
<html> | <html> | ||
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<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> | <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> | ||
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+ | <div align="center"> | ||
+ | <img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/results/new/basic-structure-of-ppk2-nadk.png"> | ||
+ | <p> | ||
+ | Figure 3: Basic structure of <i>PPK2</i>-<i>NADK</i> | ||
+ | </p> | ||
+ | </div> | ||
+ | </html> | ||
+ | <html> | ||
+ | <div align="center"> | ||
+ | <img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/engineering/pbbr1mcs-terminator-ppk2-nadk.png"> | ||
+ | <p> | ||
+ | Figure 4: Plasmid profile of PBBR1mcs-Terminator-PPK2-NADK | ||
+ | </p> | ||
+ | </div> | ||
+ | </html> | ||
<partinfo>BBa_K5034217 SequenceAndFeatures</partinfo> | <partinfo>BBa_K5034217 SequenceAndFeatures</partinfo> | ||
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===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>.(Fig. | + | 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.5) |
<html> | <html> | ||
<div align="center"> | <div align="center"> | ||
<img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/modelling.png"> | <img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/modelling.png"> | ||
<p> | <p> | ||
− | Figure | + | Figure 5: Experimental modelling proves that importing <i>PPK2</i> and <i>NADK</i> simultaneously is better than importing <i>PPK2</i> or <i>NADK</i> separately |
</p> | </p> | ||
</div> | </div> | ||
</html> | </html> | ||
− | 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. | + | 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. |
− | < | + | Since the <i>PPK2</i> gene is approximately 1.1 kb and the <i>NADK</i> gene is about 1.0 kb, the <i>PPK2-NADK</i> construct should be approximately 2.1 kb. The colony PCR results show a band at about 2.1 kb, confirming that we successfully introduce the plasmid containing <i>PPK2-NADK</i> into <i>S.oneidensis</i>.(Fig.6) |
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− | </ | + | |
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<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 | + | Figure 6: 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 <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. | + | 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.(Fig.7) |
<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 | + | Figure 7: Electricity production capacity and phosphorus accumulation capacity of <i>S.oneidensis</i> with the introduction of <i>PPK2-NADK</i> |
</p> | </p> | ||
</div> | </div> | ||
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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. | 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<sup>+</sup> inside the cell were significantly increased(Fig. | + | 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.8), indicating that the metabolic level of <i>S.oneidensis</i> increased, leading to an increase in electricity production and phosphorus accumulation levels. |
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<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 | + | Figure 8: Levels of ATP and NADH/NAD<sup>+</sup> of S.oneidensis with the introduction of PPK2-NADK |
</p> | </p> | ||
</div> | </div> | ||
</html> | </html> | ||
− | 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. | + | 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.9) |
<html> | <html> | ||
<div align="center"> | <div align="center"> | ||
<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 | + | Figure 9: Cyclic voltammetry show higher redox activity in the SPPK2-NADK strain |
</p> | </p> | ||
</div> | </div> | ||
</html> | </html> | ||
− | The LSV curve indicates lower internal resistance in the MFC cells of the SPPK2-NADK strain.(Fig. | + | The LSV curve indicates lower internal resistance in the MFC cells of the SPPK2-NADK strain.(Fig.10) |
<html> | <html> | ||
<div align="center"> | <div align="center"> | ||
<img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/results/figure20.png"> | <img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/results/figure20.png"> | ||
<p> | <p> | ||
− | Figure | + | Figure 10: LSV curve indicates lower internal resistance in the MFC cells of the SPPK2-NADK strain |
</p> | </p> | ||
</div> | </div> | ||
</html> | </html> | ||
− | 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. | + | 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.11). |
<html> | <html> | ||
<div align="center"> | <div align="center"> | ||
<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 | + | Figure 11: Output power of the PPK2-NADK strain |
</p> | </p> | ||
</div> | </div> | ||
</html> | </html> | ||
+ | |||
+ | Experimental manipulation: | ||
+ | |||
+ | <html><a href="https://2024.igem.wiki/nanjing-china/experiments#s16">Electricity production:</a>Using half-cell reaction(electrochemistry) We use half-cell experiment to measure the electricity production ability.</html> | ||
+ | |||
+ | <html><a href="https://2024.igem.wiki/nanjing-china/experiments#s11">Capacity to absorb phosphorus:</a>Conducting molybdate assays to determine Pi concentration.</html> | ||
+ | |||
+ | <html><a href="https://2024.igem.wiki/nanjing-china/experiments#s13">Determination of ATP levels:</a>We use enhanced ATP Assay Kit.</html> | ||
+ | |||
+ | <html><a href="https://2024.igem.wiki/nanjing-china/experiments#s14">Determination of NAD<sup>+</sup>/NADH levels:</a>We use NAD+ /NADH Assay Kit with WST-8.</html> | ||
+ | |||
+ | <html><a href="https://2024.igem.wiki/nanjing-china/experiments#s17">Full cell experiment:</a>We use MFC system of a two-chamber electrochemical reactor.</html> | ||
+ | |||
+ | <html><p>More Details of all experiments can be found at the <a href="https://2024.igem.wiki/nanjing-china/experiments">Experiments section on the Wiki.</a></p></html> | ||
===Chassis and genetic context=== | ===Chassis and genetic context=== | ||
− | We express this | + | We express this composite part on <i> Shewanella oneidensis</i> MR-1. |
===Potential Applications=== | ===Potential Applications=== | ||
− | In bioelectrochemical systems, we can utilize PolyP and NADP in microbial fuel cells for further | + | In bioelectrochemical systems, we can utilize PolyP and NADP in microbial fuel cells for further electron transfer improvement 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.(Fig. | + | 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.(Fig.12) |
<html> | <html> | ||
<div align="center"> | <div align="center"> | ||
<img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/hardware/figure13.jpg"> | <img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/hardware/figure13.jpg"> | ||
<p> | <p> | ||
− | Figure | + | Figure 12: Hardware about its application |
</p> | </p> | ||
</div> | </div> |
Latest revision as of 11:53, 2 October 2024
PolyP <->Pi, Poly P -> NADP
Contents
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), and the NADK enzyme converts PolyP to NADP.(Fig.1-2) Importing them separately was successful, thus we intended to proceed with continued optimisation by their combination.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.
Figure 1: Mechanism of NADK
Figure 2: Mechanism of PPK2
Sequence and 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.
Figure 3: Basic structure of PPK2-NADK
Figure 4: Plasmid profile of PBBR1mcs-Terminator-PPK2-NADK
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.5)
Figure 5: Experimental modelling proves that importing PPK2 and NADK simultaneously is better than importing PPK2 or NADK separately
Since the PPK2 gene is approximately 1.1 kb and the NADK gene is about 1.0 kb, the PPK2-NADK construct should be approximately 2.1 kb. The colony PCR results show a band at about 2.1 kb, confirming that we successfully introduce the plasmid containing PPK2-NADK into S.oneidensis.(Fig.6)
Figure 6: 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.(Fig.7)
Figure 7: Electricity production capacity and phosphorus accumulation capacity of S.oneidensis with the introduction of PPK2-NADK
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.8), indicating that the metabolic level of S.oneidensis increased, leading to an increase in electricity production and phosphorus accumulation levels.
Figure 8: Levels of ATP and NADH/NAD+ of S.oneidensis with the introduction of PPK2-NADK
Figure 9: Cyclic voltammetry show higher redox activity in the SPPK2-NADK strain
Figure 10: LSV curve indicates lower internal resistance in the MFC cells of the SPPK2-NADK strain
Figure 11: Output power of the PPK2-NADK strain
Experimental manipulation:
Electricity production:Using half-cell reaction(electrochemistry) We use half-cell experiment to measure the electricity production ability.
Capacity to absorb phosphorus:Conducting molybdate assays to determine Pi concentration.
Determination of ATP levels:We use enhanced ATP Assay Kit.
Determination of NAD+/NADH levels:We use NAD+ /NADH Assay Kit with WST-8.
Full cell experiment:We use MFC system of a two-chamber electrochemical reactor.
More Details of all experiments can be found at the Experiments section on the Wiki.
Chassis and genetic context
We express this composite part on Shewanella oneidensis MR-1.
Potential Applications
In bioelectrochemical systems, we can utilize PolyP and NADP in microbial fuel cells for further electron transfer improvement 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.(Fig.12)
Figure 12: Hardware about its application
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.