Difference between revisions of "Part:BBa K5034206"
 
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<partinfo>BBa_K5034206 short</partinfo>
 
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===Basic Description===
 
===Basic Description===
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This basic part encodes the NADK gene which is initially from Mycobacterium tuberculosis H37Rv and we performed codon optimization on, is expressed in the PYYDT plasmid. This basic part is designed to facilitate the conversion of inorganic polyphosphate (PolyP) to nicotinamide adenine dinucleotide phosphate (NADP). The NADK enzyme is crucial for the phosphorylation of NAD to NADP, which is essential for various metabolic processes. NAD kinase is regarded as a key enzyme in NADP synthesis and, hence, in numerous cellular processes such as anabolic/biosynthetic pathways and protection against oxidative stress.
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This basic part encodes the <i>NADK</i> gene which is initially from Mycobacterium tuberculosis H37Rv and we performed codon optimization on, is expressed in the PYYDT plasmid. This basic part is designed to facilitate the conversion of inorganic polyphosphate (PolyP) to nicotinamide adenine dinucleotide phosphate (NADP). The NADK enzyme is crucial for the phosphorylation of NAD to NADP, which is essential for various metabolic processes. NAD kinase is regarded as a key enzyme in NADP synthesis and, hence, in numerous cellular processes such as anabolic/biosynthetic pathways and protection against oxidative stress.
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In a sentence, it can  convert Poly p to NADP. For the first time, we expressed this element in a strain of Shewanella and conducted codon optimization based on Shewanella.
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In a sentence, it can  convert Poly p to NADP. For the first time, we expressed this element in a strain of <i>S. oneidensis</i> and conducted codon optimization based on <i>S. oneidensis</i>.
  
 
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<div style="text-align: center;">
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<img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/engineering/mechanism-of-nadk.png">
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<img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/engineering/mechanism-of-nadk.png">  
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Figure 1: Basic function of NADK
 
Figure 1: Basic function of NADK
 
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===Sequence and Features===
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<partinfo>BBa_K5034206 SequenceAndFeatures</partinfo>
  
 
===Chassis and Genetic Context===
 
===Chassis and Genetic Context===
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Successfully expressed in Escherichia coli DH5α and BL21(DE3) strains.
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Chassis：<i>Shewanella oneidensis</i> MR-1.
  
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===Construct features(only coding sequence included in basic part)===
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The gene can be expressed and function properly in <i>S. oneidensis</i>.
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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.
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===Construct features===
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* Promoter: Constitutive promoter for continuous expression. We use tac promoter in our experiment.
  
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NADK Coding Sequence: Encodes the NAD kinase enzyme.
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* RBS: Strong ribosome binding site for efficient translation. We use BBa-B0034 which shows the strongest translation in our experiment.
  
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Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use T7Te terminator in our experiment.
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* <i>NADK</i> Coding Sequence: Encodes the NAD kinase 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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<img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/engineering/fig17.png">
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<img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/engineering/fig17.png">  
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<p>
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<p style="text-align: center;">
 
Figure 2: PCR of target genes PCR before plasmids construction (The extra small fragment in the picture is primer dimer)
 
Figure 2: PCR of target genes PCR before plasmids construction (The extra small fragment in the picture is primer dimer)
 
</p>
 
</p>
 
</div>
 
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</html>
 
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The coding sequence of the NADK enzyme has approximately 924 base pairs, consistent with our electrophoretic results.
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===Origin (Organism)===
 
===Origin (Organism)===
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The NADK gene was sourced from Mycobacterium tuberculosis H37Rv strain.  
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The <i>NADK</i> gene was sourced from Mycobacterium tuberculosis H37Rv strain.  
  
 
===Experimental Characterization and results===
 
===Experimental Characterization and results===
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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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We first determined the change in the electroproduction capacity of <i>S. oneidensis</i> compared to the wild type after introducing the SNADK enzyme ("S" means that we introduced the enzyme into <i>S. oneidensis</i>, and "NADK" denotes the name of the enzyme we introduced) (e.g., fig3)
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Capacity to polymerize phosphorus: Conducting molybdate assays to determine Pi concentration.
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The expression of NADK showed relatively high phosphorus accumulation and electricity generation ability. Also, the ATP level is considerably enhanced.
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<img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/current.png">
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<img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/current.png">  
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Figure 3: statistical data on electricity production capacity of Shewanella with the introduction of different hydrolases
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Figure 3: statistical data on electricity production capacity of <i>S. oneidensis</i> with the introduction of different hydrolases
 
</p>
 
</p>
 
</div>
 
</div>
 
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We found that <i>S. oneidensis</i> showed a significant increase in electricity production capacity compared to the wild type after the introduction of NADK, which is good news!
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We then measured the phosphorus aggregation capacity of <i>S. oneidensis</i> after introduction of the enzyme(fig4), and found that it was also significantly increased compared to the wild type.
  
 
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<img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/engineering/pi-of-nadk.png">
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<img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/engineering/pi-of-nadk.png">  
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<p style="text-align: center;">
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Figure 4: statistical data on the phosphorus accumulation capacity of Shewanella with PPK2
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Figure 4: statistical data on the phosphorus accumulation capacity of <i>S. oneidensis</i> with NADK
 
</p>
 
</p>
 
</div>
 
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We also determined the changes in ATP levels in <i>S. oneidensis</i> after introduction(fig5). We found that the ATP level of <i>S. oneidensis</i> increased significantly after the introduction, which is a strong evidence that the metabolic level of <i>S. oneidensis</i> is in a high state at this time.
  
 
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<img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/atp.png">
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<img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/atp.png">  
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Figure 5: ATP level in Shewanella with the introduction of different hydrolases
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Figure 5: ATP level in <i>S. oneidensis</i> with the introduction of different hydrolases
 
</p>
 
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</div>
 
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Overall, the ability of <i>S. oneidensis</i> to generate electrical energy and the ability to aggregate phosphorus were significantly enhanced after the introduction of NADK, and presumably due to the enhanced metabolic strength. Given this excellent result, after combining it with the dry experiment, we intend to enter it into our next round of engineered <i>S. oneidensis</i> optimisation experiments!
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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>
  
 
===Potential Applications===
 
===Potential Applications===
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K5034206 SequenceAndFeatures</partinfo>
 
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<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 13:26, 1 October 2024

Poly P -> NADP


Basic Description

This basic part encodes the NADK gene which is initially from Mycobacterium tuberculosis H37Rv and we performed codon optimization on, is expressed in the PYYDT plasmid. This basic part is designed to facilitate the conversion of inorganic polyphosphate (PolyP) to nicotinamide adenine dinucleotide phosphate (NADP). The NADK enzyme is crucial for the phosphorylation of NAD to NADP, which is essential for various metabolic processes. NAD kinase is regarded as a key enzyme in NADP synthesis and, hence, in numerous cellular processes such as anabolic/biosynthetic pathways and protection against oxidative stress. In a sentence, it can convert Poly p to NADP. For the first time, we expressed this element in a strain of S. oneidensis and conducted codon optimization based on S. oneidensis.

Figure 1: Basic function of NADK

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]

Chassis and Genetic Context

Chassis：Shewanella oneidensis MR-1.

The gene can be expressed and function properly in S. oneidensis.

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.
  • NADK Coding Sequence: Encodes the NAD kinase 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)

The coding sequence of the NADK enzyme has approximately 924 base pairs, consistent with our electrophoretic results.

Origin (Organism)

The NADK gene was sourced from Mycobacterium tuberculosis H37Rv strain.

Experimental Characterization and results

We first determined the change in the electroproduction capacity of S. oneidensis compared to the wild type after introducing the SNADK enzyme ("S" means that we introduced the enzyme into S. oneidensis, and "NADK" denotes the name of the enzyme we introduced) (e.g., fig3)

Figure 3: statistical data on electricity production capacity of S. oneidensis with the introduction of different hydrolases

We found that S. oneidensis showed a significant increase in electricity production capacity compared to the wild type after the introduction of NADK, which is good news!

We then measured the phosphorus aggregation capacity of S. oneidensis after introduction of the enzyme(fig4), and found that it was also significantly increased compared to the wild type.

Figure 4: statistical data on the phosphorus accumulation capacity of S. oneidensis with NADK

We also determined the changes in ATP levels in S. oneidensis after introduction(fig5). We found that the ATP level of S. oneidensis increased significantly after the introduction, which is a strong evidence that the metabolic level of S. oneidensis is in a high state at this time.

Figure 5: ATP level in S. oneidensis with the introduction of different hydrolases

Overall, the ability of S. oneidensis to generate electrical energy and the ability to aggregate phosphorus were significantly enhanced after the introduction of NADK, and presumably due to the enhanced metabolic strength. Given this excellent result, after combining it with the dry experiment, we intend to enter it into our next round of engineered S. oneidensis optimisation experiments!

Details of all experiments can be found at the Experiments section on the Wiki.

Potential Applications

In bioelectrochemical Systems, utilizing NADP in microbial fuel cells for improved electron transfer and energy production. Also can be utilized in metabolic engineering, stress response studies, and biotechnological applications where enhanced NADP production is beneficial.

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.