Difference between revisions of "Part:BBa K5034225"
 
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<partinfo>BBa_K5034225 short</partinfo>
 
<partinfo>BBa_K5034225 short</partinfo>
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<b>Basic Description:</b>
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===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 NADK gene which is initially from Mycobacterium tuberculosis H37Rv and we performed codon optimization on, is expressed in the <html><body><a href="https://parts.igem.org/Part:BBa_K5034201">pBBR1MCS-terminator</a></body></html>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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<html>
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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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    <p style="text-align: center;">
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        Figure 1: Basic function of NADK
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    </p>
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</div>
 +
</html>
  
  
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Figure 1: Basic function of NADK
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===Sequence and Features===
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<partinfo>BBa_K5034225 SequenceAndFeatures</partinfo>
  
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<b>Chassis and Genetic Context:</b>
 
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Successfully expressed in Escherichia coli DH5α and BL21(DE3) strains.
 
  
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<b>Construct features :</b>
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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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* 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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NADK 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.
+
  
 +
* 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.
  
 +
<html>
 +
<div style="text-align: center;">
 +
    <img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/engineering/fig17.png">
 +
    <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)
 +
    </p>
 +
</div>
 +
</html>
  
 +
<html>
 +
<div style="text-align: center;">
 +
    <img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/engineering/mechanism-of-nadk.png">
 +
    <p style="text-align: center;">
 +
        Figure 3: Construction of NADK part
 +
    </p>
 +
</div>
 +
</html>
  
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<html>
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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/pyydt-nadk.png">
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    <p style="text-align: center;">
 +
        Figure 4: Construction of NADK plasmid
 +
    </p>
 +
</div>
 +
</html>
  
  
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Figure 2: PCR of target genes PCR before plasmids construction (The extra small fragment in the picture is primer dimer)
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===Origin (Organism)===
 +
The NADK gene was sourced from Mycobacterium tuberculosis H37Rv strain. The <html><body><a href="https://parts.igem.org/Part:BBa_K5034201">pBBR1MCS-terminator</a></body></html>plasmid  backbone is a standard vector used for gene expression in synthetic biology applications.
  
 +
===Experimental Characterization and results===
  
 +
We first performed Colony PCR on the target gene in S. oneidensis, and the length of NADK was 924 base pairs, which was consistent with the results obtained from the experiments.
  
 +
<html>
 +
<div style="text-align: center;">
 +
    <img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/results/figure13.png">
 +
    <p style="text-align: center;">
 +
        Figure 5: Colony PCR indicating that different plasmids can replicate in <i>S. oneidensis</i>
 +
    </p>
 +
</div>
 +
</html>
  
 +
Then, we 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., fig6)
  
 +
<html>
 +
<div style="text-align: center;">
 +
    <img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/current.png">
 +
    <p style="text-align: center;">
 +
        Figure 6: statistical data on electricity production capacity of <i>S. oneidensis</i> with the introduction of different hydrolases
 +
    </p>
 +
</div>
 +
</html>
  
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Figure 3: Basic construction of NADK plasmid
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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!
  
 +
We then measured the phosphorus aggregation capacity of <i>S. oneidensis</i> after introduction of the enzyme(fig7), and found that it was also significantly increased compared to the wild type.
  
 +
<html>
 +
<div style="text-align: center;">
 +
    <img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/engineering/pi-of-nadk.png">
 +
    <p style="text-align: center;">
 +
        Figure 7: statistical data on the phosphorus accumulation capacity of <i>S. oneidensis</i> with NADK
 +
    </p>
 +
</div>
 +
</html>
  
 +
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.
  
 +
<html>
 +
<div style="text-align: center;">
 +
    <img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/atp.png">
 +
    <p style="text-align: center;">
 +
        Figure 8: ATP level in <i>S. oneidensis</i> with the introduction of different hydrolases
 +
    </p>
 +
</div>
 +
</html>
  
 +
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!
  
 +
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>
  
  
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Figure 4: Construction of NADK plasmid
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===Chassis and Genetic Context===
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Chassis：<i>Shewanella oneidensis</i> MR-1.
  
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Figure 5: Bacterial PCR indicating that different plasmids can replicate in Shewanella
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The gene can be expressed and function properly in <i>S. oneidensis</i>.
  
−
<b>Origin (Organism):</b>
+
===Potential Applications===
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The NADK gene was sourced from Mycobacterium tuberculosis H37Rv strain. The PYYDT plasmid backbone is a standard vector used for gene expression in synthetic biology applications.
+
−
 
+
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<b>Experimental Characterization and results:</b>
+
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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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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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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 NADK
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Figure 8: ATP level in Shewanella with the introduction of different hydrolases
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<b>Potential Applications:</b>
+
 
In bioelectrochemical Systems, utilizing NADP in microbial fuel cells for improved electron transfer and energy production.
 
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.
 
Also can be utilized in metabolic engineering, stress response studies, and biotechnological applications where enhanced NADP production is beneficial.
  
−
<b>References:</b>
+
===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.  
 
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.  
  
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K5034225 SequenceAndFeatures</partinfo>
 
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<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 13:39, 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 pBBR1MCS-terminatorplasmid. 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.

Figure 1: Basic function of NADK


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]


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)

Figure 3: Construction of NADK part

Figure 4: Construction of NADK plasmid


Origin (Organism)

The NADK gene was sourced from Mycobacterium tuberculosis H37Rv strain. The pBBR1MCS-terminatorplasmid backbone is a standard vector used for gene expression in synthetic biology applications.

Experimental Characterization and results

We first performed Colony PCR on the target gene in S. oneidensis, and the length of NADK was 924 base pairs, which was consistent with the results obtained from the experiments.

Figure 5: Colony PCR indicating that different plasmids can replicate in S. oneidensis

Then, we 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., fig6)

Figure 6: 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(fig7), and found that it was also significantly increased compared to the wild type.

Figure 7: 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 8: 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.


Chassis and Genetic Context

Chassis：Shewanella oneidensis MR-1.

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

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.