Difference between revisions of "Part:BBa K3492002"

 
 
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<partinfo>BBa_K3492002 parameters</partinfo>
 
<partinfo>BBa_K3492002 parameters</partinfo>
 
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<h3>Biology</h3>
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This part encodes NAD synthetase which induces NAD synthesis in Pseudomonas aeruginosa. Up-regulation of this gene will increase the NAD level in cells. In our project, we use this gene to improve the efficiency of Microbial fuel cells (MFCs).
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<h3>Usage</h3>
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Team Nanjing_NFLS added nadE on pBBR1MCS-5 to form the expression system. Restriction endonuclease digestion result (Figure 1) showed the gene is successfully bind to the plasmid.
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<center>https://2020.igem.org/wiki/images/a/ab/T--Nanjing_NFLS--2002_1.jpg</center>
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<center><h5>Figure 1. Identification of pBBR1MCS-5- nadE by restriction endonuclease digestion</h5></center>
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                    <p>Moreover, NAD synthase activity assay was performed to test the activity of nadE. Different concentrations of NADH standard solution (0.3~1 mM) was prepared, and the absorbance was determined at 340 nm at different concentrations. Then the standard curve shown as y = 2.514x + 0.064 (R2=0.994) , y for the absorbance of the sample, and x for the concentration of NADH in the corresponding sample (mM). According to the standard curve the results of enzymatic activities analysis (Figure 2). The efficiency of nadE is 2~3 times higher in the engineered cells than the normal cells. </p>
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                    <center><table border="1px">
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                        <tr><td>strain</td><td>NADH (mM)</td><td>Total protein concentration (mg/mL)</td><td>Specific enzyme activity (U/g)</td></tr>
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                        <tr><td>PAO1</td><td>0.113</td><td>66.760</td><td>2.738</td></tr>
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                        <tr><td>PAO1-nadE</td><td>0.228</td><td>47.135</td><td>5.372</td></tr>
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                    </table></center>

Latest revision as of 23:29, 27 October 2020


nadE

NAD synthetase which induces NAD synthesis in side Pseudomonas aeruginosa. Studies showed that over expression of this gene lead to the efficiency increase of Pseudomonas aeruginosa Microbial Fuel Cells(MFCs).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 394
    Illegal XhoI site found at 661
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 178
    Illegal NgoMIV site found at 399
    Illegal NgoMIV site found at 469
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 325
    Illegal SapI.rc site found at 405


Biology

This part encodes NAD synthetase which induces NAD synthesis in Pseudomonas aeruginosa. Up-regulation of this gene will increase the NAD level in cells. In our project, we use this gene to improve the efficiency of Microbial fuel cells (MFCs).

Usage

Team Nanjing_NFLS added nadE on pBBR1MCS-5 to form the expression system. Restriction endonuclease digestion result (Figure 1) showed the gene is successfully bind to the plasmid.

T--Nanjing_NFLS--2002_1.jpg
Figure 1. Identification of pBBR1MCS-5- nadE by restriction endonuclease digestion

Moreover, NAD synthase activity assay was performed to test the activity of nadE. Different concentrations of NADH standard solution (0.3~1 mM) was prepared, and the absorbance was determined at 340 nm at different concentrations. Then the standard curve shown as y = 2.514x + 0.064 (R2=0.994) , y for the absorbance of the sample, and x for the concentration of NADH in the corresponding sample (mM). According to the standard curve the results of enzymatic activities analysis (Figure 2). The efficiency of nadE is 2~3 times higher in the engineered cells than the normal cells.

strainNADH (mM)Total protein concentration (mg/mL)Specific enzyme activity (U/g)
PAO10.11366.7602.738
PAO1-nadE0.22847.1355.372