Difference between revisions of "Part:BBa K3492003"

 
 
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<partinfo>BBa_K3492003 parameters</partinfo>
 
<partinfo>BBa_K3492003 parameters</partinfo>
 
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<h3>Biology</h3>
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This part encodes phenazine-specific methyltransferase inside Pseudomonas aeruginosa. This gene is related to the electron transduction in cells. Up-regulation of this gene will increase the rate of electron transduction. 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 phzM gene 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/9/9c/T--Nanjing_NFLS--2003_1.jpg</center>
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<center><h5>Figure 1. Identification of pBBR1MCS-5-phzM by restriction endonuclease digestion</h5></center>
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<p>Moreover, Methyltransferase activity assay was performed. Prepare standard solutions (0, 0.01, 0.02, 0.03, 0.04, 0.05, 0.08, 0.1 μ mol/mL), and the absorbance was measured at 313 nm. And the enzyme activity (E) of methyltransferase: E = (∆A_313+0.089)/(∆t×0.000578) . (t (min) for absorbance changing time, E for the methyltransferase activity (U/mL) contained in each mL of supernatant).<br>
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                    According to the standard curve for pyocyanin E = (∆A_313+0.089)/(∆t×0.000578), and the specific enzyme activity: SE = E/m . (E for the methyltransferase activity (U/mL) contained in each mL of supernatant, SE for the specific enzyme activity of the methyltransferase (U/mg)), the results of enzymatic activities analysis (Figure 2). It can be seen that methyltransferase was highly expressed in engineered cells, which is 2.8~3.04 times higher than the control group.
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                    </p>
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                    <center><table border="1px">
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                        <tr><td>strain</td><td>Enzyme activity (U/mL) </td><td>Total protein concentration (mg/mL)</td><td>Specific enzyme activity (U/mg)</td></tr>
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                        <tr><td>PAO1</td><td>33.58</td><td>21.38</td><td>1.57</td></tr>
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                        <tr><td>PAO1-phzM</td><td>83.65</td><td>17.55</td><td>4.77</td></tr>
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                    </table></center>

Latest revision as of 23:26, 27 October 2020


phzM

This gene synthesis phenazine-specific methyltransferase inside 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 BglII site found at 206
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 424
    Illegal NgoMIV site found at 428
    Illegal NgoMIV site found at 787
  • 1000
    COMPATIBLE WITH RFC[1000]


Biology

This part encodes phenazine-specific methyltransferase inside Pseudomonas aeruginosa. This gene is related to the electron transduction in cells. Up-regulation of this gene will increase the rate of electron transduction. In our project, we use this gene to improve the efficiency of Microbial fuel cells (MFCs).

Usage

Team Nanjing_NFLS added phzM gene 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--2003_1.jpg
Figure 1. Identification of pBBR1MCS-5-phzM by restriction endonuclease digestion

Moreover, Methyltransferase activity assay was performed. Prepare standard solutions (0, 0.01, 0.02, 0.03, 0.04, 0.05, 0.08, 0.1 μ mol/mL), and the absorbance was measured at 313 nm. And the enzyme activity (E) of methyltransferase: E = (∆A_313+0.089)/(∆t×0.000578) . (t (min) for absorbance changing time, E for the methyltransferase activity (U/mL) contained in each mL of supernatant).
According to the standard curve for pyocyanin E = (∆A_313+0.089)/(∆t×0.000578), and the specific enzyme activity: SE = E/m . (E for the methyltransferase activity (U/mL) contained in each mL of supernatant, SE for the specific enzyme activity of the methyltransferase (U/mg)), the results of enzymatic activities analysis (Figure 2). It can be seen that methyltransferase was highly expressed in engineered cells, which is 2.8~3.04 times higher than the control group.

strainEnzyme activity (U/mL) Total protein concentration (mg/mL)Specific enzyme activity (U/mg)
PAO133.5821.381.57
PAO1-phzM83.6517.554.77