Part:BBa_K3492003

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.

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.

strain	Enzyme activity (U/mL)	Total protein concentration (mg/mL)	Specific enzyme activity (U/mg)
PAO1	33.58	21.38	1.57
PAO1-phzM	83.65	17.55	4.77