Part:BBa_K5291004

NqrF

NqrF is a subunit of NADH dehydrogenase in P. aeruginosa.

Usage and Biology

The introduction of the pAB1-pS-nqrf plasmid into P. aeruginosa induces the production of NADH dehydrogenase, which catalyzes the transformation of NADH into and electrons, increasing the amount of electrons in P. aeruginosa and thus promoting electron shuttles (PYO), which indirectly transmit electrons to R. palustris. We have consturcted the plasmid pAB1-pS-nqrf.


Fig.1 Plasmid construction of pAB1-pS-nqrf.

Fig.2 The agarose gel electrophoresis analysis of pAB1-pS-nqrf.

SDS-PAGE analysis of the induced cells after the construction of the expression vectors showed that the Nqr(46.40 kDa) proteins had the expected molecular weights.

Fig.2 SDS-PAGE analysis of the expression of pAB1-pS-nqrf in PAO1.

And we have finished several experiments to verify the functions of nqrf.
1. Calculation of NAD⁺ / NADH amount
We used NADH Assay Kit with WST-8 to measure the amount of NAD⁺/ NADH in P. aeruginosa and P. aeruginosa/pAB-pS-nqrf. By comparing the proportion of NADH in the control group and the experimental group, we can find that NADH/(NADH + NAD⁺) in experimental group is lessen than control group, which means P. aeruginosa with pAB-pS-nqrf plasmid can product more NADH reductase and electronics as shown in the Fig.3 and Table 1.

Fig.3 NADH, NADH + NAD⁺ amount of PAO1/pAB-pS-nqrf, PAO1.


Table 1 The NADH + NAD⁺ amount, NADH amount, NADH/(NADH + NAD⁺) of PAO1/pAB-PS-nqrf, PAO1.
2. MFC Testing
We measured the conductivity of the bacterial fluid by measuring the MFC experiment and found that the voltage of the bacterial fluid with the introduction of plasmid pilA was significantly higher than the voltage of the original bacteria over time. This proves that our plasmid is effective in enhancing the electron conduction function of P. aeruginosa, and this experiment met our expectations. Therefore, compared to P. aeruginosa that did not import plasmids, plasmid-infiltrated P. aeruginosa had more advantages as electrical donator and were more conducive to extracellular transfer between R. palustris. (You can refer to the function of pilA in the part BBa_K5291045)


Fig.4 The output voltage of PAO1,PAO1/nqrf+.

In conclusion, the function of nqrf is verified.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 976
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 976
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 976
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 976
  Illegal NgoMIV site found at 150
  Illegal NgoMIV site found at 154
  Illegal NgoMIV site found at 493
  Illegal NgoMIV site found at 847
  Illegal NgoMIV site found at 890
  Illegal AgeI site found at 1054
  
• 1000
  COMPATIBLE WITH RFC[1000]