Part:BBa_K5291045

PaPilA1-61M3
PaPilA1–61M3 is a modified PilA protein in P. aeruginosa with enhanced conductivity.

Usage and Biology

We have successfully constructed the plasmid with pilA as well as introducing it into the engineered bacteria.


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


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

Next we conducted the function verification of pilA. We stained the flagella using silver staining, which was used to verify the validity of our plasmid pAB-pS-pilA. After staining, we found that the flagellum length of the experimental group was longer and larger than that of the control group, which was in line with our experimental expectations.


Fig.2 The light microscopy to visualize the bacterial surface flagella PAO1 and PAO1/pAB1-pS-pilA, magnification: 40×10.

SDS-PAGE analysis of the induced cells after the construction of the expression vectors showed that the pilA(12.08 kDa) proteins had the expected molecular weights.

Fig.3 SDS-PAGE analysis of the expression of pAB1-pS-pilA in PAO1.

We also 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 nqrf in the part BBa_K5291004)


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

In conclusion, the function of pilA is verified.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 1
  
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 1
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 1
  
• 1000
  COMPATIBLE WITH RFC[1000]