Part:BBa_K3964000
transcriptional regulator RhlR
RhlR is the transcriptional regulator in Pseudomonas aeruginosa PAO1, it is one of parts of PAO1 quorum sensing system. And in our system, by stimulating the quorum sensing system, the microbial fuel cells (MFCs) system we build can have a improved function and can better degarde wasted antibiotics.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 240
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 715
Biology
This part is one of the two regulatory genes in the Rhl quorum sensing (QS) system in Pseudomonas aeruginosa PAO1. Up regulation of this gene will lead to increased electroactive biofilm formation in MFCs. We use this gene to improve MFC efficiency to better degrade antibiotics.
Usage
Team Nanjing_NFLS added rhlR 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-rhlR by restriction endonuclease digestion
Marker: DL 5000; fragments digested by Hind III and Pst I
In addition, to test increased biofilm formation, we analyzed the anodic electroactive biofilm biomass and biofilm EPS content.
The protein content indicates the biomass on the biofilm. As shown in figure 2, the protein content with rhlR overexpression was 1.38 times of the control in the middle stage and 1.32 times in the late stage. As shown in figure 3, the EPS content after rhlR overexpression was calculated to be 1.51 times higher compared with the control group. The results indicate that he biofilms formation is significantly increased.
Figure.2 Anodic Electroactive Biofilm Biomass Analysis in middle and late term of overexpressed rhlI, rhlR and control biofilm
Moreover, we analyzed the MFC efficiency after the overexpression by measuring cyclic voltammetric curves (CV), Electrochemical Impedance Spectra (EIS), and Polarization and Power Density Curves.
Figure.3 Biofilm EPS Content Analysis of overexpressed rhlI, rhlR and control biofilm
Cyclic voltammetry (CV) is often used to represent the redox properties of MFC electrode biofilms. As shown in figure 4, the catalytic currents of rhlR was increased compared with the control group by 61.17%.
EIS can be used to characterize the internal resistance of mass transfer of MFC. As shown in figure 5, the Charge transfer resistance (Rct) of the control MFC were 20.37 Ω; the Rct of the rhlR overexpressing MFC were 13.71 Ω, which was 12.69 Ω lower than that of the control.
The power density of MFC is an indicator to judge how fast the battery works. As shown in figure 6, the maximum power density of the MFC overexpressing rhlR increased 55.76% relative to the control.
The results indicate hat the efficiency of MFCs are improved after the overexpression of rhlI.
Figure.4 Cyclic Voltammetric Curve of overexpressed rhlI, rhlR and control biofilm
Figure.5 Electrochemical Impedance Spectroscopy (EIS) Diagram of overexpressed rhlI, rhlR and control biofilm
Figure.6 E Polarization and Power Density Curves of overexpressed rhlI, rhlR and control MFC
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