Team:NTHU Taiwan/Engineering


Successfully Gene Cloning

In order to initially verify our cloning result. We use chloramphenicol as our C antibiotics to test the plasmid functionality. By the method, we are able to diminish the effect by other types of bacteria or contamination. After growing in the incubator for 24 hours. A significant amount of colony indicate the success of cloning.

Fig1. (L)cada-yhaO Plasmid JM109,(R)JM109(control) on Ampicillin agar plate

Fig2. (L)cada-mnth Plasmid JM109,(R)JM109(control) on Ampicillin agar plate

Fig3. (L)cada-MT2 Plasmid JM109,(R)JM109(control) on Ampicillin agar plate

Cadmium Ion Transportation Ability of MntH protein

In design part, we attach promoter cada with red fluorescence protein to detect the curve of fluorescence intensity (excited by wavelength 590 nanometer, emission of wavelength 630 nanometer versus time), the figure shows us the cada combine with RFP shows much higher FLU value when compare to JM109 control group. Furthermore, when cada also ligase with RFP and MntH, the increase amount of cadmium ions trigger the promoter to produce even higher amount of RFP. Result in the greater FLU value.

Fig4. The fluorescence value of different kinds of E.coli(Cd ion concentration: 0.1mM). Blue: JM109(control group), Orange: cada-RFP Plasmid JM109, Gray: cada-RFP/cada-MntH Plasmid JM109

We introduce gene MntH as the channel protein for cadmium ions as well as balance the concentration between inner and outer membrane. The figure below shows the growth curve of E.coli contain MntH is getting lower with increase of cadmium concentration. Indicating the MntH gene is function with transport more cadmium ions into the cell.

Fig5. The growth cruve of cada-MntH Plasmid JM109 in different Cd ion concentration

Cadmium Ion Tolerance Ability of MT2 peptide

MT2 are proved to lower the toxicity cause by cadmium ions. With detail figure below, we can observe O.D.600 value is relatively higher than those without MT2.

Fig6. The growth cruve value of cada-MT2 Plasmid JM109 in different Cd ion concentration