Difference between revisions of "Team:Nanjing-China/Results"

As is shown in figure1, the whole sequence of our formaldehyde pathway is about 1500 base-pairs while the vector is 2000 base-pairs. SDS-PAGE analysis(figure 2) also shows the expression of the regulator, protein FRMR, around 15kd. Therefore, we moved forward to further property study.

Figure3 illustrates the fluorescence intensity change induced by formaldehyde along with an interval of 2 hours. The peak value occurs after 6 hours, which means the detecting results can be seen with naked-eyes after only 6 hours. As is shown in figure 4, compared to the blank control, experimental group with formaldehyde induction turns to pink apparently, meaning the designed reporter pathway has worked.

Moreover, in order to set up the corresponding relationship between the quantity of formaldehyde and the fluorescence value, we prepared a series of concentrations of formaldehyde(figure5a.). We found out that from the concentration of 300 micromole to 600 micromole, a preferable equation of linear regression could be obtained(figure5b.), which laid the cornerstone for creating precise and sensitive detecting devices.

Figure5.Fluorescence measurement of E.coli cells containing the formaldehyde-induced RFP expression plasmid after gradient concentrations of formaldehyde induction and re-suspended in PBS buffer(pH7.4)

Then we incubated our recombinant E. coli for more than 10 hours to test their reponse growth rates(figure 6a) and tolerance(figure 6b) to different concentrations of formaldehyde.

Finally, we tested the selectivity of our formaldehyde pathway by inducing the recombinant E. coli with acetaldehyde, DMSO and 6 other aldehydes. The results(figure 7) demonstrated good selsctivity of our recombinant plasmids.

We first analyzed the product by dual-enzyme digestion and electrophoresis(figure8). As can be seen, our hydrogen sulfide sensing sequence is over 3,000 base pairs.

Next, we conducted a plate sensitive assay to measure the S^2– tolerance of E. coli cells with constructed probe pathway. All plates were incubated at 37℃ for 18 h before reading. No significant influence appeared to the growth of E. coli at a concentration lower than 10mmol/L.

Cells were then grown to midlog phase under aerobic conditions and 0 ~ 250 μM Na₂S. Cells were harvest after 17h and assayed for fluorescence intensity. Error bars indicate SD of the mean.

Figure10 a)RFP responsiveness of the detector system.

Figure10 b) A visible photograph of a).

Finally, we examined the plasmid's selectivity against SO₄^2-, SO₃^2- and 4 other chemical reagents(figure 11).

First we succefully detected the protein expression by SDS-Page(figure 12a) analysis and Western blot(figure 12b) analysis.

Fluorescence intensity remains stationary when IPTG is added. Whereas, it increases in a low hydrogen atmosphere. When the amount of hydrogen goes to an even higher level, fluorescence intensity increases apparently, meaning the designed report pathway works as expected(figure 13).