Team:Nanjing-China/Results
In the part of lab work, we have designed three biosensor sequences and improved an old part, BBa_J23100. What's more, all the three designs have been demonstrated by us.
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. |
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Figure1.Whole-cell sequence dual-enzyme digestion |
Figure2.SDS-PAGE analysis of recombinant E.coli expressing FrmR |
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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. |
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 Figure 3. Influence of Formaldehyde Induce Time on Fluorescence Expression |
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Figure4.A photograph of E.coli cells containing the formaldehyde-induced RFP expression plasmid, or without formaldehyde induction, and re-suspended in PBS buffer(pH7.4) |
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| It is worth to be mentioned that the team OUC help us validate the result. | ||
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. |
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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) |
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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. |
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Figure6a.Response growth curve for recombinant bioluminescent Escherichia coli BL21 to different concentration of formaldehyde | ||
Figur6b.The tolerance of recombinant bioluminescent Escherichia coli BL21 to various concentration 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. | ||
Figure7.Fluorescence test of various aldehydes using recombinant bioluminescent Escherichia coli BL21 | ||
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. |
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Figure8.Whole-cell sequence dual-enzyme digestion |
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Next, we conducted a plate sensitive assay to measure the S2– 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. |
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 Figure 9. Tolerance test |
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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. |
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Figure10 a)RFP responsiveness of the detector system.  | |
Figure10 b) A visible photograph of a).  |
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Finally, we examined the plasmid's selectivity against SO42-, SO32- and 4 other chemical reagents(figure 11). |
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Figure 11.Test of selectivity. |
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First we succefully detected the protein expression by SDS-Page(figure 12a) analysis and Western blot(figure 12b) analysis. |
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Figure12a. Coomassie Brilliant Blue R-250-stained SDS-Page analysis of recombinant E.coli expressing hoxABCJ-terminator-hoxp-gfp |
Fingure 12b. Western blot analysis of recombinant E.coli expressing his-hoxA |
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). |
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Figure 13. Influence of H2 concentration on fluorescence expression |
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Nanjing University
Nanjing, Jiangsu Province
P.R. of China
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