In the part of lab work, we have designed three biosensor sequence and improved an old part, J23100. What's more, all the three design have been demonstrate by us.
As is shown to all of us, the whole sequence is about 1500 base-pairs while the vector is 2000 base-pairs. SDS-PAGE analysis also showed 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 |
This diagram illustrates the fluorescence intensity change induced by formaldehyde along with interval time 2 hours. The peak value occurs after 6 hours, that is, only requiring 6 hours, the detecting results can be seen with naked-eyes. Compared to the blank control, experimental group with formaldehyde induction turns to pink apparently, meaning the designed reporter pathway have worked. |
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Figure 3. Influence of Formaldehyde Induce Time on Fluorescence Expression |
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) |
Moreover, in order to set up the corresponding relationship between the quantity of formaldehyde and the fluorescence value, we prepared a series of gradient concentrations of formaldehyde. We found out that from the concentrations of 300 micromole to 600 micromole, a preferable equation of linear regression could be obtained, 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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Figure6. Optical density(600nm) of (a) Escherichia coli BL21 and (b) recombinant bioluminescent Escherichia coli BL21 harboring frmR-RFP fusion after 10 hours’ incubation with 800uM different aldehydes | |
Figure7.Response growth curve for recombinant bioluminescent Escherichia coli BL21 to different concentration of formaldehyde | |
Figure8.Fluorescence test of various aldehydes using recombinant bioluminescent Escherichia coli BL21 |
Figure9.The tolerance of recombinant bioluminescent Escherichia coli BL21 to various concentration of formaldehyde |
It is worth to be mentioned that the team OUC help us demonstrate the result. |
A plate sensitive assay measuring S2– tolerance of E. coli cells with constructed probe pathway. All plates were incubated at 37℃ for 18 h before being read. No significant influence appeared to the growth of E. coli at a concentration lower than 10mmol/L. |
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We analysised the product by dual-enzyme digestion and electrophoresis. |
Figure1.Whole-cell sequence dual-enzyme digestion |
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Figure2.a)RFP responsiveness of the detector system. |
RFP responsiveness of the detector system. Cells were 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. |
Now we’ve succefully detected the protein expression by SDS-Page analysis and Western blot analysis. |
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Figure1. Coomassie Brilliant Blue R-250-stained SDS-Page analysis of recombinant E.coli expressing hoxABCJ-terminator-hoxp-gfp |
Fingure 2. Western blot analysis of recombinant E.coli expressing his-hoxA |
Fluorescence intensity remains stationary when IPTG is added. |
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Figure 3. Influence of H2 concentration on fluorescence expression |