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 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 |