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

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             <td colspan="2"><p>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.</p></td>
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             <td colspan="2"><p>A plate sensitive assay measuring S<sup>2–</sup> 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.</p></td>
 
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             <td colspan="2"><p>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.</p></td>
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             <td colspan="2"><p>RFP responsiveness of the detector system. Cells were grown to midlog phase under aerobic conditions and 0 ~ 250 μM Na<sub>2</sub>S. Cells were harvest after 17h and assayed for fluorescence intensity. Error bars indicate SD of the mean.</p></td>
 
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               <td colspan="2"><div align="center"><img src="https://static.igem.org/mediawiki/2017/6/69/T-Nanjing-China-h2-9.png" width="500" height="250" />
 
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               <p><font size="-1">Figure  3. Influence of H2 concentration on fluorescence expression</font></p>
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               <p><font size="-1">Figure  3. Influence of H<sub>2</sub> concentration on fluorescence expression</font></p>
 
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Revision as of 13:58, 1 November 2017

Team:Nanjing-China - 2017.igem.org

 

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.

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.

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.

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)

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.

We analysised the product by dual-enzyme digestion and electrophoresis.

Figure1.Whole-cell sequence dual-enzyme digestion

a)

b)

c)

Figure2.a)RFP responsiveness of the detector system.
b) A visible photograph of a).
c) Test of selectivity.

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.

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.
And Fluorescence intensity increases in a low hydrogen atmosphere.
When the amount of hydrogen goes to a higher level Fluorescence intensity increases apparently. meaning the designed reporter pathway have worked.

Figure 3. Influence of H2 concentration on fluorescence expression

Address

Life Science Department
#163 Xianlin Blvd, Qixia District
Nanjing University
Nanjing, Jiangsu Province
P.R. of China
Zip: 210046

Email

nanjing_china@163.com

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