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

Line 1: Line 1:
 +
<html xmlns="http://www.w3.org/1999/xhtml">
 
<head>
 
<head>
 
<meta http-equiv="Content-Type" content="text/html; charset=utf-8" />
 
<meta http-equiv="Content-Type" content="text/html; charset=utf-8" />

Revision as of 10:30, 29 October 2017

Team:Nanjing-China - 2017.igem.org

In the part of lab work, we have designed three biosensor sequence and improved an old part, J23000. What's more, all the three design have been demonstrate by us.

We have designed a formaldehyde sensor sequence, which is a part of our team .

The sequence is composed of PfrmR, gene frmR, flag tag, PfrmAB, gene RFP.

When the pathway works, we can see that the E.coli turns to red with naked eyes at the presence of formaldehyde.

 

 

In order to demonstrate the design, a lot of experiments have been done.

Figure 3. Influence of Formaldehyde Induce Time on Fluorescence Expression

  

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

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.

As to the hydrogen sulfide sensor, we also designed a whole-cell biocatalytic system, displaying the concentration of hydrogen sulfide by the compound’s influence on specific genes’ expression in modified E.coli. 

In our design, we use red fluorescent protein as the indicator.When hydrogen sulfide exits, the gene transcription is activated, and the bacteria turns red.
In the experiment, we proved that the sequence worked well and was useful to detect hydrogen sulfide

Figure1.Whole-cell sequence dual-enzyme digestion

a)

b)

c)

Figure 2.a)RFP responsiveness of the detector system.
b) A visible photograph of a).
c) Test of selectivity.

There is a composite of hydrogen sensor full length sequence.

The order of the elements is: HoxA-HoxB-HoxC-HoxJ-terminator-HoxP-EGFP. The sequence of HoxABCJP comes from Ralstonia eutropha H16 megaplasmid pHG1. The hole sequence acts as an hydrogen sensor.

When the amount of hydrogen goes to a higher level, Fluorescence intensity increases apparently.

The sequence was a good detecter in the lab work.

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

Social media

Twitter button:
iGEM Nanjing-China@nanjing_china