Team:INSA-Lyon/Results

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RESULTS

What about chelation ?
Nickel(II) chelated for each of the constructions (WT, HIS1, HIS2) is evaluated by using dimethylglyoxime (DMG) as the precipitating reagent. This is achieved by using absorbing properties of DMG-Ni(II) pink-colored complex (peak absorption at 554nm).
Here are the outcomes.

To get rid of biosafety issues linked with GMO, we worked on destroying our bacteria after letting them grow in a biofilm. Curli proteins being very resistant to environmental changes, our goal was to obtain a biomaterial made out of modified Curli able to chelate nickel.

To find the best way to degrade bacteria and DNA, the following protocol was used to test the influence of UV light and temperature separately :

• Wells containing M63 cultures of strain 227 were put under UV light / at 60 or 70°C for different lengths of time. Well contents were then gradually transferred into Eppendorf and diluted (100, 300, 900 and 2700 times).
  
• LB plates (without antibiotic) corresponding to UV/temperature exposure times (+ one plate for control) were then spotted with s227 different concentrations in order to be able to count survival bacteria after incubation at 37°C.
  
• Genomic DNA was extracted from s227 concentrated culture. From the solution obtained, Curli promoter(750 bp) was amplified by PCR with Q5 polymerase and designed primers.
  
• Epifluorescence observations were made after Back Light coloration with 200µL s227 liquid cultures.
  
  
  

UV light influence

5 images LB plates
No bacteria grew on LB plate after 15 minutes UV light exposure.
-> Bacterian growth can be stopped this way.

image gel PCR
Bacterian DNA seemed to be degraded after 10 min UV light exposure.
-> In consequence, UV light can be used to destroy DNA.

3 images 40X Back Light
Still some green-colored bacteria could be seen after 20 min UV exposure.
-> UV light isn’t enough to kill bacteria.

Temperature influence

4 images LB plates 60°C
Bacteria grew on LB plates even 45 min after being heated to 60°C.
-> The temperature isn't enough high to kill bacteria.

4 images LB plates 70°C
No more bacteria on LB plate after 15min at 70°C
-> Bacterian growth can be stopped as well as with UV light.

image gel PCR
No DNA degradation at all.
-> In consequence, temperature doesn't enable to destroy DNA, in contrary to UV light.

4 images 40X Back Light
No difference of coloration was observed between the control and the samples heated at 70°C : indeed a lot of green-colored bacteria remained after 45 min of heating.
-> Temperature isn’t enough to kill bacteria just like UV light.

To solve this last problem, bacteria were put in contact with ethanol absolute. The Back Light coloration gives the following picture.
image benjamin

These numerous experiments lead us to developp a protocol in three steps, illustrated by the drawing below :
schéma bilan