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RESULTS

• Curli characterization

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• For the Confocal Laser Scanning Microscopy biofilm acquisitions, all the strains were cultivated in 96-wells microplate in M63 Mannitol during 16H at 30°C (See Protocole for details). See results in Figure 3.

  

  Figure 3: Engineered bacteria biofilm characterization and quantification using Confocal Laser Scanning Microscopy

  All the strains used are constitutively fluorescent to allow detection with confocal laser microscopy (ZEISS LSM510 META, 40X/1.3OILDIC, laser Argon 4 lines 30 W 458 nm, 477 nm, 488 nm, 514 nm, see Protocole). Positive control/CsgA+ (Wild-type E.coli curli producing strain); Negative control/CsgA- (csgA-knockout E.coli strain); BBa_CsgA (BBa_K1404006); BBa_CsgAHis1 (BBa_K1404007); BBa_CsgAHis2 (BBa_K1404008). A) Biofilm sections obtained by Z-stack acquisitions. B) Biofilm 3D reconstruction using IMARIS® from acquisitions in A). C) Bio-volume quantification and maximum of thickness measurement using COMSTAT2 (ImageJ). The strain marked with a star is significantly different from all others (Tukey’s test, p<0.05). As no strains carrying our parts show a significant difference with the positive control, our parts insertion doesn’t modify the biofilm formation property. The His-Tag and His2-Tag engineered CsgA doesn’t disturb the curli formation.

• Nickel chelation

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• What about chelation ?
  
  Nickel(II) chelated for each of the constructions (Strain CsgA- with part BBa_K1404006, Strain CsgA- with part BBa_K1404007 and Strain CsgA- with part BBa_K1404008) 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).
  
  Firstly, a calibration set of Nickel and DMG was done.
  Then, strains were assayed for biofilm nickel absorption on liquid cultures using the calibration set, after measuring the OD of the complex formed for each strain at 554nm.
  This technique is more qualitative than quantitative due to the spectrometer precision.
  
  Here are the outcomes.

  
  

  
  
  
  

  Nickel-DMG complex colorimetry measurement follows a linear regression from a concentration of 20uM to 100uM, linked to the gradient from transparency (at [20uM]) to pink (at [100uM]). This visual method allows us to compare the Ni chelation between our strains. The more the color is pale, the more Ni has been chelated. That’s why, the complex formed with bacteria from strain CsgA- with part BBa_K1404008 is less colored than the others, chelating bacteria remained in the pellet with nickel fixed to their curlis.
  These results show that the part BBa_K1404008 confers increased chelation to strain CsgA-. It is shown that it chelates more than part BBa_K1404006 and part BBa_K1404007.
  
  
  A second method has been used, more quantitative and more precise: mass spectrometry assays have been realized. The quantity of chelated nickel for each strain has been compared to the quantity of curlis formed by each strain.
  Here are the results :
  

  

  
  
  
  Taken together, these results show that the constructed Strain CsgA- with part BBa_K1404008 chelates twice more than strain CsgA- with part BBa_K1404007. That means that two Histags on C-term are twice more effective than one. Moreover, one histag allows a better chelation than none, but not a really significative one. Significant differences are indicated using lowercase letters, and different letters indicate significant differences (Tukey’s test, p < 0.05). Error bars represent standard deviations.
  

• Survival after UV and high temperature exposure

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• 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

  
  

  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

  
  

  3 autres images LB plates 60°C
  Bacteria grew on LB plates even 45 min after being heated to 60°C.
  -> 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 :
  

• Promoter optimization and characterization

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• Biobrick for promoter characterization
  The principal of the construction is shown in the figure below.
  
  
  

  On a pKK backbone, two essential parts have been assembled: a promoter and a reporter.
  The reporter in this case is always the same: gfp. However, the promoter is different for each construction.
  The first promoter is P70, sequence found in the Resgistry. This construction is called p70::gfp
  The second promoter is PCurli, obtained from a PCR, sequence coding for the inter-genic regulation for curli production. This construction is called pcurli::gfp.