Team:Braunschweig/Notebook

From 2013.igem.org

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<b>Investigators: Kerstin, Laura </b><br>
<b>Investigators: Kerstin, Laura </b><br>
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Pre-cultures of chromoprotein constructs were mixed in one main culture in order to see how they behave during cultivation over several hours. OD<sub>520</sub> of pre-cultures was measured in order to inoculate the main culture with  33% of each strain with a final OD<sub>520</sub>=0.3 For the main culture, 25 ml 2xYT containing chloramphenicol were inoculated with the three different strains and grown at 37°C and 250 rpm in a non-baffled flask. Samples from the culture were taken at several time points, diluted and plated on 2xYT agar-plates containing chloramphenicol. Agar plates where incubated at 37°C over night.<br>
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Pre-cultures of chromoprotein constructs were mixed in one main culture in order to see how they behave during cultivation over several hours. OD<sub>520</sub> of pre-cultures was measured in order to inoculate the main culture with  33% of each strain to a final OD<sub>520</sub>=0.3. For the main culture, 25 ml 2xYT containing chloramphenicol were inoculated with the three different strains and grown at 37°C and 250 rpm in a non-baffled flask. Samples from the culture were taken at several time points, diluted and plated on 2xYT agar-plates containing chloramphenicol. Agar plates were incubated at 37°C over night.<br>
During the day we noticed that the 2xYT medium used for the pre-cultures yesterday showed contamination. Thus, there is a high possibility  of contamination in the pre-culture as well as in the main culture. Therefore we decided to repeat the experiment. New pre-cultures of the three chromoprotein expression cassettes where inoculated in 50 ml 2xYT containing chloramphenicol directly from -80°C.<br>
During the day we noticed that the 2xYT medium used for the pre-cultures yesterday showed contamination. Thus, there is a high possibility  of contamination in the pre-culture as well as in the main culture. Therefore we decided to repeat the experiment. New pre-cultures of the three chromoprotein expression cassettes where inoculated in 50 ml 2xYT containing chloramphenicol directly from -80°C.<br>
In order to have our constructs available in different <i>E. coli</i> strains for the fluorescence microscopy experiments, the eforRed expression cassette was transformed into <i>E. coli</i> Top10F' by electroporation. Transformed cells were plated on 2xYT agar containing chloramphenicol and incubated over night at 37°C.<br>
In order to have our constructs available in different <i>E. coli</i> strains for the fluorescence microscopy experiments, the eforRed expression cassette was transformed into <i>E. coli</i> Top10F' by electroporation. Transformed cells were plated on 2xYT agar containing chloramphenicol and incubated over night at 37°C.<br>
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A continuous cultivation of the Rhl inducible construct in <i>E. coli</i> Top10F' prepared, including preparation of pre-cultures of our inducible construct in 50 ml 2xYT containing chloramphenicol and grown at 37°C and 250 rpm over night.<br>
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A continuous cultivation of the P<sub>Rhl</sub> inducible in <i>E. coli</i> Top10F' was prepared, including preparation of pre-cultures of our inducible construct in 50 ml 2xYT containing chloramphenicol and grown at 37°C and 250 rpm over night.<br>
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Pre-culture of erforRed expression cassette in <i>E. coli</i> Top10F' in 50 ml 2xYT containing chloramphenicol was inoculated from the  agar plate and grown at 37°C and 250 rpm overnight.<br>
Pre-culture of erforRed expression cassette in <i>E. coli</i> Top10F' in 50 ml 2xYT containing chloramphenicol was inoculated from the  agar plate and grown at 37°C and 250 rpm overnight.<br>
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For the reproduction of the growth curve a pre-culture of the Rhl inducible construct in <i>E. coli</i> Top10F' in 30 mL 2xYT containing Chloramphenicol and incubated at 37°C overnight. <br>
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For the reproduction of the growth curve a pre-culture of the P<sub>Rhl</sub> inducible construct in <i>E. coli</i> Top10F' in 30 mL 2xYT containing chloramphenicol and incubated at 37°C overnight. <br>
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To test the production of AHLs by our final constructs we prepared a pre-culture of each reporter strain in 30 ml 2xYT containing Ampicillin and incubated them overnight at 37°C and 250 rpm. We also made glycerol cell stocks of the reporter strains.<br>
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To test the production of homoserine lactones (HSL) by our final constructs we prepared a pre-culture of each reporter strain in 30 ml 2xYT containing ampicillin and incubated them overnight at 37°C and 250 rpm. We also made glycerol cell stocks of the reporter strains for further use.<br>
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Cells containing the finale constructs were grown in 30 ml 2xYT containing Ampicillin as well at 37°C and 250 rpm. We inoculated these cultures with a high cell density as growth depends on the leakiness of the inducible promoters. These cultures were grown over 24 h in order to reach a high AHL concentration in the culture broth. As negative controls strains bearing constructs with the inducible promoters but not the AHL synthesis were grown in 30 ml 2xYT over night at 37°C as well.<br>
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Cells containing the finale constructs were grown in 30 ml 2xYT containing ampicillin as well at 37°C and 250 rpm. We inoculated these cultures with a high cell density as growth depends on the leakiness of the inducible promoters. These cultures were grown over 24 h in order to reach a high HSL concentration in the culture broth. As negative controls strains bearing constructs with the inducible promoters but not the HSL synthesis were grown in 30 ml 2xYT over night at 37°C as well.<br>
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The first attempt to cultivate regulated and unregulated mixed cultures in continuous culture was made today. For regulated growth Ampicllin was added to the medium, for unregulated growth Chloramphenicol was used as selection marker. Samples o9f each culture were taken at several time points. OD<sub>520</sub> was measured and dilutions of samples were plated on 2xYT agar plates containing Chloramphenicol.  
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The first attempt to cultivate regulated and unregulated mixed cultures in continuous culture was made today. For regulated growth ampicllin was added to the medium, for unregulated growth chloramphenicol was used as selection marker. Samples of each culture were taken at several time points. OD<sub>520</sub> was measured and dilutions of samples were plated on 2xYT agar plates containing chloramphenicol.  
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We had problems to find the right dilution of the agar plates (too many or too little colonies on plates). The results of these cultivations were therefore not statistically relevant and no conclusions about the regulation of growth by our constructs could be drawn.<br> </p>
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We had problems to find the right dilution of cell suspension for the agar plates (too many or too little colonies on plates). The results of these cultivations were therefore not statistically relevant and no conclusions about the regulation of growth by our constructs could be drawn.<br> </p>
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The eforRed expression cassette in <i>E.Coli</i> TOP10F’ was miniprepped and glycerol stocks of this strain were made.<br>
The eforRed expression cassette in <i>E.Coli</i> TOP10F’ was miniprepped and glycerol stocks of this strain were made.<br>
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The main cultures (induced and not induced) of the final Rhl inducible construct were inoculated to a start OD<sub>520</sub> of 0.05 in 75 ml 2xYT containing Ampicillin with cells of the pre-culture. In order to induce the expression of <i>ampR</i> N-3-buturyl homoserine lactone was added to a final concentration of 1 µM. Samples were taken at appropriate times depending on the growth phase until the induced culture reached the stationary phase. OD was determined at 520 nm to avoid absorptions by chromoproteins.<br>
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The main cultures (induced and not induced) of the final P<sub>Rhl</sub> inducible construct were inoculated to a start OD<sub>520</sub> of 0.05 in 75 ml 2xYT containing ampicillin with cells of the pre-culture. In order to induce the expression of <i>ampR</i> N-3-buturyl homoserine lactone was added to a final concentration of 10 µM. Samples were taken at appropriate times depending on the growth phase until the induced culture reached the stationary phase. OD was determined at 520 nm to avoid absorptions by chromoproteins.<br>
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To verify production of AHLs by our constructs the pre-cultures containing the finale constructs as well as the negative controls were centrifuged for 10 min at 6000 rpm and 4°C. Supernatant was transferred to a new Falcon tube and sterilized by filtration. <br>
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To verify production of HSLs by our constructs the pre-cultures containing the finale constructs as well as the negative controls were centrifuged for 10 min at 6000 rpm and 4°C. Supernatant was transferred to a new Falcon tube and sterilized by filtration. <br>
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Dilution series of the supernatants and the synthetic AHLs as standards were pipetted in 96-well microtiter plates. Wells were inoculated with the corresponding reporter strain and grown for 3 h at 37°C. Bioluminescence produced by the <i>luxCDABE</i> of the reporter strains was detected by a microplate reader. We were able to show that our constructs produced the specific AHLs. However due to the high background especially in the N-3-oxododecanoyl-HSL producing strain we need to modify the experimental layout in order to get a stronger signal.<br><br><br></p>
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Dilution series of the supernatants and the synthetic HSLs as standards were pipetted in 96-well microtiter plates. Wells were inoculated with the corresponding reporter strain and grown for 3 h at 37°C. Bioluminescence produced by the <i>luxCDABE</i> of the reporter strains was detected by a microplate reader. We were able to show that our constructs produced the specific HSLs. However due to the high background especially in the N-3-oxododecanoyl-HSL producing strain we might need to modify the experimental layout in order to get a stronger signal.<br><br><br></p>
    
    
<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px"><b>References:</b><br>
<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px"><b>References:</b><br>

Revision as of 18:04, 3 October 2013

Labjournal

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This is the documentation of our lab work. An overview on how we approached this project can be found under Approach. For detailed protocols of certain procedures please refer to Protocols. Attributions are given for each day, however please check our Attributions section for efforts beyond the lab work.

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