Team:DTU-Denmark/Achievements/Interlab study

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Revision as of 13:31, 17 October 2014

Interlab Study

Construct strains

Create multiple strains expressing GFP from different promoters from the Andersen Library. Constructs were prepared with Standard Assembly. (see protocol). Plasmid were purified with Zyppy™ Plasmid Miniprep Kit from Zymo research following manufacturer's protocol. Competent DH5α cells were prepared (see protocol). Purified plasmids were transformed into competent DH5α cells (see protocol).

Flourescence measurement on cultures

We used cell culture tubes (10 mL) containing 2 mL LB medium (with 0.5% NaCl) and 25 μg/mL chloramphenicol for each strain. To each of the tubes, we inoculated cells from a single colony with a sterile inoculation loop and left it overnight at 37°C, shaking at 350 rpm. The wells of the sterile BioLector plate (48 well FlowerPlate) was prepared with 1450 μL (1500μL for controls) of LB (0.5% NaCl) containing 25 μg/mL chloramphenicol. The following day we transferred 10 μL of each culture into an Eppendorf tube with 990 μL of sterile physiological salt water (creating a 100-fold dilution). We inoculated the individual BioLector wells with 10 μL of the dilution (creating an additional 150-fold dilution). Each strain was grown in triplicates to make sure we had 3 independent measurements and a more precise result. We incubated the plate in the BioLector at 37°C and 1000 rpm for 12.5 hours (stationary phase was reached). During the growth experiment we measured biomass and GFP fluorescence every 5 minutes. Biomass was measured by light scatter at 620 nm, while fluorescence was measured at 520 nm, with an excitation wavelength of 488 nm.

Single cell measurements

2 mL LB (0.5% NaCl) was inoculated with cells from a plate and grown overnight. The cultures were washed and resuspended in PBS. The cell suspensions were diluted in PBS to a concentration where the flow cytometer could detect 1000-1500 events per second.

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 <br><br>
+<h2>Construct strains</h2>
+Create multiple strains expressing GFP from different promoters from the Andersen Library. Constructs were prepared with Standard Assembly. (see protocol).
+Plasmid were purified with Zyppy™ Plasmid Miniprep Kit from Zymo research following manufacturer's protocol.
+Competent DH5α cells were prepared (see protocol).
+Purified plasmids were transformed into competent DH5α cells (see protocol).
+<h2>Flourescence measurement on cultures</h2>
+We used cell culture tubes (10 mL) containing 2 mL LB medium (with 0.5% NaCl) and 25 μg/mL chloramphenicol for each strain. To each of the tubes, we inoculated cells from a single colony with a sterile inoculation loop and left it overnight at 37°C, shaking at 350 rpm.
+The wells of the sterile BioLector plate (48 well FlowerPlate) was prepared with 1450 μL (1500μL for controls) of LB (0.5% NaCl) containing 25 μg/mL chloramphenicol. The following day we transferred 10 μL of each culture into an Eppendorf tube with 990 μL of sterile physiological salt water (creating a 100-fold dilution). We inoculated the individual BioLector wells with 10 μL of the dilution (creating an additional 150-fold dilution). Each strain was grown in triplicates to make sure we had 3 independent measurements and a more precise result. We incubated the plate in the BioLector at 37°C and 1000 rpm for 12.5 hours (stationary phase was reached). During the growth experiment we measured biomass and GFP fluorescence every 5 minutes. Biomass was measured by light scatter at 620 nm, while fluorescence was measured at 520 nm, with an excitation wavelength of 488 nm.
+<h2>Single cell measurements</h2>
+mL LB (0.5% NaCl) was inoculated with cells from a plate and grown overnight.
+The cultures were washed and resuspended in PBS.
+The cell suspensions were diluted in PBS to a concentration where the flow cytometer could detect 1000-1500 events per second.