Team:DTU-Denmark/Achievements/Interlab study

From 2014.igem.org

(Difference between revisions)

Revision as of 14:33, 17 October 2014

Interlab Study

Construct strains

We created multiple strains expressing GFP from different promoters from the Andersen Library. 12 out of the 15 promoters we intended to use were successfully transformed into DH5α These 12 constructed strains were applied in the further analysis of the relatively promoter strength. J23111, J23117 J23109 was not succesfully cloned into E. coli. We did not detect any fluorescence signal from these strains and it was confirmed from the sequencings. we excluded these strains. E0240: Uden promoter GFP E0206: Canamycin I20260.

Fluorescence measurement on cultures

Fluorescence was measured on the O/N cultures with our constructed strains in the BioLector. Biomass and fluorescence were measured during growth. Fluorescence signal through the growth was normalized by dividing by OD600. The average of the fluorescence is illustrated in the bar chard below. Together with the expected values relative to the J23100 promoter. Orange is the expected values and gray indicates our measured fluorescence signal.

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.

For more detailed information on the instruments used, settings and the measured quantities see the filled out interlab form.

@@ Line 10: / Line 10: @@
 <h2>Construct strains</h2>
-Create multiple strains expressing GFP from different promoters from the Andersen Library. Constructs were prepared with Standard Assembly. (see protocol).
+We created multiple strains expressing GFP from different promoters from the Andersen Library.
-Plasmid were purified with Zyppy™ Plasmid Miniprep Kit from Zymo research following manufacturer's protocol.
+out of the 15 promoters we intended to use were successfully transformed into DH5&alpha; These 12 constructed strains were applied in the further analysis of the relatively promoter strength. J23111, J23117 J23109 was not succesfully cloned into E. coli. We did not detect any fluorescence signal from these strains and it was confirmed from the sequencings. we excluded these strains.
+E0240: Uden promoter GFP
-Competent DH5α cells were prepared (see protocol).
+E0206: Canamycin
+I20260.
-Purified plasmids were transformed into competent DH5α cells (see protocol).
+ <h2>Fluorescence measurement on cultures</h2>
+Fluorescence was measured on the O/N cultures with our constructed strains in the BioLector. Biomass and fluorescence were measured during growth.
+Fluorescence signal through the growth was normalized by dividing by OD600. The average of the fluorescence is illustrated in the bar chard below. Together with the expected values relative to the J23100 promoter. Orange is the expected values and gray indicates our measured fluorescence signal.
+<img src="https://static.igem.org/mediawiki/2014/b/bf/DTU-Denmark_IL_barchard.png" width=400 />
-<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>
@@ Line 27: / Line 28: @@
 The cell suspensions were diluted in PBS to a concentration where the flow cytometer could detect 1000-1500 events per second.
-For more detailed information on the instruments, settings and the measured quantities see the filled out <a href="https://2014.igem.org/Team:DTU-Denmark/Interlab_form">interlab form</a>.
+<br>
+<br>
+For more detailed information on the instruments used, settings and the measured quantities see the filled out <a href="https://2014.igem.org/Team:DTU-Denmark/Interlab_form">interlab form</a>.
 </maincontent>