Difference between revisions of "Team:Paris Saclay/Interlab Study"
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=Introduction= | =Introduction= | ||
| − | While we were at the laboratory developing our project, we also participated to the 2016 Interlab Study. The interlab study consists in measuring the fluorescence level of constructions provided by the iGEM Measurement Committee in order to compare results obtained by worldwide iGEM teams and thus study the variations of measurements among each experiments. This year | + | While we were at the laboratory developing our project, we also participated to the 2016 Interlab Study. The interlab study consists in measuring the fluorescence level of constructions provided by the iGEM Measurement Committee in order to compare results obtained by worldwide iGEM teams and thus study the variations of measurements among each experiments. This year it consisted on measuring the fluorescence of three test devices composed of a Green Fluorescent Protein (GFP) coding sequence under the control of promoters of different strengths. The measurements could be proceeded using a plate reader or flow cytometry. We chose to use flow cytometry which was available at the laboratory where our team worked. |
==Constructions== | ==Constructions== | ||
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==Methods== | ==Methods== | ||
| − | At the beginning of the Interlab study, we had a problem with device 1, the | + | At the beginning of the Interlab study, we had a problem with device 1, the tube received was empty. We waited to receive another tube from iGEM. |
Constructions test devices 2 and 3 and the two controls were transformed into competent DH5α ''E.coli'' strand using a [[Team:Paris_Saclay/Experiments#heat-shocktransformation|heat shock transformation protocol]]. Transformed bacteria were plated on solid LB medium containing 30 μg/mL chloramphenicol. Petri dishes were incubated at 37°C overnight. For each device, a colony was used to inoculate 3 mL of liquid LB medium containing 30 μg/mL chloramphenicol. The cultures were incubated at 37°C at 180 rpm overnight. Then a glycerol stock was made from these overnight cultures and stored at -80°C. When we received the device 1, we used the same protocol to clone it into the DH5α E.coli strand. | Constructions test devices 2 and 3 and the two controls were transformed into competent DH5α ''E.coli'' strand using a [[Team:Paris_Saclay/Experiments#heat-shocktransformation|heat shock transformation protocol]]. Transformed bacteria were plated on solid LB medium containing 30 μg/mL chloramphenicol. Petri dishes were incubated at 37°C overnight. For each device, a colony was used to inoculate 3 mL of liquid LB medium containing 30 μg/mL chloramphenicol. The cultures were incubated at 37°C at 180 rpm overnight. Then a glycerol stock was made from these overnight cultures and stored at -80°C. When we received the device 1, we used the same protocol to clone it into the DH5α E.coli strand. | ||
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==Assessment== | ==Assessment== | ||
| − | The size of cells (FSC) should be the same for every sample as it is the same bacterial | + | The size of cells (FSC) should be the same for every sample as it is the same bacterial strain and only the fluorescence emission level (FL1) should vary. We expected fluorescent emission to be correlated to promoter strength for each construction as the promoter strength has an influence over the expression level of the GFP gene and fluorescence is proportional to GFP quantity in the cell. However it is important to keep in mind that even if GFP level in the cell might be correlated to promoter strength, it exists stochasticity on such expression level (Elowitz, 2002). |
=Results= | =Results= | ||
Revision as of 14:35, 19 October 2016
