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Team:EPF-Lausanne/Notebook/July2011
From 2011.igem.org
Notebook: July 2011
Friday, 1st of July 2011
Alina and Lilia did MITOMI on His-tetR expressed from linear template, it was loaded on chip in ITT expression mix. DNA was spotted on June 29 in different concentrations for both: consensus (tetO1) sequence and a random sequence ((-)control).
Images: Green fluorescence comes from green-lysine and red fluorescence comes from DNA (cy-5 labeled).
Although we don't observe much protein bound to the anti His-tag antibody, we can still see that it does bound TetO1 sequence and did not bind any (-)control sequence. Some possible reasons for low protein fluorescence under the buttons: the expression yield from the linear template with T7 promoter might be too low, the His-tag to antibody binding is not strong enough or the concentration of antibody is too low.
Friday, 1st of July 2011
Alina and Lilia amplified the His-tetR linear template. After PCR sample was loaded on 1% agarose gel with Rad Safe, “1Kb Plus DNA Ladder” was used.
We also prepared about thirty LB-agar plates with ampicilin, they are in the BM fridge.
Tween experiment
Clara and Henrike tested the right dose of Tween 20 that can be used for the "chemostat" experiment without affecting the cell growth. Tween is a detergent that would prevent the cells from sticking and thus clogging the microfluidic channels. We tested 12 different dilutions of Tween 12 both for GFP and RFP e-coli which can be potentially used for the "chemostat" experiment. We read the OD(absorption @600nm) and fluorescence every 10 minutes, for 18 hours at 37°C.
Growth curve for all the different concentrations vs time
Calculated growth factor for the previous curves
We observe tween did not have a major effect on the cell's growth.
I suggest we use 0.075% tween in media for the experiments
