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From 2013.igem.org

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Revision as of 18:14, 24 September 2013

{ "date" : "2013-8-30", "author" : "fabio", "title" : " new experiments on blue light induction: great!!! ", "content" : " In these days I tried so many times to induce transformed cells in order to have great, clean, results, and finally I found the key: I developed a standard procedure that allowed me to see difference between samples. I transformed NEB10b cells with the entire construct (first I tested different strains of E. coli in order to see which strain had the best behavior and noticed that NEB10b cells work better than any other). Then I made some inocula O/N using LB broth (I also tried to compere LB broth to M9 minimal medium, but I didn’t see any difference in the outcome) and diluted the next morning in 20 ml of liquid broth (1:50) waiting until they reached OD = 0.7. The thing is, cultures grew really slowly, in fact they reached the required concentration after 6-8 hours. Then I split them into 5ml samples that I exposed to different condition. The first time that I performed this experiment I tested different light sources in order to establish the most powerful inducing condition: I used blue LEDs, a blue bulb light and normal white light. I finally picked out the LED and the normal light for further experiments. Now let’s go back to the different samples!! I used glass tubes for overnight induction, heating the samples at 37 degrees with stirring. Previously I tested different materials (glass and plastic) tubes and different temperatures conditions: the best way to grow my bacteria was definitely in glass at 37 degrees. I put one sample in the dark wrapping it up with an aluminum foil and placed under a box to be sure that no light could pass. The second sample was illuminated using a blue LED. Instead the third one was exposed to normal white light because I previously saw that even white light induces the circuit. Experiments lasted all night long and everytime I saw the results in the morning. Finally I got my results right in front of my eyes: the dark control in almost every experiments didn’t show any sign of amilCP production. At the other hand the other two samples were blue. To obtain quantitative measurements I used the spectrometer. First I diluted the pellets in 2 ml of PBS and then sonicated the samples for 10 seconds. Fluorescence spectra reveals what we already got from visual results. Great!! ", "tags" : "blue_light-" }