From 2013.igem.org
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| | { | | { |
| - | "date" : "2013-08-30", | + | "date" : "2013-08-15", |
| - | "author" : "fabio", | + | "author" : "Michele-Viola", |
| - | "title" : " <html> new experiments on blue light induction: great!!! </html> ", | + | "title" : "Ferragosto in the lab... Impossibile!!", |
| - | "content" : " <html> | + | "content" : "<html>Today we continued to work on Bacillus. The plates that we did yesterday were not so good so we decided to wait for another day to obtain a better growth. However we did a stock of competent Bacillus by starting from inocula did yesterday. These inocula were diluited in new and fresh transforamtion medium and then, when they reached OD about 0.9, glycerol was added and aliquote of 1 ml were done. So if everything will go as expected, finally we have obtained a stock of Mr Bacillus already competent. </html>", |
| - | 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.
| + | "tags" : "Bacillus" |
| - | 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!!</html> ",
| + | |
| - | "tags" : "blue_light-" | + | |
| | } | | } |
Latest revision as of 09:15, 3 October 2013
{
"date" : "2013-08-15",
"author" : "Michele-Viola",
"title" : "Ferragosto in the lab... Impossibile!!",
"content" : "Today we continued to work on Bacillus. The plates that we did yesterday were not so good so we decided to wait for another day to obtain a better growth. However we did a stock of competent Bacillus by starting from inocula did yesterday. These inocula were diluited in new and fresh transforamtion medium and then, when they reached OD about 0.9, glycerol was added and aliquote of 1 ml were done. So if everything will go as expected, finally we have obtained a stock of Mr Bacillus already competent. ",
"tags" : "Bacillus"
}
"
