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Team:Sheffield/Notebook
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25/08 Cultured RU1012 in LB
Streaked RU1012 out on LB agar plates 3.00 pm Spread 200ml overnight culture RU1012 on two LB agar with X-gal and Ampicillin, Chloramphenicol and Kanamycin and incubate at 37℃ for 18 hours, with one plate in dark and one plate exposed to broad spectrum light. We should expect blue precipitate in the one in dark, but less precipitate in the one exposed.
9am – The plate exposed in the light has excessive precipitate and was very blue - the plate covered in foil has obviously less precipitate (opposite to the expected results) Grow RU1012 with only Kanamycin plasmid in LB Broth Restreaked on new plates and incubate on LB agar plates for 12 hours at 37℃
9am – Experiment repeated to ensure the last result was systematic. The results were the same, opposite to what would be expected. We suspect that the wavelength that activates the phytochrome is more specific, and therefore broad spectrum does not inactivate phytochrome completely.
Repeated experiments under balmer lamp(instead of broad spectrum light) in the dark room in 25C. Incubate RU1012 with all 3 antibiotics covered in foil and without foil for 12 hours Incubate control strain with Kanamycin with foil and without foil for 12 hours
The illuminated sample still produced more precipitate than the one in dark. Phytochrome was not inactivate, therefore we made Agar plates with L-arabinose to induce the 2 gene (haem-PCB) pathway, to hope to improve the inactivation.
Then cells were spread on 2 Agar plates with L-arabinose, one under a balmer lamp and the other covered in foil for 12 hours at room temperature. The wavelength of the balmer lamp is a range up to 632nm.
The illuminated sample still produced more pigment than the sample in the dark.
After contacting the authors of the article (Christopher A. Voigt), we have decided to try varying the intensity of the light shine on it, and to vary the wavelength that was shone on the plate, the phytochrome was suggested to have optimum wavelength and intensity for its activation. Also this time, S-gal (patented alternative to X-gal) was used as the substrate (it produced black precipitate instead of blue precipitate).
We put RU1012 on to agar plates with S-gal in it and wrapped the plate in different colour filter paper; black(no light), blue, red, green. With both test and control plates for a time series. - We also left a LB plate with X-gal but with no organism in to see if X-gal itself is light sensitive or not.
- Experiments shows that the wavelength does have effect on the expression of the phytochrome, red filter paper seem to inactivate the expression best. - The experiment shows that X-gal is not light sensitive - We repeated the experiment with X-gal LB agar plates, with black and red. With test and control plate. And varying the light intensity through different heights.
To quantitatively assess the activity of the beta-galactocidase, we started preparing the new experiment to carry out the miller assay. LB cultures with the RU1012 were placed in an incubator, sealed off from light sources other than the led light lamp in the incubator. Cultures were placed at the following light intensities uEins/m2/s: 2.5, 6, 7, 10 At each intensity, 4 sets of culture were placed: 3 experimental and 1 control. Samples were taken from each set of culture at 3, 6, 9 and 12 hours.
The miller assay was carried out and OD measurements taken.
Miller units were worked out and the results tabulated and put in graphs |
