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Team:Evry/Biology/CellCharacterization/Antibiotics
From 2014.igem.org
Sentivity to antibiotics
After having succeeded to make our bacteria grow up, we tested their resistance to different antibiotics. To know their resistance to antibiotics is very important, because it will allow us to finalize protocols of selection after tranformation.
We chose to test the most commonly used antibiotics. We included the three antibiotics used in iGEM (Kanamycin, Chloramphenicol and Ampicilin), plus the Erythromycin and the Tetracyclin. We chose the erythromycin to test a conjugation protocol which required this antibiotic for E.Coli, and we chose to test tetracyclin because it is quite often used for inducibles systems.
Table 1: Concentrations tested for each antibiotic
We tested several concentrations of each antibiotics (cf: Table 1), and we add a supplementary concentration for erythromycin because it is known to be not very effective on GRAM- bacteria.
The sensitivity tests were performed in four differents conditions (cf: Figure 1). We tested on Marine Broth(MB) 1X plates, MB 0.5X plates (used for conjugation) and M9 1X (+3% NaCl). We aslo tested in liquid M9 1X (+3% NaCl) to have a test in liquid culture, but unfortunately we cannot test on liquid MB cultures because this media is no usable for the spectrometer, so we cannot have a good information about the cells' growth.
The cells' sensitivity was mesured on our strain Pseudovibrio denitrificans, E. Coli, and different clones of E.Coli transformed with plasmids and carrying an acquired resistance AmpR, KanR, CamR ou ErmR.
Figure 1: Protocol of antibiotics' tests.
We let all the media in incubator at 30°C (with shaking for liquid cultures), and they have been checked at day 1, day 2 and day 3. In the following figures, find the results obtained after three days of cultures on plates and one day of liquid cultures.
Figure 2: Results of antibiotics' tests on MB plates.
On the left: Results on MB 1X plates.
On the right: Results MB 0.5X.
Figure 3: Results of antibiotics' tests for M9 media.
On the left: Results on M9 1X plates.
On the right: Results in liquid M9 1X.
Unfortunately, results for tetracyclin on MB media are not usefull. However, this year, biobricks have to be cloned in PSB1C3, and we have very good results for clhoramphenicol.
According to these results, we know that we can select transformed Pseudovibrio denitrificans on a concentration of chloramphenicol adjusted to the ratio 1/1000 (cf: Table 2).
Table 2: Concentrations whiwh can be used for selection of Pseudovibrio denitrificans
