strain: Rosetta(DE3,plysS)
plasmid: pet28a
medium: M9 medium with 50ug/ml kanamycin and 34ug/ml chloramphenicol
After incubation in LB(kanamycin, chloramphenicol) at 37℃ over night，transfer 10ml into 1L M9 medium（Molecular Cloning） 。
In the beginning, it grows normally. When OD600~0.8, a large number of bubbles produced ,and then as the pictures shows....
Dear Bin Liu,
I think that the OD you are reading is not representative of a proper cell growth. It seems caused by the aggregates that I can see in the pictures. For this reason I think probably, you have not E. coli in your medium at the moment.
Can you confirm a proper growth of E. coli like, after the overnight inoculum?
I suppose that you used Kanamicin as a selective antibiotic in the previous step in M9, so you are sure that all the cells have the correct plasmid. Did you confirm the presence af the correct insert by sequencing before this step? Did you use Kanamicin in the sub-inoculum?
Sometimes it could happen that due to an absence of plasmid in the cells and a inhomogeneities of the antibiotic in the LB agar, there are colonies in the Petri, but they are not what we suppose to be, so when you continue the cloning procedure, probably you have different cells instead of the hoped E. coli.
I hope to be helpful.
Dear Bin,
I think that if all the previous steps are ok, it could be any contaminant in the actual medium, interfering with the growth of the cells. Is the medium new and autoclaved? You can repeat the sub-inoculum in another kind of medium like LB, just to check if the colonies are ok.
Dear Bin,
I was thinking about the protein. Did you already perfom the expression it in the past. It could be toxic fro the cells, and probably lead to the aggregate formation that you have in the flask.
Hi Salvatore
I have already expressed for 1 year. It should be not toxic. Besides, the phenomenon is more ovbious, when the larger bottles (2L,5L) used. However, it usually seems normal in 50ml(10ml medium) tube.
Medium are autoclaved or filter freshly.
It's unstable in LB medium. Sometimes normal, sometimes aggregate.
Thanks for your help!
Dear Bin, have you just tried to take a microscope look? This could lead you to find or to exclude contaminations.
Actually, phage It is a possibility, and what you see is the resulting debris of phage lysis.
Hi Bin,
what a pity, something was wrong with the cells indeed!
Besides, you know the reason of that aggregates now, so you can plan your next steps.
Good luck with your future experiments!
Best wishes
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