Currently I am working on beta-lactamases. I am using pET-28a vector(present kanamycin resistance gene) for cloning purpose, and transformed in E.coli BL21(DE3) bacterial cells. During purification, primary culture looks fine and there is uniform bacterial growth but in secondary culture(both times use kanamycin at concentration 50ug/ml according to CLSI guideline) filamentous type of growth is coming, surprisingly OD increases very slowly that time. I have rechecked the autoclave procedure, and freshly made kanamycin. Even after that same circumstance.
What could be the possible reasons?
What is different between the primary and secondary cultures? I'm guessing that you use a larger culture flask and larger volume for the secondary container compared to the primary. Any change in the shaker? We have a giant floor shaker for large volume cultures but it's a bit finicky on holding temperature.
The filamentous growth sounds like possible contamination. Have you taken a look at the cells and/or streaked some out to see if you get different colonies? I had an issue one time after the person who made the competent cells did so when sick and we got Staph contamination in the E. coli!
An easy fix would be to re-wash and re-autoclave the secondary culture containers.
Thank you so much Prof. Klocko for your answer.
I have used the same shaker for primary and secondary culture with the same rpm and what I have noticed temperature is almost fixed during the entire incubation time.
I have already attempted several times with rewashed and reautoclaved secondary culture containers.
No, I have not yet streaked but keeping your words in mind, I will do it.
I agree with Amy Klocko that it sounds like lysis debris perhaps, perhaps phage contamination. Or does this occur after IPTG induction? Since beta-lactamase is a secreted periplasmic protein you may be inducing too high a level of expression and this can be toxic to E. coli.
Thank you, Prof. Benedik, for your answer.
The thread-like contamination occurred before IPTG induction.
Even after repeatedly re-washing/re-autoclaving the containers and making fresh kanamycin, the bacterial culture still got contaminated.
I have done spread plating in LB Agar of that contaminated bacterial culture (photo attached here).
The bacterial plate looks fine to me, although contamination is there. Hence, I could not make any decision to see the plate.
Could anyone suggest me about this?
I agree that this plate does not look contaminated. I still think it most likely you are getting phage infections in your culture.
I agree with Michael J. Benedik that the plate looks good, no obvious signs of a second bacterial strain. If it is phage contamination, you will likely need to retransform new competent cells.
Thank you so much Prof. Benedik for your suggestion.It will be very helpful for my further proceedings.
Thank you so much, Prof. Klocko, for your suggestion. It will assist me in my further proceedings.
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