Hi, I have a peculiar problem which persists, so my strains BL21DE3 and Rosetta both have my GOI cloned in pET vectors with Ampicillin and Kanamycin resistance. I have grown them in shakeflasks ranging volumes from 50 ml to 500ml didnt have growth issues but when i scale up to bioreactors of volumes 2L it would first grow for 1st hour and then OD comes done to initial OD (0.07).Then after 12 -16hr it would grow at a very unpredicted rate
No issues in expressing the protein in shakeflask/
Please help me I have tried everything not sure where is the problem
Dear Ankita K Rao I'm not sure in what context you are referring the growth unpredictable, but kindly ensure all the parameters such as growth time , Induction time, Temperature, RPM of agitation, pH, D.O., most importantly aeration are taken care of. Improper optimization of even one parameter can affect the growth and expression of your protein.
Kindly go through some literature to refer for optimization of these parameters.
Good luck!
It sounds to me like you may have gotten a bacteriophage infection in your bioreactor. Improper growth parameters would explain why a culture doesn't grow, but if the OD goes up and then down again that suggests lysis.
You can try nitrogen gas purging, or else maintain the biological load reduction by securing a proper cotton plug needs to be ensured.
Thank you all for your inputs. I was able to confirm it was bacteriophage infection. Any suggestions on how to prevent any future infections
Scale up means more water or media you are using; make sure that the water is free of contaminants.
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