Problem with E.coli growing in LB - unexpected growth (Jul/26/2007 )
Since a last few trial like say a month, I have been trying to grow E.coli in a 10ml LB broth with 100ug/ml Amp. Usually after overnite growth, the culture of the broth remains a li'l yellowish, but now everytime its turning white. expression of my target protein is there but another protein is also getting expressed as a dark band.
well, starting with the obvious;
Is your starter culture contaminated with a second bacteria species? Can you streak out your colonies to singles on a plate and pick single colonies?
Does the change in colour only occur when the cells are induced,@ is white due to your protein or some nasty bug.
How are your culturing condition in your test (which I presume were okay) different from the current experiment? What culturing containers did you use and what volume of media was use in the test. How does it compared to the test?
Growth condition, @ aeration, temperature, media compositioni. Can change protein expression characteristics.
As for this second band? Are you sure it is not a multimer of your desire protein? Protein expression can sometimes.. well often really, throw a wobbly and you get insoluble protein, multimer and stuff. Alternatively are you over expression a chaperon to fold your desire protein? Does that senerio work?
Finally do you have his Tag or Mic tags or something similar which will allow you to pull your protein away? Sometimes to get around a problem, one ignores it.
Yup, when faced with a wall, one can use strenght to break it, inteligence to go under it, foresight to go around it, or just be blind and ignore it.