Manish, In practice, we have found that it is very helpful to grow up an overnight starter culture in minimal media to acclimatize the cells for growth under minimal conditions. (Cells transferred from LB to M9 do not perform well for overexpression). We inoculate 1 L of modified M9 (see below) with 10 mL of an overnight culture (centrigued, washed, and ressuspended in M9) in the same medium.
In addition--and even though it should not be required for many strains of E. coli--we have found that he inclusion of thiamin, 5-10 ug/L, greatly enhances the rate of cell growth, but it may still takeup to 8 hr for cells to grow to OD600 = 0.6 for induction. Overexpression overnight (12-18 hr) may be required to get optimal cell density and overexpression yield. Using this protocol we get excellent overexpression yields of protein. Cheers, ___________________________________________ Roger S. Rowlett Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: [EMAIL PROTECTED] -----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of [EMAIL PROTECTED] Sent: Thursday, April 19, 2007 10:31 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Protein expression in Minimal media (M9) Manish, Assuming that you need the minimal medium for Se, have you tried the regular heavy atom derivatives? You already have diffracting crystals... M9 medium can be slightly difficult. However, no one says that you *have* to use minimal medium such as M9 (again, assuming that you're doing Se-Met). There are numbers of defined media compositions out there, many with very good growth characteristics. Finally, you can spike the M9 with a small quantity of yeast extract. This will give your culture a decent initial boost and the amount of normal methionine in the extract should be relatively small so it will all get eaten up while the cells are still in early log phase. Regards, Artem > Hello everybody, > > Sorry for an offtopic question. I am trying to express a protein in > M9 minimal media for Selenomet incorporation. When grown in LB this > protein expressed very well and got good crystals. Diffraction was > upto 2 A. I am having a hard time expressing the same protein in > Minimal media. It took nearly 24 hours for the OD600 to reach ~ 0.4 > before inducing with IPTG in minimal media and eventually got no > protein expression. It looks like the cells are not growing or taking > very long to grow. The cell line I am using is RossettaBlue (DE3) and > the plasmid is pET19b based (Novagen). > > It expressed poorly in BL21 (DE3) when grown in LB and thus decided to use > RosettaBlue (DE3). It worked very well in LB, but having a hard time > while expresing the same in minimal media using Rosetta Blue. Has > anybody tried expression in minimal media using Rosetta Blue cell > line? I am planning to try overnight induction. > > Any suggestions would be greatly appreciated. > > Thanks, > > Manish > > > > > > > > > ************************************************* > Manish B. Shah, PhD. > Postdoctoral Fellow > Hauptman-Woodward Medical Research Institute > 700 Ellicott Street > Buffalo, NY 14203. > ************************************************* >
