I need a good substitute for M9 minimal medium to do expression work of a B. su membrane protein. I have to grow my experimental cultures at 20 oC to slow growth and prevent/ reduce inclusion body formation (also why Im using M9). I was using M9, but I get a precipitate after overnight growth. All suggestions appreciated :)
Hi Maria, There is an oldie paper (Marley et al. journal of biomolecular NMR, 2001) where they, if I remember right, perform a double mark of a membrane protein (among others) using a particular technique. I'm not sure if will serve you for your purpose but maybe you can get some ideas, I hope to serve you :)
Most E. coli strains will not grow in M9, you have to supplement with the vitamins d-Biotin and Thiamine (as a minimum). If you are really seeing precipitate it is probably calcium phosphate which is not usually a problem. If you are adding the CaCl2 post autoclaving then this is rare. However, I assume you are seeing E. coli clumped together which looks like precipitate. This could be phage infection, or they just died for some reason. If you provide more details.....
We have been using Davis minimal medium for growth and overexpression of membrane proteins in E. coli. Here is the recipe:
Dissolve the following in 1 liter of DDW:
3 g KH2PO4
7 g K2HPO4
0.5 g Sodium Citrate
0.1 g MgSO4•7H2O
1 g (NH4)2SO4
Sterilize by filtration through a 0.22-µm filter.
Davis Minimal Medium (1 L)
Prior to use:
Add 10 ml of 2 M glucose solution to 1 liter of Davis minimal salts.
I hope this helps.
The MgSO4 gives similar problems as the CaCl2. Add both after autoclaving
As a last resort after everything you've tried doesn't work, may be you could use a different strain all together which is better suited for expression at low temperatures? Something like the Arctic Express strains (you have to buy them! from Agilent)
Minimal medium A supplemented with 0.5% glucose, 0.01% m/v MgSO4 and 2.5 µg/ml thiamine works well with E. coli.
Hi Maria.
Are you producing your protein in E. coli, right? If yes, as well as changing medium, you could try to employ an E. coli strains optimized for the expression of membrane proteins, such as C41.
Anyway, even if no strain or no media can lead you to a soluble protein, you should try a denaturing and refolding protocol designed for membrane proteins,
Have a look here:
http://www.ncbi.nlm.nih.gov/pubmed/20826347
http://www.sciencedirect.com/science/article/pii/S0022283607014970
Hi all,
Thank you for your very helpful answers. I am using T7 express E. coli (from NEB) with pLys, and I am doing some functional analyses on the membrane (so proper folding and insertion is important). I do autoclave the M9 following which MgSO4, CaCl2, antibiotic and inducer are added. The precipitate is definitely a salt (CaPO4 as suggested) and not clumps of cells. Cells do grow but since I perform 96 well assays, the precipitate is problematic for the spec readings.
I will definitely be using 20oC for my expression work to slow expression of the membrane protein and other proteins involved; and am looking forward to trying the Davis Minimal Medium - Thank you Daniel!
Well, a fast calculation with MINTEQ indicates the formation of Ca4H(PO4)3·3H2O at room temperature and additional MgHPO4·3H2O when the modelling is performed at 99.999° C (maximum for the program). Off course, this numbers correspond to an equilibrium state, but the reactions involved are speeded up by an increasing temperature. And with the experimental experience, I can say that it happens when autoclaving.
So, even if the calculations say it will precipitate at RT, it could take years to reach equilibrium.
I used a similar mineral medium for long time (see reference) and I never had problems adding the Ca and Mg source separately. Not only after autoclaving but after the medium has cooled down
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Hi Patrick, Are you suggesting that the problem is with the water I use -it (water) is distilled and deionized on site - Although I am not fully familiar with this process, shouldnt it remove excess pyrophosphate?
Also, I should mention the precipitate forms overnight when medium is stored at 4oC and over 2-3 days if stored at RT.
And thanks again for all the help everyone!
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