Avoidance of leaky expression in Terrific Broth - did I need to add 1% Glucose (Apr/25/2011 )

Hi All,

I think of trying different expression medium for some of my proteins.
Since I have power of Terrific Broth (sigma-aldrich), I decided to give it a try.

However for my construct, there are some leaky expression in LB medium, and I managed to reduce it by adding glucose.
If I were to use Terrific broth, did I need to add glucose like I did for LB medium?

Many thanks.

Adrian

Hola Adrian, as you know the difference between LB and LB glucose are the cAMP levels, High without and low with. In the case of TB alone the cAMPlevels are high, but with glucose, probably bacteria spent it first and after when the glycerol is being spent an scape of expression will be produced. But we are talkink of levels of 0.5g/l of glucose will give you absorbances of 4-6 units at 600nm before starting the glycerol comsumptionand reach about 10-12 units.I think that glycerol freely diffuses to incorporate as glycerol-3-phopsphate, probably glucose inhibits this step sintetase enzyme. Make a try Checking expression and remember that glucose levels above 1g/l gives an incomplete oxidation and produces acetic acid that affects to grow as yeast produce alcohol althought it has oxigen presence. Buena suerte

Hello protolder,
muchas gracias

But I never see in any publication people supplemented TB with glucose, or uses TB as recombinant protein expression, and that worries me.
Anyone had any experience in using TB as production of recombinant protein?

Thanks.

Yes i have worked with it and withouth glucose works as LB but gets high od(about 10 ud )Lb gets 3-4 units. Te deseo un buen dia

protolder on Tue Apr 26 07:53:19 2011 said:

I wish you a nice day too..
You really make me learn Spanish...haha

Thanks for the information.

Rgs,
Adrian

if your protein is going to be toxic then i would avoid TB since it contains high levels of lactose (bacto tryptone = digested casein).

If you want to be in control and avoid leaky expression try to use minimal medium with glucose.

Regards,
p

adrian kohsf on Tue Apr 26 14:16:10 2011 said: