Hi

i need to mention first that I haven't worked with Pseudomonas, only with ecoli. But anyways, Inductions is usually done at mid log phase. So if you have a regular growth curve recorded you can estimate the right time frame. About the concentration, the pBad system is tightly regulated. So you should try different concentrations in small scale experiments. 0.005% to 0.02% for example should be sufficient. 

Good luck

Thankyou Tobias for your answer. Will give it a try using the method that you have suggested.

1.    Both answers are correct however  I am not sure that “ tight” is proper word to describe  araB-pBAD expression system.

(i) even in the absence of L-arabinose  araB promoter is “leaky”.

 (ii) As Andrea said AraB system suffers from cell-to-cell heterogeneity when expression levels vary greatly within the cell population, making  difficult to tightly titrate expression  of given gene  and thus  making physiological interpretations problematic. Since induction of promoter yielded mixed populations of uninduced and fully induced cells pBAD system  does not allow regulatable expression araB-controlled gene  and cannot  provide  a graded and  a homogeneous/uniform cell-to-cell expression  over a wide range of  L-arabinose  concentration .

2. The problem with  pBAD and many common regulated expression systems is that high affinity active transport of inducer and variable levels of the regulatory proteins result in a very steep induction curve and generally an all-or-none induction of an increasing number of cells rather than a homogeneous increase in all cells. Contrary PLtetO-1 is TIGHTLY repressible by the Tet repressor in the absence of inducer and  can be induced with non-toxic inducer, anhydrotetracycline (Tc) which has an increased affinity for  repressor TetR and thus can  provide  tight, graded and homogeneous cell-to-cell expression in the presence of variable inducer concentrations.

3.    I think that does not matter how much of inducer (L-arabinose) you are adding   it is important how long does induction lasts.

  I suggest to inoculate the cells from an overnight culture (1% (v/v) or OD600 =0.05), then  outgrow the cells until OD600 of 0.2 and then dilute 1:1 in LB containing 0.4% (w/v) arabinose ( most commonly used final concentration 0.2%) to induce  expression “your” membrane protein by  allowing the cells to grow for 2 mass doublings (until an OD600 of 0.4–0.5).

4. How “big” is “your” membrane  protein? How many multispans/TMDs?

From my experience  with E.coli the long/prolongated  induction with 0.2% arabinose can  lead to  appearance of misfolded/ misoriented population of overexpressed   membrane protein (by unknown reason: due to overloading/”jam” of translocon/insertase?) (my unpublished data)

Hi Mikhail,

Thank you for your answer. Really helped me a lot. My membrane protein is 82.9kDa in size.  Im trying to optimize its expression in Pseudomonas. Thanks. I will try my best

Hi

I havent used these plasmids for overexpression in Pseudomonas, but i have used them in E. coli. They worked very fine for me. You can induce the cells at OD 0.5-0.8 with 0.2% Arabinose for three hours(Thats what i use), but people even have used much lower concentrations of arabinose, upto 0.02%.).  If the protein is to be expressed to the outer membrane then you can induce the cells at OD 0.5-0.8 but if they are to be expressed in inclusion bodies, you can even induce them at OD 1.0. 

Best

Hi everyone ,

Could any one suggest which E.coli strain will be useful for the membrane protein expression with pBAD vector?

please share any article available for reference.

Thanks

Nitesh