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Protein expression declines after overnight incubation - (Feb/08/2011 )

Hi!

I induced GFP protein expression with different IPTG concentrations(1mmol/l and 0,1 mmol/l) on two different samples and measured the O.D for 8 consecutive hours and then in the next morning. During the measured 8 hours, the AU of the 0,1mmol/l was bigger, and the culture with 1mmol/l had better protein expression. Left both samples to incubate overnight(about 16 h). The weird part is, when i tested again next morning, AU of the 1mmol/l was larger and protein expression lesser than that of the 0,1mmol/l sample. I cant think of a reason why that is. Please help me.
Bacterium used was e.coli BL21 and transferred plasmid pET42

Thank you in advance.

-mica-

Toxic build up of GFP killing the cells? the cells expressing more gfp using up much of the nutrients in solution to make the gfp?

-bob1-

mica on Tue Feb 8 12:23:46 2011 said:


Hi!

I induced GFP protein expression with different IPTG concentrations(1mmol/l and 0,1 mmol/l) on two different samples and measured the O.D for 8 consecutive hours and then in the next morning. During the measured 8 hours, the AU of the 0,1mmol/l was bigger, and the culture with 1mmol/l had better protein expression. Left both samples to incubate overnight(about 16 h). The weird part is, when i tested again next morning, AU of the 1mmol/l was larger and protein expression lesser than that of the 0,1mmol/l sample. I cant think of a reason why that is. Please help me.
Bacterium used was e.coli BL21 and transferred plasmid pET42

Thank you in advance.

Hola, the Bobs answer has consistence,of course, but think in the growth of bacteria withouth plasmid if the antibiotic has dissapears at all, by hydrolysis along the time, specially if its ampicillin. In fact, in long fermentations at high ODs, new dosis of antibiotics have to be added, by hidrolysis and by the increase of cell number. So these new grown bacteria without plasmid increase OD, but the total recombinant protein stay constant, because, you know, that sometimes at the induction moment the OD stays constant during the recombinant protein syntesis and depending of the protein and the inducer concentration, the culture lyses after +- 4h. at 37C. If you let induced the culture long time think in to low temperature at 25C, and add one more dosis of antibiotic one hour before harvest, checking if the OD falls. Buena suerte

-protolder-


Hola, the Bobs answer has consistence,of course, but think in the growth of bacteria withouth plasmid if the antibiotic has dissapears at all, by hydrolysis along the time, specially if its ampicillin. In fact, in long fermentations at high ODs, new dosis of antibiotics have to be added, by hidrolysis and by the increase of cell number. So these new grown bacteria without plasmid increase OD, but the total recombinant protein stay constant, because, you know, that sometimes at the induction moment the OD stays constant during the recombinant protein syntesis and depending of the protein and the inducer concentration, the culture lyses after +- 4h. at 37C. If you let induced the culture long time think in to low temperature at 25C, and add one more dosis of antibiotic one hour before harvest, checking if the OD falls. Buena suerte

The antibiotic used was kanamycin. Does that make any difference?

-mica-

The antibiotic used was kanamycin. Does that make any difference?

Hola, the fact of a plasmid resistence means that the bacteria harbouring it can grow because is able to destroy it, so after some hours the amount of antibiotic in the medium drops, and non harboring plasmid bacteria can grow. Buena suerte

-protolder-