hi to all,
i was wandering if it is enough to pellet cells (e.coli) from the protein induction medium containing iptg and resuspend them in a new medium without inductor to stop the protein expression.
is it possible that cells continue to express the protein also if i remove IPTG from the medium through this rinsing step?
if the protein that I am expressing has a periplasmic localisation, is it possible that in this step it the protein that has been accumulates in the citosol continue to translocate in the periplasm?
thank you
What is the experimental setup? It is possible to partially (because IPTG is non-metabolizable) cease the protein expression by removing IPTG and resuspending cells in fresh medium but then it can also be done by storing the cells at 4C or below. Perhaps certain protein synthesis inhibitors such as Rifamycin or Tetracycline could also be used. It all depends on exactly what you are trying to achieve.
You could try several ways
Decreasing the temperature
Chang the carbon source
But the problem here you will increase the probability of cell death. Because after the bacteria reaches the OD where you induce protein expression the cells will start to enter in the stationary phase then after the death will start. So reusing the cell at this point would not be beneficial.
IPTG inducible promoters (especially tac) are 'leaky' i.e. there will be residual expression in the absence of IPTG. Some of this can be suppressed by addition of glucose to the culture (at around 2% w/v of glucose) but it is never entirely repressed.
If residual expression is a problem, the best promoter to use is the arabinose-inducible promoter pBAD (http://jb.asm.org/content/182/24/7029.abstract).
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