Spontaneous Cell lysis after IPTG induction - (Dec/05/2008 )

Hello,

We have spontaneous cell lysis after IPTG induction of large scale (0.5 L or more) cultures occurring in our lab for some time now.
To investigate this, we have induced a 1 L culture with 0.2-1 mM IPTG and split it into three parts. One part survived while the other two died, so we know that the cells, growth medium and IPTG is not a problem. Is it the glassware or can it be anything else? If it is the glassware, then how can we get around this problem?

Thanks in advance,
Alex.

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This sounds like the protein you are trying to express is toxic to your cells. This is difficult to get around, but there are techniques. Perhaps you can clone a region of interest (kinase domain, binding domain, etc.) rather than the whole protein, induce with 0.1mM IPTG for a much longer time (o/n), and try inducing at 30 degrees.

I'm not sure I understand the splitting into 3 parts -- were these all induced with different IPTG concentrations? If so, can you just use the concentration in which the cells did not die?

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if it is the glassware then just make sure that you thoroughly clean and rinse the glassware before use.

if you don't rinse well then detergent that may be left behind may aid in the lysis of your cells.

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I am not quite sure that I understand the "split into three parts" either. Anyhow, regarding the glassware....we once had problems with cultures as well and it turned out the the dishwasher didn't remove all the soap residues from the glassware. After a while of shaking during incubation it killed everything in the media. I am not sure how that would relate to IPTG induction, though.

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do you have a IPTG-negative control?
btw i guess the problem is more likely by the toxicity of your protein expressed

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