Re: [ccp4bb] smeared bacteria pellets in protein expression

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

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Subject: problems with hydrogen names in PDB file from Shelx refinement
From: Alice Dawson A {- dot -} X {- dot -} Dawson {- at -} DUNDEE {- dot -} AC {- dot -} UK
Date: 2010-05-12

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Subject: Re: problems with hydrogen names in PDB file from Shelx refinement
From: Tim Gruene tg {- at -} SHELX {- dot -} UNI-AC {- dot -} GWDG {- dot -} DE
Date: 2010-05-12

Subject: Re: smeared bacteria pellets in protein expression
From: Atlanta Cook cook {- at -} BIOCHEM {- dot -} MPG {- dot -} DE
Date: 2010-05-12

We have had problems with phage contamination, either through
contaminated plasmid stocks or more sporadic infections. Even if the
cultures don't look clear, you can usually spot the contamination if
the E. coli pellets are slimy and spread all the way up the centrifuge
bottles. We have had good experiences with T7 Express cells for
cleaning up contaminated plasmids. The last phage contamination was
sporadic for several weeks before blowing up into an epidemic, so
maybe give all your shakers a good scrub with bleach and be sure to
decontaminate affected glassware as some phages can still survive
through glass washing and autoclaving.

If you think there might be a problem with inefficient glassware
washing (it can happen and can also cause cell lysis if your glass
ware is contaminated with detergent), try shaking your "clean" flasks
for a few hours just with water at 37 degrees before autoclaving.

Atlanta

On 12 May 2010, at 11:16, Martin Hallberg wrote:

> Dear Hannes,
>
> I am afraid it can still be phage problems. There are many kinds of
> phages that
> infect E. coli and some of them behave like you describe: not always
> lysed cells but from time to time and coming and going. The phage
> may anyway be present
> but in its lysogenic state and only entering the lytic pathway under
> stressful conditions.
>
> Try some strains that lack or have mutated form of a common phage
> receptor: fhuA.
> I know that for example NEB sells a BL21(DE3) alternative (T7
> Express) with a mutated fhuA (fhuA2).
> This strain (T7 express) also has the benefit of lacking the lambda
> prophage so that if your expressed
> proteins (esp. the inclusion bodies) trigger the SOS response you
> will not have lambda phage-caused lysis at least.
>
> Best regards,
>
> Martin
>
> PS. I am not affiliated to NEB in any way....
>
>
> On May 12, 2010, at 10:29 AM, Hannes Uchtenhagen wrote:
>
>> Dear ccp4bb,
>>
>> We used to routinely overexpress a number of mammalian proteins to
>> inclusion bodies in E.coli. Lately however we get from time to time
>> smeared and small or no pellets after spinning down the cultures
>> (lysed I assume).
>> I have read (with some horror) previous threads pointing at phage
>> contamination and toxic proteins. I am doubtful about the phages as
>> the problem is not expanding and very frustratingly it appears
>> seemingly random, coming and going. Finally the one time I checked
>> I did not see any plaque formation on a e.coli plate tested with a
>> lysed culture.
>> We have expressed the proteins before many times to inclusion
>> bodies without any problems. I still could imagine that they could
>> be toxic when soluble. But why would suddenly happen since we did
>> not change any conditions that we are aware of?
>>
>> It would be really great to get some of your ideas on what else
>> could cause this kind of problem (IPTG, aeration, leftovers from
>> glassware cleaning, media) or if I missed something with respect to
>> the phages.
>>
>> Many thanks for your time!
>> hannes uchtenhagen
>>
>>
>> --
>> Hannes Uchtenhagen
>> Karolinska Institutet
>> Center for Infectious Medicine (CIM)
>> Karolinska University Hospital Huddinge, F59
>> SE-141 86 Stockholm, Sweden
>>
>> Office: +46-(0)8-524 86981
>> Mobile: +46-(0)7-36901461

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Next message:
Subject: Re: problems with hydrogen names in PDB file from Shelx refinement
From: Tim Gruene tg {- at -} SHELX {- dot -} UNI-AC {- dot -} GWDG {- dot -} DE
Date: 2010-05-12