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Slimy recombinant E. coli pellet

MacGregor, Barbara via methods%40net.bio.net (by bmacgreg from unc.edu)
Fri Nov 10 12:27:09 EST 2017


Hello Megha,

My first thought also was contamination (how does your culture look on a plate or under the microscope?), hadn’t thought of cell lysis. Another possibility - perhaps your strain has acquired a mutation that allows it to better tolerate the protein overproduction?

For either the mutation scenario or contamination, can you go back and reintroduce the gene in the strain, see if this happens again?

Barbara


From: Wolfgang Schechinger <hubahopp from gmx.de<mailto:hubahopp from gmx.de>>
Subject: Re: Slimy recombinant e.coli pellet
Date: 9 November 2017 10:13:58 GMT-5
To: Megha Goyal <mgbiotec from gmail.com<mailto:mgbiotec from gmail.com>>, methods from magpie.bio.indiana.edu<mailto:methods from magpie.bio.indiana.edu>


Looks like contamination, Megha. Do you find your protein in the slime?

Regards,
Wo

On 09.11.2017 14:11, Megha Goyal wrote:
Dear All,

We are doing fermentation for recombinant protein using E.coli culture.
Earlier we used to obtain compact cell pellet on harvesting (3 Hrs after
IPTG Induction). But recently we observed that the cell pellet harvested
are of slimy consistency and they do not homogenise in the washing buffer
(tris-nacl-edta). We observe thread like consistency and no matter of
stirring it do we get a homogeneous suspension.

Can someone guide me what is the reason for such phenomenon.

Thanks

megha
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From: dk from no.email.thankstospam.net<mailto:dk from no.email.thankstospam.net> (DK)
Subject: Re: Slimy recombinant e.coli pellet
Date: 9 November 2017 19:21:24 GMT-5
To: methods from net.bio.net<mailto:methods from net.bio.net>


In article <mailman.110.1510233337.15473.methods from net.bio.net<mailto:mailman.110.1510233337.15473.methods from net.bio.net>>, Megha Goyal <mgbiotec from gmail.com<mailto:mgbiotec from gmail.com>> wrote:
Dear All,

We are doing fermentation for recombinant protein using E.coli culture.
Earlier we used to obtain compact cell pellet on harvesting (3 Hrs after
IPTG Induction). But recently we observed that the cell pellet harvested
are of slimy consistency and they do not homogenise in the washing buffer
(tris-nacl-edta). We observe thread like consistency and no matter of
stirring it do we get a homogeneous suspension.

Can someone guide me what is the reason for such phenomenon.

Partial cell lysis. "Slime" = chromosomal DNA.

DK




From: dk from no.email.thankstospam.net<mailto:dk from no.email.thankstospam.net> (DK)
Subject: Re: Gel Solubilization Buffer Composition
Date: 9 November 2017 19:25:54 GMT-5
To: methods from net.bio.net<mailto:methods from net.bio.net>


In article <3a598d49-8251-49e6-a147-99e9bb3e5f0f from googlegroups.com<mailto:3a598d49-8251-49e6-a147-99e9bb3e5f0f from googlegroups.com>>, angusrbishop from gmail.com<mailto:angusrbishop from gmail.com> wrote:
On Saturday, 9 January 2010 18:42:04 UTC, wattne  wrote:
On 9 Jan., 16:40, Jagadish Katam <jagadish... from hotmail.com<http://hotmail.com>> wrote:
Hi,

I am working on purifying the DNA by gel extraction procedure and are using
the commercially available kits for this purpose. A yellow color gel
solubilization buffer is used to dissolve the sliced gel.

Here i would know the composition of this gel solubilization buffer.

Any suggestions will be greatly appreciated.

Thanks and Best Regards,
Jag

Hi Jag!
Sounds like the buffer from QIAGEN, right? Why aren't you more
precise?? How should anyone help you if we don't even know what kind
of stuff you are using???
You can bet that the buffer composition is secret because otherwise
QIAGEN couldn't sell their kit anymore; what do you think? Try a
Google, and you will either find out or not. Why should we know the
exact formulation????
Thanks and Best Regards,
watnne

Hi Watnne

I found all the other replies to Jag's question helpful and constructive. Yours
felt fairly malicious. Please refrain from comments like this in the future,
its a waste of time and damages the community feeling of places like this.

I don't know about thje feelings but here is the composition:
5.5 M guanidine thiocyanate, 20 mM Tris-HCl, pH 6.6

DK








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Barbara MacGregor
University of North Carolina
Department of Marine Sciences
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