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Phage Display Woes

Phage Display Woes - Protocols and Methods Forum

Phage Display Woes - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 12-08-2005, 08:14 AM
hubahopp@gmx.de
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Default Phage Display Woes



Dear experts,

A student in my lab is doing phage display screening of a
SingleChainAntibody (ScFv) library with the Tomlinson I+J libraries.
It's her (and my) first phage display project and we follow strictly
the protocols supplied with the library.

For those who are not familiar with (this) phage display system, here
is a short description:

It's a library of M13 derived phages displaying a diversity of ScFv on
the surface. The library is supplied as plasmids in E.coli, converted
into infectious phages with a helper phage.
Phages are screened against immobilized antigens, rescued (i.e.
converted into bacterial colonies carrying the plasmid) with E. coli
TG1 (a strain susceptible to M13 phage infection), converted to phage
again with helper phage, again used for panning/screening and so on.

My student prepared working stocks of the original library (which we
have received about 10 month ago), everything is stored in a -80degC
freezer. In the beginning, everything worked fine (ampfilication and
titering of phages, sequencing of individual phage clones).

About 2 month the troubles have started:

1) Abi Sequencing of the inserts in rescued phages did not work anymore
(no data, just noise with ABI big dye chemistry, done in two indpendent
external labs) while the control PCRs with the sequencing primer and
another one still is working.

2) Now also titering of the phages seems not to be possible. She either
gets no plaques a all or just a few really big ones (1cm in diameter)
at very high phage concentrations.

Even control experiments with samples of the original stocks resp. the
1st generation working stocks do not work, too.

We're quite puzzled now and have set up several hypotheses.

1) Somehow, bacteria and or phages have gone dead due to unknown
reasons (In my opinion very unlikely, they should be stable at -80)

2) There is at least one factor that inhibits phage infection of
bacteria. Unfortunately, we changed many things within a very short
time (new batches of bacterial media, new plastic plates), so we are
not able to trace this issue well.

3) During dishwasher cleaning (also new batches of cleansing agents) of
the glassware some inhibitor is introduced.

4) during autoclaving of the glassware and the media inhibitors are
introduced as the autoclave also is used for sterilizing microbial
infectious waste.

Before we start buying everything new and start from scratch even with
a new library, I'd like to read your comments. Which of our hypotheses
is likely and which is not? Which other factors might influence the
stability of phages and infectability of bacteria? Which conditions are
really critical to phages?

Thanks for your input!

Wolfgang Schechinger
Endocrine Research
Bochum BG&University Hospital
Germany

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  #2  
Old 12-08-2005, 09:34 AM
Duncan Clark
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Default Phage Display Woes

Historians believe that in newspost
<1134029660.473823.7900@o13g2000cwo.googlegroups.c om> on Thu, 8 Dec
2005, [Only registered users see links. ] penned the following literary masterpiece:

Have you tried plating a standard M13mp18 phage or similar? If that
doesn't work .........

M13 should never give 1cm plaques! That sounds like something else.

Check your E.coli really is F' or pick a.n.other which has an antibiotic
resistant F' and try that.

The fact that it doesn't sequence is very suspicious. Can you PCR from a
plaque or template using standard M13 forward and reverse sequencing
primers - assuming the M13 is a derivative of the normal M13mp range .
Or use some primers that should PCR across your library. If the PCR will
not work then it again suggests no M13.

Duncan
--
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.
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  #3  
Old 12-09-2005, 12:22 PM
Nick Theodorakis
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Default Phage Display Woes

On Thu, 8 Dec 2005 09:34:02 +0000, Duncan Clark
<[Only registered users see links. ].com> wrote:



I'm really straining my memory, but in the "old days" ISTR that we
used to start TG-1 cells from a colony grown on a *minimal* media
plate, rather than rich media. It was said that they tend to lose the
F' episome in rich media.

Nick


--
Nick Theodorakis
[Only registered users see links. ]
contact form:
[Only registered users see links. ]
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  #4  
Old 12-09-2005, 02:47 PM
Allison
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Default Phage Display Woes

Nick Theodorakis wrote:
We have used the pCANTAB system from Amersham (NAYY). In the manual for
the expression module they say just that - streak TG1 cells from a
glycerol stock onto a mimimal medium plate.

If you're interested the manuals for their system are online at:
[Only registered users see links. ]
There is a troubleshooting section at the end of the expression module
manual that might help you.

Allison
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  #5  
Old 12-17-2005, 02:52 AM
hubahopp@gmx.de
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Default Phage Display Woes

Hi all,

thanks for your comments.

It seems that

a) our TG1 cells have gone bad and
b) found some hints that TG1 sometimes produce unsequencable plasmids

thus
1) we ordered new TG1 and try JM109 in parallel (similar genotype)
2) prep plasmids for sequencing from JM101

Thanks, all the best and some quiet x-m{a,es}s days with lotsa input
and thoughts different from science.

Wo

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