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
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!
Bochum BG&University Hospital