Dear Community,
I would like to know in phage display library construction, after transforming host cells (e.g. E. Coli SS320) with phagemid and helper phage (M13K07) and overnight culturing to collect the phage particles in medium, can we save the host cells by frozen for re-culturing in future ? The reason is because the construction of antibody library is tedious and expensive, it would be great that can be able to re-culture the host cells and re-collect the phage particles for future selection. Thanks.
hon
Sure you can freeze the cells by adding glycerol or DMSO and storing at -80. Just be sure to use a substantial aliquot for the next culture to be sure you are covering the entire library size adequately.
I would point out however that you may somewhat bias the library in favor of some clones and against other clones the more generations it goes through. But this might not be a major problem for you depending upon the library and the applications.
Thanks so much for your answer.
If my library application is for scFv antibody screening, do you think that would matter? Why there would cause bias to some clones in this way?
Alternatively, do you think it may be better to just fronzen down some phage particles and then use them to reinfect the bacteria hosts in next screening, would this reduce the bias issue?
The bias issues comes from the fact that some phages grow faster or more robustly than others due to the insert.
I don't have any data that would make me discern whether keeping infected cells frozen or reinfecting with phage would be better. Maybe someone else on here can answer that.
You normally store the phages, not the infected cells:
From: Article Isolation of Antibodies to Heparan Sulfate on Glypicans by P...
"Store the phage at 4°C for short-term storage (up to a few weeks).
The phage library can also be stored in PBS with 15 % v/v glycerol for longer term at −70°C."
Similar storage protocols are described in various protocols for commercial phage libraries.
Normally, when you have generated a phage library, you amplify the library to the desired level, and freeze it into working aliquots to avoid multiple freeze/thaw cycles.
Ideally, you should have many stocks of the input library already in your host cells. These library aliquots are used for each round of initial phage production. Attempting to reuse your cells from the first round of production will certainly lead to growth bias and a skew in your clone representation.
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