6 replies to this topic

[xabiarias]

Hi guys,

Is it normal that, performing a phage display library and after X-gal selection, in the amplification reaction appears more fragments without the insert (around 240 bp) than with the insert (around 290 bp)?? in which step could the insert be broken away?

Regards,

[BioMiha]

It depends on the library you are selecting from. If your library is poor and the selection process is not favouring higher affinity binders then it is quite normal to have insertless phage after selection/amplification. The insert is not breaking away, the insertless phage are already in your library and they have a survival advantage over the phage with the insert => therefore they amplify with more success and in a few rounds overtake the library.
What library are you using - NEB PhD?

[xabiarias]

Thanks for the replay. The research is conducted by a PhD student and I just joined her in the PCR amplification step. The library is Ph.D.-12 Phage Display Library. So, do you think that the problem is that the affinities are not high enough?

[BioMiha]

I remember when I did some experiments with this library (and I also asked the NEB technical assistance staff) that about 15% of the clones in the initial library are insertless. Which in my opinion is a shitty library but it's basically the only one available unless you make your own. When we made our own libraries, we tried to minimize the percentage of wild type phage but you can never completely get rid of the problem. If you are selecting high affinity phage, these will dominate selection and you will gradually remove the wt phage in subsequent rounds of selection/amplification. If however, the selection process is not selective enough you will have a lot of wt phage because they replicate more efficiently, especially in M13 phage display, because the peptide is expressed on p3, which is involved in the infection process. After how many rounds of selection did you observe this phenomenon? Did you do any ELISA or blot to see if you have any positive binders? What is the target protein you are selecting on (if you can tell me that)?
Cheers,
Miha

[xabiarias]

Your explanations are very interesting. As I mentioned, I have missed all the previous steps and do not really know in which way they have done them. I think that they did ELISA assays but I do not have other relevant information. Even the target protein is unknown for me...

How is it posible that in a library like this appear to be such amount of insertless phages?? maybe a stupid question..

[BioMiha]

Because of the library preparation procedure. You have a phage vector to which you add your insert - which is very small. You then ligate and transform the host bacteria. If the vector ligates without the insert, you get insertless phage.
There is a very good paper on this subject that I recommend to anyone = Levitan - Stochastic modeling and optimization of phage display. Otherwise, I did a big part of my PhD with phage display, however the successes were few and far between.

[xabiarias]

thank you again!