If all of your plaques are white (colorless), it is possible that the Xgal/IPTG plates were incorrectly prepared. Test your plates by titering the naive unpanned library, using 109 and 1010 dilutions. If both the unpanned library and your selected phage produce white plaques, then the plates are defective and should be carefully re-prepared. Also, the bacterial strain used for plating must be capable of a-complementation (lacZDM15 or equivalent), such as the supplied strain ER2738, in order for blue/white screening to work. The most likely explanation for white plaques is that the pool of phage became contaminated with an environmental M13-like phage during panning and amplification. Display of foreign peptides as N-terminal fusions to the infectivity protein pIII, as in the Ph.D. libraries, slightly attenuates infectivity of the library phage relative to wild-type M13. As a result, there is an in vivo selection for the contaminating phage during the amplification steps between rounds of panning. In the absence of a correspondingly strong in vitro binding selection during panning, even vanishingly small levels of contamination can result in a majority of the phage pool being wild-type phage after 3 (or especially 4) rounds of panning. The Ph.D.-C7C library is particularly susceptible to contamination, since phage infectivity is further attenuated by displayed cysteine-containing peptides. Contamination is an extremely common problem with any phage display system, but fortunately there are a few things you can do to minimize this problem:
- Use Xgal/IPTG plates for all titering steps, and if white plaques are evident, pick only blue plaques for sequencing.
- Use aerosol-resistant pipet tips and cotton-plugged pipets for all protocols described in the Manual.
- If contamination problems persist, all of the solutions used for panning should be autoclaved, with the exception of BSA-containing solutions which should be filter sterilized. Solutions used for phage display should not be used for anything else. Pipettors should be disassembled, the barrel autoclaved, and the internal plunger machinery soaked overnight in a detergent solution such as Count-Off™.
- Since wild-type phage are preferentially amplified during the amplification steps, pick plaques for sequencing directly after the 3rd round elution step. Do not amplify the 3rd round eluate and carry out a 4th round unless the third round sequences show no clear consensus.
- If all or most of the plaques are white (colorless) after 3 rounds of panning, it is possible that the library simply does not contain any clones that bind tightly to the target. The ideal ligand sequence may not be statistically represented in the library, or the target simply is not capable of binding to a short peptide sequence. In the case of the C7C library, where all the peptides are constrained in a disulfide loop, a ligand sequence where the imposed constraint allows a productive binding conformation will bind more tightly than the same linear sequence due to improved binding entropy. However, if the imposed constraint does not allow a productive binding conformation, than that sequence will likely not bind to the target at all. In this case either of our linear libraries may yield better results.