The effect of lac-i inactivation. The peptide recognized by the mAb CG10 was expressed on Protein 8 of fth1 phage. Protein 3 of the same phage contained the BT. As is illustrated in the dot-blot above, mAb CG10 binds 2X serial dilutions of the phages (column 2) as opposed to the total lack of binding of the control phage fth1 (column 1). Expression of the phages in DH5alpha cells transformed with pBIRAcm dramatically reduces the expression of the CG10 epitope (column 3). Addition of IPTG to the cells induces the expression of the recombinant Protein 8 (column 4). Inactivation of the lac-i gene by digestion with Apa1 and self-ligation dramatically improves the expression of the recombinant Protein 8 without the need of IPTG (column 5). Reaction of a duplicate filter with anti-M13 shows that the concentration of phages for the 5 samples is the same. The in vivo biotinylation of Protein 3 was measured by ELISA and samples 3-5 gave equal signals (1.8-2.0 OD) as the promoter of Protein 3 is not repressed by lac-i (not shown).
Smelyanski and Gershoni Virology Journal 2011 8:495 doi:10.1186/1743-422X-8-495