XL1-Blue MRF’

The RecA– E. coli host strain XL1-Blue MRF´ is supplied with theZAP Express cDNA synthesis kit.3 Because the pBK-CMV phagemid vectordoes not require a supF genotype, the amplified library grows veryefficiently on the XL1-Blue MRF´ strain. In addition, use of the correct hoststrain is important when working with the pBK-CMV phagemid vector asthe F´ episome present in the XL1-Blue MRF´ strain serves three purposes.First, the ΔM15 lacZ gene present on the F´ episome is required for theβ-galactosidase-based nonrecombinant selection strategy. When cDNA ispresent in the polylinker, expression from the lacZ gene is disrupted andwhite plaques are produced. In contrast, without insert in the polylinker, theamino terminus of β-galactosidase is expressed and nonrecombinants can bescored visually by the presence of blue plaques. To produce anenzymatically active β-galactosidase protein, two domains are required: theα-region expressed by the vector and the ΔM15 lacZ domain expressed bythe F´ episome. These two domains fold to form a functional protein, theα-region complementing the missing amino acids resulting from theΔM15 mutation. Therefore, in order to utilize the nonrecombinant selectionstrategy, the correct host strain must be used to produce a functionalβ-galactosidase protein.Second, the F´ episome expresses the genes forming the F´ pili found on thesurface of the bacteria. Without pili formation, filamentous phage (i.e., M13or f1) infection could not occur. Because the conversion of a recombinantZAP Express clone to a pBK-CMV phagemid vector requires superinfectionwith a filamentous helper phage, the F´ episome is required for in vivoexcision (see In Vivo Excision of the pBK-CMV Phagemid Vector from theZAP Express Vector).Third, the F´ episome contains the lac repressor (lacIq gene), which blockstranscription from the lacZ promoter in the absence of the inducer IPTG.This repressor is important for controlling expression of fusion proteinswhich may be toxic to the E. coli. Because the presence of thelacIq repressor in the E. coli host strain can potentially increase therepresentation or completeness of the library, XL1-Blue MRF´ is useful forscreening the amplified library.

Note

The strains used for the Lambda gt11 vector (i.e., Y1088, Y1089,and Y1090) are not suitable for use with the ZAP Express vectorbecause these strains contain the plasmid pMC9, a pBR322derivative, which contains many of the same sequences as thosefound in the phagemid portion of the ZAP Express vector. TheSURE strain and the SOLR strain are not compatible with the ZAPExpress system, since these strains contain the kanamycinresistancegene found in the pBK-CMV phagemid vector. Usingthese strains with the ZAP Express vector could result inrecombination between the homologous sequences.