The RecA– E. coli host strain XL1-Blue MRF´ is supplied with the
ZAP Express cDNA synthesis kit.3 Because the pBK-CMV phagemid vector
does not require a supF genotype, the amplified library grows very
efficiently on the XL1-Blue MRF´ strain. In addition, use of the correct host
strain is important when working with the pBK-CMV phagemid vector as
the 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 the
β-galactosidase-based nonrecombinant selection strategy. When cDNA is
present in the polylinker, expression from the lacZ gene is disrupted and
white plaques are produced. In contrast, without insert in the polylinker, the
amino terminus of β-galactosidase is expressed and nonrecombinants can be
scored visually by the presence of blue plaques. To produce an
enzymatically active β-galactosidase protein, two domains are required: the
α-region expressed by the vector and the ΔM15 lacZ domain expressed by
the F´ episome. These two domains fold to form a functional protein, the
α-region complementing the missing amino acids resulting from the
ΔM15 mutation. Therefore, in order to utilize the nonrecombinant selection
strategy, the correct host strain must be used to produce a functional
β-galactosidase protein.
Second, the F´ episome expresses the genes forming the F´ pili found on the
surface of the bacteria. Without pili formation, filamentous phage (i.e., M13
or f1) infection could not occur. Because the conversion of a recombinant
ZAP Express clone to a pBK-CMV phagemid vector requires superinfection
with a filamentous helper phage, the F´ episome is required for in vivo
excision (see In Vivo Excision of the pBK-CMV Phagemid Vector from the
ZAP Express Vector).
Third, the F´ episome contains the lac repressor (lacIq gene), which blocks
transcription from the lacZ promoter in the absence of the inducer IPTG.
This repressor is important for controlling expression of fusion proteins
which may be toxic to the E. coli. Because the presence of the
lacIq repressor in the E. coli host strain can potentially increase the
representation or completeness of the library, XL1-Blue MRF´ is useful for
screening 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 vector
because these strains contain the plasmid pMC9, a pBR322
derivative, which contains many of the same sequences as those
found in the phagemid portion of the ZAP Express vector. The
SURE strain and the SOLR strain are not compatible with the ZAP
Express system, since these strains contain the kanamycinresistance
gene found in the pBK-CMV phagemid vector. Using
these strains with the ZAP Express vector could result in
recombination between the homologous sequences.