Mycobacterial expression signals were cloned using specially constructed gene fusion shuttle plasmid probes carrying a truncated Escherichia coli lacZ (β-galactosidase) gene which lacked a promoter, a ribosome binding site, and an ATG start codon. Libraries of mycobacteriophage Bxb1, L1 and TM4 DNAs were constructed, and introduced by electroporation into Mycobacterium smegmatis and the 'bacille Calmette-Guerin' (BCG). Clones carrying mycobacterial expression sequences were detected by their blue colour or characteristic fluorescence when plated on media containing chromogenic or fluorogenic substrates. Varying degrees of β-galactosidase expression were observed, and one Bxb1 expression signal was identified where β-galactosidase expression is repressed in phage lysogens.
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