Identification of expression signals of the mycobacteriophages Bxb1, L1 and TM4 using the Escherichia-Mycobacterium shuttle plasmids pYUB75 and pYUB76 designed to create translational fusions to the lacZ gene.
Mycobacterial expression signals were cloned using specially constructed gene fusion shuttle plasmid probes carrying a truncated Escherichia coli lacZ (beta-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-Guérin' (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 beta-galactosidase expression were observed, and one Bxb1 expression signal was identified where beta-galactosidase expression is repressed in phage lysogens.
Barletta
R G
RG
Howard Hughes Medical Institute, Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461.
Kim
D D
DD
Snapper
S B
SB
Bloom
B R
BR
Jacobs
W R
WR
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D007763:Lac Operon; D008242:Lysogeny; D009160:Mycobacteriophages / Q000235:genetics*; D009161:Mycobacterium / Q000235:genetics*; D009163:Mycobacterium bovis / Q000235:genetics*; D010957:Plasmids; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D001616:beta-Galactosidase