A plasmid vector and quantitative techniques for the study of transcription termination in Escherichia coli using bacterial luciferase.
We have developed a plasmid expression vector for the study of transcription terminators in Escherichia coli that utilizes the lux genes coding for the enzyme luciferase of the bioluminescent marine bacterium, Vibrio harveyi. The pBR322-derived plasmid, called pHV100, contains the E. coli lac promoter, the polylinker regions from the plasmid vector pUC18, and the V. harveyi lux genes. Insertion of transcription termination sites into the polylinker region results in decreased luciferase expression. Because the bioluminescence genes are not indigenous to E. coli, their expression can be studied in virtually any host strain without the complications of background activity. This facilitates sensitive measurements of terminator efficiency in hosts containing termination factor mutations. Bioluminescence can be easily monitored with high sensitivity, using a rapid photographic technique or a more quantitative photometric assay.
Peabody
D S
DS
Department of Cell Biology, University of New Mexico School of Medicine, Albuquerque 87131.
Andrews
C L
CL
Escudero
K W
KW
Devine
J H
JH
Baldwin
T O
TO
Bear
D G
DG
D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005809:Genes, Regulator; D005822:Genetic Vectors; D008156:Luciferases / Q000235:genetics*; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic; D014733:Vibrio / Q000201:enzymology / Q000235:genetics*
D004269:DNA, Bacterial; D008156:Luciferases