PCR based gene engineering of the Vibrio harveyi lux operon and the Escherichia coli trp operon provides for biochemically functional native and fused gene products.
The polymerase chain reaction (PCR) was applied to clone the luxA and luxB genes from Vibrio harveyi, and the trp poL (promoter operator leader) region and the trpB and trpA genes from Escherichia coli. PCR-derived luxA/B and trpB/A genes were shown to express bacterial luciferase and tryptophan synthase respectively, when introduced into E. coli on a plasmid cloning vehicle. The trp poL was used successfully to control the expression of lac alpha, luxAB, trpB and trpA. PCR was also used to construct a functional luxAB translational fusion protein. Primers for this were designed to facilitate precise gene fusion and to provide a silent mutation within an EcoRI site in the luxB gene. Production of functional genes was verified in vitro and in vivo using polyacrylamide gel electrophoresis (PAGE) analysis of transcription-translation products and crude cell extracts, and by monitoring enzyme activity.
Hill
P J
PJ
Department of Applied Biochemistry and Food Science, University of Nottingham School of Agriculture, Sutton Bonington, Leicestershire, UK.
Swift
S
S
Stewart
G S
GS
D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D009876:Operon; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D014367:Tryptophan Synthase / Q000235:genetics* / Q000378:metabolism; D014733:Vibrio / Q000201:enzymology / Q000235:genetics*
D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D008156:Luciferases; D014367:Tryptophan Synthase