Reporter gene fusions Operon fusion: transcriptional Promoter from foreign gene Translational sequence from reporter gene BIOL 533 Lecture 4
BIOL 533 2 Lecture 4 Reporter Gene Fusions • Operon fusion: transcriptional – Promoter from foreign gene – Translational sequence from reporter gene
Reporter gene fusions Gene fusion translational Promoter and translational signal from foreign gene Initiation signal/+NH, coding sequence Translational signal also from reporter gene Initiation sequence and part of NH sequence of reporter gene missing but COOH region still present Two Coding sequences must be in frame BIOL 533 Lecture 4
BIOL 533 3 Lecture 4 Reporter Gene Fusions • Gene fusion: translational – Promoter and translational signal from foreign gene • Initiation signal/+NH2 coding sequence – Translational signal also from reporter gene • Initiation sequence and part of NH2 sequence of reporter gene missing, but COOH region still present – Two coding sequences must be in frame
Technique Use transposon vector containing lac fusion Carries promoterless lacz gene and selectable antibiotic marker Selection for antibiotic marker generates set of random insertions in chromosome Colonies carrying transposon screened for expression under conditions tested BIOL 533 Lecture 4
BIOL 533 4 Lecture 4 Technique • Use transposon vector containing lac fusion – Carries promoterless lacZ gene and selectable antibiotic marker – Selection for antibiotic marker generates set of random insertions in chromosome – Colonies carrying transposon screened for expression under conditions tested
Foreign Gene Expression Assay for production of Bgal Higher temperature —Iron etc Locate unknown gene BIOL 533 Lecture 4
BIOL 533 5 Lecture 4 Foreign Gene Expression • Assay for production of βgal – Higher temperature – +— iron – etc. • Locate unknown gene
lacz Fusion Used to find regulatory genes that control expression of virulence gene lac fusion strain is mutagenized(chemical or another transposon) Resulting colonies screened for aberrant regulation BIOL 533 Lecture 4
BIOL 533 6 Lecture 4 lacZ Fusion • Used to find regulatory genes that control expression of virulence gene – lacZ fusion strain is mutagenized (chemical or another transposon) – Resulting colonies screened for aberrant regulation