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
lacz Fusion Aberrant regulation: Colonies are not Lact in presence of inducing conditions o Loss of activator Colonies are Lact under both inducing and non-inducing conditions Loss of repressor BIOL 533 7 Lecture 4
BIOL 533 7 Lecture 4 lacZ Fusion • Aberrant regulation: – Colonies are not Lac+ in presence of inducing conditions • Loss of activator – Colonies are Lac+ under both inducing and non-inducing conditions • Loss of repressor
lacz Fusion Gene tagged by transposon can be cloned Segment of dNa adjacent to transposon Can be used as hybridization probe to clone wild type locus Can also be cloned by complementation Cloned dna can be sequenced BIOL 533 Lecture 4
BIOL 533 8 Lecture 4 lacZ Fusion • Gene tagged by transposon can be cloned – Segment of DNA adjacent to transposon: • Can be used as hybridization probe to clone wildtype locus • Can also be cloned by complementation • Cloned DNA can be sequenced
phoA Gene Fusions Virulence genes are small subset of total genes, so random mutagenesis will affect many more non-vIrulence genes Way to optimize number of virulence gene mutations takes advantage of fact that most virulence genes are on bacterial surface or excreted into medium BIOL 533 Lecture 4
BIOL 533 9 Lecture 4 phoA Gene Fusions • Virulence genes are small subset of total genes, so random mutagenesis will affect many more non-virulence genes • Way to optimize number of virulence gene mutations takes advantage of fact that most virulence genes are on bacterial surface or excreted into medium
phoa gene fusions带 Translational protein fusion Alkaline phosphatase is expressed only in periplasm Foreign gene product has to be expressed outside of cytoplasm In vector, signal sequence mutations block export of alkaline phosphatase and render it inactive BIOL 533 Lecture 4
BIOL 533 10 Lecture 4 phoA Gene Fusions • Translational protein fusion • Alkaline phosphatase is expressed only in periplasm • Foreign gene product has to be expressed outside of cytoplasm • In vector, signal sequence mutations block export of alkaline phosphatase and render it inactive
phoa gene fusions带 Export and activity restored by fusing in frame a restriction fragment to truncated phoa gene; i.e lacking signal sequences No AUG start codon No ribosome binding site Restriction fragment has portions of NH2 terminal genes encoding signal sequences of heterologous proteins, such as OmpF or LamB BIOL 533 Lecture 4
BIOL 533 11 Lecture 4 phoA Gene Fusions • Export and activity restored by fusing in frame a restriction fragment to truncated phoA gene; i.e., lacking signal sequences – No AUG start codon – No ribosome binding site • Restriction fragment has portions of NH2 - terminal genes encoding signal sequences of heterologous proteins, such as OmpF or LamB
