from these cells and the yeast genomic fragments are isolated by digestion with an appropriate restriction enzyme. So now we have a library of yeast genomic fragments each of which has the transposon inserted; these genomic fragments can be transformed into s cerevisiae cells that are ura3-. Each Ura+ transformant colony will have recombined a Tn7 transposon-containing genomic dNa into its genome. This essentially gives us a library of yeast with transposons randomly integrated into it genome Note that the lacz gene in the transposon does not carry its own/Promoter transcription or a translation start site, but if the transposon inserts hmmm in the correct orientation downstream of a yeast gene promoter, and in the correct triplet codon reading frame, the lacz gene comes under the one in two insertions will be in the incorect orientation and will not produce a Lacz-fusion control of that promoter and when transcription is activated from that promoter a Lacz-fusion only one in three correct orientation protein is expressed, and most nsertions can produce a Lacz-fusion proten Lacz-fusion proteins display .At most, only one in six insertions produce a robust B-galactosidase activity. functional Lacz-fusion proten Promoter of gene X Yeast cells expressing B-galactosidase activity can easily be detected by growth in the presence of 5-bromo-4-chloro 3-indolyl-beta-D-galactopyranoside better known as x-gal. Lacz cleaves x gal to release a chemical moiety that has Fusion protein N- c a brilliant blue color and so the colo turn bright blue Fusion Protein has B-galactosidase activity There are at least two useful things to come out of such a collection of yeast trains: (1) Any transposon that integrated into a gene will essentially disrupt that gene and is likely to cause a null mutation (2) For transposons that integrate into a yeast gene such that the lacz gene is in frame with the genes coding region, the level of B-galactosidase activity in these cells therefore becomes a reporter for the transcription f that genefrom these cells and the yeast genomic fragments are isolated by digestion with an appropriate restriction enzyme. So now we have a library of yeast genomic fragments each of which has the transposon inserted; these genomic fragments can be transformed into S. cerevisiae cells that are ura3-. Each Ura+ transformant colony will have recombined a Tn7 transposon-containing genomic DNA into its genome. This essentially gives us a library of yeast with transposons randomly integrated into it genome. romoter, and in the correct -fusion t . brilliant blue color…and so the colonies c d u such that the lacZ gene n, the level of β-galactosidase activity in these cells therefore eporter for the transcription Note that the lacZ gene in the transposon does not carry its own transcription or a translation start site, but if the transposon inserts in the correct orientation downstream of a yeast gene Tn7TR lacZ URA3 tet Tn7TR Promoter of gene X Tn7TR lacZ URA3 tet Tn7TR Promoter of gene X Yeast cells expressing β-galactosidase activity can easily be detected by growth in the presence of 5-bromo-4-chloro- 3-indolyl-beta-D-galactopyranoside, better known as X-gal. LacZ cleaves X￾gal to release a chemical moiety that has •Only one in three correct orientation insertions can produce a LacZ-fusion proten •One in two insertions will be in the incorrect orientation and will not produce a LacZ-fusion protein •At most, only one in six insertions produce a functional LacZ-fusion proten •Only one in three correct orientation insertions can produce a LacZ-fusion proten •One in two insertions will be in the incorrect orientation and will not produce a LacZ-fusion protein •At most, only one in six insertions produce a functional LacZ-fusion proten p RT Tn7 t te A3 UR Zcal RT7 Tn triplet codon reading frame, the lacZ gene comes under the control of that promoter and when transcription is activated from that promoter a LacZ protein is expressed, and mos LacZ-fusion proteins display robust β-galactosidase activity Promoter of gene X Transcription stop Tn7TR lacZ URA3 Transcription start Translation start mRNA AUG Promoter of gene X Transcription stop Tn7TR lacZ URA3 Transcription start Translation start Transcription stop Tn7TR lacZ URA3 Transcription start Translation start Tn7TR lacZ URA3 Transcription start Translation start mRNA AUG a turn bright blue! ome out of such a collection of yeast into a gene will essentially disrupt that ll mutation. into a yeast gene There are at least two useful things to strains: (1) Any transposon that integrate gene and is likely to cause a n (2) For transposons that integrate is in frame with the genes coding regio Fusion protein N- -C Gene X encoded amino acids Mini-Tn7 encoded amino acids LacZ encoded amino acids Fusion Protein has β−galactosidase activity Fusion protein N- -C Gene X encoded amino acids Mini-Tn7 encoded amino acids LacZ encoded amino acids Fusion Protein has β−galactosidase activity becomes a r of that gene