Gene Regulation in Yeast be manipulated and studied, and we will begin with an example of examine can In the next few lectures we will consider how eukaryotic genes and genomes how genes are regulated in S cerevisiae. First, let's figure out how to use some neat genetics to identify some regulated genes, and in the next lecture we will igure out how one can use genetics to dissect the mechanism of that regulation Characterizing function and regulation of S cerevisiae genes: We are going to combine a few neat genetic tools that you learned about in Prof Kaisers lectures for this, namely a library of yeast genomic fragments cloned into a bacterial plasmid, a modified transposon(mini-Tn7),and the lacz gen embedded within the transposon. In this experiment the lacz gene is going to be used as a reporter for transcriptional activity of yeast genes. Mini-In7 In E col TTIR UR:3 In yeast Requiredfor Reparterdf action in Selection in Required for Ecol transposition Yeast genomic DNA The mini-Tn7 is introduced into a population of E coli that harbor a plasmid library of the s cerevisiae °○-(0○+°○ genome; i.e., each E. coli cell is home to a plasmid that contains a different un7 donor segment of the S cerevisiae genome, Random yeast such that the whole geneome is represented many times over in this population of E. coli. The mini-Tn7 is lowed to transpose by integrating into either the plasmid DNA or the bacterial ○○ DNA; the original dna that carries the mini-Tn7 can not replicate, but cells that have integrated the mini-Tn7 into the lasmid or e. coli chromosome are selected as tetracycline resistant colonies. Plasmid DNA is purified from these transformants and retransformed into tetracycline sensitive E. coli; the resulting tetracycline resistant bacteria harbor only plasmids that have an integrated mini-Tn7 transposon. plasmid is isolatedGene Regulation in Yeast In the next few lectures we will consider how eukaryotic genes and genomes can be manipulated and studied, and we will begin with an example of examining how genes are regulated in S. cerevisiae. First, let’s figure out how to use some neat genetics to identify some regulated genes, and in the next lecture we will figure out how one can use genetics to dissect the mechanism of that regulation. Characterizing function and regulation of S. cerevisiae genes: We are going to combine a few neat genetic tools that you learned about in Prof. Kaiser’s lectures for this, namely a library of yeast genomic fragments cloned into a bacterial plasmid, a modified transposon (mini-Tn7), and the lacZ gene embedded within the transposon. In this experiment the lacZ gene is going to be used as a reporter for transcriptional activity of yeast genes. Tn7TR lacZ URA3 tet Tn7TR Reporter of transcription Selection in yeast Selection in E. coli Required for transposition Required for transposition Mini-Tn7 Tn7TR lacZ URA3 tet Tn7TR Reporter of transcription Selection in yeast Selection in E. coli Required for transposition Required for transposition Mini-Tn7 Tn7TR lacZ URA3 tet Tn7TR Tn7TR lacZ URA3 tet Tn7TR In E. coli Tn7TR lacZ URA3 tet Tn7TR In yeast Yeast genomic DNA Tn7TR lacZ URA3 tet Tn7TR Tn7TR lacZ URA3 tet Tn7TR In E. coli Tn7TR lacZ URA3 tet Tn7TR Tn7TR lacZ URA3 tet Tn7TR In E. coli Tn7TR lacZ URA3 tet Tn7TR In yeast Yeast genomic DNA he + Random yeast insertion library Tn7 donor Yeast genomic plasmid library Tn7 E. coli + Random yeast insertion library Tn7 donor Yeast genomic plasmid library Tn7 + Random yeast insertion library Tn7 donor Yeast genomic plasmid library Tn7 Tn7 donor Yeast genomic plasmid library Tn7 E. coli The mini-Tn7 is introduced into a population of E.coli that harbor a plasmid library of the S. cerevisiae genome; i.e., each E. coli cell is home to a plasmid that contains a different segment of the S. cerevisiae genome, such that the whole geneome is represented many times over in this population of E. coli. The mini-Tn7 is allowed to transpose by integrating into either the plasmid DNA or the bacterial DNA; the original DNA that carries the mini-Tn7 can not replicate, but cells that have integrated the mini-Tn7 into t plasmid or E. coli chromosome are selected as Tetracycline resistant colonies. Plasmid DNA is purified from these transformants and retransformed into tetracycline sensitive E. coli; the resulting tetracycline resistant bacteria harbor only plasmids that have an integrated mini-Tn7 transposon. Plasmid is isolated