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BIMM100 Problem Set 2_JL_TAs

the mcs of puc18 is embedded near the beginning of

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Unformatted text preview: pontaneously KpnI NotI 4,000bp 3,000bp 2,000bp 1,000bp 500bp assemble to make an active enzyme. The pUC18 plasmid carries coding information for lacZ!. The MCS of pUC18 is embedded near the beginning of the coding region of 4,000bp lacZ! in such a way that the lacZ! reading frame is not disrupted. Thus although extra amino acids are introduced by translation of the MCS, adding a few extra residues near 3,000bp the N-terminus of lacZ! does not affect its structure, its ability to assemble with lacZ", or the enzymatic activity of the assembled !-galactosidase 2,000bp pUC18 is protein. When transformed into an E. coli strain carrying the lacZ" gene, active !-galactosidase is produced, which can cleave X-gal, a substrate that is included in the agar plate. X-gal is 1,000bp colorless, but when cleaved by !-galactosidase, it produces a product with an intense blue color. Thus, plasmids with an intact lacZ! fragment will500bp blue bacterial colonies produce when transformed into bacteria grown on an X-gal plate. c) To allow expression of the human protein in E. coli, inserted into the MCS of pUC18, it almostcoding region If DNA has been would you amplify the human always destroys the lacZ! gene p that it can by Reverse a lacZ! protein that human by PCR from human genomic DNA, or from cDNAso reparedno longer produceTranscription ofassembles with acZ" (3) mRNA? In two sentences or less, explain lwhy. to produce active !-galactosidase. Thus when a pUC18 plasmid containing an insert is transformed into the E. coli strain carrying the lacZ" gene, no active !galactosidase is produced, so the resulting colonies are white. A. (4.5 points) In order for blue/white selection to work, the presence of an MCS lacking an inser must not affect the function of the lacZ! gene. Aside from being relatively small, how is it that insertless MCS does not disrupt the lacZ! gene (i.e., does not prevent the gene from encodin...
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