Discussion section 0530

Discussion section 0530 - Discussion section#8 Regulation...

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Discussion section#8 Regulation of Gene Expression in Bacteria 05/30/08
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lac operon: lac repressor and CAP trp operon: trpL RNA 2° structure phage lambda
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Lactose metobolism requires galactoside permease beta- galactosidase lac to s e  induc e s  the ir s ynthe s is  (g e ne  e xpre s s io n)
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lactose isomer non-degradable  analog Inducers of the System
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Blue-white screen The vector (e.g. pBluescript) encodes the α subunit of LacZ protein with an internal muliple cloning site(MCS) The chromosome of the host strain(E.coli) encodes the remaining Ω subunit to form a functional beta-galactosidase enzyme. The MCS can be cleaved by different restriction enzymes so that the foreign DNA can be inserted within the lacZ α gene, thus disrupting the production of functional beta-galactosidase. The chemical required for this screen is X-gal , a colourless modified galactose sugar that is metabolized by β-galactosidase to form an insoluble product which is bright blue, and thus functions as an indicator. I IPTG, which functions as the inducer of the Lac operon, can be used in some strains to enhance the phenotype. The hydrolysis of colourless X-gal by the β-galactosidase causes the characteristic blue colour in the colonies; it shows that the colonies contain unligated vector. White colonies indicate insertion of foreign DNA and loss of the cells' ability to hydrolyse the marker.
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Lac repressor tetramer HTH motif
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CAP: catabolite (cAMP) gene activator protein Dimeric protein, HTH motif cAMP-CAP complex binds DNA stimulates txn lac operon:   e ve n in a bs e nc e  o f la c I, pro m o te r is  we a k hig h g luc o s e : lo w c AMP le ve ls lo w g luc o s e  -> c AMP le ve ls  ris e -> c AMP-C AP b inds  la c  o pe ro n -> m o re  RNA po l -> m o re  la c  e nzym e  e xpre s s io n Mechanism c AMP-C AP b e nds  DNA b y 90°  inc re a s e s  affinity o f la c  o pe rato r fo r RNA po l m a y a ls o  dire c tly re c ruit RNA po l
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Trp operon
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Discussion section 0530 - Discussion section#8 Regulation...

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