MBIO 3410 lecture Oct 11 2007

MBIO 3410 lecture Oct 11 2007 - Last lecture overview...

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1 Last lecture - overview Selection of recombinants Growth of transformant for analysis Gel analysis Analysis of a clone MID-TERM CUT-OFF Vectors E. coli host bacteria E. coli F factor manifestations Vector characteristics Plasmids Relaxed plasmids Stringent plasmids E. coli plasmid ColE1 bacteriocin, colicin E1 ( cea ) bacteriocin resistance gene ( imm ) RNAI RNAII ROP protein pBR322 vectors Twin antibiotic resistance Replica plating pUC Plasmids High copy number Blue-white screening A multiple cloning site or polylinker pUC vs. pBR322
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2 Selectable Markers
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3 Selectable Markers Ampicillin Binds to and inhibits a number of enzymes in the bacterial membrane that are involved in the synthesis of the gram- negative cell wall Prevents proper cell replication The ampicillin resistance gene ( bla ) codes for the enzyme β - lactamase Secreted into the periplasmic space of the bacterium where it catalyzes hydrolysis of the β -lactam ring of the ampicillin Ampicillin β -lactam ring penicillin (core) http://commons.wikimedia.org/wiki/Image:Penicillin-core.png http://commons.wikimedia.org/wiki/Image:Ampicillin-2D-skeletal.png www.elecuter.co.uk/.../md/penicillin.gif
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4 Tetracycline Binds to a protein of the 30S subunit of the ribosome Inhibits binding of aminoacyl-tRNA to ribosomal A-site This interferes with ribosomal translocation along the mRNA preventing protein translation The tetracycline resistance gene ( tet R ) encodes an outer membrane associated protein of gram-negative cells that prevents the antibiotic from entering the cell The drug resistance gene does not destroy the antibiotic www.sigmaaldrich.com/Area_of_Interest/The_Americas/Canada.html
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5 Chloramphenicol Binds to the ribosomal 50S subunit Inhibits translation on the 50S ribosomal subunit at the peptidyltransferase step (elongation inhibition) The chloramphenicol resistance gene ( cmr ) codes for chloroamphenicol acetyltransferase (CAT) CAT is a tetrameric cytosolic protein that, in the presence of acetyl coenzyme A catalyzes the formation of a derivative of chloramphenicol that are unable to bind to the ribosome www.sigmaaldrich.com/Area_of_Interest/The_Americas/Canada.html
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6 Kanamycin and neomycin Bind to ribosomal components Inhibit protein synthesis The kan R gene codes for a protein that is secreted into the periplasmic space and interferes with the transport of these antibiotics into the cell Kanamycin Neomycin www.labsupplymall.com/.../Biotech/images/kan.gif www.bio-world.com/images/714179.jpg
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7 Streptomycin Inhibits prokaryote protein synthesis Causes mRNA misreading and inhibits chain initiation Binds to S 12 protein of 30S ribosomal subunit, preventing the transition from initiation complex to chain-elongating ribosome, causing miscoding or inhibiting initiation Many other antibiotic resistance genes are also used in molecular biology (vector specific) Erythromycin, Fusidic acid, Puromycin, Bleomycin, Zeocin, Hygromycin, etc.
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