Cloning of chemically synthesized lactose operators. II. EcoRI-linkered operators. |
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| Authors: | J R Sadler J L Betz M Tecklenburg D V Goeddel D G Yansura M H Caruthers |
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| Institution: | Department of Biophysics and Genetics, University of Colorado Medical Center, Denver, Colo. 80262, USA;Department of Chemistry, University of Colorado, Boulder, Colo. 80302 U.S.A. |
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| Abstract: | A 40 base, mainly duplex DNA segment, with the following sequence pAATTCCACATGTGGAATTGTGAGCGGATAACAATTTGTT (3') GGTGTACACCTTAACACTCGCCTATTGTTAAACACCTTAAp (5') has been synthesized by combination of chemical and enzymatic methods. It consists of a wild-type lactose operator sequence (boxed) bracketed by "linker" sequences which permit excision of the segment from plasmid vehicles by the EcoRI restriction endonuclease. This segment has been ligated into the pMB9 plasmid and the resulting operator plasmids used to transform E. coli K-12. Among the transformant products were strains carrying plasmids with one, two, three, or four operator segments in tandem. Derepression of the lactose operon effected by these plasmids in vivo as well as the lifetimes of complexes formed between repressor and these plasmids in vitro increase with increasing numbers of operators per plasmid. |
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| Keywords: | plasmid β-galactosidase constitutivity oligo operators BCIG bp base pair(s) IPTG TEAB triethylammonium bicarbonate TES buffer 50 mM Tris pH 7 5 at 37°C 100 mM NaCl 3 mM EDTA |
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