Plasmids containing many tandem copies of a synthetic lactose operator |
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| Authors: | J R Sadler M Tecklenburg J L Betz |
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| Institution: | Department of Biophysics and Genetics, University of Colorado Health Sciences Center Denver, CO 80262 U.S.A. |
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| Abstract: | Up to 12 tandem copies of the lactose operator sequence AATTCCACATGTGGAATTGTGAGCGGATAACAATTTGTGG (3') GGTGTACACCTTAACACTCGCCTATTGTTAAACACCTTAA (5') have been cloned in the EcoRI site of plasmid pMB9. A 12-operator plasmid is about 8% operator by weight and represents a rich source of this DNA segment. A procedure for the rapid and convenient isolation of operator in mg quantities is presented. The lifetimes of complexes formed between repressor and oligo-operator plasmids increased with increasing numbers of tandem operators per plasmid. Evidence is presented indicating that only one tetrameric repressor molecule binds strongly to a segment of four (or fewer) tandem operators, but that two repressor molecules can be accommodated on segments containing at least six tandem operators. |
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| Keywords: | Recombinant DNA pMB9 plasmid DNA cloning BCIG 5-bromo-4-chloro-3-indolyl-β-D-galactoside bp base pair(s) IPTG isopropyl-β-D-thiogalactoside SET buffer 12 5 mM Tris pH 7 5 at 37°C 50 mM NaCl 1 mM EDTA |
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