Search for the optimal sequence of the ribosome binding site by random oligonucleotide-directed mutagenesis

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Title
Search for the optimal sequence of the ribosome binding site by random oligonucleotide-directed mutagenesis
Author(s)
Kyung Tai Min; Kim Myung Hee; Dae Sil Lee
Bibliographic Citation
Nucleic Acids Research, vol. 16, no. 11, pp. 5075-5088
Publication Year
1988
Abstract
Synthetic DNA duplexes corresponding to the ribosome binding site (RBS) were synthesized through the phosphite method on solid support. The synthetic RBS DNA with partial random sequences was inserted into an appropriate site between the lpp-lac promoter and the beta-galactosidase structural gene in plasmid pMKT2. The level of beta-galactosidase expression was correlated with the color intensity of the recombinant colonies on X-gal plates. The bluest colonies were isolated and characterized with respect to beta-galactosidase enzyme activity and RBS sequence. There was good correlation between color intensity and the level of the enzyme activity, and this provided a reliable phenotypic screening method in the search for the optimal regulatory sequences. Novel RBS sequences obtained here show not only the unique nucleotide distribution, but also strong complemetarity to the 3' end region of 16S rRNA, from which could be deduced a generalized RBS sequence, the position of the SD region, and the 16S rRNA position mediated during translation initiation.
ISSN
0305-1048
Publisher
Oxford Univ Press
DOI
http://dx.doi.org/10.1093/nar/16.11.5075
Type
Article
Appears in Collections:
1. Journal Articles > Journal Articles
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