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Nucleic Acids Research, 1988, Vol. 16, No. 11 5075-5088
© 1988

Articles

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

Kyung Tai Min, Myoung Hee Kim and Dae-Sil Lee*

Genetic Engineering Center, Korea Advanced Institute of Science and Technology PO Box 131, Cheongryang, Seoul, Korea

*To whom correspondence should be addressed

Received February 29, 1988. Revised May 12, 1988. Accepted May 12, 1988.

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 ß-galactosidase structural gene in plasmid pMKT2. The level of ß-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 ß-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 hero show not only the unique nucleotide distribution, but also strong complementarity 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.


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