Tuning the Expression Level of a Gene Located on a Bacterial Chromosome.
Katashkina, J. I.; Skorokhodova, A. Yu.; Zimenkov, D. V.; Gulevich, A. Yu.; Minaeva, N. I.; Doroshenko, V. G.; Biryukova, I. V.; Mashko, S. V.
[Miscellaneous] Molecular Biology. 39(5):719-726, September 2005.

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A new method of constructing a set of bacterial cell clones varying in the strength of a promoter upstream of the gene of interest was developed with the use of Escherichia coli MG1655 and lacZ as a reporter. The gist of it lies in constructing a set of DNA fragments with tac-like promoters by means of PCR with the consensus promoter Ptac and primers ensuring randomization of the four central nucleotides in the -35 region. DNA fragments containing the tac-like promoters and a selective marker (CmR) were used to replace lacI and the regulatory region of the lactose operon in E. coli MG1655. Direct LacZ activity assays with independent integrant clones revealed 14 new promoters (out of 44 = 256 possible variants), whose strength varied by two orders of magnitude: LacZ activity in the corresponding strains gradually varied from 102 Miller units with the weakest promoter to 104 Miller units with consensus Ptac Sequencing of the modified promoters showed that randomization of three positions in the -35 region is sufficient for generating a representative promoter library, which reduces the number of possible variants from 256 to 64. The method of constructing a set of clones varying in expression of the gene or operon of interest is promising for modern metabolic engineering.

(C)2005 Kluwer Academic Publishers