High-level expression of RNAs and proteins: the use of oligonucleotides for the precise fusion of coding-to-regulatory sequences

We show that the fusion between regulatory sequences present on expression vectors and coding sequences can be efficiently achieved by oligonucleotide-directed mutagenesis. We have constructed single-stranded (ss) expression vectors that facilitate this process. These plasmids derive from vectors that have been used for the synthesis of quantities of proteins in Escherichia coli or RNAs in vitro. By inserting the origin of replication of the ss phage f1 into these plasmids it became possible to package their ss DNA into phage rods. Deletion of unwanted sequences or simple base changes can then be obtained by oligonucleotide-directed mutagenesis using the vector ss DNA as a template. We discuss the results of several experiments where this technique was applied to our expression vectors and we demonstrate the construction of a plasmid which efficiently synthesizes in vitro a regulatory RNA molecule that is involved in the control of plasmid copy number.