Schematic diagram outlining the general approach adopted to alter the amino acid (aa) sequence by site-directed mutagenesis. The DNA fragment encoding the wild-type (unaltered) protein of interest is cloned into a plasmid vector. An appropriate oligonucleotide (oligo) is designed, the ends of which are complementary to the nucleotide (nt) sequence of the gene, while the central portion of the oligo contains an altered nt sequence corresponding to the desired change in the protein (marked with a star). The dsDNA is denatured to make the template strand available for hybridization with the mutagenic oligo (Step 1). The addition of dNTPs, DNA polymerase, and DNA ligase enables DNA synthesis, forming closed circular molecules (Step 2). The mutated plasmid is selected, and the desired change is verified (by sequencing). The mutant plasmid is selected and transformed into a final host strain (Step 3), where large amounts of the mutant protein can be produced using standard recombinant methods.
3. Select mutant (verify by sequencing) and transform into final host
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