Base changes in the rpoB gene leading to an amino change of a conserved histidine gives rise to rifampicin resistance. a. Local alignment of RpoB from four LAB strains that highlights a conserved histidine (highlighted by a single black dot above the sequence) which has been found to yield rifampicin resistance in these LAB strains as well as in Bacillus subtilis and E. coli when another amino acid is substituted in its place. Asterisks (*) below the alignment indicate a single conserved residue, a colon (:) represents residues which share strongly similar properties and a period (.) indicates residues that have weak similar properties. b. An example of how to design an oligonucleotide that will incorporate four consecutive mismatches while only altering the histidine residue. The leading and lagging strands are indicated while the oligonucleotide is shown below in bold. - indicates the nucleotide found in the lagging strand is identical to the nucleotide in the oligo and the base mismatches are indicated in bold letters. The successful incorporation of the oligonucleotide into the chromosome will eventually lead to the alteration of the serine codon from TCA to AGC, which still codes for serine while the histidine codon (CAC) is changed to AAC (asparagine).