The structural gene for rusticyanin from Thiobacillus ferrooxidans: cloning and sequencing of the rusticyanin gene

The rusticyanin gene from the acidophilic chemolithotroph Thiobacillus ferrooxidans has been cloned and sequenced. A central portion of the gene was identified by PCR reactions utilising primers optimised for codon bias followed by nested PCR with degenerate primers. The 5' and 3' ends of the rusticyanin gene were then cloned using degenerate primers to each end and anchor sequences to the known internal sequence. The entire gene was amplified using Tli DNA polymerase and specific primers to the 5' and 3' ends and the sequence confirmed after cloning into Bluescript and transformation of XL-1 Blue Escherichia coli.