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 c odon bias followed by nested PCR with degenerate primers, The 5' and 3 ' ends of the rusticyanin gene were then cloned using degenerate prime rs to each end and anchor sequences to the known internal sequence, Th e entire gene was amplified using Tli DNA polymerase and specific prim ers to the 5' and 3' ends and the sequence confirmed after cloning int o Bluescript and transformation of XL-1 Blue Escherichia coli.