Recombinant protein expression and site-directed mutagenesis of target genes have demonstrated an increasing importance in the fields of molecular biology, biochemistry, biotechnology, and medicine. By using the flavodoxin of the model cyanobacterium Anabaena sp. PCC 7120 as a laboratory tool, we designed a comprehensive laboratory practice encompassing several well-established molecular biology techniques and procedures in order to fulfill two main objectives: (1) overexpression and immunodetection of Anabaena flavodoxin in recombinant Escherichia coli cell extracts, and (2) site-directed mutagenesis of the Anabaena flavodoxin gene isiB. This lab practice provides undergraduate students the possibility to perform by themselves several essential techniques in the field. With the aid of professors, students are stimulated to think, to interpret, and to discuss the results based on what they had learned in previous theoretical courses. © 2018 by The International Union of Biochemistry and Molecular Biology, 46(5):493-501, 2018.