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Updated: Jul 15, 2026
Functional Complementation Analysis (FCA): A Laboratory Exercise Designed and Implemented to Supplement the Teaching of Biochemical Pathways
Published on: June 24, 2016
Complementation of an E. coli cysteine auxotrophic mutant for the structural modification study of
Jong-Joo Cheong1, Ingyu Hwang, Sangkee Rhee
1School of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University, Seoul, Korea. cheongjj@snu.ac.kr
Abstract:
The Arabidopsis AHL gene encodes a 3'(2'),5'-bisphosphate nucleotidase (BPNTase) involved in the reductive sulfate activation pathway. A bacterial expression vector containing AHL cDNA was randomly mutagenized with hydroxylamine and transformed into the E. coli cysteine auxotrophic mutant cysQ. Bacterial colonies that did not show evidence of complementation, i.e. those that exhibited slower growth on cysteine-free medium, were selected for further study. Sequencing of the AHL cDNA in one such clone revealed the conversion of cytosine 635 (C635) to thymine, resulting in an Alanine (A212) to Valine substitution. This microbial complementation procedure is useful in BPNTase structure-activity studies for biotechnological applications.
