pGLO mutagenesis: A laboratory procedure in molecular biology for biology students

• Published in: Biochemistry and molecular biology education 2011-11, Vol.39 (6), p.432-439
• Main Author: Bassiri, Eby A.
• Format: Article
• Language: English
• Subjects: College Science; Cytology; DNA sequencing; DNA Transposable Elements - genetics; Education, Professional - methods; Genetics; Laboratory Experiments; Laboratory Procedures; Molecular Biology; Molecular Biology - education; Mutagenesis; Mutagenesis, Insertional; Plasmids - genetics; restriction digestion analysis; Science Instruction; Science Laboratories; Science Process Skills; screening for mutants; Sequence Analysis, DNA; transformation; transposon
• ISSN: 1470-8175; 1539-3429
• DOI: 10.1002/bmb.20538

A five‐session laboratory project was designed to familiarize or increase the laboratory proficiency of biology students and others with techniques and instruments commonly used in molecular biology research laboratories and industries. In this project, the EZ‐Tn5 transposon is used to generate and screen a large number of cells transformed with mutagenized pGLO plasmid. EZ‐Tn5 carries the kanamycin resistance (KanR) gene, and the pGLO plasmid carries the β‐lactamase gene for ampicillin resistance (AmpR), the gene encoding green fluorescent protein (GFP) and the arabinose operon repressor (araC). Insertion of the Tn5 transposon into pGLO occurs randomly, and any gene into which it inserts is knocked out. By screening cells transformed with mutagenized pGLO with kanamycin, ampicillin, arabinose and/or for GFP expression in different combinations, pGLO plasmids with mutations in different genes are identified. The locations of these insertions are then mapped approximately by restriction fragment analysis and precisely by sequence analysis of the pGLO plasmid. BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION Vol. 39, No. 6, pp. 432–439, 2011