A protocol for functional assessment of whole-protein saturation mutagenesis libraries utilizing high-throughput sequencing

Michael A. Stiffler, Subu K. Subramanian, Victor H. Salinas, Rama Ranganathan

Research output: Contribution to journalArticlepeer-review

4 Scopus citations


Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 β-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to β-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

Original languageEnglish (US)
Article numbere54119
JournalJournal of Visualized Experiments
Issue number113
StatePublished - Jul 3 2016


  • Antibiotic resistance
  • High throughput sequencing
  • Issue 113
  • Molecular Biology
  • Mutagenesis
  • Next generation sequencing
  • Orthogonal primers
  • Saturation mutagenesis
  • TEM-1 beta-lactamase

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)


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