High-throughput robotically assisted isolation of temperature-sensitive lethal mutants in Chlamydomonas reinhardtii

Michal Breker, Kristi Lieberman, Frej Tulin, Frederick R. Cross*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

Abstract

Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the conventional approaches of permanent gene deletion cannot be applied to essential genes. We have pioneered a unique collection of ~70 temperature-sensitive (ts) lethal mutants for studying cell cycle regulation in the unicellular green algae Chlamydomonas reinhardtii1. These mutations identify essential genes, and the ts alleles can be conditionally inactivated by temperature shift, providing valuable tools to identify and analyze essential functions. Mutant collections are much more valuable if they are close to comprehensive, since scattershot collections can miss important components. However, this requires the efficient collection of a large number of mutants, especially in a wide-target screen. Here, we describe a robotics-based pipeline for generating ts lethal mutants and analyzing their phenotype in Chlamydomonas. This technique can be applied to any microorganism that grows on agar. We have collected over 3000 ts mutants, probably including mutations in most or all cell-essential pathways, including about 200 new candidate cell cycle mutations. Subsequent molecular and cellular characterization of these mutants should provide new insights in plant cell biology; a comprehensive mutant collection is an essential prerequisite to ensure coverage of a broad range of biological pathways. These methods are integrated with downstream genetics and bioinformatics procedures for efficient mapping and identification of the causative mutations that are beyond the scope of this manuscript.

Original languageAmerican English
Article numbere54831
JournalJournal of Visualized Experiments
Volume2016
Issue number118
DOIs
StatePublished - 5 Dec 2016
Externally publishedYes

Bibliographical note

Publisher Copyright:
© 2016 Journal of Visualized Experiments.

Keywords

  • Chlamydomonas
  • Complementation testing
  • Genetics
  • Genomics
  • Issue 118
  • Mutant isolation
  • Robotics
  • Temperature-sensitive mutants

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