Blasticidin S Deaminase: A New Efficient Selectable Marker for Chlamydomonas reinhardtii.

Félix De Carpentier,Stéphane D Lemaire,Pierre Crozet,Jeanne Le Peillet,Antoine Danon,Nicolas D Boisset

doi:10.3389/fpls.2020.00242

Abstract

Chlamydomonas reinhardtii is a model unicellular organism for basic or biotechnological research, such as the production of high-value molecules or biofuels thanks to its photosynthetic ability. To enable rapid construction and optimization of multiple designs and strains, our team and collaborators have developed a versatile Chlamydomonas Modular Cloning toolkit comprising 119 biobricks. Having the ability to use a wide range of selectable markers is an important benefit for forward and reverse genetics in Chlamydomonas. We report here the development of a new selectable marker based on the resistance to the antibiotic blasticidin S, using the Bacillus cereus blasticidin S deaminase (BSR) gene. The optimal concentration of blasticidin S for effective selection was determined in both liquid and solid media and tested for multiple laboratory strains. In addition, we have shown that our new selectable marker does not interfere with other common antibiotic resistances: zeocin, hygromycin, kanamycin, paromomycin, and spectinomycin. The blasticidin resistance biobrick has been added to the Chlamydomonas Modular Cloning toolkit and is now available to the entire scientific community.

Highlights

Chlamydomonas reinhardtii is a model microalga widely used for basic and biotechnological research such as photosynthesis, cilia/flagella, production of biofuels, or other molecules of interest (Georgianna and Mayfield, 2012; Barahimipour et al, 2016; Salomé and Merchant, 2019)
This concentration is slightly higher than the efficient concentration reported for V. carteri (Ortega-Escalante et al, 2018) or diatoms (Buck et al, 2018), but remains comparable with other antibiotics used in Chlamydomonas (Crozet et al, 2018)
We successfully engineered BSR coding sequence to adapt it to Chlamydomonas and no addition of intron was necessary for efficient expression, contrary to what was reported for V. carteri (Ortega-Escalante et al, 2018)

Summary

Introduction

Chlamydomonas reinhardtii is a model microalga widely used for basic and biotechnological research such as photosynthesis, cilia/flagella, production of biofuels, or other molecules of interest (Georgianna and Mayfield, 2012; Barahimipour et al, 2016; Salomé and Merchant, 2019). In the last decades Chlamydomonas has been shown to be amenable to powerful genetic approaches including CRISPR-Cas gene editing (Jiang et al, 2014; Greiner et al, 2017; Kao and Ng, 2017). A wide range of molecular tools for engineering of the nuclear genome are available in Chlamydomonas, most of which have been grouped in a Modular Cloning toolkit (Chlamy MoClo toolkit). This collection contains 119 biobricks (promoters, terminators, reporter genes, selectable markers, targeting peptides, antibiotic resistance genes, riboswitch, miRNA backbone, etc.), which can be assembled through Golden Gate cloning (Crozet et al, 2018).

Methods

Results

Conclusion