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

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.

SUBMITTER: de Carpentier F

PROVIDER: S-EPMC7066984 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:'Marker-free' strategies for creating transgenic microorganisms avoid the issue of potential transmission of antibiotic resistance genes to other microorganisms. An already-established strategy for engineering the chloroplast genome (=plastome) of the green microalga Chlamydomonas reinhardtii involves the restoration of photosynthetic function using a recipient strain carrying a plastome mutation in a key photosynthesis gene. Selection for transformant colonies is carried out on minimal media, such that only those cells in which the mutated gene has been replaced with a wild-type copy carried on the transgenic DNA are capable of phototrophic growth. However, this approach can suffer from issues of efficiency due to the slow growth of C. reinhardtii on minimal media and the slow die-back of the untransformed lawn of cells when using mutant strains with a limited photosensitivity phenotype. Furthermore, such phototrophic rescue has tended to rely on existing mutants that are not necessarily ideal for transformation and targeted transgene insertion: Mutants carrying point mutations can easily revert, and those with deletions that do not extend to the intended transgene insertion site can give rise to a sub-population of rescued lines that lack the transgene. In order to improve and accelerate the transformation pipeline for C. reinhardtii, we have created a novel recipient line, HNT6, carrying an engineered deletion in exon 3 of psaA, which encodes one of the core subunits of photosystem I (PSI). Such PSI mutants are highly light-sensitive allowing faster recovery of transformant colonies by selecting for light-tolerance on acetate-containing media, rather than phototrophic growth on minimal media. The deletion extends to a site upstream of psaA-3 that serves as a neutral locus for transgene insertion, thereby ensuring that all of the recovered colonies are transformants containing the transgene. We demonstrate the application of HNT6 using a luciferase reporter.

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

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