| Summary: | ‘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 <i>Chlamydomonas reinhardtii</i> 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 <i>C. reinhardtii</i> 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 <i>C. reinhardtii</i>, we have created a novel recipient line, HNT6, carrying an engineered deletion in exon 3 of <i>psaA</i>, 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 <i>psaA-3</i> 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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