Abstract
Chlamydomonas reinhardtii has many attractive features for use as a model organism for both fundamental studies and as a biotechnological platform. Nonetheless, despite the many molecular tools and resources that have been developed, there are challenges for its successful engineering, in particular to obtain reproducible and high levels of transgene expression. Here we describe a synthetic biology approach to screen several hundred independent transformants using standardised parts to explore different parameters that might affect transgene expression. We focused on terminators and, using a standardised workflow and quantitative outputs, tested 9 different elements representing three different size classes of native terminators to determine their ability to support high level expression of a GFP reporter gene. We found that the optimal size reflected the median size of element found in the C. reinhardtii genome. The behaviour of the terminator parts was similar with different promoters, in different host strains and with different transgenes. This approach is applicable to the systematic testing of other genetic elements, facilitating comparison to determine optimal transgene design.
Highlights
These include well-established basic laboratory protocols for culturing and molecular analysis, excellent genetics [7], fully sequenced genomes for the nucleus, mitochondrion and chloroplast [8], and efficient transformation protocols for all three genomes [9]. This is via electroporation [10], but simple vortexing with glass beads is possible [11]. Such resources have led directly to the generation of an impressive array of sequences to enable transgene expression, including selectable markers, reporter genes [12,13,14] and promoter elements, as well as gene silencing techniques that utilise RNA interference (RNAi) [15] or artificial microRNAs [16] and gene editing via CRISPR/Cpf1 [17]
HSP70A/RBCS2 chimeric promoter (AR promoter; [37]) and RBCS2 30 UTR/terminator, a combination that has been widely used in the literature [18]
We found that the terminators from PSAD and CA1 gave the best results in all experiments
Summary
It is often referred to as a model alga, and this designation has helped to galvanise efforts to develop several essential resources, which are enabling the use of C. reinhardtii for biotechnology [4,5,6] These include well-established basic laboratory protocols for culturing and molecular analysis, excellent genetics [7], fully sequenced genomes for the nucleus, mitochondrion and chloroplast [8], and efficient transformation protocols for all three genomes [9]. This is via electroporation [10], but simple vortexing with glass beads is possible [11]. The implementation of synthetic biology approaches, most notably the adoption of standard parts and modular cloning methods, has enabled much more rapid generation of constructs for transformation and effective comparison between different designs, culminating in the generation of a kit of over 100 standardized elements [18], available at the Chlamydomonas
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