Abstract
The synthetic yeast genome constructed by the International Synthetic Yeast Sc2.0 consortium adds thousands of loxPsym recombination sites to all 16 redesigned chromosomes, allowing the shuffling of Sc2.0 chromosome parts by the Cre-loxP recombination system thereby enabling genome evolution experiments. Here, we present L-SCRaMbLE, a light-controlled Cre recombinase for use in the yeast Saccharomyces cerevisiae. L-SCRaMbLE allows tight regulation of recombinase activity with up to 179-fold induction upon exposure to red light. The extent of recombination depends on induction time and concentration of the chromophore phycocyanobilin (PCB), which can be easily adjusted. The tool presented here provides improved recombination control over the previously reported estradiol-dependent SCRaMbLE induction system, mediating a larger variety of possible recombination events in SCRaMbLE-ing a reporter plasmid. Thereby, L-SCRaMbLE boosts the potential for further customization and provides a facile application for use in the S. cerevisiae genome re-engineering project Sc2.0 or in other recombination-based systems.
Highlights
The synthetic yeast genome constructed by the International Synthetic Yeast Sc2.0 consortium adds thousands of loxPsym recombination sites to all 16 redesigned chromosomes, allowing the shuffling of Sc2.0 chromosome parts by the Cre-loxP recombination system thereby enabling genome evolution experiments
Upon red light illumination PhyBNT undergoes a conformational switch from the Pr to the Pfr form, which is reversible by illumination with far-red light[28]
In the pLH_Scr[15] system, phytochrome interacting factor 3 (PIF3)-CreC protein is expected to be nuclearly localized in the dark, while PhyBNT-Cre protein (CreN) is predicted to be located in the cytoplasm
Summary
The synthetic yeast genome constructed by the International Synthetic Yeast Sc2.0 consortium adds thousands of loxPsym recombination sites to all 16 redesigned chromosomes, allowing the shuffling of Sc2.0 chromosome parts by the Cre-loxP recombination system thereby enabling genome evolution experiments. We present L-SCRaMbLE, a lightcontrolled Cre recombinase for use in the yeast Saccharomyces cerevisiae. The budding yeast Saccharomyces cerevisiae is one of the most intensively studied eukaryotic model organisms It is a single cell organism with a relatively small (12 Mb) nuclear genome, has a short generation time, and can be cultured and genetically manipulated. It allows efficient protein expression with high productivity making it relevant for industry-scale production of proteins, enzymes and low-molecular-weight substances[1]. In contrast to natural loxP sites of the bacteriophage P1 Cre-loxP system, loxPsym sites contain a symmetric spacer region within the 34 bp long Cre recombination site[12,13,14]. As a proof of concept, we scrambled a plasmid, harboring four loxPsym-flanked genes of the β-carotene biosynthesis pathway, in a light-controlled manner[22]
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