L-SCRaMbLE as a tool for light-controlled Cre-mediated recombination in yeast

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.

Fig. 1

Schematic overview of L-SCRaMbLE. a Mode of action in cells harboring pLH_Scr15. In darkness or far-red light (λ = 740 nm), PIF3-CreC is located in the nucleus due to an incorporated NLS, while PhyBNT-CreN is located in the cytoplasm. Upon red light illumination (λ = 660 nm) PhyBNT-CreN binds PIF3-CreC in the cytoplasm (not shown), thereby reconstituting Cre recombinase from its split halves. Reconstituted Cre moves to the nucleus where it induces recombination between the loxPsym sites. b Mode of action in cells harboring pLH_Scr16. In darkness or far-red light (λ = 740 nm) PIF3-CreC and PhyBNT-CreN are separately located in the nucleus. Upon red light illumination (λ = 660 nm) PhyBNT-CreN binds PIF3-CreC, thereby reconstituting Cre recombinase from its split parts, allowing recombination at loxPsym sites

Fig. 2

Recombination experiment with incubation times of 4 h, 16 h or 24 h. Light-Cre1, Light-Cre2 and EST-Cre1 cells were grown for 6 h in darkness. Light-Cre1 and Light-Cre2 cells were grown in media containing 25 μM PCB. After induction with a 5-min red light pulse or by addition of β-estradiol (1 µM final concentration), samples were grown for further a 4 h, b 16 h, and c 24 h at 30 °C and 230 rpm. One ml of each culture was pelleted, diluted and plated on appropriate synthetic complete (SC) drop-out media. Each experiment was performed in three independent replicates. Bar blots show the mean values of the percentages of white and yellow colonies in induced and uninduced samples for strains Light-Cre1, Light-Cre2 and EST-Cre1. Grey, white colonies; yellow, yellow colonies. Error bars indicate standard deviation (SD)

Fig. 3

Restriction patterns of pLM494 after Cre-mediated recombination. Plasmids were isolated from different yeast colonies of induced and uninduced Light-Cre1 and EST-Cre1 cultures (16 h) and after passage through E. coli digested with PstI and SacI. a Gel electrophoresis of digested plasmids. Numbers shaded in yellow show patterns of plasmids isolated from yellow yeast colonies, while numbers shaded in white show patterns of plasmids isolated from white colonies. ´2-Log´, DNA Ladder (NEB). Kb: Kilobases. b Overview of the recombination events. Gene names are given. Numbers on the right refer to the corresponding plasmid restrictions shown in a. ´s´ indicates sequence-verified plasmids. Green diamonds indicate loxPsym sites

Fig. 4

L-SCRaMbLE-mediated recombination at varying PCB concentrations. Light-Cre1 cells were grown in SC-Ura-Leu media with a 0, b 15, c 25, d 50 and e 100 µM PCB for 6 h in darkness. Induced cells were treated with a 5-min red light pulse, followed by 16 h growth with 10-s red light pulses every 5 min. Uninduced samples were grown for 16 h in darkness. Around 100 µl of each culture were pelleted, diluted and plated on SC-Ura-Leu. Bar blots show the means of the percentages of white and yellow colonies in induced and uninduced samples. Grey, white colonies; yellow, yellow colonies. Data are the means of three independent replications ± SD

Fig. 5

Measurement of recombination efficiency. a Recombination efficiency was determined using plasmid pLH_Scr18, which contains a loxP-flanked double terminator cassette upstream of the yEGFP CDS. Recombination between the loxP sites, shown as green triangles, results in deletion of the terminators allowing yEGFP expression. Plasmid pLH_Scr19 served as positive control. b Wild-type and pLH_Scr18-transformed BY4742 cells served as negative controls for yEGFP signal, while BY4742 cells containing plasmid pLH_Scr19 served as positive control. EST-Cre3 cells contain plasmids pLH_Scr18 and pLM006, while Light-Cre3 cells contain pLH_Scr18 and pLH_Scr15. After 6 h of preculture, EST-Cre3 cells were induced with 1 µM β-estradiol, and Light-Cre3 cells with red light pulses. After 24 h of induction the number of yEGFP-positive cells was determined by flow cytometry. Uninduced samples as well as controls were grown in darkness. Data are the means of three independent replications ± SD