Artificial nondirectional site-specific recombination systems

Abstract

Site-specific recombination systems (SRSs) are widely used in studies on synthetic biology and related disciplines. Nondirectional SRSs can randomly trigger excision, integration, reversal, and translocation, which are effective tools to achieve large-scale genome recombination. In this study, we designed 6 new nondirectional SRSs named Vika/voxsym1-4 and Dre/roxsym1-2. All 6 artificial nondirectional SRSs were able to generate random deletion and inversion in Saccharomyces cerevisiae. Moreover, all six SRSs were orthogonal to Cre/loxPsym. The pairwise orthogonal nondirected SRSs can simultaneously initiate large-scale and independent gene recombination in two different regions of the genome, which could not be accomplished using previous orthogonal systems. These SRSs were found to be robust while working in the cells at different growth stages, as well as in the different spatial structure of the chromosome. These artificial pairwise orthogonal nondirected SRSs offer newfound potential for site-specific recombination in synthetic biology.

Keywords: Bioengineering; Biological sciences; Biological sciences research methodologies; Biotechnology; Genetic engineering; Synthetic biology.

Graphical abstract

Figure 1

Establishment of non-directional SRSs (A) Sequence alignment of the directional sites and symmetrical sites. Nucleotides that have changed compared to the directional recombination site are colored in red. Nucleotides in vox that break the perfect palindrome with 13 bp sequences on both sides are colored in blue. Recombination sites with perfect palindrome structure are indicated by arrows. (B) Schematic diagram on the principles of directional recombination system and nondirectional recombination system. (C) The nondirectional recombination system can both reverse and delete the sequence among recombination sites. Agarose gel electrophoresis diagram of colony PCR before and after recombination. M, marker; Trans2K Plus Ⅱ DNA Marker, TransGen Biotech, C, Control: before recombination, L, loxPsym, V1, voxsym1, V2, voxsym2, V3, voxsym3, V4, voxsym4, R1, roxsym1, R2, roxsym2.

Figure 2

Efficiency of the designed nondirectional SRSs (A) Experimental flow chart of the efficiency determination of the recombination systems. The efficiency characterization cassettes (symmetrical recombination site-P_ura-Ura-T_ura- symmetrical recombination site) were both integrated into the HO locus of S. cerevisiae BY4742 and pRS415 plasmid. The SSR expression plasmid, fused with the EBD binding domain induced by the Gal1 promoter, was transferred to this strain (His was used to screen the SSR expression plasmid). The strain was then induced by galactose and β-estradiol to start the recombination. After 8 h, the genome recombination was completed and the deletion efficiency was first quantified by phenotype. PCR was performed on the colonies without a deletion event. To accurately quantify the inversion efficiency, the strains showing the Ura cassette inversion in the first PCR result were streaked and purified. Four single colonies were randomly selected from each purified strain, and PCR was performed again to calculate the final inversion efficiency. (B) The deletion and inversion efficiency of the recombination systems, error bars indicate SD (n = 3). A total of 96 colonies were screened after each group of recombination experiments. (C) Schematic overview of rapid quantitative method of recombination system efficiency. The “Ura-T_ura-T_leu-Leu” module was randomly deleted or inverted under the action of SSR. Without deletion and inversion, Ura gene was expressed, kanMX gene was expressed when deletion occurs, and Leu gene was expressed when inversion takes place. (D) A rapid quantitative efficiency method using dilution coating and colony counting. yJYW0052 (Cre, loxPsym, and plasmid) was subjected to recombination experiments under the induction of galactose and β-estradiol. Two OD yeast cells were collected, resuspended in 1 mL water and serially diluted. Finally, an appropriate amount of yeast cells was taken and coated on solid media SC, SC-URA, SC-LEU, and SC+G418 (before the formal verification of efficiency, gradient dilution was used to determine the optimum volume of yeast solution for coating on each plate). The total number of cells spread on each solid plate is marked above each picture. The total number of cells spread on each solid plate and the count of single colonies on each plate (blue numbers) are marked on the top of each picture. The deletion and inversion efficiency and non-recombination of the Cre/loxPsym recombination system were calculated (numbers in the upper left corner). (E) The deletion and inversion efficiency of the Cre/loxPsym and Dre/roxsym1 systems on chromosome and plasmid,error bars indicate SD (n = 3). The total number of yeast cells collected in each group of parallel experiments is 2OD.

Figure 3

Orthogonality of the designed non-directional SRSs (A) Schematic overview on the development of orthogonal non-directional SRSs. The LEU expression cassette flanked by loxPsym sites can only be deleted or inverted under the action of Cre. The URA expression cassette flanked by voxsym1-4 sites can only be deleted or inverted under the action of Vika, while the URA expression cassette flanked by roxsym1/2 sites can only be deleted or inverted under the action of Dre. (B) The orthogonality of Cre/loxPsym and Dre/roxsym2 recombination systems. After the recombination experiment of strain yJYW0038 (Cre, loxpsym, roxsym2) and yJYW0034 (Dre, loxpsym, roxsym2), 96 colonies were randomly selected and spread on SC-URA+G418 and SC-LEU+G418 plates to determine whether the URA and LEU genes were deleted. Two pairs of primers (JYW115, JYW117, and JYW118, JYW119) were then used for colony PCR to determine whether URA and LEU genes were inverted. Galactose and estrogen induction were absent in the control group. A clearer gel electrophoresis image is shown in Figure S4. Primers for characterization of the recombination events in this study are listed in Table S1. (C) A heatmap was used to represent the orthogonality of Cre/loxPsym and the six non-directional SRSs developed in this study.

Figure 4

Robustness of the designed non-directional SRSs (A) The effect of the growth stages on the efficiency of the designed non-directional SRSs. We quantified the recombination efficiency of strains at different stages under the Cre/loxPsym and Dre/roxsym2 systems. As the inversion efficiency of the Cre/loxPsym system is too low, we used a dual axis to express the system's deletion (left axis) and inversion efficiency (right axis).Error bars indicate SD (n = 3). (B) The effect of chromosome spatial structure on the efficiency of the recombination system. We selected 6 sites on the genome with different interaction strengths to quantify the recombination efficiency of the Dre/roxsym2 system.Error bars indicate SD (n = 3).