GoKawiilBacterial strains and culture conditions
All cloning was performed in E. coli Trans1-T1 chemically competent cells (TransGen Biotech). Genetic circuit validation used E. coli DH10B (TSC-C09, Tsingke). Cultures were grown in lysogeny broth (LB) at 37 °C with shaking at 220 rpm. Plasmid maintenance was ensured by supplementing LB with chloramphenicol (34 μg ml−1), carbenicillin (100 μg ml−1), kanamycin (50 μg ml−1), spectinomycin (100 μg ml−1) or zeocin (100 μg ml−1), as appropriate.
Yeast strains and media
All yeast experiments used S. cerevisiae BY4741 (MATa his3Δ1 leu2Δ0 met17Δ0 ura3Δ0 sed1Δ; Horizon Discovery) as the standard chassis. Cells were cultured in YPD medium containing 1.5% (w/v) yeast extract (Angel Yeast), 2.0% (w/v) peptone (Angel Yeast) and 2% (w/v) glucose (Sangon). YPD was also used for circuit induction and growth curve measurements. Auxotrophic selection used SD minimal media (SD-Ura, SD-Leu, SD-His or SD-Ura-Leu-His) solidified with 2% agar (Coolaber). Antibiotic selection used SD supplemented with hygromycin (500 μg ml−1) or zeocin (300 μg ml−1). Snowflake yeast was generated by CRISPR–Cas9-mediated deletion of ACE2, as previously described42.
Mammalian cell culture
HEK293FT cells (Invitrogen, R70007) were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% (v/v) penicillin–streptomycin (10,000 U ml−1 penicillin, 10 mg ml−1 streptomycin; Gibco) at 37 °C and 5% CO 2 . Cells were passaged at a 1:3 ratio every two days in 10-cm dishes. CHO-K1 cells (NICR) were cultured in F-12K medium (Kaighn’s modification; M&C Gene Technology) with 10% FBS and 2% (v/v) penicillin–streptomycin under identical conditions and passaged at a 1:6 ratio every three days. For selection, media were supplemented with hygromycin (800 μg ml−1), zeocin (400–1,000 μg ml−1), blasticidin S (20 μg ml−1) or puromycin (4 μg ml−1), as required.
Plasmid construction and single-copy maintenance in E. coli
To maintain the branching device at a single copy in E. coli, we used a bacterial artificial chromosome (BAC) backbone18. The intrinsically low copy number of BAC vectors minimizes copy-number-dependent variability and stabilizes circuit behaviour. All genetic components, including fluorescent reporters, promoters, terminators and insulators, were amplified via 2 × Phanta UniFi Master Mix (P526-02, Vazyme Biotech) and assembled into the BAC backbone using one-pot Gibson assembly (CU201-02, TransGen Biotech). Recombinase expression was controlled using a tightly regulated aTc-inducible system to drive the expression of Bxb1 recombinase53. Plasmids were verified by Sanger sequencing (Tsingke) and whole-plasmid sequencing (KBSeq, Sangon Biotech). Detailed genetic parts, protein sequences, plasmid backbones and representative constructs are provided in Supplementary Tables 4–7.
Plasmid construction and genomic integration in S. cerevisiae
To ensure single-copy circuit presence in S. cerevisiae BY4741, all devices were integrated into the genome. Plasmids were assembled using the Yeast Toolkit (YTK)24 and MYT system54, both based on Golden Gate cloning. Assembly reactions contained 6.5 μl DNA inserts, 1.0 μl backbone, 1.0 μl T4 ligase buffer (B0202S, NEB), 0.5 μl T4 DNA ligase (M0202L, NEB) and 1.0 μl restriction enzyme, adjusted to a total volume of 10 μl. BsaI-HF v2 (R3733L, NEB) or BsmBI v2 (R0739L, NEB) were used. Thermocycling comprised 80 cycles of digestion (BsaI at 37 °C or BsmBI at 42 °C for 5 min) and ligation (16 °C for 5 min), followed by final digestion at 55 °C for one hour and heat inactivation at 80 °C for 10 min. Assembled plasmids were transformed into E. coli, selected on antibiotic plates and verified by Sanger and whole-plasmid sequencing. Genetic parts and protein sequences are listed in Supplementary Tables 4 and 5, and plasmid backbones and representative constructs in Supplementary Tables 6 and 7.
... continue reading