Bglii

The SPLCV V1 fragment was inserted into the pCTCON plasmid. The V1 gene in T-vector was digested with AvrII and BglII (Takara, Tokyo, Japan), and inserted between the NheI to BamHI sites of pCTCON according to the manufacturer’s instructions. The ligate was inserted into a DH5α-competent cell, and colony selection onto LB agar plate with ampicillin, colony culture selection in broth, mini-scale plasmid extraction, and sequencing analysis were performed.

To prepare the original circular DNA, we first PCR-amplified DNA fragments encoding the phi29 DNA polymerase and loxP sequence using KOD FX (Toyobo, Japan), primer 1 (5′-CGGAGATCTCGTTGTAAAACGACGGCCAG-3′), primer 2 (5′-ACGAGATCTCCGGCTCGTATGTTGTGTGG-3′), and the template plasmid (pUC-phi29DNAP-loxP) constructed in a previous study^{22 (link)}. The whole sequence of the original circular DNA is shown in Fig. S6. For clone 6 and clone 6-wt-loxP, corresponding plasmids, pUC-clone6 and pUC-clone 6-loxP-wt, respectively, were used. Preparation of these plasmids were described below. Then, we digested the DNA fragments with 0.5 U/μl BglII (TaKaRa, Japan) in the reaction mixture according to the manufacturer’s instruction for 1 h at 37 °C and self-ligated them using 1.75 U/μl T4 DNA ligase (TaKaRa) to produce the original circular DNA in the reaction mixture according to the manufacturer’s instruction for 1 h at 16 °C. For the circular DNA used for the in vitro evolution experiment, the initial PCR contained 0.12 mM alpha-S 2-deoxycytidine-5′-O-1-thiotriphosphate (dCTP; TriLink Biotechnologies). A DNA fragment encoding GFP under the control of the T7 promoter was prepared by amplifying the template plasmid pET-g5tag. We used PrimeSTAR HS (TaKaRa) and the primers 5′-GCGAAATTAATACGACTCACTATAGGG-3′ and 5′-GGTTATGCTAGTTATTGCTCAGCGG-3′ for amplification.

Escherichia coli BL21 and DH5α were used as host strains for biosensor development and plasmid construction. The heavy metals (Mn, As, Cd, Ni, Hg, Pb, Au, Sb, Co, Fe, Zn, and Cr) used in this study were purchased from Sigma Aldrich (Steinheim, Germany) in chloride salt form and were prepared as 1 mM stocks. All restriction enzymes, namely, NdeI, BglII, BamHI, XbaI, XhoI, and DNA T4 ligase, were purchased from Takara, Korea. Taq polymerase used for gene amplification was purchased from Takara Biomedical, and Turbu Pfu used for site-directed mutagenesis was purchased from New England Biolabs. Primer synthesis and DNA sequencing were performed by Macrogen (Seoul, South Korea). Mancozeb, which was used to prepare artificially contaminated samples, was purchased from Sigma-Aldrich.

The yellow fusion protein (YFP) construct 35S::ZjZFN1:YFP and pGBKT7-ZjZFN1 were produced by transferring the complete ZjZFN1 coding sequence (CDS) into 3302Y (Jia et al., 2016 (link)) and pGBKT7 vectors, respectively. Firstly, 3302Y and pGBKT7 vector were digested by BglII or BamHI (TaKaRa, Dalian, China), respectively, and purified using a E.Z.N.A Cycle-pure Kit (Omega Bio-Tek, Norcross, GA, United States). Then primers 3302Y-ZFN1-F/R and BD-ZFN1-F/R were used to amplify the ZjZFN1 CDS (Table 1). Amplicons were then purified and infused into the linearized 3302Y and pGBKT7 plasmids using an In-fusion HD Cloning Kit (TaKaRa, Dalian, China).
The GUS fusion construct ZjZFN1_pro::GUS contained a 1406-bp ZjZFN1 promoter region that was amplified from the plasmid containing the target sequence using primers 1391-ZFN1-F and 1391-ZFN1-R (Table 1). After digesting the pCAMBIA1391Z vector with NcoI (TaKaRa, Dalian, China), purified PCR product of ZjZFN1 promoter was infused into the digested vector using the In-fusion HD Cloning Kit (TaKaRa, Dalian, China) to produce ZjZFN1_pro::GUS.

The glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter (GenBank accession no. KL198006.1) was cloned from P. ostreatus. Then it was inserted into the expression plasmids pCAMBIA1304 (GenBank: AF234300.1) to replace its CAMV35S promoter by using the restriction sites of HindIII and NcoI. Subsequently, Pomnp6 (GenBank accession no. MF681783) and Povp3 (GenBank accession no. MF681784) were cloned from P. ostreatus using specific primers (Table S5), respectively. All amplified PCR products were purified, sub-cloned with the pMD18-T vector (Takara, China) and sequenced (Sangon, China). The PCR products were digested with BglII and SpeI (Takara), then inserted into the pCAMBIA1304-Pogpd vetor (Sharma and Kuhad, 2010 (link)). Final vector plasmids were designated as pCAMBIA1304-Pogpd-Pomnp6 and pCAMBIA1304-Pogpd-Povp3.