Clonexpress multis one step cloning kit

The rpoN (VP2670) in-frame deletion mutant strain was constructed according to a previous method using the suicide vector pDM4 (Zhou et al., 2010 (link)), the primers used were listed in Supplementary Table S2. In brief, the upstream and downstream fragments of rpoN were amplified from the genome of V. parahaemolyticus with primers rpoN up-F/R and rpoN down-F/R and cloned into pDM4 with Sac I/Sal I sites by a ClonExpress Multis One Step Cloning Kit (Vazyme, Nanjing, China). Then, the positive recombinant plasmid pDM4::ΔrpoN was transformed into the WT strain by conjugation and cultured on an LB agar plate containing Carb and Cm. Following this, the second cross-over recombination was detected in the LB agar plate with 15% sucrose. Finally, the rpoN deletion mutant strain was verified by PCR with the primers rpoN out-F/R and rpoN in-F/R and sequencing analysis.
The ORF of rpoN was amplified with primers rpoN com-F/R and cloned into the pMMB207 plasmid with Xba I/Hind III sites by a ClonExpress Multis One Step Cloning Kit (Vazyme, Nanjing, China). Then, the positive recombinant plasmid pMMB207::rpoN was transformed into the ΔrpoN strain and selected on an LB agar plate containing Carb and Cm. The complemented strain was confirmed by PCR with primers pMMB207-F/R and named rpoN⁺.

The plasmid schematic diagrams used in this study are shown in Figure 1. For pBS-EMR, the ef-1α B promoter and rap-1 terminator were amplified from the B. duncani genome, the mCherry fragment was amplified from the plasmid of pBS-PAC-mCherry, and these fragments were cloned separately into the pBluescript (pBS) backbone plasmid using the ClonExpress MultiS One Step Cloning Kit (Vazyme, China). The plasmid of pBS-EHEG was modified on the basis of plasmid pBS-EMR, replacing the mCherry gene and 3′UTR of rap-1 with the hDHFR-eGFP and 3′UTR of ef-1αB by the ClonExpress MultiS One Step Cloning Kit (Vazyme, China). The plasmid of pBS-DHFR-EGFP-TPX-1 KO was cloned using the same method. Briefly, on the basis of the plasmid pBS-EHEG, the 5′UTR of the TPX-1 gene was inserted into the upstream region of the ef-1α B promoter and the 3′UTR of ef-1αB was replaced with the 3′UTR of TPX-1. All plasmids were sequenced to confirm the accuracy of the sequence. All primers used in this study are shown in Table 1.

The design and synthesis of bta-miR-23a mimic, mimic negative control (NC), small interfering RNAs (siRNAs) for MDFIC knockdown and non-targeting siRNA NC were performed by RiboBio (Guangzhou, China). The sequence of si-MDFIC was GAATCGAAGACTTTCAGCA. The 3’-UTR region of MDFIC encompassing bta-miR-23a binding site was amplified and cloned into the XhoI/NotI restriction sites of psi-CHECK2 vectors (Promega, Madison, WI, USA). Then, site-directed mutagenesis was carried out using the Fast Site-Directed Mutagenesis Kit (TIANGEN).
To obtain the MDFIC overexpression plasmid, MDFIC open reading frame sequence was amplified and cloned into pBI-CMV3 vector (Clontech, Mountain View, CA, USA) using ClonExpress MultiS One Step Cloning Kit (Vazyme, Nanjing, China). MEF2C promoter sequence was taken out and cloned into pGL3-basic vector by using ClonExpress MultiS One Step Cloning Kit (Vazyme). The primer sequences used for plasmid construction and mutagenesis are listed in Table 2.
Cell transfection was conducted with Lipofectamine RNAiMAX reagent (Invitrogen), along with bta-miR-23a mimic, NC, si-MDFIC and si-NC. Plasmid transfection was carried out with Lipofectamine 3000 (Invitrogen). All assays were conducted as per the manufacturer’s protocols.

Restriction enzymes and DNA ligase were purchased from Fermentas (Thermo Fisher Scientific Inc. Waltham, USA). TransStart FastPfu DNA polymerase was obtained from TransGen Biotech (Beijing, CN). ClonExpress^TM MultiS One Step Cloning kit was obtained from Vazyme Biotech (Nanjing, CN). Plasmid extraction kits and DNA gel extraction kits were purchased from Qiagen (Valencia, CA). LipoFiter^TM transfection reagent was purchased from Hanbio Biotech (Shanghai, CN). TRIzol reagent was obtained from Invitrogen (Carlsbad, CA, USA). EGF was purchased from peprotech (Rocky Hill, NJ, USA). Gefitinib was from AstraZeneca (London, UK). D-Luciferin potassium was from Xenogen (Alameda, CA). Coelenterazine was purchased from Regis (Morton Grove, IL, USA).

For construction of the plasmid pUC19-Bbura5-Donor, the gpd promoter (Pgpd) fragment and the trpC terminator (TtrpC) fragment were PCR-amplified from Cordyceps militaris genomic DNA with primer pairs Pgpd-F/Pgpd-R and Ttrpc-F/Ttrpc-R, respectively. The ura5 gene was amplified from the genomic DNA of Bb252 using primers Bbura5-F and Bbura5-R. These three fragments were sequentially assembled with the ClonExpress^TM MultiS one step cloning kit (Vazyme, C113-01), and cloned into the SmaI site of the plasmid pUC19 to generate the plasmid pUC19-Bbura5-Donor. To construct dDNAs of different genes, the 5′ and 3′ flanking sequences of target genes were amplified and cloned into the SmaI site of the plasmid pUC19-Bbura5-Donor vector. The generated vectors were propagated in DH5α and purified using the Plasmid Midi Kit (Omega).

The OsUBC12^IR64 (2 kb) or OsUBC12^Koshihikari (7.9 kb, containing the inserted transposon) promoter sequence was cloned into pGreenII-0800-LUC, and subsequently transformed into rice protoplasts. Moreover, pOsUBC12^{KoshihikariΔTransposon} (pOsUBC12^Koshihikari but lacking the transposon) and pOsUBC12^{IR64+Transposon} (pOsUBC12^IR64 but containing the transposon, the transposon was artificially inserted into the same position as the pOsUBC12^Koshihikari) were constructed by ClonExpressTM MultiS One Step Cloning Kit (vazyme, C113). The primer sequences are listed in Supplementary Data 1. The Renilla luciferase (REN) gene directed by the 35 S promoter in the pGreenII 0800-LUC vector was used as an internal control. Firefly LUC and REN activities were measured using the Dual-Luciferase reporter assay kit (Beyotime) and a GloMax 20/20 luminometer (Promega). LUC activity was normalized to REN activity and LUC/REN ratios were calculated. The data presented are the averages of at least three independent replicates.

To construct the green fluorescence gene (gfp) reporter plasmids, the original plasmid pSET152 was cut with BamHI and XbaI. The scyA1 promoter (P_A1) and the coding region of scyR1 were amplified from S. cyaneogriseus ssp. noncyanogenus NMWT1 genomic DNA with primer pairs pscyA1GFPF/R and ScyR1GFPF/R, respectively. The gfp and the strong constitutive promoter SF14 were amplified from pSET152:P_sbbAgfp:SF14sbbR with primer pairs GFPF/R and pSF14F/R, respectively (He et al., 2018 (link)). First, P_A1 and gfp coding region were ligated into pSET152 using the ClonExpress^TM MultiS One Step Cloning Kit (Vazyme) to obtain pSET152:P_A1gfp (Supplementary Table 1). Secondly, the plasmid pSET152:P_A1gfp was digested with NheI and then assembled with SF14 promoter and the scyR1 coding region to generate the corresponding reporter plasmid pSET152:P_A1gfp:SF14scyR1 (Supplementary Table 1), in which scyR1 was controlled by SF14 and the gfp gene was controlled by P_A1. These two plasmids together with the control vector pSET152 were introduced into DH5α, respectively, to detect the intensity of green fluorescence (excitation at 485 nm; emission at 535 nm, Synergy H4 Multi-Mode Reader). All fluorescence values were normalized to growth rates (OD₆₀₀).