T4 dna ligase

S. cerevisiae strain BY4742 (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0)55 (link) was used as the host for DNA integration and biosynthetic pathway engineering. E. coli DH5α (Novagen, USA) was used for gene cloning. LB (Luria-Bertani broth) medium with antibiotics (100 μg ml⁻¹ of ampicillin or 50 μg ml⁻¹ of kanamycin) was used for cultivation of recombinant E. coli. YPD medium (1% yeast extract, 2% peptone and 2% glucose) and YPG medium (1% yeast extract, 2% peptone and 2% D-galactose) were used for cultivation of yeast. SD-URA (synthetic complete drop-out medium with 2% D-glucose and without uracil), SS-URA (synthetic complete drop-out medium with 2% D-sucrose and without uracil) and SG-URA (synthetic complete drop-out medium with 2% galactose and without uracil) were used for selection and cultivation of recombinant strains harbouring the pESC-URA (Stratagene)-derived plasmids. YPD medium containing 200 μg ml⁻¹ geneticin (G418) was used for selection of yeast strains with KanMX marker. SD-FOA (SD medium with 0.1% w v⁻¹ 5-fluoroorotic acid) was used for selection of yeast strains with KanMX-URA-PRB322ori marker excision. All restriction enzymes, T4 DNA ligase, DNA polymerase and primers were purchased from Sangon Biotech (Shanghai, China). The standard isoprene, antibiotics and chemicals were purchased from Sigma (Sigma Aldrich, USA).

The microRNAs, the padlock probe and DNAzyme, were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). T4 DNA ligase, phi29 DNA polymerase, exonuclease I (EXO I), ribonuclease inhibitor, BSA, (NH4)₂SO₄, and Tris-buffer solution (1 mol L⁻¹; pH 8.0) were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). dNTP mix (25 mmol L⁻¹ each) was purchased from Solarbio. Cell culture-grade ultrapure water was purchased from KeyGEN BioTECH. Magnesium chloride was purchased from Shanghai Macklin Biochemical industry (Shanghai, China). The sequences of nucleic acids employed in this study are given in Table 1 in the Supporting Information.

The P. pastoris strain X33 and the expression vector (pPIC3.5K and pPICZαA) were purchased from Invitrogen (Carlsbad, CA, USA). The E. coli strain Top 10 is routinely conserved in our laboratory. Restriction enzymes, T4-DNA ligase, and Pfu DNA polymerase were purchased from Sangon Biotech (Shanghai, China). All other chemicals used were analytical grade reagents unless otherwise stated. Yeast extract peptone dextrose (YPD) medium, buffered glycerol complex (BMGY) medium, and buffered methanol complex (BMMY) medium were prepared according to the manual of Pichia Expression Kit (Version F, Invitrogen). Fermentation Basal Salts Medium (BSM) and PTM1 Trace Salts used for fermentation were prepared according to the Pichia Fermentation Process Guidelines (Invitrogen).

Deionized water and Tris-HCl buffer solution (pH 8.0, 1 M) were purchased from Sangon Biotech (Shanghai, China). Lithium chloride was purchased from Shanghai Aladdin Bio-Chem Technology (Shanghai, China). T4 DNA ligase and 10 × T4 DNA ligase reaction buffer (400 mM Tris-HCl, 100 mM MgCl2, 100 mM DTT, 5 mM ATP, pH 7.8) were purchased from Sangon Biotech (Shanghai, China).

Aladdin Industrial Corporation (Shanghai, China) provided N-Hydroxysuccinimide sodium salt (NHSS) and Vancomycin hydrochloride,1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC). Carboxylated MBs (180 nm, 1 mg/mL) were provided by Allrun Nano Science and Technology Co. Ltd. (Shanghai, China). Sigma-Aldrich Chemical Co. Ltd. (Shanghai, China) provided bovine serum albumin (BSA). The agarose was obtained from Solarbio Technology Co., Ltd. (Beijing, China). The juice was obtained from the local supermarket. The DNA oligonucleotides (Table 1) prepared by TsingKe Biotech. Co. Ltd. (Beijing, China) were used. The dNTPs, phi29 DNA polymerase, and T4 DNA ligase were from SangonBiotech (Shanghai, China).