T4 dna ligase

FastPfu DNA polymerase and T4 DNA ligase were purchased from TransGen Biotech (China) and Thermo Fisher (United States), respectively. Restriction enzymes were purchased from Thermo Fisher (United States). Polymerase chain reaction (PCR) primers were provided by Sangon (Shanghai, China). Racemic acetoin, diacetyl and 2,3-butanediol were purchased from Sigma. (2R,3R)-2,3-butanediol (98.0%), (2S,3S)-2,3-butanediol (99.0%), and meso-2,3-butanediol (98.0%) were purchased from ACROS (The Kingdom of Belgium). NADH was purchased from Roche (United States). Yeast extract (FM902) was a kind gift from Angel Yeast Co., Ltd. (Yichang, Hubei, China). All other chemicals were of analytical grade and commercially available.

(2R,3R)-2,3-BD (98.0%), (2S,3S)-2,3-BD (99.0%), and meso-2,3-BD (98.0%) were purchased from ACROS (The Kingdom of Belgium). Racemic AC, ethyl 2-acetoxy-2-methyl-acetoacetate, and diacetyl were purchased from Sigma. NADH was purchased from Amresco. Restriction enzymes were purchased from TaKaRa Bio Inc. (China). PCR primers were prepared by Sangon (Shanghai, China). FastPfu DNA polymerase and T₄ DNA ligase were purchased from Transgen Biotech (China) and MBI (USA), respectively. All other chemicals were of analytical grade and commercially available.

All genes were cloned into the vector pEASY-T3 cloning Vector, which contains a LacZ gene suitable for TA-cloning (TransGen Biotech Company, CT301-01, Beijing, China). The vector was propagated in Trans1-T1 Phage Resistant Chemically Competent Cells with genotype F-φ80(lacZ)ΔM15ΔlacX74hsdR(rk-, mk+ )ΔrecA1398endA1tonA (TransGen Biotech CD501-01).
All oligonucleotides were obtained from BGI (Beijing, China) with standard purification. Primer sequences were designed with vector NTI software (Informax Vector NTI Suite 11.5) and are listed in Table 3. DNA sequencing to confirm cloning products was performed by Quintara Biosciences.
Genes were amplified using their respective primers (Table 3) from the template of the mutant strain of S. cerevisiae AH109 that demonstrated the highest yielding alcohol production using the pEASY-Blunt Simple Cloning Kit including high fidelity polymerase (CB111-01) and TransStart FastPfu DNA Polymerase (AP221-11). The PCR products were linked to a pEASY-T3 cloning Vector (TransGen Biotech Company) with T₄ DNA ligase (TransGen Biotech Company), and the sequences were detected by BGI Tech. Company (Beijing, China).

The SARS-CoV-2 RBD gene (GenBank: OA993881.1) and the p170 gene (GenBank: KX981911.1) were connected with a GGGGS linker and subcloned into the pET28a (+) (Thermo Fisher, USA) expression plasmid using unique the EcoRI and XhoI restriction sites to create the pET28a-p170-RBD plasmid. The p170 gene was subcloned into the pET28a expression plasmid using unique EcoRI and XhoI sites as a negative control. The ACE2 gene (GenBank: AB046569.1) was subcloned into the pcDNA3.1 (+) (Thermo Fisher) expression plasmid using unique KpnI and XhoI sites. All operations were performed in accordance with molecular experimental procedures. The general procedure was as follows: The sequence was amplified with PCR using forward and reverse primers (Table 1). After digestion with the corresponding restriction enzymes (TransGen, China), the plasmids and PCR products were recovered by a gel recovery kit (TransGen), ligated by T4 DNA ligase (TransGen) and transformed into E. coli DH5α competent cells (TransGen). Positive colonies were identified by PCR and sent for sequencing (Sangon Biotech, China).

Ni-NTA agarose resin was purchased from Qiagen (Hilden, Germany). Trans5K DNA marker, T4 DNA ligase, E. coli Trans5α and BL21(DE3) competent cells were products of TransGen Biotech (Beijing, China). NdeI and XhoI endonucleases were purchased from New England Biolabs (Ipswich, MA, USA). The substrates of pNPA, pNPB, pNPC, pNPL and pNPP were obtained from Aladdin Biotech (Shanghai, China).

Escherichia coli strains T1 (Transgen Biotech, Beijing, China) and BL21 (DE3) (Novagen, Madison, USA) were used for plasmid construction and production of PHA granules, respectively. The pACYCDuet-1 and pETDuet-1 vectors (Novagen), both with two sets of T7 promoter, were used for co-expression of target genes. These two plasmids have different replicons, thus can exist in one E. coli cell. The gene encoding organophosphorus hydrolase was amplified from the previously constructed pET-opaa4301 plasmid [22 (link)]. The PHA granule synthesis genes, phaC, phaA, phaB, and phaP, were amplified from Cupriavidus necator ATCC 17699, the most investigated poly(3-hydroxybutyrate) producer [23 (link)]. The oligonucleotide primers used in this study are listed in Additional file 1: Table S1. Gene manipulation reagents, including T4 DNA ligase, restriction endonuclease, Pfu polymerase, and seamless assembly kit, were all purchased from Transgen Biotech.