Agarose gel dna recovery kit

To further verify the candidate gene, Spo12821, we selected the parents, Sp62 and Sp39, and designed primers according to the genome sequence published on SpinachBase by using the program primer 3 plus (https://www.primer3plus.com/, accessed 25 June 2020). After 1.0% agarose gel detection, an agarose gel DNA recovery kit (TIANGEN, Beijing, China) was used to cut the gels and recover target fragments. Subsequently, the target fragments were connected and transformed with the vector using a pEASY-T5 Zero cloning kit (TransGen Biotech, Beijing, China), and the white single colonies were selected for PCR amplification and verification. The bacterial liquid sequencing was performed by the Beijing Genomics Institute (BGI, Shenzhen, China). Then, the candidate gene sequencing results were obtained after using Multalin web online tools to carry out the multiple sequence alignment (http://multalin.toulouse.inra.fr/multalin/multalin.html, accessed 26 Steptember 2020). The Expasy online web tools were used to translate the base sequence into the amino acid sequence (https://web.expasy.org/translate/, accessed 26 Steptember 2020), and Multalin was used for sequence alignment. Finally, the EBI website Interpro was used for protein structure prediction (http://www.ebi.ac.uk/interpro/, accessed 26 Steptember 2020).

pEsgRNA-eGFP and pCas plasmids are relaxation plasmids, which are used in genome engineering ways. The pEsgRNA-eGFP plasmid (Biomics Biotech Company, Kunming, China) carries the ampicillin resistance gene (Ampicillin, 60 μg/ml). The plasmid pCas (Biomics Biotech Company, Kunming, China) carries kanamycin resistance gene (Kanamycin, 50 μg/ml) and can encode Cas9 nuclease.
41 wild-type E. coli strains were isolated and identified from a Saba swine farm in Chuxiong autonomous prefecture of Yunnan province in China. E. coli DH5α strain and CVCC1565 strain (irp2⁺ bacteria) were preserved in our laboratory. Bacterial genomic DNA extraction kit, agarose gel DNA recovery kit and plasmid mini-extraction kit were purchased from Tiangen Biochemical Technology Co. Ltd. (Kunming, China). Other reagents were all domestic analytical pure products.

Each 1 L water sample was collected in triplicate at selected sites and filtered through a 0.22 μm polyethersulfone (PES) micropore filter (Millipore, Temecula, CA, USA). The filtered membranes were placed in 30 mL normal saline and then eluted in a shaker with a temperature of 37 °C and a speed of 200 rpm for 2 h. The eluents were stored at −20 °C for later use. The DNA in the eluents was extracted using a bacterial genomic DNA extraction kit (TIANGEN, Beijing, China), suspended in a final volume of 50 μL and further purified using an Agarose Gel DNA Recovery Kit (TIANGEN) to minimize inhibition of the PCR.

The 5′ flanking sequence of the SRPP promoter was obtained from the T. kok-saghyz genome of the Genome Warehouse (GWH; http://bigd.big.ac.cn/gwh/, accessed on 10 March 2021) under the accession number PRJCA000437 [17 (link)], based on sequence alignment of the TkSRPP gene using Blast 2.11.0 software (ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/, accessed on 3 January 2021). Genomic DNA was isolated from T. kok-saghyz leaves as per the method proposed by [18 (link)]. The primers (Table S1) for cloning of the promoter region were designed using Primer 5.0 software. PCR experiment was performed using the total DNA of leaf as a template, and the DNA polymerase is PrimeSTAR Max DNA Polymerase (TAKARA, Beijing, China). The PCR program was as follows: 30 cycles of 10 s at 95 °C, 20 s at 55 °C, and 2.5 min at 72 °C, with a final extension at 72 °C for 8 min. PCR products were detected by 1% agarose gel electrophoresis and recovered with the agarose gel DNA recovery kit (TIANGEN, Beijing, China). The recovered product was connected with the pMD-19T vector (TAKARA, Beijing, China) at 16 °C for 8 h according to the manufacturer’s instructions. The recombinant plasmid was then transformed into E. coli DH5α strain, and several positive single colonies were selected and sequenced for confirmation.

Restriction enzymes (NdeI and HindIII), DNA polymerase PrimerSTAR HS and rtaq, calf intestine alkaline phosphatase, agarose, and nucleic acid electrophoresis standards were purchase from Takara Biotechnology Co, Ltd. The bacterial genomic DNA extraction kit, agarose gel DNA recovery kit, and EZ-10 spin column plasmid mini-prep kit were obtained from Tiangen Biotech Co., Ltd (Beijing, China). PCR primers were synthesized by Genscript Biotech Corporation (Nanjing, China). The protein electrophoresis standards and polyacrylamide gel electrophoresis kit were obtained from Beyotime Biotechnology (Shanghai, China). α-Amylase, pullulanase, and maltogenic amylase were obtained from Novozymes (Copenhagen, Denmark). All other chemicals were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) unless otherwise indicated.

Pfu DNA polymerase, 200 bp DNA ladder, an agarose gel DNA recovery kit, pre-stained protein marker, and a plasmid extraction kit were purchased from Tiangen (China). Restriction endonucleases (NcoI and XhoI) and T4 DNA ligase were purchased from Thermo Fisher Scientific (USA). A SDS-PAGE kit and isopropyl-β-D-thiogalactoside (IPTG) were purchased from Sangon Biotech (China). Kanamycin was purchased from Bomei Biotechnology (China). Luria-Bertani (LB) medium (5 g/l yeast extract, 10 g/l tryptone, and 10 g/l NaCl) was used for plasmid propagation, seed preparation, and protein induction expression. M9Y medium (17.1 g/l Na₂HPO₄·12H₂O, 3g/l KH₂PO₄, 0.5 g/l NaCl, 1 g/l NH₄Cl, 2 g/l glucose, 1 g/l yeast extract, 0.12 g/l MgSO4, 0.0111 g/l CaCl₂) with 1-(4-hydroxyphenol)-ethanol (0.4 mM, 4 mM, and 40 mM) was used for biotransformation. The information about the strains and vectors used in this study is shown in Table 1.

Diammonium 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic sulfonate, ABTS), acid violet, alphazurine A, and methyl orange were purchased from Sigma-Aldrich (St. Louis, MO, USA). Restriction enzymes EcoR I, Kpn I, Prime STAR Max, DNA Marker, and Protein Marker all purchased from TaKaRa (Dalian China). The bacterial genomic DNA extraction kit, instant error-prone PCR kit, agarose gel DNA recovery kit, plasmid extraction kit, and fast site-directed mutagenesis kit all purchased from TianGen (Beijing, China). Ampicillin, kanamycin, and IPTG were purchased from Solarbio (Beijing, China). His-tagged protein purification kit was purchased from Kangweishiji (Beijing, China). All other chemicals were standard reagent grade.
PCR instrument (T100™ Thermal Cycler, Bio-Rad) Molecular Devices Spectra Max M2 microplate reader, Ultrasonic processor type ultrasonic crusher, E-201-C type pH composite electrode, ZWY-211C constant temperature, Constant temperature incubator, Nucleic acid electrophoresis equipment, Protein electrophoresis equipment, and other equipment. digital pH meter (PHS-25, Shanghai Instrument and Electric Scientific Instrument Co., Ltd.), E-201-C type pH composite electrode.