Uvioresistant bacillus thuringiensis strain and building method and application thereof
A technology of Bacillus aureus, construction method, applied in microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve problems such as preventing mother cell lysis and unreported research
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Embodiment 1
[0029] Example 1 Preparation and Transformation of Escherichia coli JM110 and BL21 (DE3) Strain Heat Shock Competent
[0030] Pick a single colony in 5ml LB liquid medium, culture overnight at 37°C with shaking; inoculate 1% inoculum in 100ml LB liquid medium, culture at 37°C, 220rpm for 2-2.5hr, (OD 600 =0.5-0.6); centrifuge at 4000rpm for 10min at 4°C; discard the supernatant, and add 50ml of pre-cooled 0.1mol / L CaCl to the pellet 2 Suspend the cells, put them on ice for 30min; centrifuge at 4000rpm for 10min at 4°C, recover the cells; use 2-4ml ice-cooled 0.1mol / L CaCl 2 Resuspend cells, aliquot into 200μL / 0.5mL centrifuge tubes, store at 4°C, and use up within one week. Mix 200 μL of competent cells and plasmid, ice bath for 30 min, heat shock at 42°C for 1.5 min, ice bath for 3 min, add 800 μL of LB medium, incubate at 37°C for 60 min, take 200 μL to plate, and incubate at 37°C. Preparation of Escherichia coli JM110 Heat Shock Competent
Embodiment 2B
[0031] The preparation of embodiment 2Bt electric shock competence
[0032] Pick a single colony and put it in 5ml LB liquid medium, shake and culture overnight at 30°C. Inoculate in 100ml BH liquid medium according to 1% inoculum amount, culture at 37°C, 220rpm for 3-4hr (OD 600 =3.0). Centrifuge at 4000rpm for 10min at 4°C. Discard the supernatant, add pre-cooled ultrapure water to suspend the cells, centrifuge at 4000rpm for 10 min at 4°C, repeat twice, discard the supernatant, add 1ml 40% PEG to the pellet to resuspend the cells, aliquot, and store at -70°C for later use.
Embodiment 3
[0033] The cloning of embodiment 3cwlB gene and the construction of expression vector
[0034] A pair of cwlB gene full-length primers cwlB-5 / cwlB-3 was designed and synthesized, the sequence is as follows:
[0035] cwlB-5:5'-CG GGATCC GATGCATACAGCTGTCAATCC-3'
[0036] cwlB-3:5'-ACGC GTC GAC ATATTGAGTATATTTAATA-3'
[0037] Using the total DNA of Bacillus thuringiensis strain HD73 as a template, the full-length cwlB gene was amplified by PCR using 2×Primer Star mix. The 50μL PCR reaction system is:
[0038]
[0039] PCR reaction conditions: pre-denaturation at 94°C for 5min; denaturation at 94°C for 1min; annealing at 50°C for 30s; extension at 72°C for 30s, 30 cycles; final extension at 72°C for 10min. The results showed that after PCR amplification, a full-length fragment of 669bp was obtained ( figure 1 ). The recovered and purified cwlB full-length gene was connected to the vector pET21b, and the recipient strain E.coli JM110 was transformed. After the identific...
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