Bacillus licheniformis industrial production strain genetic transformation method
A technology of Bacillus licheniformis and genetic transformation method, which is applied in the field of microbial genetic engineering and fermentation engineering, can solve the problems of unstable and difficult genetic information, and the lack of obvious advantages of Bacillus licheniformis, and achieve the effect of ensuring genetic stability
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Embodiment 1
[0044] Example 1: Identification and Cloning of the Bacillus licheniformis Methyltransferase Encoding Gene
[0045] Analysis of the genome sequence of B. licheniformis ATCC 14580 (GenBank accession number: AE017333, Veith B et al, J Mol Biotechnol, 2004), determined that there is an open reading frame in the region 4169271 to 4170800 of the genome, and its encoded gene product may be a B. licheniformis-specific type I DNA methyltransferase. Chromosomal DNA was extracted with Chromosomal DNA Extraction Kit (Qiagen Company), using this DNA as a template, chemically synthesized oligonucleotides
[0046] metP1 5′-TC AGAATTC TGAGGAACACAACCATGGCTGAATT-3′, the underlined part is the EcoRI site, and
[0047] metP2 5′-TAC GAATTC TCCTAACTTACGCTCTTCCCA-3', the underlined part is the EcoRI site, which is a gene amplification primer, and under the action of PfuDNA polymerase, the coding gene of B. licheniformis methyltransferase ( Bli-met). The PCR product was digested with EcoRI, an...
Embodiment 2
[0050] Example 2: Methylation of Exogenous DNA Molecules
[0051] Competent cells of the recombinant strain Ec(met) were prepared by the method introduced by Sambrook (Sambrook et al, Molecular Cloning: ALab Manual, 1989) and stored at -70°C. The Escherichia coli-Bacillus licheniformis shuttle plasmids pHY 300PLK, pDG148-Stu, and pHY-WZX were prepared with a plasmid purification kit (Qiagen). Using the competent method, the above plasmids were transformed into recombinant bacteria Ec(met), and the transformants were selected on LB plates with a final concentration of 100 μg / mL ampicillin, 50 μg / mL tetracycline or 5 μg / mL kanamycin. After the positive transformants are isolated and purified, they are cultured in LB medium at 37°C for 24-48 hours, and the plasmid DNA transformed into it is methylated by the expression of the met gene. The plasmid DNA molecules in the above transformants were isolated and purified using a plasmid purification kit (Qiagen Company). That is, a pl...
Embodiment 3
[0052]Example 3: Specific methylated DNA molecules are used for genetic transformation of Bacillus licheniformis
[0053] Inoculate industrial production of Bacillus licheniformis strain B2004 or B0204 in 2.5mL LB medium containing 0.5mol / L sorbitol, after overnight culture, transfer to 50mL LB medium containing 0.5mol / L sorbitol, at 37℃ Grow to A 600 0.85-0.95. The bacteria were placed in an ice-water bath for 10 minutes, and the bacteria were collected by centrifugation at 10,000 r / min at 4°C for 5 minutes. The cells were repeatedly washed 4 times with pre-cooled EP buffer (0.5mol / L sorbitol, 0.5mol / L mannitol and 10% glycerol). Suspend the cells in about 1 mL of pre-cooled EP buffer. Aliquot 60 μL of bacteria into 1.5 mL Eppendorf tubes. Take 1 μL of the methylated or unmethylated plasmid DNA (50ng / L) prepared by the above method, mix it with the cells, transfer it into a pre-cooled electroporation cell, and add 1mL recovery medium (containing 0.5mol / L sorbitol and 0.3...
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