β-alanine synthetase mutant, coding gene, genetic engineering bacteria and application
A technology of genetically engineered bacteria and coding genes, applied in the field of β-alanine synthetase mutants, can solve the problem of low yield and achieve the effects of increased yield, clear breeding goals, and good application prospects
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Embodiment 1
[0030] Example 1: Escherichia coli panD K43Y Construction of Gene Mutants
[0031](1) Acquisition of the pET28b(+)-panD plasmid containing the Escherichia coli panD gene: from the laboratory through one-step cloning of the constructed Escherichia coli BL21-pET28b(+)-panD (construction process: the plasmid PET28b(+) PCR as a template and detection of the carrier by agarose gel electrophoresis, the panD fragment shown in SEQ ID No.3 obtained by PCR was connected to the carrier PET28b (+) by a one-step cloning method to obtain PET28b (+)-panD recombinant plasmid) inoculation In LB liquid culture medium, culture at 37°C and 200rpm on a shaking table for 12h, centrifuge at 8000rpm for 5min, collect bacterial cells, and use a plasmid extraction kit to extract plasmid pET28b(+)-panD; take 2μL of plasmid DNA and use ND500 The concentration and purity of the sample were measured by an ultra-micro ultraviolet-visible spectrophotometer; the composition of the LB liquid medium was: pepto...
Embodiment 2
[0034] Example 2: Carry out pre-cultivation of the empty strain, the parent and the genetically engineered mutant strain bacteria respectively
[0035] 1. Pick single colonies from the plate and inoculate them into 5mL LB test tubes containing 50mg / L kanamycin resistance, incubate at 37°C for 6-8 hours and transfer to 50mL LB shakers containing 50mg / L kanamycin resistance. In the bottle, culture OD600=0.5-0.6, add 0.5mmol IPTG, culture on a shaker at 37°C, induce for 10 hours, and centrifuge to collect bacteria.
[0036] 2. Transfer the bacterial cells obtained in step 1 to shake flasks, and prepare β-alanine by fed-batch culture at 37° C., 180 rpm, for 36 hours. Such as figure 2 As shown, the β-alanine production of the mutant reached 5.72g / L in 24 hours, which was 1.84 times and 2.23 times higher than that of the parent and wild type under the same culture conditions.
[0037] 3. Wash and resuspend the thalline obtained in step 1 twice with PBS, and control the concentrat...
Embodiment 3
[0038] Embodiment 3: the research of the kinetic characteristic of mutant bacterial strain
[0039] In Example 1, the correct transformants, parental strains and empty strains were inserted into 5mL LB test tubes containing 50 mg / L, cultured at 37°C for 6-8 hours, and then transferred to 50 mg / L kanamycin containing 50mL LB shake flask, to OD 600 When =0.5~0.6, add 0.5mmol IPTG, culture on a shaker at 37°C, and after 10h, centrifuge at 6000rpm at 4°C for 5min to obtain three kinds of cells expressing panD. Wash the strain twice with PBS, and finally resuspend it with PBS, sonicate the cells, and centrifuge at 10000rpm at 4°C for 5 minutes to collect the supernatant, which is the crude enzyme solution, and then use the protein purification kit to purify the target protein through the NI column to obtain the final product The concentration of pure protein was measured with BCA kit. The protein concentrations of the empty strain, the parent strain (K43R) and the mutant strain (...
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