A kind of recombinant Euaerobic rosenbergii and its preparation method and application
A technology of eutrophic bacteria and starting bacteria, which is applied in the field of recombinant eutrophic bacteria and its preparation, can solve problems such as time-consuming and labor-consuming, affect industrial production, and affect other characteristics of bacteria, and achieve the effect of broadening the variety
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Embodiment 1
[0043] Example 1: Construction of a recombinant Euaeobesia rosenbergii that knocks out the upstream promoter sequence of nagR
[0044] Homologous fragments H1 and H2 were obtained by PCR amplification using the genome of Euaerobic roseri H16 (China General Microorganism Culture Collection and Management Center, CGMCC 1.7092) as a template, and plasmid pK18mobsacB (Orita, I., Iwazawa, R., Nakamura, S., Fukui, T., 2012. Identification of mutation points in Cupriavidusnecator NCIMB 11599 and genetic reconstitution of glucose-utilization ability in wild strain H16for polyhydroxyalkanoate production. J. Biosci. Bioeng. 113, 63-69) as template PCR amplification to obtain the vector Fragment, H1, H2 were connected with the vector fragment by Gibson Assembly method to obtain the recombinant plasmid pK18mobsacB-ΔPnagR. The primers used are as follows:
[0045]
[0046] The recombinant plasmid pK18mobsacB-ΔPnagR was transformed into Escherichia coli S17-1 (ATCC number: 47055, which ...
Embodiment 2
[0050] Embodiment 2: Construction of recombinant Euaerobes rosenbergii with amino acid mutations in the nagE gene
[0051] Homologous fragments H1 and H2 were obtained by PCR amplification using the Genaerobes rosenbergii H16 genome as a template, and the vector fragment was obtained by PCR amplification using the plasmid pK18mobsacB as a template, and the recombinant plasmid pK18mobsacB was obtained by connecting H1 and H2 with the vector fragment by the Gibson Assembly method -nagE. The primers used are as follows:
[0052]
[0053] The recombinant plasmid pK18mobsacB-nagE was transferred into Escherichia coli S17-1, and then transferred into the Euaerobes rosenbergii PnagR obtained in Example 1 by the conjugative transformation method. Using the characteristic that the suicide plasmid could not replicate in the host bacteria, it contained 200 μg / mL kanamycin and 100μg / mL apramycin LB plates to screen positive clones. The recombinant plasmid with homologous fragments in ...
Embodiment 3
[0059] Example 3: Recombinant bacteria RE01 grows with glucose as carbon source
[0060] Recombinant bacteria RE01 and starting bacteria Euaerobes rosenbergii H16 were cultured in LB medium at 200rpm at 30°C for 12 hours and then inoculated at 5% into 50mL MMG medium at 200rmp at 30°C for 48 hours.
[0061] After 48 hours, the bacteria were collected, and the dry cell weight and PHA content were detected, as follows:
[0062] Use a graduated cylinder to measure 30 mL of the bacterial solution into a 50 mL centrifuge tube, and centrifuge at 10,000 rpm for 10 min to collect the bacterial cells. The cells were resuspended in deionized water and washed once, centrifuged at 10,000 rpm for 10 min, and the supernatant was discarded. After the bacteria were frozen at -80°C for 1 hour, vacuum freeze-drying was carried out for more than 12 hours to completely remove water. Weigh the weight of the centrifuge tube before and after sampling, and the difference is the dry cell weight CDW....
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