Method for constructing high-yield coenzyme Q10 engineering bacteria and application of method
A technology of engineering bacteria and coenzymes, which is applied in the field of genetic engineering, can solve the problems of low yield and achieve the effects of increasing potency, promoting synthesis, and increasing yield
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Embodiment 1
[0041] Embodiment 1, the method for constructing high-yielding coenzyme Q10 engineering bacteria is as follows:
[0042](1) Select Rhodobacter sphaeroides containing the gene encoding isopentenyl pyrophosphate isomerase IDI as the parent strain, and use the genomic DNA extraction kit to extract the genomic DNA of Rhodobacter sphaeroides.
[0043] (2) The upstream primer IDIF and the downstream primer IDIR were designed by Primer5 primer design software, the upstream primer added the restriction site NcoI, and the downstream primer added the restriction site BamHI.
[0044] (3) Using the extracted genomic DNA as a template, the target gene was obtained by PCR amplification. A standard reaction system was used: 25 μL of GC buffer, 16 μL of water, 4 μL of dNTP mixture, 1.5 μL (10uM) of upstream primer IDIF, 1.5 μL (10uM) of downstream primer IDIR, 1.5 μL of extracted genomic DNA, and 0.5 μL of PrimeSTAR enzyme. The amplification program was: 30 cycles, each cycle including denat...
Embodiment 2
[0051] Embodiment 2, the method for constructing high-yielding coenzyme Q10 engineering bacteria is as follows:
[0052] (1) Select Rhodopseudomonas capsulatum containing the gene encoding isopentenyl pyrophosphate isomerase IDI as the parent strain, and use the genomic DNA extraction kit to extract the genomic DNA of Rhodopseudomonas capsulatum.
[0053] (2) The upstream primer IDIF and the downstream primer IDIR were designed by Primer5 primer design software, the upstream primer added the restriction site NcoI, and the downstream primer added the restriction site BamHI.
[0054] (3) Using the extracted genomic DNA as a template, the target gene was obtained by PCR amplification. A standard reaction system was used: 25 μL of GC buffer, 16 μL of water, 4 μL of dNTP mixture, 1.5 μL (10uM) of upstream primer IDIF, 1.5 μL (10uM) of downstream primer IDIR, 1.5 μL of extracted genomic DNA, and 0.5 μL of PrimeSTAR enzyme. The amplification program was: 30 cycles, each cycle includ...
Embodiment 3
[0061] Embodiment 3, the method for constructing high-yielding coenzyme Q10 engineering bacteria is as follows:
[0062] (1) Agrobacterium tumefaciens containing the gene encoding isopentenyl pyrophosphate isomerase IDI was selected as the parent strain, and genomic DNA of Agrobacterium tumefaciens was extracted using a genomic DNA extraction kit.
[0063] (2) The upstream primer IDIF and the downstream primer IDIR were designed by Primer5 primer design software, the upstream primer added the restriction site NcoI, and the downstream primer added the restriction site BamHI.
[0064] (3) Using the extracted genomic DNA as a template, the target gene was obtained by PCR amplification. A standard reaction system was used: 25 μL of GC buffer, 16 μL of water, 4 μL of dNTP mixture, 1.5 μL (10uM) of upstream primer IDIF, 1.5 μL (10uM) of downstream primer IDIR, 1.5 μL of extracted genomic DNA, and 0.5 μL of PrimeSTAR enzyme. The amplification program was: 30 cycles, each cycle inclu...
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