Application of pinus massoniana alpha-pinene synthetase to preparation of terpene type compounds and products including terpene type compounds
A masson pine, synthetic enzyme technology, applied in the direction of application, enzyme, lyase, etc., to achieve high synthesis efficiency, avoid pollution, and low impurity content
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Embodiment 1
[0052] RNA was extracted from the stem tissue of Pinus massoniana, and cDNA was synthesized by reverse transcription using reverse transcriptase M-MLV. Using the cDNA as a template for amplification, the primers are as follows:
[0053] The forward primer is: 5'-ACATGGGCAAGAACCCCTAT-3' (SEQ ID NO.3);
[0054] The reverse primer is: 5'-TTAAGATGGGCGAAGGCTAA-3' (SEQ ID NO.4);
[0055] Vazyme Phanta Max Super-Fidelity DNA polymerase was used for PCR amplification; PCR conditions were: 95°C, 3min; 95°C, 15sec; 56°C, 15sec; 72°C, 2min; 35 cycles; 72°C extension for 5min. The PCR product was detected by 1% agarose gel electrophoresis, and the results were as follows: figure 1 as shown, figure 1 M is the DNA marker DL2000, lanes 1 and 2 are amplification products, and the size of the target gene fragment is about 1890bp, which is in line with expectations.
[0056] Use the agarose gel electrophoresis gel recovery kit method to recover the target gene fragment, clone the target fra...
Embodiment 2
[0058] α-pinene synthase gene construction expression vector and transforming prokaryotic cells include the following steps:
[0059] The cloned target fragment was transformed into pET28a linearized vector by homologous recombination. The ligation reaction conditions are as follows: mix the reaction system and incubate at 37°C for 30 minutes, then add 5 μL of the reaction solution to 50 μL of Escherichia coli DH5α competent cells at 20°C for 1 hour, mix well and let it stand in an ice bath for 30 minutes, then take it out gently , 42°C, heat shock for 60s, immediately ice-bath for 2min, add 500μL LB medium and incubate at 37°C for 1h; take 100μL of the bacterial solution and spread it on the LB plate containing Kan resistance, and culture overnight. The positive colony obtained by antibiotic (Kan) screening was picked, and the plasmid was extracted. Transfer the prokaryotic expression vector into Escherichia coli BL21 strain, 37°C, 200rpm, wait until OD600 is 0.6, add IPTG w...
Embodiment 3
[0061] The prokaryotic expression, protein purification and protein detection of α-pinene synthase include the following steps:
[0062] Pick a single colony containing the recombinant plasmid into 3mL LB liquid medium (ampicillin resistance), culture at 37°C overnight and then store it at -20°C; pick a single colony containing the recombinant plasmid respectively and put it into 3mL LB liquid medium (ampelin resistance) medium, shake culture at 37°C until OD600 is about 0.6; take part of the bacterial liquid as the control group, add IPTG inducer (final concentration 1mM) to the rest of the bacterial liquid, and shake at 37°C for 3 hours; take 0.15mL of bacterial liquid from two groups, 12000×g After centrifugation for 2 minutes, the cell pellet was resuspended and lysed in 40 μL 1×loadingbuffer, and detected by SDS-PAGE. Take 100 μL of the bacteria stored at -20°C and inoculate in 100mL LB liquid medium (amphicillin resistant) for overnight shaking culture; take 100mL of bac...
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