A kind of coding gene of s-type 1-phenylethanol synthase and its application
A technology of phenylethanol and synthase, applied in the directions of application, genetic engineering, plant genetic improvement, etc., can solve problems such as unreported, and achieve the effects of avoiding environmental pollution, low cost and high transformation efficiency
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Embodiment 1
[0042] The S-type 1-phenylethanol synthase gene shown in SEQ ID No.2 was cloned into pET32a prokaryotic expression vector.
[0043] The S1PES-pET32a vector was transformed into Escherichia coli Rosetta. Pick a single clone and inoculate it into 100mL LB medium, at 37°C, shake at 230°C to OD 600 Approximately equal to 0.6, add isopropylthiogalactoside to a final concentration of 0.1mM. Then continue to culture at 20° C. and 180 rpm for 16 hours.
[0044] 12,000g centrifugation for 10 minutes to collect the bacteria, add 10mL lysate (50mM NaH 2 PO 4 , 300mM NaCl, 10mM imidazole, pH 8.0) resuspended and sonicated the cells. The parameters of the sonicator are: 40% power, 3 seconds of ultrasound, 5 seconds of rest. After sonication for 30 minutes, the supernatant was collected by centrifugation at 12,000 g for 30 minutes. Add 500 μL of Ni-NTA agarose gel to the supernatant, and incubate at 4°C for 1 hour at 200 rpm. Pack the mixture of protein and Ni-NTA agarose gel into th...
Embodiment 2
[0047] The S-type 1-phenylethanol synthase gene shown in SEQ ID No.2 was cloned into pET32a prokaryotic expression vector.
[0048] The S1PES-pET32a vector was transformed into Escherichia coli Rosetta. Pick a single clone and inoculate into 400mL LB medium, shake at 230°C at 37°C to OD 600 Approximately equal to 0.6, add isopropylthiogalactoside to a final concentration of 0.1mM. Then continue to culture at 20° C. and 180 rpm for 16 hours.
[0049] 12,000g centrifugation for 10 minutes to collect the bacteria, add 10mL lysate (50mM NaH 2 PO 4 , 300mM NaCl, 10mM imidazole, pH 8.0) resuspended and sonicated the cells. The parameters of the sonicator are: 40% power, 3 seconds of ultrasound, 5 seconds of rest. After sonication for 30 minutes, the supernatant was collected by centrifugation at 12,000 g for 30 minutes. Add 500 μL of Ni-NTA agarose gel to the supernatant, and incubate at 4° C. for 1 hour at 200 rpm. Pack the mixture of protein and Ni-NTA agarose gel into the ...
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