A method for improving Escherichia coli flavonoid tolerance by regulating OMPA expression
A technology of Escherichia coli and flavonoids, which is applied in the field of improving the tolerance of Escherichia coli flavonoids, can solve the problems of increasing E.coli, etc., and achieve the effect of improving the resistance to flavonoids
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Embodiment 1
[0027] The discovery of embodiment 1OmpA
[0028] Use an inoculation needle to pick a single colony on the E.coli BL21 (DE3) plate and inoculate it in 25 mL LB medium, and activate it for 12 hours. Inoculate 4 bottles of 25mL LB medium according to the inoculum amount of 1%, and place them in a shaker (200rpm) at 37°C to cultivate to a stable stage, and then add 25 μL of rutin dissolved in DMSO at a concentration of 0.1g / L to three bottles, respectively. Naringenin and resveratrol (final concentration 1g / L), another bottle was added with 25 μL DMSO as a control, after 3 hours of culture, 8000rpm low-temperature centrifugation collected the bacteria, washed with deionized water for 3 times and followed the FOCUS TM The Global Fractionation manual extracts membrane proteins, and removes ions in the protein sample with the 2-D Clean-Up kit, and finally uses the non-interfering protein concentration determination kit to measure the concentration in the sample, and finally hydrate...
Embodiment 2
[0029] Embodiment 2 Construction of flavonoid-resistant engineering bacteria
[0030]Design primers to amplify the gene sequence of ompA found on NCBI, respectively connect to the cloning vector pMD19-T for sequencing, obtain the correct transformants, perform double enzyme digestion and connect to the plasmid pACYC-Duet1, and construct the recombinant plasmid pACYC -ompA, transform the constructed expression vector into E.coli JM109, spread it on the LB medium containing chloramphenicol (yeast extract 5g / L, peptone 10g / L, NaCl10g / L, solid medium plus 20g / L L agar, sterilized at 121°C for 15 minutes), pick the transformant on the transformed plate for PCR verification, obtain the correct single colony for fermentation and extract the plasmid, transfer the plasmid into E.coli BL21 (DE3) competent, spread on On the LB medium containing chloramphenicol, pick the transformant on the transformed plate for PCR verification, and the correct single colony is the recombinant strain, th...
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