Colibacillus with high flavonoid tolerance as well as construction method and application thereof
A technology of flavonoids and Escherichia coli, applied in the directions of microorganism-based methods, biochemical equipment and methods, bacteria, etc., 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 1 MalE
[0028] Use an inoculation needle to pick a single colony on the E.coli BL21 (DE3) plate and inoculate it in 25 mL of 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 the stationary phase, 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 3 times with deionized water and followed the FOCUS TM The Global Fractionation manual extracts membrane proteins, and removes ions in protein samples with a 2-D Clean-Up kit, and finally uses a non-interfering protein concentration determination kit to measure the concentration in the sample, and finally hydrates...
Embodiment 2
[0029] Embodiment 2 Construction of flavonoid-resistant engineering bacteria
[0030]Amplify the first 140 bp of the malE gene found on ncbi and the upstream 40 bp designed primers, respectively connect to the cloning vector pMD19-T for sequencing, and obtain the correct transformant after double enzyme digestion and connect to the plasmid pRSF-PTasRNA, Construct the recombinant plasmid pRSF-PTasRNA-malE, 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 agar, sterilized at 121°C for 15min), pick the transformant on the transformed plate for PCR verification, obtain the correct single colony for fermentation and extract the plasmid, and transfer the plasmid into E.coli BL21 (DE3 ) competent state, spread on the LB medium containing chloramphenicol, pick the transformant on the plate after transformation and carry out PCR verification, and t...
Embodiment 3
[0031] Example 3 Determination of growth curve of highly tolerant flavonoids E.coli BL21 (DE3) engineered bacteria
[0032] Pick engineering bacteria and E.coli BL21 (DE3) single colonies containing empty plasmid pRSF-PTasRNA and inoculate them into M9 medium containing 1% kanamycin (13.3g / L M9CA Broth, 0.2% glucose, 1mM magnesium sulfate, After activation in 0.5 μg / mL thiamine-HCl) for 10 h, insert 1% kanamycin, 1 g / L flavonoids (rutin, naringenin, resveratrol), and different Concentration of IPTG (0, 100, 200, 400μM) in M9 medium, culture at 200rpm at 30°C, measure OD every 1h 600 until the strain grows to a stationary phase. According to the formula μ=d x / x*d t Calculate the specific growth rate map of the strain, and then obtain the maximum specific growth rate μ max , it was found that the μ of engineering bacteria in the environment with 1g / L rutin max is 0.42, while that of the original bacterial strain is 0.23, and the growth rate is increased by 1.83 times; in t...
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