Recombinant escherichia coli and its use in the synthesis of rebaudioside d
A technology for recombining Escherichia coli and Escherichia coli, which is applied in the field of bioengineering, can solve the problems of being unable to meet market needs and obtaining high-purity product yields, etc.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0062] The construction of embodiment 1 recombinant escherichia coli
[0063] 1. Cloning of glycosyltransferase gene eugt11
[0064] Rice (Oryza sativa) leaf total RNA was extracted and rice cDNA was obtained by reverse transcription. According to the eugt11 gene sequence (AccessionNo.AK121682) in the GeneBank database, PCR amplification primers were designed, and the upstream and downstream primers were respectively introduced into the BamH I and Hind III sites.
[0065] The primer sequences were: F: 5'-CGCGGATCCATGGACTCCGGCTACTCCTCC-3' (BamH I) and R: 5'-CCCAAGCTTTCAATCCTTGTAAGATTCCAATTGC-3' (Hind III).
[0066] The PCR reaction conditions were: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 s, annealing at 55°C for 15 s, extension at 72°C for 30 s, and 30 cycles; extension at 72°C for 10 min.
[0067] The electrophoresis pattern of the amplified PCR product is shown in figure 1 . from figure 1 It can be seen from the figure that the size of the product...
Embodiment 2
[0072] Obtaining and purifying of embodiment 2 recombinant protein
[0073] 1. Obtaining recombinant protein
[0074] Take E.coli BL21 (pETDuet-eugt11) to save glycerol bacteria and streak it on the LB plate containing ampicillin, pick a single clone into 2mL LB liquid medium (containing 100mg / L ampicillin), 37°C, 250r / min overnight culture. The recombinant strain E.coli BL21 (pETDuet-eugt11) was transferred to 1L of fresh LB medium (containing 100mg / L ampicillin) with 1% inoculum amount by 2-step inoculation method, and continued to culture under the same conditions until the OD600 was 0.5. Add IPTG to a final concentration of 0.1 mmol / L, induce culture at 16°C and 180 r / min for 20 h, and collect the bacteria. The fermentation broth was centrifuged at 4°C and 6000r / min for 10mm, and the obtained bacteria were used for recombinant protein purification and subsequent whole-cell transformation research.
[0075] The cells were washed twice with 0.1mol / L, pH7.5 sodium phospha...
Embodiment 3
[0084] Embodiment 3 recombinant protease catalyzed reaction synthesis RD
[0085] In a 2mL reaction system (0.1mol / L sodium phosphate buffer pH=8.0, 5mmol / L UDPG, 1mmol / LMgCl 2 , 0.25mg / mL 6His-EUGT11) was added to the substrate 1mmol / L RA (1mmol / L RA in DMSO solution), reacted at 30°C for 0.5h, and then added 5% HCl to terminate the reaction. The reaction solution was extracted four times with the same volume of n-butanol, and the organic phases were combined and concentrated to dryness. The residue was dissolved in 0.5 mL DMSO and filtered through a 0.22 μm filter membrane. The filtrate was analyzed by high performance liquid chromatography (Thermo, Ultimate3000). The chromatographic conditions are: chromatographic column TOSOHC18 (ODS, 5um, 4.6mm×250mm); gradient elution: 0min~5min25% acetonitrile, 5min~30min25%~65% acetonitrile, 30min—40min65%~100% acetonitrile; flow rate 1mL / min ; Column temperature: 30°C; UV: 205mm. The blank control is the reaction solution without ...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More