Method for producing ectoine from recombinant escherichia coli and purifying ectoine
A technology for recombining Escherichia coli and ectoine, which is applied in genetic engineering and biological fields, can solve the problems of complex operation, low yield, and insufficient removal of impurities such as proteins, and achieve high product yield, convenient collection, and product purity and quality excellent effect
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0029] Example 1 - Genome Extraction
[0030] Vibrio New Caledonia strain CJG02-2 was cultured overnight at 30°C with 2216E (Haibo Biology, Qingdao) medium, centrifuged at 8000rpm at room temperature for 5 minutes, collected the thalline, and extracted according to the takara microbial genome extraction kit (TaKaRaMiniBEST Bacteria Genomic DNA Extraction Kit Ver.3.0) was used to extract genomic DNA.
Embodiment 2- 4
[0031] Example 2 - Cloning of the ectoine synthetic gene cluster ectABC and construction of a recombinant expression vector
[0032] According to the whole genome sequencing results of Vibrio New Caledonia strain CGJ02-2, a pair of primers for cloning the ectABC gene cluster were designed: EctF-CTA GCTAGC ATGATCACATCAG CACCTTGGGTC, EctR-EctR-CCG GAATTC TTAGTCAACGAGAGG ATAAACACC (the underlined parts are NheI and EcoRI restriction sites, respectively). The whole genome DNA of Vibrio New Caledonia strain CGJ02-2 was used as a template for PCR amplification. The amplification conditions were: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 70 s, a total of 35 cycles; and finally extension at 72°C for 10 min. The product recovered from PCR gel and the expression vector pBAD were double-enzymatically digested with NheI and EcoR I, and recovered separately. The recovered product was ligated by T4DNA ligase,...
Embodiment 3-e
[0033] Recombinant expression and SDS-PAGE identification of embodiment 3-ectABC in Escherichia coli
[0034] The positive clone (verified by sequencing) containing the recombinant plasmid pBADectABC was inoculated overnight at 37° C. in LB medium containing ampicillin. Then take an appropriate amount of overnight culture and add it to LB medium at a ratio of 1:100, and culture it at 37°C for 3 hours to make it O6 600= 0.6. Then L-arabinose was added to the bacterial culture to make the final concentration 0.1%, and induced for 8 hours at 30° C. and 220 rpm.
[0035] Collect 1 ml of bacterial culture and centrifuge at 5000 x g, 4 °C for 5 min. Cells were resuspended with 50 microliters of 1×SDS loading buffer (BOSTER, AR1142) and boiled in boiling water for 10 minutes. Then the lysate was centrifuged at 12000×g for 10 minutes, and 10 microliters of the supernatant was collected to analyze the recombinant expression by SDS-PAGE. The negative controls were culture products o...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More