Team:XHD-Wuhan-Pro-China/Protocol
Protocol
1. Construction of ethanol degradation probiotics
1.1 Construction of recombinant plasmid
The purpose of this study is to construct a probiotics that can efficiently degrade ethanol and acetaldehyde. The ethanol dehydrogenase Gene sequence ADH (Gene ID in NCBI) of Saccharomyces Cerevisiae S288C was selected. 855349) and aldehyde dehydrogenase Gene ald2 (NCBI Gene ID: 855206), which were synthesized by Suzhou Jinwezhi Biotechnology Co., LTD., after codon optimization. In addition, the NAD synthase Gene sequence nadE was derived from Escherichia coli str.K-12 substr.MG1655 (Gene ID in NCBI: 946946), genomic DNA was extracted from E. coli K12, and primers F-Nade and R-Nade were used as templates for PCR amplification to obtain the gene fragment. The Gene sequence of NADH oxidase nox was derived from Lactobacillus brevis (Gene ID in NCBI is). Primers F-nox and R-nox were used as templates for PCR amplification to obtain the Gene fragment.
By Seamless Cloning technology, the recombinant plasms PSB-AA and PSB-AN were constructed using The Asy -Basic Seamless Cloning and Assembly Kit (TransGen Biotech, Beijing, China). Primers were designed according to the instructions of the seamless cloning kit, the dosage of each component in the cloning reaction system was calculated, and the reaction liquid, carrier and inserted fragments were added into the centrifugal tube, gently mixed, and reacted at 50℃ for 15 min. After the reaction, the centrifugal tube was placed on ice to cool for a few seconds before the connection was completed. Details of relevant plasmids and primers in this section are shown in Table 2.1.
1.2 Construction of engineered strains
The recombinant plasmid was transferred into the competent state DH5α by heat shock method, and the positive monoclones were selected for culture. Plasmid Mini Kit I (OMEGA Biotek, Dalian, China) is used to extract the recombinant Plasmid, and the construction result is verified by sequencing (Huada Technology Co., LTD.). The correct recombinant plasmids PSB-AA and PSB-AN were transformed into competent Escherichia coli Nissle1917, respectively, and the target genes were efficiently expressed. In addition, the empty plasmid pSB1A3 was transferred into the competent state of EScherichia coli 1917 as a control for subsequent experiments.
1.3 Culture of engineering strains
Escherichia coli 1917 containing empty plasmid, recombinant plasmid PSB-AA and PSB-AN were inoculated in 5 mL LB medium containing ampicillin, respectively. Culture at 200 RPM at 37℃ overnight. Store in glycerol tubes with a final concentration of 50% at -20℃ for future use.
Table.2.1 Strains, plasmid and primers used
2.Performance of engineered strains
2.1 Tolerance and degradation ability of the engineered strain to ethanol and acetaldehyde
The prepared engineering strains of wild type EScherichia coli 1917 were cultured with 1% inoculation amount until OD600 reached about 0.1, and the concentration of the bacteria in the medium was unified. At this time, different doses of ethanol or acetaldehyde were added to the medium, so that the concentration of ethanol in the medium reached 0%, 2%, 4%, 6%, 8% and 10%, and the concentration of acetaldehyde reached 0%, 0.1%, 0.2%, 0.3%, 0.4% and 0.5%, respectively. The medium was cultured at 37℃ and 200 RPM, and the OD600 value in each medium was recorded every 2h to compare the growth of different strains under different concentrations of ethanol or acetaldehyde.
During the culture process, samples were collected from each culture system for 0h and 14h, respectively, and the content of ethanol and acetaldehyde in the samples was detected by gas chromatography.
(1) Gas chromatography conditions
Capillary column GSBP-LNOwax-MS (30 m × 0.25 mm × 0.25 um), nitrogen flow rate: 25 mL/min, air pressure: 40 kPa, hydrogen pressure: 55 kPa. Injection port temperature :200℃, column temperature: 45℃, detector temperature :200℃, injection volume: 0.2 μL, no split injection.
(2) Prepare the standard curve
0.1 mL of standard ethanol and acetaldehyde were constant-volume in 10 mL volumetric flask with ultra pure water to make a 1% mixed standard reserve solution. Accurately absorb the mixed standard storage solution 100 μL, 200 μL, 400 μL, 600 μL, 800 μL and 1000 μL, constant volume to 10 mL with ultrapure water, It is equivalent to the standard series with acetaldehyde content of 0.0037%, 0.0074%, 0.0148%, 0.0222%, 0.0296%, 0.0370% (the coefficient of conversion from ethanol to acetaldehyde is 0.37) and ethanol content of 0.01%, 0.02%, 0.04%, 0.06%, 0.08%, 0.10%, respectively. 0.2μL samples were injected for gas chromatographic analysis under the above chromatographic conditions, and the standard linear regression curve was drawn.
(3) Determine the content of ethanol and acetaldehyde in the sample
Ethanol culture medium without bacterial solution was used as blank control. The samples were filtered by 0.45 μm membrane and analyzed by the above chromatographic conditions. The content of ethanol and acetaldehyde and the degradation rate were calculated by the standard curve.