Team:UST Beijing/Notebook
Notebook
As early as April of this year, we started to prepare for our project. After the brainstorming, we thought we had a perfect idea, and decided to take action. There have been many failures in the process of experiment, but these failures make the success even more exciting. Here records good memories of these months.
Preparation I: Refer to Literature
April to May
We searched some literature on notoginseng and glucosidase in the database and learned about these papers. We revived E. coli containing β-glucosidase and extracted the plasmid of β-glu. Then the plasmid was cut with NcoI and XhoI in the 37℃ for 1 h.After electrophoresis, we confirmed that plasmid was correct and it can be used in the next experiments.
Preparation II: Liquid fermentation
May to June
The E. coli was centrifuged and the precipitation was immersed with different osmotic pressure buffer to draw β-glu expressed by E. coli. After drawing enzyme, we added different volumes of enzyme solution and pNPG to each well on the 96-well plate and made the bulk equal with extract buffer. OD450 was measured every one hour.
Preparation III: Enzyme activity determination
April to May
E. coli expressed β-glucosidase was fermented in a 3L fermenter. And we collected a small of fermentation sample at 0, 4, 8, 12, 16, 18, 20 and 24 hours since the fermentation start. After centrifugation, β-glu was extracted from the cell. And we tested the activity of the enzyme extracted from different growth stages of E. coli on a 96-well plate with pNPG as the substrate.
Week 1
7/4-7/10
Solid fermentation: notoginseng and water were mixed in a appropriate proportion. After boiling the mixture, we put the paste of notoginseng into glass containers. Then inoculate rhizopus , 25℃, to the fermentor.
Animal experiment : 15 mices were weighed and divided into three groups. Each of group was fed different content of notoginseng.
Transformation experiment: Amplification of pSB1C3-T7RNAp plasmid. We transformed the plasmid into DN5α, and spread E. coli on an LB plate containing Chloramphenicol. After 12h, a monoclonal colony was amplification and was stored at -80 ℃ with 25 % glycerol. The plasmids were obtained from the monoclonal E. coli and stored at -20 ℃. The β-glucosidase plasmid was extracted from E. coli and stored at -20 ℃, too.
Week 2
7/11-7/17
Animal experiment: Mices were killed and serum cholesterol was measured.
Solid fermentation: The reducing sugar concentration of the notoginseng was determined by DNS solution
Transformation experiment: The T7RNAp-plasmid and β-glu-plasmid was mixed with DH5α together and was settled in ice for 30 min. Then the mixture was moved to 42 ℃ water bath for 90 s and settled in ice for 3 min . The SOC medium was added in the tube and we put the tube in the 37 ℃ shaker for 1 h. Lastly we screened the E. coli with double plasmid by LB plate containing chloramphenicol and kanamycin. The result was that there was nothing on the plate after a few days.
Week 3
7/18-7/24
Solid fermentation: Took the notoginseng sterilization by boiling and sprinkled it into the mold, yeast, probiotic bacteria, 25℃, to fermentation.
Transformation experiment: We found the possibility of transforming two plasmids simultaneously was too small. So we transformed T7RNAp into DH5α firstly. Then we transformed β-glu plasmids into the new-made competent cells and spread them on double antibiotic plate. Unfortunately there was still nothing on the surface of plate.
Week 4
7/25-7/31
Animal experiment: The hamsters were divided into four groups, and each group was fed with different high-fat diet containing notoginseng powder.
Solid fermentation: The treated and untreated notoginseng was sterilized by boiling, adding two glassware into the yeast fermentation.
Transformation experiment: After transferring the T7RNAp plasmid into the E. coli containing β-glu plasmid, we spread the E. coli on the LB plate with chloramphenicol and kanamycin. After about one or two day, surprisingly, we found a few colonies on the double antibiotic plates. Finally we got a E. coli strain that is regulated by arabinose and express β-glu theoretically.
Week 5
8/1-8/7
We extracted the two plasmid from the cell with kits, and cut them with restriction endonucleases BglⅡ,XhoⅠ,EcoRⅠrespectively. Then we found an interesting phenomenon that the two plasmids recombined to a big plasmid.
Week 6
8/8-8/14
Solid fermentation: The reducing sugar concentration of the notoginseng was determined by DNS solution. BL21(DE3) containing the double-resistant plasmid was ultrasonically disrupted. The pieces were detected by PNPG in 96-well plant.
Transformation experiment: For confirming the safety of our organism, we used LB without antibiotic to cultivate the double-plasmid E. coli for a few days. And we spread a little E. coli on antibiotic and antibiotic-free LB solid medium. About 10 days later, the antibiotic plate had 47 colonies and the antibiotic-free plate had about 12,000 colonies. The result indicated that there was only 0.4 % E.coli containing the two plasmids.
Week 7
8/15-8/21
Induced experiment : The expression of glucosidase was induced by different concentrations of lactose and arabinose and detected by PNPG . In order to ensure the repeatability of the experiment, we repeat the experiment , and the results remain the same.
Week 8
8/22-8/28
Solid fermentation: notoginseng paste, which ferment on the July 24th, boiled sterilization, was sub-installed to two conical flask. One flask added DH5α containing the RFP(Red fluorescent protein), another one added BL21(DE3) containing the double-resistant plasmid to fermentation at 25℃.
Week 9
8/29-9/4
Inoculation and culture: We Picked a BL21(DE3) monoclonal colony from solid plate containing KAN and CHM, then Inoculated into M9 liquid medium containing the same antibiotics.put it into a shaker. Conditions in Shaker: 37℃, 250r/min,12-16h. The next day we used 200uL bacteria solution inoculated in 20 mL M9(contain 15% glycerol) liquid culture.
Enzyme activity assay: We added 20ul Lac , 20uL Ara,10uL 10 mMpNPG,150uL bacteria solution in each well of a 96-well palte. Then we sealed 96-well plate and put it into a shaker, set the speed to 100r/min. We measured the A620 and A450 using microplate reader each 2 hours. The lac and ara solution was diluted by gradient from 10Mm to 0.
Inoculation and culture:We Picked a BL21(AI) monoclonal colony from solid plate containing KAN, Inoculated into LB liquid medium containing 1‰antibiotics. Put it into a shaker.Conditions in Shaker: 37℃, 150r/min,12-16h. The next day we then divided the bacteria solution into four groups, Inoculated into 20mL M9(contain 15% glycerol) liquid medium containing 0, 0.05‰,0.25‰,1‰antibiotics using 200uL bacteria solution each.
Enzyme activity assay: We added 20uL Ara,10uL 10 mMpNPG,170uL bacteria solution in each well of a 96-well palte. Then we sealed 96-well plate and put it into a shaker, set the speed to 100r/min. We measured the A620 and A450 using microplate reader each 2 hours. The ara solution was diluted by gradient from 10Mm to 0.
Animal experiment: In the pet hospital, we took blood from the hearts of 9 hamsters after anesthesia and opened the abdominal cavity to collect the liver. After handling the rest of hamsters in our lab, we centrifuge blood samples, take the upper serum, and add sodium azide to prevent bacterial contamination.
Week 10
9/5-9/11
Animal experiment: The serum samples were centrifuged again: 12000r/min, 3min. take 0.2ml into FPLC(Fast protein liquid chromatography) for protein separation and purification. The parameters were set as follows: flow rate: 0.4ml / min, UV detection wavelength: 280nm, collecting 45 centrifuge tubes, 0.5ml each, mobile phase: PBS. The collected centrifuge tube labeled, each hall were measured cholesterol and triglyceride concentration, through special reagents.
Week 11
9/12-9/18
Inoculation and culture: We Picked a BL21(AI) monoclonal colony from solid plate containing KAN, then Inoculated into M9 liquid medium containing the same antibiotics.put it into a shaker. Conditions in Shaker: 37℃, 250r/min,12-16h. The next day we used 200uL bacteria solution inoculated in 20 mL M9(contain 15% glycerol) liquid culture.
Enzyme activity assay: We added 20ul Lac , 20uL Ara,10uL 10 mMpNPG,150uL bacteria solution in each well of a 96-well palte. Then we sealed 96-well plate and put it into a shaker, set the speed to 100r/min. We measured the A620 and A450 using microplate reader each 2 hours. The lac and ara solution was diluted by gradient from 10Mm to 0.
Solid fermentation: Adsorption of notoginseng paste which was fermented on the August 23rd , with macroporous resin. After adsorption, the resin was loaded into the filter bag, and the elution was sufficiently carried out. Half of the resin was eluted with absolute ethyl alcohol, and the rest was eluted with a gradient of 50% ethanol and absolute ethanol.
Plasmid synthesis experiment: 4 DNA samples were centrifuged for 3s, 100μl of ultrapure water was added. The samples were incubated at 50 ℃ for 10min and centrifuged again for 3s.PCR sample size: 1,2,3,4 each template: 8ul; 10 * buffer: 2ul, dNTPs: 2ul; pfu polymerase: 1ul;PCR conditions: 94 ° C, 30 s; 72 ° C, 2 min; ten cycles. The two tubes were mixed and dNTPS: 4ul;pfu: 1ul. Conditions: 94 ° C, 30 s; 72 ° C, 4 min; 10 cycles. Without result.
Induced experiment : To find the relationship of lactose、arabinose and the expression of β-glu in a 96-well plate, we added different concentrations of lactose each column and different concentrations of arabinose each line. Then we added PNPG as substrate and E. coli expressing glucosidase. OD620 and OD450 were measured every one hour. (OD620 can represent E. coli cell concentration and OD450 can represent consumption of PNPG).
Week 12
9/19-9/25
Inoculation and culture: We used 10uL bacteria solution inoculated in 4mL free antibiotics LB liquid culture. Put it into a shaker. Conditions in Shaker: 37℃, 250r/min,12-16h. The next day we used 200uL bacteria solution inoculated in 20 mL M9(contain 15% glycerol) liquid culture.
Enzyme activity assay: We added 20ul Lac , 20uL Ara,10uL 10 mMpNPG,150uL bacteria solution in each well of a 96-well palte. Then we sealed 96-well plate and put it into a shaker, set the speed to 100r/min. We measured the A620 and A450 using microplate reader each 2 hours. The lac and ara solution was diluted by gradient from 10Mm to 0.
Induced experiment: Double induction experiments were carried out again through increasing the concentration of arabinose and lactose.
Plasmid synthesis experiment: 100ml of DH5α was divided into 20/20/30/30, 20ml as positive control, 30ml as PCR transformation group.The second time PCR sample size: 1,2,3,4 each template: 4ul; 10 * buffer: 2ul; dNTPs: 2ul; pfu polymerase: 1ul. PCR conditions: 94 ℃, 30 s; 55 ℃, 30 s; 72 ℃, 4 min; 20 cycles. 72 ℃ for 15min.
Induced experiment: We used 40uL bacteria solution inoculated in 4mL LB liquid culture containing KAN. Put it into a shaker.Conditions in Shaker: 37℃, 250r/min,12-16h; The next day we used 200uL bacteria solution inoculated in 20 mL M9(contain 15% glycerol) liquid culture.
Enzyme activity assay: We added 20ul hyocholic acid, 20uL IPTG, 10uL 10 mMpNPG,150uL bacteria solution in each well of a 96-well palte. Then we sealed 96-well plate and put it into a shaker, set the speed to 100r/min. We measured the A620 and A450 using microplate reader each 2 hours. The IPTG solution was diluted by gradient from 10Mm to 0. The hyocholic acid solution was diluted by gradient from 5Mm to 0.
Week 13
9/26-10/2
Plasmid synthesis experiment: Recombination of four DNA fragments was performed by using a new DNA builder kit. The final re-set experiments: 1,2,3,4 each template: 25ul; DNA mixed 50ul, 50 ° C water bath for 1 hour. After reaction, we took 20μl reaction solution for PCR. PCR conditions: 94 ℃, 30 s; 55 ℃, 30 s; 72 ℃, 4 min; 20 cycles. 72 ℃ for 15min.
