Theme: Team icebreaker
Time: 9:00-16:30, August 18, 2022
Location: Road Show Hall, 2F, Block A, 58 Xiangke Road, Pudong New Area, Shanghai
Experiments:
Introduction to the mentor team and team members
○ Ice Breaker (game)
○ Electing candidates for team captains of the wet and dry teams
Team discussion: team name, team LOGO, merchandise design, etc.
Meaning:
Bis-Vacc
The direction of this experiment is to make a safe oral vaccine for the infant and young children without endotoxin. Bis originally means "dual". The research on this vaccine can solve the problems caused by EV71 and RV viruses comprehensively, which is more economic and time-saving. The research subject is vaccine, so the suffix “Vacc” is added to “Bis”. The team’s name is based on the research direction and the basic word root. On the premise of not deviating from the topic, the team carries out certain innovation, which is novel and closely related to the theme.
Theme: LB nutrient solution configuration, PCR amplification
Time: 9:00-16:30, August 19, 2022
Place: Dart Laboratory, No. 1077 Zhangheng Road, Pudong New Area, Shanghai
Experiments:
● Laboratory safety training
● LB culture medium
● PCR amplification of vp1, vp7, IL12, GFP and GLBP genes
● Gel extraction of vp1, vp7, IL12, GFP and GLBP genes
● Overlap extension PCR of vp7 and vp1
● Overlap extension PCR of IL12 and GFP
● Culturing E.coli
Progress of the team:
Before the experiment in the morning, the coach of the wet team led the students to conduct laboratory safety training. After that, PCR amplification of vp1, vp7, IL12, GFP and GLBP genes was completed, and LB medium was prepared at the same time.
In the afternoon, students performed agarose gel electrophoresis and image analysis, and completed the DNA gel extraction. Finally, E. coli was incubated.
Theme: extraction of pET28a plasmid, gel extraction, enzyme digestion, ligation, and heat shock transformation
Time: 9:00-16:30, August 20, 2022
Place: Dart Laboratory, No. 1077 Zhangheng Road, Pudong New Area, Shanghai
Experiments:
● Plasmid pET28a was extracted
● Gel extracted of VP1-VP7 gene
● Enzyme digestion, ligation and transformation
● Plate coating screening
● Overlap extension PCR of GLBP and vp7-vp1
Progress of the team:
The students of the wet team extracted the plasmid pET28a in the morning, determined its concentration, and then used the agarose gel method to separate the sizes of vp1-vp7 DNA fragment, and then completed the DNA extraction.
In the afternoon, the plasmid pET28a and vp1-vp7 DNA fragments were digested with restriction enzymes EcoRI and XhoI, and then ligate them with T4 Ligase. Finally, the ligations were transformed into E.coli DH5α
Theme: Identification of transformants by colony PCR, selection of monoclonal culture seed liquid, and extraction of plasmids
Time: 9:00-16:30, August 21, 2022
Place: Dart Laboratory, No. 1077 Zhangheng Road, Pudong New Area, Shanghai
Experiments:
● Identification of transformants by PCR
● Select monoclonal culture
● Plasmid pET28A-vp7-vp1 was extracted and transformed into BL21(DE3)
● Culturing bacteria
● Overlap extension PCR of GLBP-vp7-vp1 and IL12-GFP
● Overlap extension PCR of GLBP-vp7-vp1 and GFP
● Enzyme digestion, ligation and transformation
● Plate coating screening
Progress of the team:
In the morning, the coach and the students of the wet team selected the E. coli colony PET28A-VP1-VP7, and then identified whether the colonies were transformed PET28A-VP1-VP7 by PCR. At the same time, we amplified GLBP-vp7-vp1-IL12-GFP, and GLBP-vp7-vp1-GFP by overlap PCR, respectively.
In the afternoon, the students transformed plasmid pET28A-vp7-vp1 into E.coli BL21(DE3). Then, plasmid pET28a, GLBP-vp7-vp1- IL12-GFP DNA fragments, and GLBP-vp7-vp1-GFP DNA fragments were digested with restriction enzymes EcoRI and XhoI, and then ligate them with T4 Ligase. Finally, the ligations were transformed into E.coli DH5α.
Topics: E. coli transformation, learning plasmid profile, learning and configuration solutions
Time: 9:00-16:30, August 22, 2022
Place: Dart Laboratory, No. 1077 Zhangheng Road, Pudong New Area, Shanghai
Experiments:
● Identification of transformants by PCR
● Select monoclonal culture
● Plasmids were extracted and transformed into BL21(DE3)
● Proliferating bacteria
● Preparation of protein expression reagent
Progress of the team:
In the morning, the coach and the students of the wet team transformed the plasmid PET28A-VP1-VP7 into E.coli BL21(DE3), and coated the plate overnight for culture. Next, we selected the E. coli colony pET28A-GLBP-vp7-vp1-IL12-GFP and pET28A-GLBP-vp7-vp1-GFP, and then identified whether the colonies were correct.
In the afternoon, the students transformed plasmid pET28A-GLBP-vp7-vp1-IL12-GFP and pET28A-GLBP-vp7-vp1-GFP into E.coli BL21(DE3). The instructor taught the students how to prepare the buffer including HisA buffer, HisB buffer, Tris-HCl buffer and electrophoretic decolorization solution in groups, and then learned to adjust the buffer pH with the pH meter, in preparation for the later purification protein experiment.
Theme: IPTG induction
Time: 9:00-16:30, August 23, 2022
Place: Dart Laboratory, No. 1077 Zhangheng Road, Pudong New Area, Shanghai
Experiments:
● Select E. coli colonies and run PCR test
● Agarose gel electrophoresis
● Analysis of electrophoresis images and results
● IPTG induced protein expression
Progress of the team:
In the morning, the students and the coach of the wet team picked up the E. coli colonies in the sterile ultra-clean table, prepared the colony PCR system, and amplified them with PCR instrument. Then nucleic acid gel was prepared, agarose gel electrophoresis was performed, and the results and images were analyzed.
In the afternoon, the correct colony was induced the expression of with IPTG, and cultured in the shaker overnight. Then the coach introduced the principle and operation steps of SDS-PAGE.
Topics: ultrasonic cell crushing, nickel column affinity chromatography
Time: 9:00-16:30, August 24, 2022
Place: Dart Laboratory, No. 1077 Zhangheng Road, Pudong New Area, Shanghai
Experiments:
● Collect bacteria
● Ultrasonic cell crushing
● Nickel column affinity purification
● SDS-PAGE
● Proliferating bacteria
Progress of the team:
In the morning, the students of the wet team collected the E. coli induced by IPTG by centrifugation, and then the cells of the bacterial solution were broken by ultrasound for 15 minutes. It was observed that the bacterial solution became clear.
In the afternoon, the students collected the supernatant of the cells for affinity purification with a nickel column. Then the coach taught the students the principle of SDS-PAGE and the configuration of Buffer. Finally, the supernatant and protein samples were loaded into wells of SDS-PAGE and the experimental data were analyzed. Last but not least, the bacteria pET28A-GLBP-vp7-vp1-GFP/E.coli BL21(DE3) were cultured in the LB medium.
Topics: SDS-PAGE protein electrophoresis, sorting experimental results and data
Time: 9:00-16:30, August 25, 2022
Place: Dart Laboratory, No. 1077 Zhangheng Road, Pudong New Area, Shanghai
Experiments:
● SDS-PAGE protein electrophoresis
● Detection of GFP fluorescence signal
● Organize the electrophoresis results and GFP data
Progress of the team:
The experiment has come to an end. The students of the wet team cultured the pET28A-GLBP-vp7-vp1-GFP/E.coli BL21(DE3) with IPTG, and detected GFP fluorescence signal every hour. Then, we carried out SDS-PAGE electrophoresis on the protein and analyzed the electrophoresis experiment results and related data.
In the afternoon, students continue to detect GFP fluorescence signals every hour and took profile photos, and the coach explained the experimental principle, precautions, and operation steps of Western Blot. The SDS-PAGE gel was stained and destained. Finally, the laboratory was cleaned up and the experimental journey was successfully completed.
