Online: Online Training, Topic Discussion - June~July
Offline:
Day 1 2021/08/05
Ice breaking: self-introduction & games
Elected 3 preliminary leaders for dry team and 2 for wet team
Designed team name and first drafts of logo and uniform, decided the types of peripheral products
Introduction of dry team coach and tasks of the day and after
Everyone wrote their self-introductions
Day 2 2021/08/06
Laboratory safety training, instrument introduction
Established experimental design framework and introduced basic experimental principles
Prepared LB solid / liquid culture medium and poured Amp / Kan antibiotics to the plates
Prepared competent solutions for Bacillus subtilis: 200 mL (LB + 0.5 M Sorbitol), 200 mL electroporation culture medium, and 10 mL RM
Plasmid transformation: transferred pET28a(+) and PHT43-His into competent E. coli DH5 α
Tried to re-activate Bacillus subtilis strain WB800n and DH5α carrying pET28a plasmid
Day 3 2021/08/07
Transformed Bacillus subtilis plasmid WB800n into competent cells of E. coli
Extracted and purified pET28a plasmid, which was digested overnight with enzyme BamHI
Inoculated the re-activated Bacillus subtilis into the culture medium for competence preparation and cultured it overnight
Day 4 2021/08/08
Successfully prepared Bacillus subtilis competent cells
Transformed pET28a-VP7-LTB into E. coli competent cell line BL21 (DE3)
Small scale of PHT43-His plasmid extraction
Used PCR to amplify the DNA sequence of VP7 and VP7-LTB, confirmed with agarose gel electrophoresis, sent to a company to be sequenced
First round PCR and second round PCR (did the second round twice)
Recovered enzyme digestion linearization vectors by gel-cutting
Day 5 2021/08/09
PHT43-His plasmid small extraction
Electroporation: transferred PHT43-His plasmid to Bacillus subtilis to detect whether the competence preparation and electroporation system are feasible
Prepared of E. coli and Bacillus subtilis induced expression culture medium (TM medium)
Recovered VP7 fragment and VP7-LTB by gel cutting
Day 6 2021/08/10
One day off
Day 7 2021/08/11
Optimized primers, completed PCR amplification of the target gene of each expression vector
Transformed pET28a non-loaded plasmid into E. coli competent cells as a negative control for induced expression
IPTG induced expression in E. coli, examined ITPG concentration
Explored the use of the ultrasonic crusher
Plasmid digestion, homologous recombination, vector construction
Explained the experimental principles and precautions of SDS-PAGE and Western-blot
Expanded the culture of positive monoclonal antibody of Bacillus subtilis and tried colony PCR to further eliminate false positive
Day 8 2021/08/12
Inducible expression of VP7-LTB growing in BL21 using IPTG, establish bacterial growth curve by measuring OD600, and collect samples with different IPTG concentration at different time (0, 2, 4, 5, and 6 hrs)
Optimize the electroporation system of B. Subtilis
SDS-PAGE gel preparation
construct plasmids with target genes: pET28a-VP7, PHT43-His-VP7, PHT43-His-VP7-LTB, using homologous recombination
Day 9 2021/08/13
Extraction of protein of VP7-LTB
SDS-PAGE preparation
Establish bacterial growth curve by measuring OD600
B. Subtilis inducible expression preliminary experiment
Expansion culture of pET28a-VP7，PHT43-his-VP7，PHT43-his-VP7-LTB, and DNA purification for transformation
Day 10 2021/08/14
SDS-PAGE: VP7-LTB 1nM IPTG
Establishment of all carriers
Inducible expression of VP7 in BL21 using IPTG
B. Subtilis electroporation
Personal and group photo
Day 11 2021/08/15
SDS-PAGE: VP7-LTB 0.5 nM, 2 nM IPTG, VP7 1nM IPTG
Observe the positive single colony of WB800n
Summary of the project
Presentation preparation: power point
Day 12 2021/08/16
Periodic report
