Team:Shanghai high school/Notebook

Online: Online Training, Topic Discussion - June~July


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


  • 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