Notebook
2022.7.21
Team ice-breaking
Introduction to the project
Project discussion
Introduction to the project
Project discussion
2022.7.22
Laboratory safety training
Culture medium preparation
Inoculation of E. coli strains containing plasmids pET22b and pRSFDeut1.
Culture medium preparation
Inoculation of E. coli strains containing plasmids pET22b and pRSFDeut1.
2022.7.23
PCR to amplify target genes
Plasmid Extraction and double-enzyme digestion
Gel electrophoresis and gel extraction
Link vector and target gene by Gibson-assembly
Recombinant vector transport target gene into competent cells
Plasmid Extraction and double-enzyme digestion
Gel electrophoresis and gel extraction
Link vector and target gene by Gibson-assembly
Recombinant vector transport target gene into competent cells
2022.7.24
Colony PCR to identify the correct colony
Gel electrophoresis test
Inoculation of correct recombinant E. coli strains
Gel electrophoresis test
Inoculation of correct recombinant E. coli strains
2022.7.25
Plasmid Extraction
Plasmids digestion verification
Send the correct plasmids to the company for Sanger sequencing
Plasmids digestion verification
Send the correct plasmids to the company for Sanger sequencing
2022.7.26
Transport recombinant plasmids into BL21(DE3)
Plate on the agar plate
Plate on the agar plate
2022.7.27
Cultivate the bacteria
Prepare IPTG for inducing the protein expression
Prepare buffer A and buffer B for protein purification
Site-direct mutation of SbPETase by PCR and transform DH5α
Prepare IPTG for inducing the protein expression
Prepare buffer A and buffer B for protein purification
Site-direct mutation of SbPETase by PCR and transform DH5α
2022.7.28
Check the transformation and inoculate the strain into LB liquid medium
Inoculate strains containing SbPETase mutants
Inoculate strains containing SbPETase mutants
2022.7.29-30
Protein purification
SDS-PAGE
Mutant plasmids extraction and send for Sanger sequencing
SDS-PAGE
Mutant plasmids extraction and send for Sanger sequencing
2022.7.31
In vitro platform to identify the activity of SbPETase towards PET and BHET
HPLC experiments of SbPETase degrade PET and BHET materials
Transport recombinant mutant plasmids into BL21(DE3)
HPLC experiments of SbPETase degrade PET and BHET materials
Transport recombinant mutant plasmids into BL21(DE3)
2022.8.1
Cultivate the bacteria
Prepare IPTG for inducing the protein expression
Prepare buffer A and buffer B for protein purification
Prepare IPTG for inducing the protein expression
Prepare buffer A and buffer B for protein purification
2022.8.2-3
Protein purification
SDS-PAGE
SDS-PAGE
2022.8.4-5
In vitro platform to identify the activity of SbPETase towards PET and BHET
HPLC experiments of SbPETase degrade PET and BHET materials
Collect the data
HPLC experiments of SbPETase degrade PET and BHET materials
Collect the data