7.23
- Participated in a lecture on experiment procedures
- Familiarize with the construction of the designed plasmid
- Went through the local laboratory’s safety protocol and iGEM official safety guideline
- Familiarize with the operation of equipment
7.24
- Preparing pipette tips and both liquid/solid medium
- Disinfected pipette tips and the mediums
- Prepared the antibiotics
- Prepared petri dish with a solid medium
- Practiced the operation under aseptic conditions
- Inoculated bacteria with biosensor sequences and pHY300PLK backbone
- Cultivated the bacteria overnight at 37°C
7.25
- Processed plasmid extraction for the cultivated bacteria
- Prepared the enzyme restriction system for extracted plasmid (biosensor and pHY300PLK)
- Processed enzyme restriction for both biosensor sequence and pHY300PLK backbone
- Ran the gel electrophoresis for both plasmids
- Extracted the gel
- Ligate biosensor sequences and pHY300PLK through both DNA ligase and Gibson Assembly
7.26
- Transformed ligation product to the amplified bacteria E.coli DH5α
- Waited for DH5α colony to grow
- Researched for a heavy metal involved experiment (operation, safety, material handling)
7.27
- Inoculated the developed single colony of DH5α for tests
- Cultivated the bacteria overnight at 37°C
- Prepare the gradient concentrated heavy metal solutions
7.28
- Processed recombinant plasmid pHY300PLK-pVeg-Biosensor extraction for cultivated bacteria DH5α
- Prepared and processed the enzyme restriction for the recombinant plasmid
- Run the gel electrophoresis for identifying recombinant plasmid
- Transformed plasmids pHY300PLK-pVeg-Biosensor and pHY300PLK-pCadA-Biosensor to the bacteria Bacillus
subtilis, respectively
- Cultivated the bacteria ArsA, ArsD, and ArsR overnight at 37°C
7.29
- Test the green fluorescence of the reaction product of bacteria and arsenic solution
- Waited for Bacillus subtilis colony to grow
7.30
- Cultivated the engineered Bacillus subtilis colony
- Express ferulic acid esterase protein in pHY300PLK-pVeg-Biosensor/Bacillus subtilis
- Test the protein expression of ferulic acid esterase in pHY300PLK- pCadA-Biosensor/Bacillus
subtilis with cadmium and Zinc solution, respectively
7.31
- Detection of the enzymatic activity of ferulic acid esterase
8.1-8.5
- Data processing and analysis (shown in WiKi: Result)
- Take a group photo
8.6-8.15
- Functional test of cadmium biosensor (pHY300PLK-PcadA-Biosensor / Bacillus subtilis) and repeat at
least three times
- Collect and analysis data
- Added to the experimental results of the wiki
