Day1 15/08/23
Team Ice-breaking
Brainstorm Meeting on team logo, etc.
Day2 16/08/23
PCR, uses PCR instruments to amplify the AocI gene, FAD gene and pAO815 vector gene.
Prepare agarose gel and use this gel to do gel electrophoresis.
Gel Extraction means extracting the PCR product.， which is the AocI gene and FAD gene.
Prepare culture medium (LB).
Day3 17/08/23
Gel Extraction means extracting the PCR product. which is the pAO815 vector gene.
Homologous recombination pAO815- AocI and pAO815- AocI-FAD.
Plasmid extraction of pAO815 and pAO815- AocI .
Heat shock response, the purpose is to put the extracted plasmid (pAO815- AocI & pAO815- AocI-FAD) into E.coli (DH5α)， then apply amp+ to a culture dish, and then let it stand overnight for culture.
Shake bacteria of pAO815-AocI and pAO815-AocI-FAD on the shaker.
Day4 18/08/23
PCR testing, this purpose is to verify whether the plasmid was successfully constructed and transferred into DH5α.
Preparation before yeast transformation， firstly， preparing culture medium (YPD) and SDS-His solution. Secondly, uses PCR instruments to amplify the pAO815- AocI and pAO815-AocI-FAD.
Product purification, the purpose of this step is to increase the concentration of plasmid.
Yeast transformation was carried out by using a yeast transformation promoter in an ultra-clean bench.
Plasmid extraction of pAO815- AocI-FAD and pAO815- AocI.
Prepare protein extract solution.
PCR testing for yeast, the purpose is to identify whether the recombinant plasmid goes into yeast cells.
Day6 21/08/23
Enlarged culture was carried out according to different methanol induction concentrations.
Prepared ‘transparent circle’ experiments and the result was observed next day.
Day7 22/08/23
Observed transparent circle results.
The crude protein was extracted by cell disruption with an ultrasonic cell disruption instrument. Then, the crude protein concentration was measured.
SDS-PAGE gel electrophoresis (crude protein supernatant)
His protein purification.
Protein gel electrophoresis after purification.
Day8 25/08/23
Activate strains pAO815-AocI as well as pAO815-AocI-FAD.
Configure 200ug/ml histamine solution and set aside.
Configure 10 bottles of 300 mL LB medium and set aside.
Day9 26/08/23
1% inoculum was transferred to the activated bacterial solution, and Amp+ and histamine solution were added.
Five temperature gradients were set up, respectively 20, 25, 30, 37, 45℃; five sampling times were set up, respectively 12, 24, 36, 48, 72h, according to the different treatments, sampling was done on time for 72h.
Day10 30/08/23
The samples were centrifuged to remove the supernatant, labelled and sent to the company for HPLC testing to detect changes in histamine concentration.
Day11 07/09/23
HPLC results analysis.
