Week 1 (4.14-4.21)

We performed single enzyme digestion, electro transformation, plasmid extraction, and nucleic acid gel electrophoresis, DNA purification, coating, and bacterial shaking.

Week 2 (4.22-4.28)

We performed electroporation of bifidobacterium, prepared 1XTE buffer, PCR identification of recombinant bacteria and gel electrophoresis. At the same time, we carried out plasmid extraction and PCR identification of GS115 and KM71H control group.

Week 3 (4.29-5.5)

In this week's experiment, we performed WB electrophoresis on the previously induced samples and observed the bands by Coomassie bright blue staining.

Week 4 (5.6-5.13)

We performed enzyme digestion, linearization, agarose gel electrophoresis verification and plasmid purification of the newly constructed plasmid. At the same time, Pichia pastoris and Escherichia coli were transformed, and the bacterial culture was amplified. Bacterial culture and plasmid extraction were performed.

Week 5 (5.14-5.20)

In this week, we identified the positive clones of the early bacteria by PCR, ran the nucleic acid gel gel, and purified the samples by nickel column.

Week 6 (5.21-5.27)

The samples purified by nickel column in the previous stage were then subjected to WB electrophoresis, followed by membrane transfer and double antibody incubation to obtain the bands we needed.

Week 7 (5.28-6.3)

In the seventh week, we extracted new plasmids, performed plasmid cleavage linearization, verified by agarose gel electrophoresis, and the results showed that the linearization was successful. Then, the linearized plasmids were purified and the concentration was measured.

Week 8 (6.4-6.10)

We performed the transformation of GS115 yeast receptive cells, KM71H receptive cells, and extended culture of E. coli.

Week 9-12 (6.11-7.7)

We summarized and reflected on the two-month experiment, and improved and perfected our experiment during this period, laying a foundation for the later experiment.

Week 13 (7.8-7.14)

In this week, we performed PCR amplification of the new sample, WB electrophoresis, part of the membrane transfer, part of the Coomasi bright blue staining, in order to observe and get the target band.

Week 14 (7.15-7.21)

We cultured a batch of new colonies, amplified by PCR, performed agarose gel electrophoresis, combined with target bands, and induced culture of the bands in the later stage, and expressed them on Pichia pastoris.

Week 15 (7.22-7.28)

In this week's experiment, we preserved the previous bacteria and extracted the new plasmid, which was subjected to single enzyme cleavage and verified by agarose gel electrophoresis. Subsequently, we performed induction culture on the sample bacteria.

Week 16 (7.29-8.5)

In order to further express the protein, we purified the supernatant of the induced culture bacterial solution by nickel column this week, ran the glue by WB electrophoresis, and transferred the film to obtain the target band.

Week 17 (8.6-8.13)

In the early stage, we expressed the protein. In this week, we did a lot of culture on the bacteria, hoping to express the protein. We carried out experiments and carried out WB electrophoresis gel verification.