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We did

Week 1:

Brainstorming to focus on man-made breast milk

Week 2:

Paper work to focus on four main proteins encoded by CSN2, CSN3, LALBA, LTF

Week 3:

Because of the COVID-19, we could not receive the kit from hq, so we asked bio-company to synthesis our gene this year and today we receive plasmids and solution from company. We begin to inoculate and culture in liquid medium in order to preserve them in glycerol the next week.

The blast consequence of old and new CSN3

Week 4:

Transfect the plasmid into E.coli and pichia pastoris, the former for more plasmid, the latter for expression. We also use glycerol to preserve the bacteria. So this week we focus on four projects:

1. plasmid extraction
2. transformation
3. agarose gel electrophoresis
4. glycerol preservation

Week 5

1. We help other teams with their project, especially KEYSTONE iGEM team. We mentored each other during the molecular cloning project.
2.We have prepared the culture medium for pichia pastoris, not only the cultivate culture medium, but the expression induction medium.
3.We keep on trying to transfect the plasmid into pichia pastoris, but it haven’t succeeded up to now.

Week 6

1.We are still trying to use pichia pastoris to express our protein. We have succeeded once but the culture medium seems to be polluted.
2.We asked KEYSTONE iGEM team again for their project, and we are thinking about exchange the carrier from pichia pastoris to E.coli, so we started a new term of brainstorming.

Week 7

1.The yeast still doesn’t work well.
2.We finally move on to E.coli, we want to put our four genes on the pET28a(+) backbone. We cut the backbone and designed the primers for each gene.
3.We want to use infusion method and restriction enzyme ligation method to ligate and we successfully construct the four plasmids.

Yeast culture solution

Week 8

1.We transfect the four plasmid into E.coli.
2.We induce the expression of the four plasmids and use SDS-PAGE to test the expression.
3.The yeast still couldn’t be cultivated.
4.We also helped KEYSTONE with their project during this period.