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We did

Based on the background described above, we made our mind to focus on man-made breast milk. The primary purpose of our project was to produce a beverage wich has a same function as breast milk. So we began to work out the major component of breast milk, which are listed below:

-Aqueous true solution 87%
-Casein body solution 0.3%
-Fat emulsion 4%
-Adipose capsule and living cells 8.7%

However, after we read more papers and expanded each part in breast milk, we found natural breast milk is a sooooo big and complex mix. Proteins, cells, fat, polysaccharides and so many things are included.
So we started a new section of brainstorming and we decided to move on to produce protein components to mimic breast milk, which consist the major part of nutrients.
In the scientific paper we cited1, the total amount of protein in mature breast milk(in contrast to colostrum, which is secreted days after parturition) is 9g/L. And the major protein component are:

-β-casein: 4.4g/L in mature breast milk
-κ casein: 1.3g/L in mature breast milk
-α lactalbumin: 3.26g/L in mature breast milk
-lactoferrin: 1.94g/L in mature breast milk

So we wondered whether the four proteins above could be the major component in our man-made breast milk.

The blast consequence of old and new CSN3

The blast consequence of old and new CSN2

The blast consequence of old and new LALBA3

The blast consequence of former and new LTF

Based on Uniprot website(https://www.uniprot.org/) and NCBI website(https://www.ncbi.nlm.nih.gov/), we found four genes are responsible for encoding those proteins and they are CSN2, CSN3, LTF and LALBA genes. So we finally decided to synthesis β-casein (encoded by CSN2), κ casein (encoded by CSN3), lactoferrin (encoded by LTF), and α lactalbumin (encoded by LALBA) using synthetic biology method.

To solve the problems

We used both Chemical transformation method and electrotransformation method to transfect pPIC9K-CSN2, pPIC9K-CSN3, pPIC9K-LTF and pPIC9K-LALBA vectors into pichia pastoris. And then put them in the 30℃ incubator to grow for 2days. After sequencing to verify the transfect result, we pick the fungus and cultivated in liquid medium.

Yeast culture solution

We also transferred the four genes into E.coli for amplification, which allows the limited number of genes to be multiplied, we use glycerinum to store them. Now we get our pichia pastoris with pPIC9K-CSN2, pPIC9K-CSN3, pPIC9K-LTF and pPIC9K-LALBA vectors in it and store the plasmids carefully!

Thirdly, in order to make our production more efficient, we also think of E.coli to play a role in our system. E.coli could grow fast and is also widely used. So we firstly chose 2 protein(β-casein and κ-casein) to express in E.col, as the two is the dominant protein existing in human breast milk..

Yeasts are used as expression pattern in our experiments primarily because the public accept yeast to be a safe organism. We chose pichia pastoris in our expression system, as this kind of yeast is properly modified and widely used.
The main principle of constructing carriers is molecular cloning. Specifically in our project, through the restriction cloning. We synthesized four genes through biological company and ligated them in the pichia pastoris expression vector pPIC9K.
There are four detailed differences in our design:
- Due to our project's purpose is to produce humanized proteins in yeast, we did codon optimization to make it more suitable for expressing in yeast, so what is important for us is only the amino acid sequence, the nucleotide sequence is newly designed.
-Due to our project's purpose is to produce proteins, we designed to tandem the protein with α secretion factor, which can make yeasts secrete proteins outside the cells.
-Due to our project's purpose is to have clean proteins, we designed to tandem his-tag both in N-terminal and C-terminal of the protein in order to to purify them.
-We are not only trying to use yeast, we also put the gene on pET28a(+) banckbone. We wonder whether bacteria could play a more efficient role in production.
The processes of our experiment are illustrated below:
1. For yeast: Transfect pPIC9K-CSN2, pPIC9K-CSN3, pPIC9K-LTF and pPIC9K-LALBA vectors into yeasts. For E.coli: Transfect pET-28a(+)-CSN2, pET-28a(+)-CSN3, pET-28a(+)-LTF and pET-28a(+)-LALBA vectors
2. For yeast: Cultivate yeasts in a 30℃ incubator For E.coli: Cultivate bacteria in a 37℃ incubator
3. Yeasts or E.coli produce proteins mentioned before under a proper inducing condition.
4. Concentrate the supernatant and identify target protein.
5. Using SDS-Page to examine whether proteins are produced successfully.
6. Purify target protein with Ni-NTA.
7. Find a suitable buffer to dissolve the proteins.
[1]郗文政, & 袁则. (2002). 人乳的组成及其功能. 中国妇幼健康研究, 13(006), 243-245.

(Left pannel: pET28a(+) cut by NotⅠand EcoRⅠ; Right pannel: pPIC9K-CSN3 PCR product cut by NotⅠand EcoRⅠ)

(Blast consequence between predicted result and real result)

After sequencing, we have successfully constructed pET28a(+)-CSN3, but not pET28a(+)-CSN2. Now, we added a new E.coli DH5α with pET28a(+)-CSN3! Fourthly, we did SDS-Page to verify the expression of CSN3, which encodes κ-casein of around 30kDa. We use pET28a(+) empty vector as a negative control, and set gradient concentration of IPTG(0mM, 0.2mM, 0.5mM, 1.0mM) to induce the expression of κ-casein.
In our result, we could see a sharp band between 35kDa and 25kDa. With the increase of IPTG concentration, the band are more clearly.
Based on this, we verified our part and proved it could work properly.

(Blast consequence between predicted result and real result)

Because of the COVID-19, we could only enter to the lab in limited time and with limited number of people. The first time we went into the lab we know nothing, we are trained to do molecular cloning and have already got the results listed above. We are moving on slowly, but are still trying our best to move on.

We have listed the future plans and hope we could finish them:

1.We are thinking of creating a proper inducing condition for yeasts and E.coli to produce proteins. We have noticed the conventional method for yeast is to use methyl alcohol as a inducer. We wonder whether other component are available as methyl alcohol is toxic and not proper to be used in food industry.
2.For yeast, because of the presence of α-secretion signal, we could concentrate the supernatant and identify target protein directly. But for E.coli, to get the protein, we need to ultrasonicate the solution. We are searching a proper peptide which could use for bacteria secretion.
3.We want to using SDS-Page to examine whether proteins are produced successfully. Because our protein has his-tag in N-terminal and C-terminal, we could use his antibody or specific protein antibody to test.
4.If the protein could be seen on the gel, we would then enlarge the cultivate system and purify target protein with Ni-NTA.
5.We also think of finding a suitable buffer to dissolve the proteins. We hope to work out a beverage mimic the real human breast milk.
6.From the research and interview, we found that carbohydrates also play an important role in infant appetite regulation, breastfeeding patterns and body composition, so including carbohydrates will be our step to refine our product. Study has shown that there are nearly 200 unique oligosaccharides structures that help stimulate to build up the Lactobacillus bifidus, a kind of probiotic bacteria in the gut, while inhibiting the growth of E.coli and other negative bacteria. Compared to cow’s milk, human milk contains a nearly ten times more oligosaccharides, making oligosaccharides a special and beneficial component in human milk. Besides oligosaccharide, we also plan to include polysaccharides and monosaccharides along all other important component in human breast milk.