Team:BJ101HS/Results - 2020.igem.org

We did

Firstly, in order to make the protein expressed properly in yeast and bacteria, we optimized the codon, so the newly used model organism could transcript and translate well. As shown in our design, what is important for us is only the amino acid sequence, the nucleotide sequence is newly designed. So we mainly focus on the amino acid sequence and changed up to 20% of the nucleotides.

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

Secondly, we synthesized the four new genes from biological company, using the primary name CSN2, CSN3, LALBA, and LTF. What they give us is dry powder. Nest, we use restriction cloning method to construct four carriers, in which the four genes CSN2, CSN3, LALBA, and LTF were connected to the pPIC9K.

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..

To do this, we used the pET28a(+) backbone to express our gene. We designed Forward and Reverse primer containing NotⅠand EcoRⅠrespectively and did a PCR experiment to get the gene from pPIC9K-CSN2, pPIC9K-CSN3 vector. We also cut the pET28a(+) with NotⅠand EcoRⅠat the same time. Then we ligated them with T4 DNA ligase and transformed into DH5α. E.coli are cultivated in a 37℃ incubator overnight.

(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.

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.