Team:Shanghai Metro HS/Proof Of Concept


Proof of Concept
In recent years, the development husbandry in China has been preoccupied with great opportunities. Still, the energy feed is relatively lacking, which makes animal production development face severe supply and demand challenges in the future. Silage has been a good choice for ruminants feeding, whereas, the cellulose contained in silage causes potential problems in ruminants’ digestive system. To address this problem, we are planning to develop feeding additives containing cellulase, which will be compatible with silage, thereby enhancing ruminants’ digestion. With this efficient uptake of feed, the feeding costs can be reduced while improving the yield and quality of ruminants. In our design, the cellulase gene is extracted from Pseudomonas aeruginosa, P. aeruginosa, PKC-001. The gene is connected to vector pET-25b and then transformed to E.coli (BL21) and to construct plasmid pET25b-PKC-OP. The engineered E.coli is used to test protein expression conditions, and then used to conduct enzyme activity test, e.g. the degrading capacity of our engineered strain of cellulose.
In the future plan, the E.Coli(BL21) will be replaced by other probiotic, such as Nissle 1917 to make further test about protein expression and enzyme activity.
Supporting Experiment Results
Figure 1 E. coil having the desired pET-25b and PKC-OP
The pET25b-PKC-OP was constructed.
The plate shows monoclonals of pET25b-PKC-OP constructs
Figure 2. The result of Apa1 enzyme digestion validation
Column “marker” is a column that is used to show the position of different lengths of genes. Number 1 to 10 is the result for recombinant plasmid pET25b-PKC-OP after Apa1 enzyme digestion. We get two bands of 4712bp and 1894bp. It further indicates that the obtained monoclonals were positive monoclonals containing the recombinant plasmid.
Figure 3. SDS-PAGE analysis of culture supernatant under different induction conditions
Figure 4. Protein molecular weight result of PKC-op by Snapgene
We designed several different induction conditions for protein expression. As the vector we chose to construct the plasmid contains a pelB signal peptide, the engineered strain would secret the protein into the medium. Therefore, we collected the culture supernatant after induction and ran SDS-PAGE for verification.
According to the calculation by Snapgene, the expected protein molecular shall weigh 45.6 KDa. As seen from the SDS-PAGE map (Fig. 4), there is a wide band just above 40 KDa and it indicates that we have obtained the PKC-op protein.
Our experiment proved the potential success of our design, e.g. the PKC-op protein could be acquired by genetic engineering on the chassis of E.coli (BL21). In the future, we will move on with the experiment to test the protein’s degradation capacity of cellulose, consolidating the research foundations for future development of our feeding additives.