Team:Shanghai Metro HS/Engineering


In China, animal husbandry in many places has become a pillar industry of the rural economy and the main source of increasing farmers’ income. With the continuous expansion of the scale of animal husbandry production, the demand for high-quality feed has also increased significantly. Silage is a kind of natural plant feed. After cutting the stems and leaves of plants with a moisture content of 65%-75%, under airtight hypoxia conditions, the fermentation effect of anaerobic lactic acid bacteria is used to inhibit the reproduction of various miscellaneous bacteria. And it is mainly used to feed ruminants (such as cattle, sheep, alpacas, and deer). Compared with fresh feed, silage is more durable in storage, has stronger nutrients than dry feed, and is rich in nutrients, which is conducive to long-term preservation. It is an excellent source of feed for livestock.
Silage contains a lot of cellulose, which is the main structural component of plant cell walls. It is usually combined with hemicellulose, pectin and lignin, which is difficult to digest and absorb. Enzymes that can degrade cellulose can be added to the silage to improve the ruminant's ability to obtain monosaccharides. We can prepare cellulase through experiments and explore the best conditions for cellulose degradation. Use it as a silage additive and add it to feed to promote appetite of livestock, increase and supplement the content of various digestive enzymes in the animal’s body, prevent and treat some common gastrointestinal diseases of livestock and improve the quality of livestock products.
The alkaline cellulase gene from Pseudomonas aeruginosa PKC-001 was selected and inserted into the pET-25b vector which contains a pelB signal peptide. The recombinant plasmid then was transformed it to E. coli BL21(DE3) strain to produce cellulase.
The pET-25b-PKC expression plasmid was successfully constructed by homologous recombination PCR technology (Figure 1).
Figure 1. Schematic map of pET-25b-PKC expression plasmid.
Different induction conditions were tested for protein expression. As the pET-25b vector 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 (Figure 2).
Figure 2. SDS-PAGE analysis of culture supernatant under different induction conditions.
The theoretical molecular weigh of the PKC cellulase is 45.6 kDa. As seen from the SDS-PAGE (Figure 2), there is a wide band just above 40 kDa and it indicates that we have obtained the PKC cellulase.
In this experiment, we successfully prepared genetically engineered bacteria which produce PKC cellulase.