Our team focuses on the study of feed additives products for increasing feed degradation rate. To carry forward the concept of igem, while providing data support for future teams, we specially searched the base part BBa_K4096002 from the iGEM Biological Parts library. But we can only find some basic sequence and plasmid construction information from BBa_K4096002. Our team did experiments from multiple aspects, such as protein expression, and added data from enzyme activity tests to test protein expression. This data information can be a good reference for future iGEM teams working on feed degradation.
In addition, we added new information about a new composite part BBa_K4277006 to the iGEM Biological Parts library. It provides more choices of Feed degradation for future iGEM teams.
What's more, we proposed a new idea-multi-enzyme combination. We combined the xylanase xynA gene from Bacillus subtilis with the β-xylosidase xynA gene to make a mixed feed additive to improve the utilization rate of silage and grain mixed feed. A new idea-multi-enzyme combination also provides more ideas for future iGEM teams to feed degradation.

In the iGEM2021_Shanghai_Metro_HS team, the team only was constructed for the parts BBa_K4096002, and attempted protein expression, but the enzymatic activity was not detected. Compared with 2021, we not only completed the construction of BBa_K4277006, pET28a-PKC, and protein expression but also successfully measured enzyme activity to add enzyme activity data for this part and provide some experimental data reference for the future iGEM team.

We amplify PKC by PCR, double-enzyme digestion, and inserted into the NheI and HindIII site of pET28a (+) carrier, respectively, to obtain the plasmids pET28a-PKC. Then the pET28a-PKC transform into DH5α, the colony PCR identification results show that the construction of pET28a-PKC is successful (Figure 1).

We send the constructed recombinant plasmid to a sequencing company for sequencing. The returned sequencing comparison results showed that there were no mutations in the ORF region (Figure 2), and the plasmidpET28a-PKC was successfully constructed.

The plasmid pET28a-PKC was extracted from E. coli DH5α, and transformed it into E. coli BL21(DE3). The colony PCR identification results showed that it was successful (Figure 3).

In order to obtain the protein PKC, we transferred the plasmids into E. coli BL21(DE3), expanded the culture in the LB medium, and added IPTG to induce protein expression when the OD600 reached 0.3-0.5. After overnight induction and culture, we collected the cells and ultrasonic fragmentation of cells to release the intracellular proteins. Next, we used nickel column purification to purify the protein we wanted.
At this point, we obtained the protein solutions we wanted.

The molecular weights of PKC were 48.27 KD. Referring to the marker in Figure 4, we found the protein PKC in lane S, indicating that it was successfully expressed in E. coli BL21 (DE3). The protein PKC was also found in lane P, possibly due to the inactivation of a small number of proteins.

Determination of reducing the sugar by DNS method: The absorbance OD 540 value of the purified enzyme solutions (PKC) was measured after color reaction with DNS. The activity of the enzyme can be converted by the amount of sugar consumed and the working time.

The enzyme activity of PKC is about 0.26 U/mL (Figure 5). The results indicated that the protein PKC was successfully expressed, and the enzyme activity of PKC was active. It also proved that PKC had the function of degrading cellulose.The enzyme activity of PKC is about 0.26 U/mL (Figure 5). The results indicated that the protein PKC was successfully expressed, and the enzyme activity of PKC was active. It also proved that PKC had the function of degrading cellulose.

We have added the new pSIP403-PUS-xynA-xyl3A, number BBa_K4277010, and provided some information about the part. The xylanase xynA gene from Bacillus subtilis has the function of degrading xylan. A novel family of β-xylosidases (xyl3A) from Humicola insolens Y1 is highly tolerant to D-xylose. We ligated the gene xyl3A and xynA with peptide PUS and inserted it into the vector pSIP403 to obtain a pSIP403-PUS-xynA-xyl3A（Figure 6). The fusion plasmid can be expressed simultaneously with the gene xyl3A and xynA to degrade xylan and cellulose, and improve the utilization ratio of mixed feed of silage and grain.

We provide the new idea of a mixed feed additive, which is added to the feed as a feed additive. Through the combined action of multiple enzymes, the complex xylan and cellulose components in the feed are rapidly decomposed, thereby improving the digestion and utilization ability of animals, and the quality of animal husbandry products. Our innovative idea of multi-enzyme combination can provide new ideas for future IGEM teams.We provide the new idea of a mixed feed additive, which is added to the feed as a feed additive. Through the combined action of multiple enzymes, the complex xylan and cellulose components in the feed are rapidly decomposed, thereby improving the digestion and utilization ability of animals, and the quality of animal husbandry products. Our innovative idea of multi-enzyme combination can provide new ideas for future IGEM teams.