We design four plasmids: The DNA sequences of PKC, xynA, CcxynA, and xyl3A were inserted into the pET-28a (+) vector, respectively. In order to build our plasmids, we let the synthetic company synthesize two target gene fragments, PKC and xynA integrate it into the pUC57 vector. And the target gene fragments CcxynA and xynl3A integrate them into the pET28a vector.
The certificate of synthesize analysis are as follows:

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 DH5α, and transformed it into BL21(DE3). The colony PCR identification results showed that it was successful (Figure 3).

We amplify xynA by PCR, double-enzyme digestion, and inserted into the XbaI and BamHI site of pET28a (+) carrier, to obtain the plasmids pET28a-xynA. Then the pET28a-xynA transform into DH5α, the colony PCR identification results show that the construction of pET28a-xynA is successful (Figure 4).

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 5), and the plasmid pET28a-xynA was successfully constructed.

The plasmid pET28a-xynA was extracted from DH5α, and transformed into BL21(DE3). The PCR identification results showed that the plasmid pET28a-xynA was successful (Figure 6).

Because the gene synthesis company delivered a tube of plasmid, we transformed the pET28a-CcxynA into E.coli BL21(DE3) for expressing proteins. The PCR identification results showed that the transformation of plasmid pET28a-CcxynA was successful.

As a result, we can find that when the length of inserted gene is around 3.5k, we still achieved gene-editing with casposons. What’s more, as the length of the inserted gene increased, the number of colonies decreased. However, casposons is still an excellent tool we could use in future research for gene editing.

We transformed the pET28a-xyl3A into E.coli DH5α for amplification. Next, the plasmid pET28a-xynA was extracted from DH5α, then transformed into BL21(DE3). The PCR identification results showed that the transformation of plasmid pET28a-xyl3A was successful (Figure 8).

In order to obtain the four proteins(PKC, xynA, CcxynA, xyl3A), we transferred the recombinant 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 four proteins solutions we wanted.

The molecular weights of xynA, PKC, xyl3A, and CCxynA were 22.87 KD and 48.27 KD, respectively. 84.93KD and 57.0KD; referring to the marker in Figure 1, we found the four proteins (PKC, xynA, CcxynA, xyl3A) in lane S, indicating that they were successfully expressed in E. coli BL21 (DE3). The four proteins (PKC, xynA, CcxynA, xyl3A) were 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 OD540 value of the four purified enzyme solutions (xynA, PKC, xyl3A, and ccxynA) 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.
Table 1. Enzyme activity of four solutions (xynA, PKC, xyl3A, and ccxynA)

The results showed that the activity of ccxynA enzyme is the highest activity of the four enzymes. The enzyme activity of xyl3A is higher than that of xynA and PKC. The results indicated that all four proteins were successfully expressed，and the enzyme activity of four solutions (xynA, PKC, xyl3A, and ccxynA) was active. It indicated that all four proteins were successfully expressed. According to the different activity values of four enzymes, we can adopt the strategy of multi-enzyme synergistic degradation to accelerate the degradation rate of cellulose or xylan in feed, which can be added to feed as a feed additive to improve the ability of animals or digest and utilize feed, promote animal appetite and improve the quality of animal husbandry products. It can solve the bottleneck problem of low efficiency and high cost of cellulase hydrolysis.

Firstly, we designed the pSIP403-PUS-xynA. We let the xynA to light the plasmid pSIP403-PUS to obtain the pSIP403-PUS-xynA plasmid.

Next, the xynA and pSIP403-PUS plasmids were digested with HindIII. Figure12 is shown that the plasmid pSIP403-PUS-xynA was successfully constructed.