Part:BBa_K4277007

pET28a-xynA

pET28a-xynA

BBa_K4277007

Name: pET28a-xynA

Base Pairs: 5069 bp

Origin: Bacillus subtilis strain E20

Properties: endo-1,4-beta-xylanase

Usage and Biology

BBa_K4277001 is a coding sequence of endo-1,4-beta-xylanase (endo-β-1,4-xylanase, EC 3.2.1.8). It hydrolyses the hemicellulose in to a mixture of xyloligosaccharides. according to the CAZy classification system, xylanases belong to GH family 5, 8,10, 11 and 43. among the GH family, the G11 xylanases tolerate a high pI (>9.0) with under 30 Kd molecular weight.

Construct design

xynA gene fragment was inserted into the pET28a and the protein expressed in the BL21. The addition of xylanase will improve the substrate solubility (figure 1).

Figure 1. map of pET28a-xynA.

Experimental approach

1.1 The colony PCR of pET28a- xynA in competent cells DH5α

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 2).

Figure 2.Colony PCR result of pET28a-xynA M:2000 kd marker; Lane 1. pET28a-xynA (DH5α).

1.2 Sequencing result of plasmid pET28a- xynA

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 3), and the plasmid pET28a-xynA was successfully constructed.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 3), and the plasmid pET28a-xynA was successfully constructed.

Figure 3. Sequencing result of pET28a-xynA.

1.3 The colony PCR of pET28a-xynA in competent cells BL21

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 4).

Figure 4. Colony PCR of pET28a-PKC in competent cells BL21.

Functional assay

2.1 Protein expression of PKC

In order to obtain the protein PKC, we transformed the plasmid pET28a-PKC into E. coli BL21(DE3). The colony was inoculated and cultured in the LB medium, and added IPTG to induce protein expression when the OD600 reached 0.3-0.5. After overnight induction and cultivation, the cells were collected and lysed by ultrasonication to release the intracellular proteins. Next, we used nickel resin to purify the interested protein. The molecular weights of PKC were 48.27 KD.

Figure 5.SDS-page M: maker; S: supernatant; P: precipitant; E: elution.

The molecular weights of xynA is 22.87 KD; referring to the marker in Figure 5, we found the proteins (xynA and ccxynA) in lane S, indicating that they were successfully expressed in E. coli BL21 (DE3).

2.2 Determination of PKC enzymatic activity

Determination of reducing the sugar by DNS method. The absorbance OD540 value of the enzyme solutions xynA 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.

Figure 6. The enzymatic activity of PKC.

The enzyme activity of xynA is 0.14U/mL. The results indicated that the proteins were successfully expressed, and the enzyme activity of xynA was active. The enzyme activity of xynA is 0.14U/mL. The results indicated that the proteins were successfully expressed, and the enzyme activity of xynA was active.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 4402
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2622
  Illegal NgoMIV site found at 2782
  Illegal NgoMIV site found at 4370
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 4916
  Illegal SapI.rc site found at 4970