Part:BBa_K4277009

pET28a-xyl3A

pET28a-xyl3A

BBa_K4277009

Name: pET28a-xyl3A

Length: 6761 bp

Origin: Humicola insolens Y1

Properties: β-xylosidase and α-arabinfuranosidase activities

Usage and Biology

BBa_K4277009 is a coding sequence of xyl3A. The enzyme is identified as β-xylosidase which belongs to geycosyl hydrolase (GH) family. xyl3A is capable to produce xylose in fermentation.

Construct design

Due to the T7 promoter and T7 RNA polymerase having strong ability in translation and usually being used as protein expression, we choose pET28a-vector and E.coli BL21(DE3), to express our target protein xyl3A. Moreover, we inserted the DNA sequences of xyl3A into the NheI and HindIII sites of the pET-28a vector and transferred the plasmid into E.coli BL21(DE3) for protein expression (Figure 1).

Figure 1. The plasmid of pET28a-xyl3A.

Experimental approach

1.1 The colony PCR of pET28a-xyl3A in competent cells DH5α

In order to build our plasmids, we let the company synthesize the xyl3A integrate it into the pET28a vector. We transform the plasmid pET28a-xyl3A into E.coli DH5α. Figure 2 showed that the plasmid pET28a-xyl3A was successfully transform.

Figure 2. Colony PCR result of pET28a-xyl3A.

M:2000 kd marker; Lane 15-16. pET28a- xyl3A (DH5α);

1.2 Sequencing result of plasmid pET28a-PKC

We send the plasmid pET28a-xyl3A to biological company for sequencing. The returned sequencing comparison results showed that there were no mutations in the ORF region (Figure 3.)

Figure 3. Sequencing result of pET28a-PKC.

Functional assay

2.1 Protein expression of xyl3A

We 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 xyl3A. Next, we used nickel column purification to purify the protein xyl3A we wanted.

Figure 4. SDS-page.

M: maker; S: supernatant; P: precipitant; E: elution

The molecular weights of xyl3A were 84.93KD, referring to the marker in Figure 4, we found the proteins xyl3A in lane S, indicating that proteins 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 purified enzyme solutions (xyl3A) was measured after color reaction with DNS. The activity of the enzyme can be converted by the amount of sugar consumed and the incubation time.

Figure 6. The enzymatic activity of PKC.

The enzyme activity of xyl3A is 0.3U/mL. Figure 5 indicated that protein xyl3A was successfully expressed, and the enzyme activity of xyl3A was active. It indicated that the xyl3A has the function of decomposing xylan.

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
  Illegal BglII site found at 4528
  Illegal BamHI site found at 4905
  Illegal XhoI site found at 5034
  Illegal XhoI site found at 6423
  Illegal XhoI site found at 6585
  
• 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
  Illegal NgoMIV site found at 5022
  Illegal NgoMIV site found at 5745
  Illegal AgeI site found at 6201
  
• 1000
  COMPATIBLE WITH RFC[1000]