Part:BBa_K4762005

T7_Promoter-RBS-CAT-T7_Terminator

CAT expressed in E. coli can break down hydrogen peroxide (H2O2) into water and oxygen, thereby protecting cells from the toxic effects of hydrogen peroxide.

Usage and Biology

It catalyzes hydrogen peroxide (H2O2) to produce water and oxygen from peroxisome oxidase, thereby protecting cells from the toxic effects of hydrogen peroxide. Promote the growth of T cells, B cells, bone marrow leukemia cells, melanoma cells, mast cell tumor cells and normal and transformed fibroblasts.

Experimental Result Diagram

Figure 1. The recombinant plasmid pET-28a(+)-CAT was transformed into DH5α.

Figure 2. The recombinant plasmid pET-28a(+)-CAT was transformed into BL21.

Figure 3. Agarose gel electrophoresis of recombinant plasmid pET-28a(+)-CAT.

We detected the successful expression of CAT in BL21. After gray scale analysis, we found that the expression of CAT was the highest at 6h.

Figure 4. SDS-PAGE of recombinant plasmid pET-28a(+)-CAT transformed into BL21. The two control groups were BL21 induced by IPTG for 12 h and the supernatant of BL21 introduced with pET-28a(+)-CAT without induction for 12 h, respectively. The experimental group was the supernatant of the recombinant strain CAT broken at different time periods.

We successfully expressed CAT protein in BL21 strain, and the activity of CAT protein was high. Click to view the video-NWU-CHINA-A: Detection of CAT activity in E.coli BL21(DE3) transformed by pET-28a(+)-CAT

Figure 5. The diagram of the activity of CAT protein expressed in E. coli BL21 transformed with recombinant plasmid pET-28a(+)-CAT. From left to right, the control group were LB medium, BL21 bacterial solution without plasmid introduction and BL21 bacterial solution without induction of CAT expression. And the experimental group were the BL21 bacterial liquid that was induced and cultured for 2, 6, 10, 14 and 18 h.

Figure 5. Growth curve of BL21 transformed by pET-28a(+)-CAT in OD600.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1260
  Illegal PstI site found at 1093
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1260
  Illegal NheI site found at 1075
  Illegal PstI site found at 1093
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1260
  Illegal BamHI site found at 57
  Illegal BamHI site found at 1510
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1260
  Illegal PstI site found at 1093
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1260
  Illegal PstI site found at 1093
  Illegal AgeI site found at 127
  
• 1000
  COMPATIBLE WITH RFC[1000]