The co-expression of these three enzymes further improves the therapeutic ability of IBD.
Figure 1. The function diagram of CAT-SOD-Elafin.
Figure 2. CAT-SOD-Elafin gene circuit.
Experimental Results
Three proteins were expressed in BL21
Figure 3. The recombinant plasmid pET-28a(+)-T5-CAT-SOD-Elafin was transformed into DH5.
Figure 4. Agarose gel electrophoresis of recombinant plasmid pET-28a(+)-T5-CAT-SOD-Elafin.
Figure 5. The recombinant plasmid pET-28a(+)-T5-CAT-SOD-Elafin was transformed into BL21.
We successfully expressed CAT, SOD and Elafin in BL21, and the expression of 6-8h was more.
Figure 6. The experimental group was to induce the expression of recombinant strains in BL21 at different time periods. The two control groups were BL21 which was induced for 10 h and BL21 which was introduced into pET-28a(+)-T5-CAT-SOD-Elafin without induction for 10 h.
We successfully expressed CAT protein in BL21 strain, and the activity of CAT protein was high
Figure 7. The diagram of the activity of CAT protein expressed in E. coli BL21 transformed with recombinant plasmid pET-28a(+)-CAT-SOD-Elafin. From left to right, the control group was LB medium, BL21 bacterial solution without plasmid introduction and BL21 bacterial solution without induced expression of CAT. The experimental group was induced to culture BL21 bacterial solution for 4,8,10,12,14 and 18 h.
We expressed active SOD in BL21, and the activity was the highest at 2h.
Figure 8. The plasmid pET-28a(+)-T7-SOD measured by pyrogallol method was transformed into BL21 strain and IPTG was added to induce the activity of SOD protein for 0-20 hours. The abscissa represents the time we induce expression, and the ordinate represents the inhibition rate.
Figure 9. The diagram of the activity of Elafin expressed in E. coli BL21 transformed with recombinant plasmid pET-28a(+)-CAT-SOD-Elafin. From left to right, the control groups were no Protease K, Protease K. The experimental group was induced for 14 h after the bacteria liquid broken supernatant and Protease K（Details refer to video link: https://video.igem.org/w/3qspwMZ2Xb8hsPijepGu7n）
Figure 10. Growth curve of BL21 transformed by pET-28a(+)-T5-CAT-SOD-Elafin in OD600.
Three proteins were expressed in EcN
Figure 11. The recombinant plasmid pET-28a(+)-T5-CAT-SOD-Elafin was transformed into EcN.
We successfully expressed CAT, SOD and Elafin in EcN, and the expression level of 6-8h was higher.
We successfully expressed CAT protein in EcN, and the activity of CAT protein was high
Figure 12. The diagram of the activity of CAT protein expressed in EcN transformed with recombinant plasmid pET-28a(+)-CAT-SOD-Elafin. From left to right, the control group was LB medium, EcN bacterial solution without plasmid introduction and EcN bacterial solution without induced expression of CAT. The experimental group was induced to culture EcN bacteria solution for 4, 8, 12, 16, 18h.
We expressed active SOD in EcN ,and the activity was the highest at 4h.
Figure 13. The recombinant plasmid pET-28a(+)-CAT-SOD-Elafin was transformed into EcN strain to express SOD activity.
It can be clearly seen from the video(https://video.igem.org/w/aEYiF2mF6qLZypKPDM7tRY)that the egg white of the 20 h experimental group was still sticky after the reaction, indicating that our bacterial supernatant inhibited the activity of protease K. It was proved that we expressed the active Elafin protein in EcN.
Figure 14. The diagram of the activity of Elafin expressed in E. coli EcN transformed with recombinant plasmid pET-28a(+)-CAT-SOD-Elafin. From left to right, the control groups were no Protease K, Protease K. The experimental group was induced for 20 h after the bacteria liquid broken supernatant and Protease K.
Figure 15. Growth curve of EcN transformed by pET-28a(+)-CAT-SOD-Elafin in OD600.
Sequence and Features
Assembly Compatibility:
10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 1286 Illegal XbaI site found at 2337 Illegal PstI site found at 1119
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 1286 Illegal NheI site found at 1101 Illegal NheI site found at 2634 Illegal PstI site found at 1119
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 1286 Illegal BamHI site found at 83 Illegal BamHI site found at 1536
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 1286 Illegal XbaI site found at 2337 Illegal PstI site found at 1119
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 1286 Illegal XbaI site found at 2337 Illegal PstI site found at 1119 Illegal AgeI site found at 153 Illegal AgeI site found at 1943
