loading..
Result
Plasmid Construction
Firstly, we constructed two plasmids pET-28a(+)-T7-CAT and pET-28a(+)-T7-SOD.
Figure 1-The plasmid profiles of pET-28a(+)-T7-CAT and pET-28a(+)-T7-SOD
After they were transferred to DH5α, the colony growth on LB medium was observed. The bacteria containing recombinant plasmids pET-28a(+)-T7-CAT and pET-28a(+)-T7-SOD were successfully grown in the plate.
Figure 2-Left image: The DH5α colonies containing pET-28a(+)-CAT on LB solid culture medium ; Right image: The DH5α colonies containing pET-28a(+)-SOD on LB solid culture medium
We performed agarose gel electrophoresis to verify the successful introduction of the plasmid.
Figure 3-The agarose gel electrophoresis result of the recombinant plasmids pET-28a(+)-T7-CAT and pET-28a(+)-T7-SOD transformed into DH5α.
Electrophoresis results showed that pET-28a(+)-T7-CAT and pET-28a(+)-T7-SOD were successfully transferred into E.coli DH5α.
Then, we constructed two plasmids, pET-28a(+)-T5-CAT-SOD and pCDFDuet TM-1-Elafin.
Figure 4-The plasmid profiles of pET-28a(+)-T5-CAT-SOD and pCDFDuet TM-1-Elafin.
After we transferred them to DH5α respectively, their growth on LB medium was observed. The bacteria containing recombinant plasmids pET-28a(+)-CAT-SOD and pCDFDuet-1-Elafin were successfully grown in the plate.
Figure 5-Left image: The DH5α colonies containing pET-28a(+)-CAT-SOD on LB solid culture medium ; Right image: The DH5α colonies containing pCDFDuet-1-Elafin on LB solid culture medium.
We performed agarose gel electrophoresis to verify the successful introduction of the plasmid.
Figure 6-The agarose gel electrophoresis results of the recombinant plasmids pET-28a(+)-CAT-SOD and pCDFDuetTM-1-Elafin transformed into DH5α.
Electrophoresis results showed that pET-28a(+)-T5-CAT-SOD and pCDFDuet™-1-Elafin were successfully transferred into E.coli DH5α.
Finally, in order to make the three proteins play a role at the same time and enhance the ability of the engineering bacteria to alleviate intestinal inflammation, we cut the Elafin gene on the plasmid pCDFDuet™-1-Elafin, and connected it to the downstream of the gene SOD in the plasmid pET-28a(+)-CAT-SOD. The three genes were integrated into a vector to form pET-28a(+)-T5-CAT-SOD-Elafin.
Figure 7-The plasmid profile of pET-28a(+)-T5-CAT-SOD-Elafin.
We transferred it into DH5α, and then its growth on LB medium was observed. The bacteria containing the recombinant plasmid pET-28a(+)-CAT-SOD-Elafin were successfully grown in the plate.
Figure 8-The DH5α colonies containing pET-28a(+)-CAT-SOD-Elafin on LB solid culture medium.
We performed agarose gel electrophoresis to verify the successful introduction of the plasmid.
Figure 9-The agarose gel electrophoresis result of the recombinant plasmid pET-28a(+)-CAT-SOD-Elafin transformed into DH5α.
We see the correct size of the stripe. The electrophoresis result showed that we successfully transformed pET-28a(+)-CAT-SOD-Elafin into DH5α.
Expression of plasmid
Firstly, we separately transformed the recombinant plasmids pET-28a(+)-T7-CAT and pET-28a(+)-T7-SOD into E.coli BL21(DE3), and the transformed colonies on LB medium were observed.
Figure 10-The left image: the BL21 coated LB solid plate transformed by pET-28a(+)-CAT was coated with LB solid plate by heat shock method ; right picture: The BL21 transformed by pET-28a(+)-SOD was coated on LB solid plate by heat shock method.
We performed agarose gel electrophoresis to verify that the plasmids were successfully introduced.
Figure 11-The agarose gel electrophoresis results of the recombinant plasmids pET-28a(+)-T7-CAT and pET-28a(+)-T7-SOD transformed into BL21 respectively.
The results of electrophoresis showed that pET-28a(+)-T7-CAT and pET-28a(+)-T7-SOD were successfully transferred into BL21 .
We measured the growth curves of three strains. As we have seen, the growth curves of all these bacteria experienced lag phase, logarithmic phase, stationary phase and decline phase.
Figure 12-The growth curves of BL21 transformed with recombinant plasmids pET-28a(+)-T7-CAT and pET-28a(+)-T7-SOD and BL21 without plasmid in OD600(the
dotted line in the figure is the trend line).(Raw data)
We exogenously added IPTG to induce the expression of related proteins, and broken the cells to take the supernatant. We observed the correct size of the band by SDS-PAGE.
Figure13-The SDS-PAGE map of BL21 where the recombinant plasmid pET-28a(+)-CAT transferred.The two control groups were BL21 induced by IPTG for 12h and the supernatant of BL21 introduced with pET-28a(+)-CAT without induction for 12h, respectively. The experimental groups were the supernatant of the recombinant strain CAT broken at different time periods.
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 14-The SDS-PAGE map of BL21 where the recombinant plasmid pET-28a(+)-SOD transferred. The control group was the supernatant of BL21 with pET-28a(+)-SOD without induction for 12h, and the experimental groups were the
supernatant of recombinant strain SOD in BL21 induced at different time periods.
We detected the successful expression of SOD in BL21. After gray analysis, we found that the expression of SOD protein was the highest at 6h.
Then, we transformed the recombinant plasmid pET-28a(+)-T5-CAT-SOD into EcN, and observed the recombinant plasmid transformed colonies on LB medium.
Figure 15-The EcN colonies containing pET-28a(+)-CAT-SOD on LB solid culture medium
We performed agarose gel electrophoresis to verify its successful transfer.
Figure 16-The agarose gel electrophoresis result of the recombinant plasmids pET-28a(+)-T5-CAT-SOD transformed into EcN .
Secondly, we ligated the Elafin gene sequence to the downstream of the vector pET-28a(+)-T5-CAT-SOD to construct pET-28a(+)-T5-CAT-SOD-Elafin to achieve their co-expression. We transformed the recombinant plasmid pET-28a(+)-T5-CAT-SOD-Elafin into BL21, and observed the recombinant plasmid transformed colonies on LB medium.
Figure 17-The BL21 colonies containing pET-28a(+)-CAT-SOD-Elafin on LB solid culture medium
We measured the growth curve of the strain. As we can see, the growth curves of all these bacteria experienced lag phase, logarithmic phase, stationary phase and decline phase.
Figure 18-The growth curves of BL21 transformed with recombinant plasmids pET-28a(+)-T5-CAT-SOD-Elafin(induced expression and not induced expression)and BL21 without plasmid in OD600(the dotted line in the figure is the trend line).
(Raw data)
We exogenously added IPTG to induce the expression of related proteins, then broke the cells and took the supernatant for SDS-PAGE.
Figure 19-The SDS-PAGE map of BL21 where the recombinant plasmid pET-28a(+)-T5-CAT-SOD-Elafin transferred. The two control groups were BL21 which was induced for 10h and BL21 which was introduced into pET-28a(+)-T5-CAT-SOD-Elafin without induction for 10h.The experimental groups were to induce the expression of recombinant strains in BL21 at different time periods.
We observed the correct bands of CAT, SOD and Elafin, which proved the successful expression of the three. And the expression of CAT in the bacterial solution of 6-8h was more.
Finally, we transformed the recombinant plasmid pET-28a(+)-T5-CAT-SOD-Elafin into EcN, and the transformed colonies on LB medium were observed.
Figure 20-The EcN colonies containing pET-28a(+)-CAT-SOD-Elafin on LB solid culture medium
We measured the growth curve of the strain. As we can see, the growth curves of all these bacteria experienced lag phase, logarithmic phase, stationary phase and decline phase.
Figure 21-The growth curves of EcN transformed with recombinant plasmid pET-28a(+)-T5-CAT-SOD-Elafin(Induced expression and not induced expression)and
EcN without plasmid in OD600(the dotted line in the figure is the trend line)
We exogenously added IPTG to induce the expression of related proteins, then broke the cells and took the supernatant. SDS-PAGE showed a band of the correct size.
Figure 22-The SDS-PAGE map of EcN where the recombinant plasmid pET-28a(+)-T5-CAT-SOD-Elafin transferred. The two control groups were EcN introduced into pET-28a(+)-T5-CAT-SOD-Elafin with EcN induced for 8h and without induction for 8h.The experimental groups were to induce the expression of recombinant strains in EcN at different time periods.
We successfully expressed CAT, SOD and Elafin in EcN, and the expression of 6-8h was more.
Activity determination
1.The recombinant plasmid pET-28a(+)-T7-CAT and pET-28a(+)-T7-SOD were transformed into BL21 strain to express CAT and SOD activity.
Detecting the activity of CAT in BL21 by bubble method.
Firstly, we conducted a preliminary experiment to explore the relationship between the amount of bacteria and hydrogen peroxide and the amount of bubbles produced, so as to determine the amount of bacteria and hydrogen peroxide added in our formal experiment.
(a)
(b) (c)
Figure 23-The diagram of the activity of CAT protein expressed in E.coli BL21(CAT) transformed with recombinant plasmid pET-28a(+)-CAT.(a)From left to right, 5ml bacterial solution was cultured with IPTG for 4h, 8h, 12h, 16h and 18h, and 1ml volume fraction of 7.5 % hydrogen peroxide was added to it at the same time.(b)From left to right, the control groups : the supernatant of LB medium and BL21(CAT) bacterial solution without IPTG after centrifugation ; the experimental groups : The supernatant of BL21(CAT) bacterial solution centrifuged at 4h, 8h, 12h, 16h and 18h was cultured with IPTG. Each group of 5ml was added with 1ml volume fraction of 7.5 % hydrogen peroxide at the same time.(c)From left to right, the control groups : LB liquid medium, supernatant after centrifugation of BL21(CAT) cultured without IPTG, sediment resuspension after centrifugation of BL21(CAT) cultured without IPTG, BL21(CAT) cultured without IPTG, supernatant after centrifugation of BL21(CAT) cultured with IPTG, sediment resuspension after centrifugation of BL21(CAT) cultured with IPTG, and BL21(CAT) cultured with IPTG ;
each group of 6ml, while adding 1ml 7.5 % hydrogen peroxide.
Analysis :(a) A large number of bubbles were generated after the bacterial solution was directly added with hydrogen peroxide. It shows that the bacterial solution containing CAT or hydrogen peroxide destroys the cells to release CAT. We expressed active CAT. The supernatant after centrifugation of the bacterial solution produced bubbles after hydrogen peroxide, but the amount was very small.(b)The bubbles generated by directly adding hydrogen peroxide to the bacterial solution were much more than those generated by adding hydrogen peroxide to the supernatant after centrifugation, indicating that 7.5 % hydrogen peroxide could destroy BL21 cells.(c)The amount of oxygen produced by adding hydrogen peroxide after the centrifugal precipitation of the bacterial solution was re-suspended was less than the amount of oxygen produced by adding hydrogen peroxide directly to the bacterial solution, but it was greater than the amount of oxygen produced by adding hydrogen peroxide to the supernatant of the bacterial solution by centrifugation. It can be seen from the results that BL21 without induction culture can produce CAT itself, but the amount is relatively small. It shows that CAT protein is mainly present in the cell and rarely secreted outside the cell.
The results of preliminary experiments showed that we successfully expressed CAT protein in BL21. The preliminary experiment provides a reference for our formal experiment. We chose to add 1ml 7.5 % hydrogen peroxide to 4ml bacterial solution.
Figure 24-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 groups were LB medium, BL21(CAT) bacterial solution without plasmid introduction and BL21(CAT) bacterial solution without induction of CAT expression. And the experimental groups were the BL21(CAT) that was induced and cultured for 2, 6, 10, 14 and 18h. (Video: NWU-CHINA-A: Detection of CAT activity in E.coli BL21(DE3)transformed by pET-28a(+)-CAT(2023)[English] - iGEM Video Universe)
The results showed that we successfully expressed CAT protein in BL21, and the activity of CAT protein was higher.
Detecting the activity of SOD by pyrogallol solution solution method.
We reflected the SOD content in the sample by the inhibition rate of pyrogallol solution solution autoxidation rate. It was verified that we successfully expressed active SOD. The calculation formula and the change of inhibition rate with the induced expression time are as follows:
Figure 25-The diagram about the determination of SOD activity after the recombinant plasmid pET-28a(+)-T7-SOD was transformed into BL21(SOD). The activity of SOD protein induced by IPTG for 0-24 hours was measured by pyrogallol solution solution method. The abscissa represents the time we induce expression,
and the ordinate represents the inhibition rate. (Raw data)
The results showed that our recombinant strain could express active SOD protein, and the activity was the best at 8 hours.
2.The recombinant plasmid pET-28a(+)-T5-CAT-SOD-Elafin was transformed into BL21 to express CAT, SOD and Elafin activity determination.
Detecting the activity of CAT in BL21 by bubble method.
We judged the activity of CAT by adding hydrogen peroxide at the same time to observe bubble production. It was verified that we successfully expressed active CAT.
Figure 26-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 groups were LB medium, BL21(CAT) bacterial solution without plasmid introduction and BL21(CAT) bacterial solution without induced expression of CAT. The experimental groups were induced to culture BL21(CAT) bacterial solution for 4,8,10,12,14 and 18h.(NWU-CHINA-A: Detection of CAT activity in E. coli BL21(DE3)transformed by pET-28a(+)-CAT-SOD-Elafin.(2023)[English] - iGEM Video Universe)
The bubbles generated in the experimental groups were much higher than those in the control groups, and the CAT activity was high. It was verified that we successfully expressed active CAT.
Detecting the activity of SOD by pyrogallol solution solution method.
We reflected the SOD content in the sample by the inhibition rate of pyrogallol solution solution autoxidation rate. It was verified that we successfully expressed active SOD. The calculation formula and the change of inhibition rate with the induced expression time are as follows :
Figure 27-The diagram about the determination of SOD activity after the recombinant plasmid pET-28a(+)-T5-CAT-SOD-Elafin was transformed into BL21. The plasmid pET-28a(+)-T7-SOD measured by pyrogallol solution solution method was transformed into BL21(SOD) 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.(Raw data)
The results showed that our recombinant strain could express active SOD protein, and the activity was highest at 2 hours.
Determination of Elafin Activity by Egg White Assay
According to the principle that protease can hydrolyze protein, and Elafin can inhibit the activity of protease, we designed the reaction experiment of egg white and protease K. After the reaction, the viscosity of the solution was added to the BL21 bacterial solution that we induced and cultured for 8, 10 and 14h, respectively, to react, and observed the viscosity after the reaction.
The experimental results showed that the viscosity of BL21 bacterial solution at 14h was higher and the Elafin activity was higher. It is proved that we have expressed the active Elafin protein in BL21.
3.The recombinant plasmid pET-28a(+)-T5-CAT-SOD-Elafin was transformed into EcN to express CAT, SOD and Elafin activity determination.
Detecting the activity of CAT in BL21 by bubble method.
We judged the activity of CAT by adding hydrogen peroxide at the same time to observe bubble production. It was verified that we successfully expressed active CAT.
Figure 28-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 groups were LB medium, EcN(CAT) bacterial solution without plasmid introduction and EcN(CAT) bacterial solution without induced expression of CAT. The experimental groups were induced to culture EcN(CAT) bacteria solution for 4,8,12,16,20h.(NWU-CHINA-A: Detection of CAT activity in EcN transformed by pET-28a(+)-CAT-SOD-Elafin.(2023)[English] - iGEM Video Universe)
The bubbles generated in the experimental groups were much higher than those in the control groups, which verified that we successfully expressed active CAT.
Detecting the activity of SOD by pyrogallol solution solution method.
We reflected the SOD content in the sample by the inhibition rate of pyrogallol solution solution autoxidation rate. It was verified that we successfully expressed active SOD. The calculation formula and the change of inhibition rate with the induced expression time are as follows :
Figure 29-The diagram about the determination of SOD activity after the recombinant plasmid pET-28a(+)-T5-CAT-SOD-Elafin was transformed into EcN. The recombinant plasmid pET-28a(+)-CAT-SOD-Elafin was transformed into EcN to express SOD activity. The abscissa represents the time we induce expression, and the
ordinate represents the inhibition rate.(Raw data)
The results showed that we successfully expressed the active SOD in the EcN, and the activity was highest at 4 hours.
Determination of Elafin Activity by Egg White Assay
According to the principle that protease can hydrolyze protein, and Elafin can inhibit the activity of protease, we designed the reaction experiment of egg white and protease K. After the reaction, the viscosity of the solution was added to the BL21 bacterial solution that we induced and cultured for 14,18 and 20 h, respectively, to react, and observed the viscosity after the reaction.
The experimental results showed that the viscosity of EcN bacterial solution at 20 h was higher and the activity of Elafin was higher. It was proved that we expressed the active Elafin protein in EcN.