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Proof of concept
Overview
We successfully prepared an engineering probiotics. The probiotics can express catalase (CAT), superoxide dismutase (SOD), and Elafin, in which CAT and SOD can eliminate reactive oxygen species and alleviate inflammation. Elafin can attenuate several key processes in the inflammatory cascade by inhibiting elastase and protease-3. Therefore, the expression of these three enzymes in E. coli Nissle 1917 (EcN) can alleviate intestinal inflammation. We have experimentally demonstrated that they are active and can work together.
Plasmid construction
Firstly, we constructed two recombinant plasmids to verify the expression and activity of CAT and SOD, respectively. The CAT and SOD genes were inserted into the pET-28a(+) vector to construct two recombinant plasmids pET-28a(+)-T7-CAT and pET-28a(+)-T7-SOD, and the two plasmids were transformed into E. coli DH5α for amplification.
Figure 1-pET-28a(+)-T7-CAT plasmid map
Figure 2-pET-28a(+)-T7-SOD plasmid map
Then, we constructed the recombinant plasmid pET-28a(+)-T5-CAT-SOD and pCDFDuet TM-1-Elafin, and ligated the Elafin of the latter to the downstream of the SOD gene of the former plasmid to form pET-28a(+)-CAT-SOD-Elafin.
Figure 3-pET-28a(+)-T5-CAT-SOD plasmid map
Figure 4-pCDFDuet™-1-Elafin plasmid map
Figure 5-pET-28a(+)-T5-CAT-SOD-Elafin plasmid map
Methods
Our ultimate goal is to construct an engineered strain EcN that can simultaneously express CAT, SOD, and Elafin. We first studied on E. coli BL21 to confirm whether these genes can be normally expressed in prokaryotes and whether they have activity. After confirming the normal expression and function of this part in E. coli BL21, we transformed the three genes into EcN at the same time to construct a recombinant EcN that can alleviate intestinal inflammation.
Firstly, the two recombinant plasmids pET-28a(+)-T7-CAT and pET-28a(+)-T7-SOD were transformed into E. coli BL21(DE3), respectively, and verified by agarose gel electrophoresis. Then, we added IPTG to induce the expression of the related protein, and then detected its correct expression by SDS-PAGE, and detected its activity by various methods. Finally, in order to make the three proteins play a role at the same time and enhance the ability of engineering bacteria to alleviate intestinal inflammation, we connect CAT, SOD, and Elafin into a vector to form pET-28a(+)-T5-CAT-SOD-Elafin, and then verified the expression and detection activity.
Figure 6-Experimental design diagram
Experimental verification
Construction of plasmid
Construction of pET-28a(+)-T7-CAT and pET-28a(+)-T7-SOD plasmids.
(1) Construction of strain
The pET-28a(+)-T7-CAT and pET-28a(+)-T7-SOD were introduced into DH5α competent cells by chemical transformation (heat shock method).
Figure 7-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.
(2) Agarose gel electrophoresis
In order to verify whether the transformed strain contains the correct pET-28a(+)-T7-CAT and pET-28a(+)-T7-SOD plasmids, we extracted DNA from the transformed BL21 strain and performed agarose gel electrophoresis.
Figure 8-The agarose gel electrophoresis result of the recombinant plasmids pET-28a(+)-T7-CAT and pET-28a(+)-T7-SOD transformed into DH5α. Electrophoresis result showed that pET-28a(+)-T7-CAT and pET-28a(+)-T7-SOD were successfully transferred into E. coli DH5α.
Construction of pET-28a(+)-T5-CAT-SOD-Elafin plasmid
(1) Construction of strain
The recombinant plasmids pET-28a(+)-T5-CAT-SOD and pCDFDuetTM-1-Elafin were transformed into DH5α strain by chemical transformation (heat shock method).
Figure 9-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.
(2) Agarose gel electrophoresis
Figure 10-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.
We extracted the DH5α plasmid pCDFDduet-1-Elafin as a template for PCR amplification, digested with NotI-HF and HindIII-HF restriction endonucleases, and connected the Elafin gene to the downstream of the SOD gene in pET-28a(+)-T5-CAT-SOD by T4 DAN ligase to construct pET-28a(+)-T5-CAT-SOD-Elafin plasmid.
It was introduced into DH5α, and their growth on LB medium was observed. The bacteria containing the recombinant plasmid pET-28a(+)-CAT-SOD-Elafin were successfully grown on the plate.
Figure 11-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 12-The agarose gel electrophoresis result of the recombinant plasmid pET-28a(+)-CAT-SOD-Elafin transformed into DH5α.
Detection of successful expression of engineering bacteria
Expression of recombinant plasmids pET-28a(+)-T7-CAT and pET-28a(+)-T7-SOD.
(1) Strain construction
We verified that the two plasmids were successfully transformed and then transformed into BL21 competent cells by chemical transformation (heat shock method).
Figure 13-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.
(2) Agarose gel electrophoresis
Figure14-The agarose gel electrophoresis results of the recombinant plasmids pET-28a(+)-T7-CAT and pET-28a(+)-T7-SOD transformed into BL21 respectively.
Figure15-The agarose gel electrophoresis result of the recombinant plasmids pET-28a(+)-T7-SOD transformed into BL21. The results of electrophoresis showed that pET-28a(+)-T7-CAT and pET-28a(+) -T7-SOD were successfully transferred into BL21 strain.
(3) SDS-PAGE
We used SDS-PAGE to detect the expression of CAT, SOD, and Elafin.
Figure16-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.
Figure17-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 was the highest at 6h.
Expression of recombinant plasmid pET-28a(+)-CAT-SOD-Elafin
We transformed the recombinant plasmid pET-28a(+)-T5-CAT-SOD-Elafin into BL21, and observed the recombinant plasmid transformed colonies on LB medium.
Figure 18-The BL21 colonies containing pET-28a(+)-CAT-SOD-Elafin on LB solid culture medium.
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 10 h 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 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 21-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 BL21, and the expression of 6-8h was more.
Activity detection
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.
(1) Pre-experiment: 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.
First of all, we directly added 5ml of CAT bacteria liquid for 4, 8, 12, 16, and 18 hours of induction culture to each of the five test tubes at the same time and added 1 ml of 7.5 % hydrogen peroxide to each of them at the same time.
Figure 22 - 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.
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.
Then, we added 5ml LB medium in each of the seven test tubes, 5ml of the supernatant after centrifugation of the 24h CAT bacterial solution without induction culture, 5ml of the supernatant after centrifugation of the 4, 8, 12, 16, 18h CAT bacterial solution with induction culture, and added 1ml volume fraction of 7.5 % hydrogen peroxide to it.
Figure 23 - From left to right, the control groups were the supernatant of LB medium and BL21(CAT) bacterial solution without IPTG after centrifugation. The experimental groups were the supernatant of BL21(CAT) bacterial solution centrifuged at 4h, 8h, 12h, 16h, and 18h cultured with IPTG.
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.
Finally, we added 6 ml LB liquid medium in seven test tubes, centrifuged the supernatant of the bacterial solution without induction culture, centrifuged the suspension of the bacterial solution without induction culture, centrifuged the suspension of the bacterial solution without induction culture, centrifuged the supernatant of the bacterial solution with induction culture, centrifuged the suspension of the bacterial solution with induction culture, centrifuged the suspension of the bacterial solution with induction culture, and added 1 ml 7.5 % hydrogen peroxide to the bacterial solution with induction culture. The induction time of bacterial solution was more than 24 hours.
Figure 24 - From left to right, the control groups were the 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.
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 strain. The preliminary experiment provides a reference for our formal experiment. We chose to add 1 ml 7.5 % hydrogen peroxide to 4 ml bacterial solution.
(2) Formal experiment: 4 ml of LB medium, BL21 bacterial solution without plasmid, BL21 bacterial solution without inducing expression of CAT, and BL21 bacterial solution induced for 2, 6, 10, 14, and 18 hours were added respectively, and 1 ml of 7.5 % hydrogen peroxide was added at the same time. The results are as follows:
Figure 25 - 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 18 hours.
The results showed that we successfully expressed CAT protein in BL21 strain, and the activity of CAT protein was high.
Detecting the activity of SOD by pyrogallol solution method.
Phloroglucinol can rapidly self-oxidize under alkaline conditions, releasing superoxide anion radicals( O2- ) to produce intermediate products with special absorption at 325 nm. The addition of SOD enzyme solution can catalyze the disproportionation reaction of O2- to inhibit the self-oxidation of pyrogallol solution, remove superoxide anion free radicals, prevent the accumulation of intermediate products, and reduce the absorbance at 325 nm. The inhibition rate of pyrogallol solution autoxidation rate can reflect the content of SOD in the sample. The calculation formula is as follows:
Figure 26 - The diagram about the determination of SOD activity after the recombinant plasmid pET-28a(+)-T7-SOD was transformed into BL21 strain. The activity of SOD protein induced by IPTG for 0-24 hours was measured by pyrogallol 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 added 4 ml LB medium, BL21 bacteria solution without plasmid, BL21 bacteria solution without inducing expression of CAT, and BL21 bacteria solution induced for 2, 6, 10, 14, and 18 hours to 8 test tubes respectively, and added 1 ml 7.5 % hydrogen peroxide at the same time. We judge the activity of CAT by observing the generation of bubbles.
Figure 27 - 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 18 hours.
The bubbles generated in the experimental group were much higher than those in the control group, and the CAT activity was higher at 14 hours. It was verified that we successfully expressed active CAT.
Detecting the activity of SOD by pyrogallol solution method.
We reflected the SOD content in the sample by the inhibition rate of pyrogallol 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 28 - The diagram about the determination of SOD activity after the recombinant plasmid pET-28a(+)-T5-CAT-SOD-Elafin was transformed into BL21 strain. The plasmid pET-28a(+)-T7-SOD measured by pyrogallol solution 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. (Raw data)
The results showed that our recombinant strain could express active SOD protein, and the activity was high in each time period.
Determination of Elafin Activity by Egg White Assay.
According to the principle that protease can hydrolyze the protein and change the viscosity, we designed the reaction experiment of egg white and protease K, and added the BL21 bacterial solution that we induced and cultured for 14 hours to react, and observed the viscosity after the reaction. The specific design is as follows:
First, 1 ml egg white was added to a 1.5 ml centrifuge tube. In the experimental group, 10 μL of 20 mg/ml protease K and 100 μL of pET-28a(+)-CAT-SOD-Elafin EcN bacterial solution induced for 14 hours were fully mixed and reacted at 37 °C for five minutes. Then the mixture was added to 1 ml egg white and reacted at 55 °C for 20 minutes. In the control group, 10 μL 20 mg/ml protease K and 100 μL double distilled water were fully mixed at 37 °C for five minutes, and then the mixture was added to 1 ml egg white and reacted at 55 °C for 20 minutes. Blank control: 110 μL double distilled water was reacted at 37 °C for five minutes, and then the mixture was added to 1 ml egg white and reacted at 55 °C for 20 minutes. Finally, the viscosity was observed at the end of the reaction.
The experimental results showed that the viscosity of BL21 bacterial solution at 14 hours 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 directly added LB medium, the supernatant of EcN bacterial solution without plasmid, the supernatant of EcN bacterial solution without inducing expression of CAT, and 4 ml of CAT bacterial solution induced for 4, 8, 10, 12, 14, and 18 hours respectively in nine test tubes, and added 1 ml of 7.5 % hydrogen peroxide at the same time. We judged the activity of CAT by adding hydrogen peroxide at the same time to observe bubble production.
Figure 29 - 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, 20 hours.
The bubbles generated in the experimental group were much higher than those in the control group, which verified that we successfully expressed active CAT.
Detecting the activity of SOD by pyrogallol solution method.
We reflected the SOD content in the sample by the inhibition rate of pyrogallol 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 30 - The diagram about the determination of SOD activity after the recombinant plasmid pET-28a(+)-T5-CAT-SOD-Elafin was transformed into EcN strain. The recombinant plasmid pET-28a(+)-CAT-SOD-Elafin was transformed into EcN strain 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 strain, and the activity was high in each time period.
Determination of Elafin Activity by Egg White Assay.
We took 1 ml egg white and added it to a 1.5 ml centrifuge tube. In the experimental group, 10 μL of 20 mg/ml proteinase K was fully mixed with 100 μL of pET-28a(+)-CAT-SOD-Elafin EcN bacterial solution induced for 20 hours, and reacted at 37 °C for 5 min. Then the mixture was added to 1 ml egg white and reacted at 55 °C for 20 minutes. In the control group, 10 μL 20 mg/ml proteinase K and 100 μL double distilled water were fully mixed at 37 °C for five minutes, and then the mixture was added to 1 ml egg white and reacted at 55 °C for 20 minutes. Blank control: 110 μL double distilled water was reacted at 37 °C for 5 minutes, and then the mixture was added to 1 ml egg white and reacted at 55 °C for 20 minutes. We observe the viscosity after the re-reaction. The video of the experimental results is as follows.
The experimental results showed that the viscosity of EcN bacterial solution at 20 hours was higher and the activity of Elafin was higher. It was proved that we expressed the active Elafin protein in EcN.
Realization
In order to popularize public awareness of intestinal health, we have jointly developed the Intestinal Alliance Handbook with other universities, surveyed community residents and students, and guided the public's attention to intestinal health. At the same time, we promoted the knowledge of Love in Prolonging Inflammatory Bowel Disease Foundation (CCCF) and inflammatory bowel disease on the 99th Public Welfare Day, and also helped more iGEM intestinal field teams to make full use of our value. (If you would like to learn more about our user manual, please visit the Gut Alliance page).