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Inflammatory bowel disease (IBD) is a group of diseases that cause long-term inflammation in the colon and small intestine, including ulcerative colitis and Crohn 's disease, which may further induce more serious and fatal diseases, such as colorectal cancer. Reactive oxygen species (ROS) plays an important role in the progression of IBD[1][2]. CAT (catalase) and SOD (super-oxide dismutase) can eliminate reactive oxygen species and alleviate inflammation[3][4]. The inhibition of elastase and protease-3 by Elafin can attenuate several key processes in the inflammatory cascade[5]. Our ultimate goal is to construct a recombinant Escherichia coli Nissle 1917 (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 that, we transformed three genes into EcN to express and construct recombinant EcN that can alleviate inflammatory bowel disease.
The first iteration:
Design: pET-28a(+) is a commonly used vector, and we are familiar with it, so we decided to use it as a vector to verify the feasibility of our design. We inserted CAT and SOD genes into pET-28a(+) plasmid respectively , and verified their expression in E. coli.
Build: Using pET-28a(+) as a vector, pET-28a(+)-CAT and pET-28a (+)-SOD expression vectors were constructed respectively.
Figure 1- The plasmid map of pET-28a(+)-CAT
Figure 2- The plasmid map of pET-28a(+)-SOD
Test:
1.Two plasmids were transformed into E. coli DH5α, respectively.
Figure 3-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
Figure 4- The agarose gel electrophoresis results of plasmid pET-28a(+)-CAT and pET-28a(+)-SOD
2.These two plasmids were transformed into BL21 respectively.
Figure 5- Left image: The BL21 colonies containing pET-28a(+)-CAT on LB solid culture medium ; Right image : The BL21 colonies containing pET-28a(+)-SOD on LB solid culture medium
3.(1) The two plasmids were transformed into BL21 respectively, and the expression of different time periods was verified after IPTG induction.
Figure 6-The two control groups were BL21 induced by IPTG for 12 h and the supernatant of BL21 containing pET-28a(+)-CAT plasmid without induction for 12 h, respectively. The experimental group was the supernatant of the engineering strain BL21 containing pET-28a(+)-CAT plasmid broken at different time periods.
The results showed that we successfully expressed CAT in BL21 strain. After gray scale analysis, we found that the expression of CAT was the highest at 6h.
Figure 7-The control group was the supernatant of BL21 with pET-28a(+)-SOD without induction for 12 h. The experimental group was the supernatant of the engineering strain BL21 containing pET-28a(+)-SOD plasmid broken at different time periods.
The results showed that we successfully expressed SOD in BL21 strain. After gray analysis, we found that the expression of SOD protein was the highest at 6h.
(2) The activity of CAT and SOD was detected:
Figure 8-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 bacterial solution without plasmid introduction and BL21(CAT) bacterial solution without induction of CAT expression. And the experimental groups were the BL21(CAT) bacterial liquid 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)
We expressed active CAT, and the activity was higher at 14 hours.
Pyrogallol solution can be rapidly self-oxidized under alkaline conditions, releasing superoxide anion radicals (O2-), generating intermediate products with special absorption at 325 nm. The addition of SOD enzyme solution can scavenge superoxide anion free radicals, prevent the accumulation of intermediate products, and reduce the absorbance at 325 nm.
Figure 9-The calculation formula of SOD activity measured by Pyrogallol solution method
Figure 10- The results of SOD activity were measured by Pyrogallol solution method, and the inhibition rate of Pyrogallol solution reaction was induced by IPTG at different time.
Figure 11-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.
Conclusion: The active SOD protein was expressed, and the activity of SOD protein was the highest at 8h.
Learn: Considering that plasmid incompatibility occurs when two homologous plasmids are introduced into a bacteria at the same time, we want to co-express the two genes by inserting them on the same vector.
The second iteration
Design: The CAT and SOD genes were simultaneously inserted on the pET-28a(+) vector, so that they can be expressed simultaneously and our engineered probiotics have a stronger role in alleviating IBD.
Build: We added the SOD gene sequence downstream of the CAT gene sequence. (Both CAT and SOD are preceded by RBS sequences.). Considering that this vector needs to play a role in EcN, and the absence of T7 RNA polymerase in EcN makes T7 promoter unable to work, so we changed the promoter of the vector from T7 promoter to T5 promoter. The vector pET-28a(+)-T5-CAT-SOD was obtained.
Figure 12-The plasmid map of pET-28a(+)-T5-CAT-SOD
Test：The plasmid was transformed into E. coli DH5α.
Figure 13- The DH5α colonies containing pET-28a(+)-CAT-SOD on LB solid culture medium
Figure 14- The agarose gel electrophoresis results of plasmid pET-28a(+)-CAT-SOD
Learn：Because we finally plan to connect the three genes together, this iteration does not perform protein expression detection.
The third iteration
Design: In order to enhance the role of our engineered bacteria, we introduced another peptide, Elafin, which can alleviate inflammatory bowel disease. At the same time, in order to avoid the problem of incompatibility of homologous plasmids, we selected pCDFDuet-1 from different sources of pET-28a(+) as the expression vector of Elafin.
Build: We selected the plasmid vector pCDFDuet-1 with different sources from pET-28a(+) as the expression vector of Elafin. The amino acid sequence of Elafin protein was obtained on UniProt, and its nucleic acid sequence was obtained by reverse transcription on NCBI, and codon optimization was performed to finally obtain pCDFDuet-1-Elafin.
Figure 15-The plasmid map of pCDFDuet-1-Elafin
Test：The plasmid pCDFDuet-1-Elafin was transformed into E. coli DH5α.
Figure 16- The DH5α colonies containing pCDFDuet-1-Elafin on LB solid culture medium
Figure 17- The agarose gel electrophoresis results of plasmid pCDFDuet-1-Elafin
We successfully transferred pCDFDduet-1-Elafin into DH5α.
Learn: Because we finally plan to connect the three genes together, this iteration does not perform protein expression detection. Next, we need to verify whether our three genes can play a role in E. coli at the same time.
The fourth iteration
Design: In order to make the three proteins play a role at the same time and enhance the ability of engineering bacteria to treat diseases, we integrated CAT, SOD and Elafin genes into a vector.
Build: 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.
Figure 18-The plasmid map of pET-28a(+)-T5-CAT-SOD-Elafin
Test：
1.The pET-28a(+)-T5-CAT-SOD-Elafin was transformed into DH5α and BL21, respectively.
Figure 19-The DH5α colonies containing pET-28a(+)-CAT-SOD-Elafin on LB solid culture medium
Figure 20-The agarose gel electrophoresis results of plasmid pET-28a(+)-CAT-SOD-Elafin
The pET-28a(+)-T5-CAT-RBS-SOD-Elafin was transformed into BL21 coated LB solid plate by heat shock method.
Figure 21- The BL21 colonies containing pET-28a(+)-CAT-SOD-Elafin on LB solid culture medium
(1) SDS-PAGE was used to detect protein expression.
Figure 22- The control group was the supernatant of BL21 with pET-28a(+)-CAT-SOD-Elafin without induction for 10h and BL21 bacterial solution without plasmid introduction for 10h. The experimental group was the supernatant of the engineering strain BL21 containing pET-28a(+)-CAT-SOD-Elafin plasmid broken at different time periods.
We successfully expressed CAT, SOD and Elafin in BL21, and the expression of 6-8h was more.
(2) The activity of CAT was detected by bubble method.
We determined 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 23-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 bacterial solution without plasmid introduction and BL21(CAT) bacterial solution without induction of CAT expression. The experimental group was induced and cultured for 4, 8, 10, 12, 14 and 18h. (video: 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 results showed that we successfully expressed CAT in BL21 strain, and It was verified that we successfully expressed active CAT.
(3) The activity of SOD was detected 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 24-The calculation method of SOD activity detected by Pyrogallol solution method
Figure 25-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.
We expressed active SOD, and the activity was the highest at 2h.
(4)Determination of Elafin Activity by Egg White Assay
Protease K is a highly efficient serine proteinase. Elafin can act on serine proteinase and inhibit its activity[5], so when we mix a certain amount of active Elafin with protease K, the effect of protease K will be inhibited, thus reducing the efficiency when acting on egg white. After a certain period of reaction, the viscosity of egg white will be lower than that of the control group. The viscosity of the egg white after the reaction can be used to determine whether Elafin is active.
We took 1ml egg white and added it to a 1.5ml centrifuge tube. In the experimental group, 10μL of 20mg / ml protease K and 100μL of pET-28a(+)-CAT-SOD-Elafin BL21 bacterial solution induced for 8, 10 and 14h were fully mixed and reacted at 37 °C for 10 minutes. Then the mixture was added to 1ml egg white and reacted at 55 °C for 25 minutes. In the control group, 10μL 20mg / ml protease K and 100μL double distilled water were fully mixed at 37 °C for 10 minutes, and then the mixture was added to 1ml egg white and reacted at 55 °C for 25 minutes. Blank control 110μL double distilled water was reacted at 37 °C for ten minutes, and then the mixture was added to 1ml egg white and reacted at 55 °C for 25 minutes. Viscosity was observed after the reaction.
It can be clearly seen from the video that the egg white of the 14 h experimental group was still sticky after the reaction, indicating that our bacterial supernatant inhibited the activity of protease K. It is proved that we have expressed the active Elafin protein in BL21.
Learn: Our expression is successful, and the use of our engineered strains should be considered next.
The fifth iteration
Design: Considering the safety of food, we transformed pET-28a(+)-T5-CAT-SOD-Elafin into probiotic EcN.
Build: The pET-28a(+)-T5-CAT-SOD-Elafin was transformed into the probiotic EcN.
Test:
Figure 26- The EcN colonies containing pET-28a(+)-CAT-SOD-Elafin on LB solid culture medium
(1) SDS-PAGE
Figure 27- The control group was the supernatant of EcN with pET-28a(+)-CAT-SOD-Elafin without induction for 8h and EcN bacterial solution without plasmid introduction for 8h. The experimental group was the supernatant of the engineering strain EcN containing pET-28a(+)-CAT-SOD-Elafin plasmid broken at different time periods.
It was proved that we successfully expressed CAT, SOD and Elafin in EcN, and the expression level of 6-8h was higher.
(2) The activity of CAT was detected by bubble method.
We determined 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 E. coli EcN transformed with recombinant plasmid pET-28a(+)-CAT-SOD-Elafin. From left to right, the control groups were LB medium, EcN bacterial solution without plasmid introduction and EcN (CAT) bacterial solution without induction of CAT expression. The experimental group was induced and cultured for 4, 8, 12, 16and 20h. (video: 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.
(3) The activity of SOD was detected 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 29-The calculation method of SOD activity detected by Pyrogallol solution method
Figure 30-The recombinant plasmid pET-28a(+)-CAT-SOD-Elafin was transformed into EcN strain to express SOD activity.
It was proved that SOD was active, and the activity was the highest at 4h.
(4) Determination of Elafin Activity by Egg White Assay
Protease K is a highly efficient serine proteinase. Elafin can act on serine proteinase and inhibit its activity[5], so when we mix a certain amount of active Elafin with protease K, the effect of protease K will be inhibited, thus reducing the efficiency of acting on egg white. After a certain period of reaction, the viscosity of egg white will be lower than that of the control group. The viscosity of the egg white after the reaction can be used to determine whether Elafin is active.
We took 1ml egg white and added it to a 1.5ml centrifuge tube. In the experimental group, 10μL of 20mg/ml protease K and 100μL of pET-28a(+)-CAT-SOD-Elafin EcN bacterial solution induced for 14,18 and 20h were fully mixed and reacted at 37 °C for 10 minutes. Then the mixture was added to 1ml egg white and reacted at 55 °C for 25 minutes. In the control group, 10μL 20mg/ml protease K and 100μL double distilled water were fully mixed at 37 °C for 10 minutes, and then the mixture was added to 1ml egg white and reacted at 55 °C for 25 minutes. Blank control 110μL double distilled water was reacted at 37 °C for ten minutes, and then the mixture was added to 1ml egg white and reacted at 55 °C for 25 minutes. Viscosity was observed after the reaction.
It can be clearly seen from the video 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.
The sixth iteration
Design: In order to enable our engineered bacteria to specifically play a role in the location of inflammatory bowel disease, we plan to change the promoter of the plasmid to HemH promoter.
Build: We obtained a hydrogen peroxide-inducible promoter (Part:BBa K1104202 - parts.igem.org) from the iGEM part. Seamless cloning primers were designed to reinsert the original T5 promoter with a hydrogen peroxide-inducible promoter. The pET-28a(+)-HemH promoter-CAT-RBS-SOD-Elafin was obtained.
Figure 31-Plasmid map of pET-28a(+)-HemH promoter-CAT-SOD-Elafin
Learn: We will finish this work in the near future.
The seventh iteration
Design: In order to prevent the engineering bacteria from leaking to the outside world and polluting the environment, we decided to set a suicide switch for the engineering bacteria. In the design of suicide switch, we decided to choose different suicide switches from previous years in order to innovate.
Build: Suicide switch has two main parts: protein synthesis inhibitor (RelE) and its antitoxin (RelB). The translation of the gene RelE was promoted by conventional RBS, while the translation of the gene RelB was promoted by temperature-sensitive RBS. The expression of RelB is completed only when the temperature reaches the body temperature. When the engineered bacteria leave the human body, the temperature drops, inhibiting the expression of the antitoxin RelB, and the bacteria die due to excessive toxin RelE to prevent pollution.
Figure 32-Action principle diagram of suicide switch
Learn: Due to limited time, we failed to complete this iteration. We will apply this suicide switch to our project in the future.
Quote
[1]Ramos, G. P., & Papadakis, K. A. (2019). Mechanisms of Disease: Inflammatory Bowel Diseases. Mayo Clinic proceedings, 94(1), 155–165.
[2]陈丽霏,张世倡,肖林,吕凯结 & 黎志晴.(2020).炎症性肠病中活性氧及抗氧化的研究进展. 中国当代医药(09),24-27.
[3]McCord JM. Superoxid edismu tase：Anenzymatic function for erythrocuprein（hem ocup rein）[J]. J Biol Chem，1969，244： 6049-6055.
[4]ZHANG Xiaoyan. Research status and application of superoxide dismutase in food[J].Journal of Yangzhou Vocational University, 2002(01):34-37.
[5]Lee S,Oliver W. Therapeutic potential of human elafin.[J]. Biochemical Society transactions,2011,39(5).