Design

The prevalence of inflammatory bowel disease (IBD) is on the rise worldwide, affecting the healthy lives of millions of people[1]. The current pathogenesis is still unclear. Although some anti-inflammatory drugs and immunosuppressive agents have been used clinically for IBD treatment, long-term use of these drugs will lead to a series of side effects and consume a lot of time and money[2].

Although the pathogenesis of IBD is complex and poorly understood, many studies have pointed out that ROS plays an important role in the progression of IBD[3,4]. Superoxide dismutase (SOD) and catalase (CAT) are two antioxidant enzymes that are ubiquitous in various organisms and are key enzymes in the biological defense system[5,6]. In addition, we found that Elafin protein can bind to neutrophil-derived serine protease elastase and protease-3, weakening several key processes in the inflammatory cascade[7]. Therefore, we chose to transform the plasmid containing SOD, CAT, and Elafin genes into probiotics this year, hoping to weaken the inflammatory response and alleviate IBD by overexpressing these three enzymes.

Construction of recombinant plasmid

We obtained the sequence of pET-28a(+) from SnapGene, the sequence of CAT and SOD from NCBI, and optimized the codon. After the optimized CAT sequence, RBS sequence (Part: BBa B0034 - parts.igem.org) and scar sequence (Help: Standards/Assembly/RFC10 - parts.igem.org) were added, and then the optimized SOD sequence was connected. We added the recognition sequences of Sac I and Hind III restriction enzymes at both ends of the constructed CAT-RBS-scar-SOD sequence, so that they were ligated to the pET-28a(+) vector. At the same time, in order to express the target gene in E.coli Nissle 1917 (EcN), we also used Bg III and Xba I to replace the T7 promoter of the plasmid vector with the T5 promoter.

Figure 1. plasmid pET-28a(+)-T5-CAT-SOD
Figure 2. plasmid profile pET-28a(+)-T5-CAT-SOD

We obtained the pCDFDuet-1 sequence from SnapGene, the amino acid sequence of Elafin from UniProt, the base sequence of Elafin by reverse transcription on NCBI, and the codon optimization of Elafin. We replaced the T7 promoter sequence with the T5 promoter upstream of the optimized Elafin sequence to obtain the pCDFDuet-1-T5-Elafin plasmid.

Figure 3. plasmid pCDFDuet-1-T5-Elafin
Figure 4. plasmid profile pCDFDuet-1-T5-Elafin

We designed the upstream and downstream primers of Elafin and amplified the Elafin sequence with RBS sequence and NotI and HindIII restriction sites from the plasmid pCDFDuet-1-T5-Elafin by Touchdown PCR. The pET-28a(+)-T5-CAT-SOD and the amplified Elafin sequence were digested with Not I and Hind III, and then ligated with T4 DNA ligase to obtain a new plasmid

pET-28a(+)-T5-CAT-SOD-Elafin.

Figure 5. plasmid pET-28a(+)-T5-CAT-SOD-Elafin
Figure 6. plasmid profile pET-28a(+)-T5-CAT-SOD-Elafin

Finding hydrogen peroxide-sensitive promoter

In order to improve the specificity and safety of our engineered probiotics, we plan to replace the T5 promoter of the recombinant plasmid

pET-28a(+)-T5-CAT-SOD-Elafin with a hydrogen peroxide-induced promoter HemH (Part: BBa K1104202 - parts.igem.org), so that our engineered probiotics only overexpress our target genes under the induction of hydrogen peroxide. After our engineered bacteria are taken by the human body, these three proteins will be expressed only after the reactive oxygen species in the intestinal tract are detected, thereby eliminating reactive oxygen species and alleviating intestinal inflammation. When the reactive oxygen species in the intestinal tract return to normal, the engineered bacteria will not express and will not destroy the intestinal environment, thus ensuring the safety of the engineered bacteria in the human body. However, due to time reasons, we have not completed this part of
the experiment. In the future, we will complete this part to make our engineered bacteria more sensitive and secure.

Figure 7. plasmid pET-28a(+)-HemH-CAT-SOD-Elafin
Figure 8. plasmid profile pET-28a(+)-HemH-CAT-SOD-Elafin

Selection of expression strains

Regarding the selection of chassis engineering bacteria, we decided to select Escherichia coli Nissle 1917 (EcN), a Gram-negative probiotic[8]. EcN can colonize in the gastrointestinal tract (GI) and exhibit better growth advantages than other bacteria in the gut, inhibiting pathogenic Enterobacter[9]. More importantly, EcN is the active ingredient of the pharmaceutical preparation Mutaflor. Mutaflor is a microbial drug that is currently approved for use in human drugs in some European countries to treat various diseases and dysfunctions of the intestine[10]. In view of its long-term and safe human drug delivery record, the application range of EcN has been greatly expanded by engineering the strain into a living drug, industrial platform, and drug delivery carrier[11]. Therefore, we used EcN as a chassis cell to overexpress our target genes this year.

Construction of the suicide switch

In order to increase safety and prevent our engineering probiotics from leaking into the environment and causing unnecessary trouble, we intend to construct the 2019 BNU-China team's toxin-antitoxin system(Team:BNU-China/Design - 2019.igem.org). The suicide switch consists of a protein synthesis inhibitor (RelE) and an antitoxin (RelB). Among them, the translation of RelE gene is promoted by conventional RBS, while the translation of RelB gene is 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 decrease in temperature will inhibit the expression of the antitoxin RelB, and the bacteria will die due to excessive toxin RelE, which can prevent the leakage of strains and genes and ensure system efficiency and biosafety.

Figure 9. Toxin-antitoxin system metabolic pathway

Reference

  1. [1] Rui W, Zhaoqi L, Shaojun L, et al. Global, regional and national burden of inflammatory bowel disease in 204 countries and territories from 1990 to 2019: a systematic analysis based on the Global Burden of Disease Study 2019.[J]. BMJ open,2023,13(3).
  2. [2] Luke J, Stephan R, K R W, et al. Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease.[J]. Nature,2012,491(7422).
  3. [3] Ramos, G. P., Papadakis, K. A. Mechanisms of Disease: Inflammatory Bowel Diseases. Mayo Clinic proceedings, 201994(1) ,155–165.
  4. [4] 陈丽霏, 张世倡, 肖林等. 炎症性肠病中活性氧及抗氧化的研究进展[J]. 中国当代医药,2020,27(09):24-27.
  5. [5] 徐靖. 超氧化物歧化酶及其应用的研究进展[J]. 食品工业科技,2013,34(12):387-391.
  6. [6] 于德玲, 王昌留. 过氧化氢酶的研究进展[J]. 中国组织化学与细胞化学杂志,2016,25(02):189-194.
  7. [7] Lee S, Oliver W. Therapeutic potential of human elafin.[J]. Biochemical Society transactions,2011,39(5).
  8. [8] U. Sonnenborn, J. Schulze The non-pathogenic Escherichia coli strain Nissle 1917 – features of a versatile probiotic Microb. Ecol. Health Dis., 21 (3-4) (2009), pp. 122-158
  9. [9] C.S. Alvarez, R. Gimenez, M.A. Canas, R. Vera, N. Diaz-Garrido, J. Badia, L. Baldoma Extracellular vesicles and soluble factors secreted by Escherichia coli Nissle 1917 and ECOR63 protect against enteropathogenic E. coli-induced intestinal epithelial barrier dysfunction BMC Microbiol., 19 (1) (2019), p. 166
  10. [10] U. Sonnenborn, J. Schulze The non-pathogenic Escherichia coli strain Nissle 1917 – features of a versatile probiotic Microb. Ecol. Health Dis., 21 (3-4) (2009), pp. 122-158
  11. [11] Lee S, Oliver W. Therapeutic potential of human elafin.[J]. Biochemical Society transactions,2011,39(5).