• Part 1: Introduction
  • Part 2: Modular Design
  • Secretion module
  • Biosafety module
  • Adhesion module
  • Part 3: Laboratory
                Management
  • Part 4: Chemical or Biological
                Substances Safety
  • References

    • Part 1: Introduction

    Our project aims to construct an effective and sustainable short peptide delivery system with high security in the intestine, which involves multiple safety aspects including modular design, laboratory management, and Chemical or Biological Substances Safety. During the progression of the project, we strictly adhered to the laboratory management regulations under the guidance of professionals and emphasized safety in the project design. Ultimately, we completed all experiments safely. Here are the efforts we have taken this year.

    Part 2:Modular Design

    Secretion module

    We used two protein and two short peptides as parts for the design of the Secretion module. Lpp'OmpA, the protein facilitating target protein's translocation to the outer membrane, lptE, lipoprotein localized in the inner leaflet of the outer membrane, are both from E.coli K-12. E.coli K-12 is a safe and non-pathogenic strain1. AQ and QEP, the short peptides often used as nutritional supplements, are proven to be safe and beneficial to health (AQ and QEP) 2,3.

    Biosafety module

    We used a type II toxin-antitoxin pair, CcdB-CcdA, and two AHL modules, LuxI/LuxR and LasI/LasR, for the design of the Quorum Sensing module. CcdB, a common toxin which inhibit bacterial growth, is harmless to eukaryotic cell, and CcdA which is used in pair with it, could neutralize CcdB's toxicity 4,5. If CcdA is lost during cultivation, then CcdB would inhibit the growth of engineered strain itself to avoid biosafety risks. Two AHL molecules, 3OC6HSL(LuxI) and 3OC12HSL(LasI), are generally harmless to human.

    Adhesion module

    We used three proteins as parts for the design of the Adhesion module. Neae, a surface presentation system containing an export tag, a LysM sequence, a β-barrel, and a Spacer sequence, has been proven to be a safe, effective, and mature E.coli outer membrane presentation system.6 LAP(Listeria Adhesion Protein) is a protein that comes from a non-pathogenic Listeria (L. innocua), which is benign because LAP exhibits virulent attributes only in pathogenic Listeria due to a lack of secretion and surface re-association of LAP on nonpathogenic species of Listeria7. Last, HSP60 is a human protein widely expressed on the surface of the human intestine. 8 In conclusion, all parts we used in the Adhesion module are non-toxic and safe.

    Part 3: Laboratory Management

    Our laboratory has strict management regulations, including but not limited to, the disposal of experimental waste, the proper use of equipment, and the storage of chemicals. Before the experiments began, the team leader and deputy team leaders had explained the basic laboratory rules and equipment usage guidelines to all wet lab members. Additionally, the lab administrator Yan Long conducted a unified training session for the wet lab members. Wet lab members take turns maintaining laboratory cleanliness and safety, ensuring all experimental waste is collected in designated bins and disposed of at the recycling station within the timeframe specified by the College of Life Sciences. The access control system of the laboratory has been updated, allowing only iGEM members to enter. The -80℃ refrigerator storage room and shared instrument platform also have corresponding access control systems.

    Part 4: Chemical or Biological             Substances Safety

    This year, we used Escherichia coli DH5α and BL21 (DE3) as our chassis organisms. According to the check-in form, they are classified as Risk Level 1 organisms, indicating they are harmless. When applying for reagents and consumables, team members must submit a detailed experimental plan specifying the types and quantities of materials. This plan is first reviewed by the team leader and then by team's instructor, Lei Bai. Upon approval a nd receipt of the necessary materials, members should follow the provided protocols or use the materia ls under the guidance of graduate students.

    References

    [1]

    Nicchi, S. et al. Decorating the surface of Escherichia coli with bacterial lipoproteins: a comparative analysis of different display systems. Microb Cell Fact 20, 33 (2021).

    [2]

    Otto, C. et al. Antidiabetic Effects of a Tripeptide That Decreases Abundance of Na+-d-glucose Cotransporter SGLT1 in the Brush-Border Membrane of the Small Intestine. ACS Omega 5, 29127-29139 (2020).

    [3]

    Zhu, J. et al. Metabolic engineering of Escherichia coli for efficient production of L-alanyl-L-glutamine. Microb Cell Fact 19, 129 (2020).

    [4]

    Balagaddé, F. K. et al. A synthetic Escherichia coli predator-prey ecosystem. Mol Syst Biol 4, 187 (2008).

    [5]

    Qiu, J., Zhai, Y., Wei, M., Zheng, C. & Jiao, X. Toxin-antitoxin systems: Classification, biological roles, and applications. Microbiological Research 264, 127159 (2022).

    [6]

    Glass, D. S., & Riedel-Kruse, I. H. (2018). A Synthetic Bacterial Cell-Cell Adhesion Toolbox for Programming Multicellular Morphologies and Patterns. Cell, 174(3), 649-658.e616. doi:https://doi.org/10.1016/j.cell.2018.06.041

    [7]

    Drolia,R.,Amalaradjou,M.A.R.,Ryan,V.,Tenguria,S.,Liu,D.,Bai,X.,Bhunia,A.K.(2020).Receptor-targeted engineered probiotics mitigate lethal Listeria infection. Nature Communications, 11(1), 6344. doi:10.1038/s41467-020-20200-5

    [8]

    Planesse C, Nativel B, Iwema T, Gasque P, Robert-Da Silva C, Viranaïcken W. Recombinant human HSP60 produced in ClearColi™ BL21(DE3) does not activate the NFκB pathway. Cytokine. 2015 May;73(1):190-5. doi: 10.1016/j.cyto.2015.01.028.