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EngineeringSuccess

Introduction

To develop an engineered probiotic bacterium that could degrade trace amounts of gluten for people with celiac disease, we adopted a collaborative research strategy that involved dividing our team into four distinct groups: promoter group, secretion group, transformation group, and enzyme group. Each group, having focused on developing a specific part of our proposed modular system, gained a detailed understanding of their biological systems that has been necessary to achieve our final product. Thus, our engineering success page is divided into four objectives to reflect how each group went through at least one iteration of the Design, Build, Test, and Learn (DBTL) cycle.

  • Objective 1: Characterize several antibiotic resistance gene promoters and test their strength in E. coli.
  • Objective 2: Measure secretion of fluorescent proteins and gluten degrading enzymes to the extracellular milieu using Sec and SRP signal peptides.
  • Objective 3: Transform GI-tract associated gram-positive bacteria with modular plasmids.
  • Objective 4: Designing selective media to assess glutenase activity of various bacteria.

These objectives were not sequentially related, but rather connected through the common goal of combining each objective's research and parts to produce our final modular system as shown in the figure below.

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Figure 1. How each objective contributes to our final modular system and chassis. Created with Biorender.com

Each group used PCR and Golden Gate Assembly (GGA)1 to construct their parts in plasmid backbones for use in various assays such as fluorescence assays, qualitative degradation assays, and transformation assays. Below are figures demonstrating each and every part type we have attempted to use and their proposed assembly into a plasmid backbone.

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Figure 2. Catalogue of biological parts we are using along with SBOL designations. Created with Biorender.com

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Figure 3. Example of our final modular system. Created with Biorender.com

References

  1. Engler C, Kandzia R, Marillonnet S (2008) A One Pot, One Step, Precision Cloning Method with High Throughput Capability. PLoS ONE 3(11): e3647. https://doi.org/10.1371/journal.pone.0003647
  2. Leonard, S. P. et al. Genetic Engineering of Bee Gut Microbiome Bacteria with a Toolkit for Modular Assembly of Broad-Host-Range Plasmids. ACS Synth. Biol. acssynbio.7b00399 (2018). doi:10.1021/acssynbio.7b00399.
  3. LaFleur, T.L., Hossain, A. & Salis, H.M. Automated model-predictive design of synthetic promoters to control transcriptional profiles in bacteria. Nat Commun 13, 5159 (2022). https://doi.org/10.1038/s41467-022-32829-5
  4. Shilling, P. J., Mirzadeh, K., Cumming, A. J., Widesheim, M., Köck, Z., & Daley, D. O. (2020). Improved designs for pet expression plasmids increase protein production yield in escherichia coli. Communications Biology, 3(1). https://doi.org/10.1038/s42003-020-0939-8
  5. Kleiner-Grote, G. R. M., Risse, J. M., & Friehs, K. (2018). Secretion of recombinant proteins from E. coli. Engineering in life sciences, 18(8), 532–550. https://doi.org/10.1002/elsc.201700200
  6. Ravn, P., Arnau, J., Madsen, S. M., Vrang, A., & Israelsen, H. (2003). Optimization of signal peptide SP310 for heterologous protein production in Lactococcus lactis. Microbiology, 149(8), 2193–2201. https://doi.org/10.1099/mic.0.26299-0
  7. Ng, D. T., & Sarkar, C. A. (2013). Engineering signal peptides for enhanced protein secretion from Lactococcus lactis. Applied and environmental microbiology, 79(1), 347–356. https://doi.org/10.1128/AEM.02667-12
  8. Biswas, I., Jha, J. K., & Fromm, N. (2008). Shuttle expression plasmids for genetic studies in Streptococcus mutans. Microbiology (Reading, England), 154(Pt 8), 2275–2282. https://doi.org/10.1099/mic.0.2008/019265-0
  9. Helmerhorst, E. J., & Wei, G. (2014). Experimental Strategy to Discover Microbes with Gluten-degrading Enzyme Activities. Proceedings of SPIE--the International Society for Optical Engineering, 9112, 91120D. https://doi.org/10.1117/12.2058730