Hero background

Engineering

Here we go through the different iterations of the iGEM engineering cycle.

Engineering Cycles

The goal of our project is to engineer the commensal gut bacterium Pseudomonas alcaligenes to autonomously produce and secrete therapeutic molecules inside the zebrafish gut. We use Danio rerio (zebrafish) larvae as our model organism, whose optical transparency and conserved gut physiology enable live imaging of colonization of our engineered bacteria. We first validate our delivery strategy using fluorescent proteins, before advancing to the microbial synthesis of L-DOPA, the main treatment for Parkinson's disease. We aim to validate our production of L-DOPA in vitro, and with HPLC, before introducing the bacteria into the zebrafish. Moreover, considerations of biosafety are also implemented in the wet lab activities of our project by the design of killswitches. We also test the colonization of our bacteria in a human intestinal transwell model, to bridge the gap between zebrafish proof of concept and a potential application in humans.

Select a section below to view the engineering design cycles for that aspect of our project. Each section contains multiple experiments that follow the Design → Build → Test → Learn cycle.

Cloning

L-DOPA Production & Quantification

Zebrafish

Transwell

  1. Bird, J. E., Marles-Wright, J., & Giachino, A. (2022). A User's Guide to Golden Gate Cloning Methods and Standards. ACS Synthetic Biology, 11(11), 3551–3563. https://doi.org/10.1021/acssynbio.2c00355
  2. Valenzuela-Ortega, M., & French, C. (2021). Joint universal modular plasmids (JUMP): a flexible vector platform for synthetic biology. Synthetic Biology, 6(1). https://doi.org/10.1093/synbio/ysab003
  3. iGEM Registry. (2003). Part:BBa_J23119 – Anderson constitutive promoter. iGEM Registry of Standard Biological Parts. https://parts.igem.org/Part:BBa_J23119
  4. iGEM Registry. (2021a). Part:BBa_K3814004 – fuGFP reporter. iGEM Registry of Standard Biological Parts. https://parts.igem.org/Part:BBa_K3814004
  5. Einhauer, A., & Jungbauer, A. (2001). The FLAG™ peptide, a versatile fusion tag for the purification of recombinant proteins. Journal of Biochemical and Biophysical Methods, 49(1–3), 455–465. https://doi.org/10.1016/S0165-022X(01)00213-5
  6. Thomas, G. (2002). Furin at the cutting edge: From protein traffic to embryogenesis and disease. Nature Reviews Molecular Cell Biology, 3(10), 753–766. https://doi.org/10.1038/nrm934
  7. Saleh, A. F., Arzumanov, A. A., Abes, R., Owen, D., Lebleu, B., & Gait, M. J. (2010). Cell-penetrating peptide conjugates of steric block oligonucleotides for splice correction: Tat-LK15 conjugates are highly effective. Journal of Controlled Release, 143(1), 10–17. https://doi.org/10.1016/j.jconrel.2009.12.021
  8. Alkotaji, M., Saleh, A. F., Pluen, A., Hallden, G., & El-Andaloussi, S. (2014). Tat-LK15: A novel cell-penetrating peptide for the efficient delivery of therapeutic molecules into mammalian cells. Biochimica et Biophysica Acta (BBA) - Biomembranes, 1838(11), 3118–3127. https://doi.org/10.1016/j.bbamem.2014.07.010
  9. iGEM Registry. (2019). Part:BBa_K3096016 – Tat-LK15 fusion peptide. iGEM Registry of Standard Biological Parts. https://parts.igem.org/Part:BBa_K3096016
  10. iGEM Registry. (2011). Part:BBa_K554002 – HlyA secretion tag. iGEM Registry of Standard Biological Parts. https://parts.igem.org/Part:BBa_K554002
  11. UniProt. (2023). P31011 – Tyrosine phenol-lyase (TPL). UniProt Knowledgebase. https://www.uniprot.org/uniprotkb/P31011
  12. UniProt. (2023). X5DSL3 – mCherry fluorescent protein. UniProt Knowledgebase. https://www.uniprot.org/uniprotkb/X5DSL3
  13. iGEM Registry. (2019). Part:BBa_K3257021 – λt0 terminator. iGEM Registry of Standard Biological Parts. https://parts.igem.org/Part:BBa_K3257021
  14. Nigam, A., Ziv, T., Oron-Gottesman, A., & Engelberg-Kulka, H. (2019). Stress-Induced MazF-Mediated Proteins in Escherichia coli. mBio, 10(2). https://doi.org/10.1128/mbio.00340-19
  15. Széliová, D., Krahulec, J., Šafránek, M., Lišková, V., & Turňa, J. (2016). Modulation of heterologous expression from P BAD promoter in Escherichia coli production strains. Journal Of Biotechnology, 236, 1–9. https://doi.org/10.1016/j.jbiotec.2016.08.004
  16. Antunes, L. C. M., Ferreira, R. B. R., Lostroh, C. P., & Greenberg, E. P. (2007). A Mutational Analysis Defines Vibrio fischeri LuxR Binding Sites. Journal Of Bacteriology, 190(13), 4392–4397. https://doi.org/10.1128/jb.01443-07
  17. iGEM Team Tsinghua. (2018). Tsinghua iGEM 2018: Neon Coli! Necessary expression only. https://2018.igem.org/Team:Tsinghua
  18. iGEM Team HZAU-China. (2019). HZAU-China iGEM 2019: Smell once more. https://2019.igem.org/Team:HZAU-China/Description
  19. Soares, J. A., & Ahmer, B. M. (2011). Detection of acyl-homoserine lactones by Escherichia and Salmonella. Current Opinion in Microbiology, 14(2), 188–193. https://doi.org/10.1016/j.mib.2011.01.006
  20. Stirling, F., Bitzan, L., O'Keefe, S., Redfield, E., Oliver, J.W.K., Way, J., Silver, P.A. (2017). Rational Design of Evolutionarily Stable Microbial Kill Switches. Molecular Cell, 68(4), 686-697. https://doi.org/10.1016/j.molcel.2017.10.033
  21. McMackin, E. A. W., Corley, J. M., Karash, S., Marden, J., Wolfgang, M. C., & Yahr, T. L. (2021). Cautionary Notes on the Use of Arabinose- and Rhamnose-Inducible Expression Vectors in Pseudomonas aeruginosa. Journal of Bacteriology, 203(16). https://doi.org/10.1128/jb.00224-21
  22. Román, R., Lončar, N., Casablancas, A., Fraaije, M. W., & Gonzalez, G. (2020). High-level production of industrially relevant oxidases by a two-stage fed-batch approach: Overcoming catabolite repression in arabinose-inducible Escherichia coli systems. Applied Microbiology and Biotechnology, 104(12), 5337–5345. https://doi.org/10.1007/s00253-020-10622-y
  23. iGEM Registry of Standard Biological Parts. Part:BBa_I0500. Accessed October 6th 2025. https://parts.igem.org/Part:BBa_I0500
  24. Guzman, L. M., Belin, D., Carson, M. J., & Beckwith, J. (1995). Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. Journal of Bacteriology, 177(14), 4121–4130. https://doi.org/10.1128/jb.177.14.4121-4130.1995
  25. Xu, D.-Y., Chen, J.-Y., & Yang, Z. (2012). Use of cross-linked tyrosinase aggregates as catalyst for synthesis of L-DOPA. Biochemical Engineering Journal, 63, 88-94. https://doi.org/10.1016/j.bej.2011.11.009
Sponsors