Contribution

We used a bidirectional promoter which is inducible in E. coli by the addition of copper. In E. coli, copper sensing and response are controlled by the two-component regulatory system. When copper ions are present, the histidine kinase detects elevated copper levels and becomes activated. CusS then autophosphorylates and transfers the phosphate group to CusR. Once phosphorylated, CusR binds to the promoter region of the cus operons, activating transcription of genes involved in copper resistance.

We demonstrated this promoter worked in expressing our chromoprotein when we added copper. We replicated the same chromoprotein gene in both directions; however, others may wish to use this bi-directional promoter to express two different genes at once. Future work could also further characterize this promoter by testing both directions with different outputs to determine if there is a difference in promoter strength in the two directions. Based on our learning when attempting synthesis of matching genes with this promoter, we recommend ensuring alternate codon optimization to create differing DNA sequences to minimize hairpin interactions.

We have registered a new composite part: BBa_25zau49i

One of the challenges in our project was identifying a reliable and user-friendly method to measure formaldehyde. We explored several protocols described in the literature but found that many were too complex, required difficult reaction conditions, or produced colour changes that were not easily distinguishable to the naked eye. These limitations made the assays difficult to reproduce and unsuitable for consistent field use.

One assay of interest to our team, developed by Thepchuay et al. (2021), reported high sensitivity and clear colourimetric results within five minutes. However, we were unable to replicate the experiment using the method as described. Consequently, we tested and refined the assay ourselves, running multiple trials over several weeks. We varied reagent concentrations, compared iron(II) sulfate and iron(III) chloride as potential catalysts, and carefully documented which conditions yielded observable gradients.

The final version of the protocol uses bromothymol blue, sodium hydroxide, hydrogen peroxide, and either FeSO₄ or FeCl₃ as the iron source. We tested a range of formaldehyde concentrations (10–400 ppm), and found that colour change is most noticeable after 7 days. Higher concentrations of hydrogen peroxide and certain iron conditions gave inconsistent results, so we recommend avoiding them.

By sharing both what worked and what did not, we hope to save future iGEM teams the time and frustration of repeating the same trial-and-error process. Our improved formaldehyde assay offers a reproducible and easy-to-use method that can be directly adopted or further refined by others. We are now able to share with the iGEM community a colorimetric assay capable of detecting formaldehyde concentrations below 100 ppm, with the most distinct colour delineation observed between 20 ppm, 100 ppm, and 200 ppm.

The results are seen clearly 5–7 days after addition of formaldehyde. We do hope in the future to be able to further improve on this to achieve the 5-minute results seen in the literature [2].

Formaldehyde Assay

Formaldehyde Detection Reagent

*Both Fe ion sources were tested.

Procedure

The best reagent-to-sample ratio was found with 150 µL reagent and 50 µL sample added to detect formaldehyde. A 1:1 ratio was not as defined in colour, and reversing the ratio (150 µL sample with 50 µL reagent) was immediately too acidic for the BTB indicator.

Concentrations of formaldehyde tested were 10, 20, 100, 200, and 400 mg/L, corresponding to 10, 20, 100, 200, and 400 ppm, or approximately 0.001%, 0.002%, 0.01%, 0.02%, and 0.04% (w/v), respectively.

Best colour differentiation was not observed until day 7. The formulation above represents the composition that provided the most reliable and reproducible colourimetric results.

Fundraising Pitch Deck

Another contribution we are proud of is the pitch deck we developed for fundraising. One of the major challenges for iGEM teams is securing sufficient funds to cover registration, travel, and project expenses. To address this, we created a series of tailored pitch decks designed specifically for synthetic biology projects, each highlighting our project’s goals, potential impact, and the value of supporting iGEM initiatives. These were instrumental in our fundraising efforts. Through targeted presentations to sponsors, industry partners, and university stakeholders, our team successfully raised $51,200, enabling five students to attend the iGEM Jamboree in Paris.

We also tailored our pitch decks to different audiences. We created a version for a more general outreach, a more technical version for researchers, and a concise, outcome-focused version for investors and industry partners. This flexibility allowed us to adjust our message depending on who we were speaking to and made our pitches more engaging and relevant.

This approach was highly effective since we raised enough funding to cover our registration fees and to send five students to Paris for the Jamboree. By making these resources available, we hope to give future iGEM teams a strong starting point for their own fundraising efforts. Adapting our pitch structures could save teams significant time and improve their chances of securing sponsorship, allowing them to focus more energy on their science.

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Resources

Pitch deck for investors — Ester Spirits

Project presentation — Macquarie University Showcase

Full pitch deck set
https://2025.igem.wiki/macquarie-australia/entrepreneurship#appendix

1. Karp, P. D., Weaver, D., Paley, S., Fulcher, C., Kubo, A., Kothari, A., Krummenacker, M., Subhraveti, P., Weerasinghe, D., Gama-Castro, S., Huerta, A. M., Muñiz-Rascado, L., Bonavides-Martinez, C., Weiss, V., Peralta-Gil, M., Santos-Zavaleta, A., Schröder, I., Mackie, A., Gunsalus, R., Collado-Vides, J., … Paulsen, I. (2014). The EcoCyc Database. EcoSal Plus, 6(1), 10.1128/ecosalplus.ESP-0009-2013. https://doi.org/10.1128/ecosalplus.ESP-0009-2013
2. Le, T.-K., Lee, Y.-J., Han, G. H., & Yeom, S.-J. (2021). Methanol dehydrogenases as key biocatalysts for synthetic methylotrophy. Frontiers in Bioengineering and Biotechnology, 9, 787791. https://doi.org/10.3389/fbioe.2021.787791