When developing molecular dynamics simulations, it is common to utilise existing files or assets found from databases. However, when modelling more specific or complex systems such as membrane proteins, it is likely that such models are not built nor characterised well. For our project, we faced many issues with existing bioinformatics tools, which made it imperative to develop our simulation. Fusion proteins, especially surface-expressed proteins containing linkers, often yield unreliable and inconclusive results due to the limitations of models like AlphaFold. So while AlphaFold helps to democratise structure prediction by generating 3D structures from merely a sequence, these outputs lack contextualisation and thus are mostly reliable as snapshots for static proteins and making them less dependable for more complex structures such as membrane proteins.

In the case of bioremediation, this issue is even more exacerbated as conditions are seldom ideal or able to be safely tested in the scope of an iGEM project. Thus, Molecular Dynamics outputs such as GROMACS simulations aim to inch closer towards providing context to engineered proteins. However, these tools face two problems:

• Computational Stress serves as a bottleneck for many teams or young undergraduate researchers
• Complex Terminal Commands that intimidate and complicate the workflow, which can create a steep learning curve

In this part, we hope to highlight our contribution to the second problem by providing our troubleshooting tips and troubleshooting errors we ran into along the way. In our modelling, we developed literature-accurate bacteria membranes for Pseudomonas putida and Escherichia coli and embedded fusion proteins into them using CharmmGUI. We also subsequently show how to import these models into GROMACS and simulate them in metal ion environments, and measure the stability.

We hope our documentation can show other teams how to do the same and overcome the many troubles we encountered during our learning process.

For more information, please visit our Model page

For our project, we worked extensively with the scarcely documented metallothionein known as PflQ2MT. We have introduced this part to the iGEM registry as a basic part and showed both our Wet Lab and Dry Lab outputs on this protein. Our results hope to illustrate that this protein is a robust protein that has promising bioremediation capabilities for cadmium. We have also documented how the part is compatible with the well-documented iGEM Part: the Lpp-OmpA System and direct PflQ2MT onto the surface. Additionally, we also provide data on the registry that suggests its ability to improve cell survivability and growth under toxic cadmium concentrations, and its ability to adsorb cadmium when surface-expressed. Thus, we hope our results can inspire other teams to explore more with our initial findings and do more things with it.

Additionally, our contribution to the registry extends towards increasing the characterisation of existing parts. Aside from the aforementioned Lpp-OmpA system, we have shown a general unreliability of the LVA Degradation Tag working with the fluorescent protein mScarlet. From our findings, it appears unable to undergo a sufficient rate of degradation in both Escherichia coli and Pseudomonas putida to cause a drop in fluorescent signal. Furthermore, our findings show that the previously documented aTc, tetR regulatory system, when paired with a cadR promoter and Toehold switch to increase the rate of expression, is not as reproducible as hoped. Thus, we hope our findings can help other iGEM teams slowly progress towards finding the genetic cassette to produce a sensitive and dynamic biosensor.

Some notable parts contributed to the iGEM community include:

Parts no.	Part Name	Description & Application
BBa_25VCEOE5	PflQ2MT	PflQ2MT is the coding sequence for a metallothionein protein from P. fluorescens, whose primary function is cadmium binding, and is the cadmium binding protein that we used in our main circuit. This protein was further characterised in our project, and we demonstrate its ability to increase the robustness of cell survivability under toxic cadmium conditions, to express in both Escherichia coli and Pseudomonas putida and also bind to cadmium ions.
BBa_25OWO9RF	Surface Expression of metallothionein for E. coli	Main Circuit for surface expression of metallothionein, specified in E. coli . This part builds upon the basic PflQ2MT part and shows its promise in expressing onto the cell surface. This demonstrates compatibility and improves the characterisation of the existing Lpp-OmpA registry part.
BBa_256ORVBQ	RiboSwitch Cadmium Biosensor for P. putida	Cadmium biosensor with RiboSwitch, aiming at glowing with cadmium persistence, with fast replenishment. Shows some promise to produce fluorescent signal under cadmium concentration ranges found within sludge.

For more information, please visit our Parts page

Pseudomonas putida KT2440 is a well-documented strain known for its more robust and enduring characteristics. However, when transforming Pseudomonas putida , we discovered a lack of conclusive protocols tailored specifically to this strain of bacteria. Through our protocol and the repeated transformation successes found in our notebook and results, we hope our protocol can serve as a guide to future teams on how to engineer and transform Pseudomonas putida KT2440.

To make it easier for future teams to organise an Elderly Education Workshop, we have provided our teaching materials as inpsiration or usage for multiple demographics (Primary, Secondary and Elderly). Furthermore, we hope our subsequent documentation and reflection under our Education page shows the feedback on how to improve these workshops based on the demographic. we hope teams can use and reference our work in the future to produce more effective education events.

High School Workshop Day 1 Material High School Workshop Day 2 Material Primary School Workshop Material Elderly Workshop Material