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Parts


Part link Description
https://parts.igem.org/Part:BBa_K5114823 Device encoding superfolder GFP under prmA. After transforming into E. coli and not observing fluorescence, it is our hypothesis that the prmA promoter does not work in E. coli, likely due to lack of transcriptional machinery specific to Rhodococcus jostii.
https://parts.igem.org/Part:BBa_K5114227 Coding sequence for human liver fatty acid binding protein conjugated with circularly permuted GFP (hlFAB-GFP or FAB-GFP).
https://parts.igem.org/Part:BBa_K5114228 Expression Device for hlFAB-GFP
TBD Coding sequence for a synthetic, estradiol-induced transcription factor that binds to the LexA operator DNA region.
TBD Expression device for the synthetic transcription factor
TBD GFP with RBS and Terminator under control of synthetic promoter bound by the synthetic transcription factor

Molecular Dynamics


Using Amber, ChimeraX, and AutoDock Vina, we engineered and tested various mutations on hlFAB to enhance its lower detection limit. To streamline the calculation of the dissociation constant (Kd) using MMPBSA, we developed an automated pipeline. This pipeline outputs an Excel-compatible data file and a PDB file from the final simulation step, allowing for easy visualization of charge contributions. It integrates all critical steps of molecular dynamics simulation, including LEaP for system parameterization, a minimization step using steepest descent, a heating phase to bring the system to 300K, a density equilibration step to stabilize the system and allows for RMSD tracking, followed by equilibration, and finally, a 10-nanosecond production run, from which data for MMPBSA analysis is extracted. The pipeline also includes MMPBSA calculations, and a custom Python script that formats the raw output data into a user-friendly format. It is available in our team's software repository along with all prerequisite files. This pipeline is free to use and can assist teams in determining the effectiveness of ligand-receptor interactions. Additionally, it is fully customizable, making it adaptable for use with other ligands beyond PFOA.

Instructions to download and use the pipeline can be found on our wiki and our software tools repository:

https://gitlab.igem.org/2024/software-tools/gcm-ky

Vcell Contribution


We have created a VCell BioModel that simulates our entire gene circuit. This model includes all components for the pRMA_GFP, FAB_GFP, and the Synt_Tran factor construct, all extremely valuable systems in synthetic biology that do not yet have a VCell model. With a few modifications, our model can be used to model different types of genetic circuits involving the LuxR-LuxI gene regulatory system.

Vcell_Contributions_Image

The models are hosted on VCell servers and are shared publicly in the “Uncurated” folder. They are completely free to use and modify for anyone with VCell.

Our most up-to-date models can be found in a table on the experiments page. These models are free for anyone to tinker with and use. Additionally, with the help of the VCell support team, we debugged VCell’s ability to export and import simulation data. This allows future users to pick up where they left off on previous simulations. A document that outlines how to do so is attached below:


How to Access Our Vcell Models

Our most up-to-date models can be found in a table on the experiments page. These models are free for anyone to tinker with and use.

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