CONTRIBUTION
OUR POTENTIAL CONTRIBUTION TO THE FUTURE
OUR POTENTIAL CONTRIBUTION TO THE FUTURE
| 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 |
| https://parts.igem.org/Part:BBa_K5114231 | Coding sequence for a synthetic, estradiol-induced transcription factor that binds to the LexA operator DNA region. |
| https://parts.igem.org/Part:BBa_K5114824 | Synthetic promoter sequence designed for maximal inducibility for the synthetic estradiol-induced transcription factor |
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-kyWe 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.
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:
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.
This is an VCell Tutorial on how to create models and run simulations.
First, create a molecule for each molecule in the model that needs to be defined.
Highlighted in red is the structure tab. This is where we can create structures or the boxes shown in our model. This is useful for creating simulations where a molecule is inputted into a cell.
Using the molecule feature highlighted in green we can create a molecule. Using the arrow highlighted in red we can create reactions between molecules. Using an arrow creates a yellow box which is the reaction. We can add reverse and forward arrows to mimic reactions. Shown in blue is the mouse feature. This can be used to stop placing reactions or molecules.
Highlighted in yellow is the feature to specify what each molecule we just created in our model is. We have created a list of molecules from earlier that we can specify from. After right clicking the box the specify molecule screen comes up.
Once you click on the reaction boxes(yellow square) you get this menu pulled up. On the far right column, you see units. Circled in blue is the annotations tab. This is basically your notepad per reaction for you to add information. The orange is where you can set a name which is shown in the reactions tab at the very top. The yellow is the forward rate constant at which the reaction happens, The mint green is the reverse rate constant at which the reaction happens.
By pressing the green button we can create a simulation or an test run. We recommend using stochastic as it is most closely related with an cell in real life. We used stochastic for all of our models.
Highlighted in yellow is the volumes of each structure. These are the compartments that were created in step 2. We can create different volumes to change size ratios of cells compared to the environment.
The yellow highlight shows this is the specifications tab of our application. This is where we can set initial starting conditions such as how many molecules of each species exist. We can do this in molecules or concentration.
This image contains our simulations tab where we run our actual simulations. This has a lot of features that come in handy. The feature circled in green is creating a new simulation. The blue highlight is where we can duplicate a simulation very handy when you need to duplicate a simulation which has custom inputs which leads to the brown circle. This is where we can edit the starting conditions of a single simulation instead of changing them at the specifications page. The red circle is the start button and once it is done running. The orange circle will have a colorful graph button. Clicking on this will show you results for the simulation. Once clicking on a simulation the pink box at the bottom shows up. This shows all parameters that were changed in starting conditions
This is for practice purposes and it is construct 1 from our project. This is to create the same model we did and ours is publicly located.
Molecules (All purple/green dots) PFAS_Outside PFAS_Inside pRMA_Operon PFOA_pRMA_Cplx GFP_gene GFP_mRNA GFP_mRNA_degr SF_GFP SF_GFP_degr SF_GFP_dimer SF_GFP_dimer_degr
1. First, create the molecules in the molecules tab as shown.
2. After creating molecules create another compartment. Make sure one is labeled cell and the other is environment.
3. Create reactions using the forward and reverse constants from the tables.
4. Run simulations using the starting conditions for Geometry: Cell Size: 3.5 um, Environment: 1000 um