iGEM 2024 — Team iit-roorkee

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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## What should this page contain?

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* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Inspirations

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* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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![](https://static.igem.wiki/teams/5493/project/eng/macbook-air-29.webp)

###### ZAP1-ITSrDNA Simulation

[Introduction](#overview)

[Cycle 1](#model1)

[Cycle 2](#model2)

Introduction

We were able to successfully carry out the docking process using
Google's AlphaFold 3 server. The server allowed us to predict
multiple conformations of the protein, each of which was then
thoroughly analyzed. We utilized the structural insights provided
by AlphaFold to evaluate the stability and accuracy of each
conformation, allowing us to identify the most probable binding
interactions. This iterative analysis enabled us to refine our
understanding of the protein's structural behavior and its
potential functional implications in the context of the docking
study.

![](https://static.igem.wiki/teams/5493/prisha/screenshot-384.png)

Cycle 1

Design

We obtained the 2-D and 3-D structures of the ZAP1 protein and its
ITS rDNA and docked them using Google's AlphaFold 3 server.
Through this process, we identified the most energetically
favorable clusters, on which we plan to conduct molecular dynamics
(MD) simulations.

Build

We generated the input files for MD simulations of the ZAP1 (1ZW8)
protein and its ITS rDNA using the appropriate tools and methods.
During this process, we set up the MD conditions with the
generated grid box and a 0.15 M KCl solution. As a result, we
obtained the GROMACS input files in .mdp format, along with the
necessary topology files, which are essential for conducting the
molecular dynamics simulations.

Test

We conducted the energy minimization run, followed by NVT and NPT
equilibration phases, and finally performed the molecular dynamics
run. Through this process, we obtained results that allowed us to
create an MD movie. Subsequently, we executed the commands for
analyses, including RMSD, RMSF, hydrogen bond, and radius of
gyration, to evaluate the dynamic behavior and stability of the
protein-DNA complex throughout the simulation.

![](https://static.igem.wiki/teams/5493/prisha/model-3.png)

Learn

The initial docking cycle provides insights into the potential
binding interactions between the ZAP1 protein and ITS rDNA. This
information guides the direction for subsequent docking cycles.

Cycle 2

Design

After the previous cycle, we learned about the simulation time for
the proteins. We picked the best clusters for the proteins and ran
the GROMACS simulations on them.

![](https://static.igem.wiki/teams/5493/prisha/model-2.png)

Build

We set the simulation time and generated the input files for MD
simulations of the 1ZW8 Protein, where we established the MD
conditions with a generated grid box and a solution of 0.15 M KCl
and 0.15 M MgCl2. Consequently, we obtained the GROMACS input
files in .mdp format, along with the topology files.

Test

We set the simulation parameters and ran the energy minimization
run, NVT, NPT, and finally the molecular dynamics run. Through
this process, we obtained results that allowed us to create an MD
movie and ran commands for RMSD, RMSF, hydrogen bond, and radius
of gyration analysis.

Learn

As we can see from the images above, simulations for the docked
structures will be carried out after obtaining results from
AlphaFold. This step is crucial, as it allows us to observe the
dynamic behavior of the protein-DNA complexes over time, offering
insights that static structures cannot provide. By simulating the
docked conformations, we can analyze how the binding interactions
evolve, including conformational changes, stability, and
flexibility of the protein and DNA components. This dynamic
modeling will help us understand the operational mechanisms
underlying the molecular interactions, revealing key details about
binding affinity and specificity. Additionally, the simulations
can identify potential allosteric sites or conformational states
that may be relevant for regulatory functions. Overall,
integrating molecular dynamics simulations with docking results
will deepen our understanding of the biological processes at play,
guiding us in optimizing the interactions for applications such as
drug design or synthetic biology.

![](https://static.igem.wiki/teams/4838/wiki/engineeringsuccess/img7.png)

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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## What should this page contain?

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Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](https://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

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![](https://static.igem.wiki/teams/5493/background-new/welab-results.png)

###### Description

[Results](#overview)

Results

The FIMO analysis revealed several prospective protein-DNA binding
motifs within the ITS rDNA sequence of C. falcatum. Among these motifs,
the transcription factor ZAP1 (Zinc-responsive activator protein 1)
emerged as the top candidate with the lowest p-value of . This p-value
indicates a strong match between ZAP1’s binding motif and the conserved
sequence within C. falcatum.

**p-Value Significance:** The p-value in FIMO refers to the probability of
observing a motif of this strength by chance. A lower p-value suggests
that the observed match is highly unlikely to have occurred randomly. In
this case, the p-value of 3.2e-6 indicates that the ZAP1 binding site
identified within the sequence is very likely to represent a true
biological interaction.

The ZAP1 binding motif was located between nucleotide positions 264 and 278 of the conserved sequence, providing a 15-base pair region where ZAP1 is most likely to interact with the DNA.

The presence of a ZAP1 binding site in C. falcatum could suggest a regulatory role in zinc metabolism, which might be important for the fungus's pathogenicity or survival.

The uniqueness of the rDNA sequence in C. falcatum adds another layer of significance to our findings. Since rDNA is one of the most conserved sequences in the genome, finding a 15 base pair sequence that matches the ZAP1 binding site outside this context is relatively rare. The specificity of the interaction is crucial because it limits the possibility of ZAP1 binding to other, unrelated DNA sequences. Given that our target sequence is unique to C. falcatum, we infer that ZAP1 would not likely bind to any other DNA sequence from different organisms or even from other regions within the C. falcatum genome.

![](https://static.igem.wiki/teams/5493/wetlab/results/result-img1.png)

![](https://static.igem.wiki/teams/5493/wetlab/results/results-img2.png)

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