iGEM 2023 — Team iit-roorkee

{% extends "layout.html" %}
{% 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?

---

* 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

---

* [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)

{% endblock %}

{% extends "layout.html" %} {% block page\_content %}

![](https://static.igem.wiki/teams/4838/wiki/project/engineering.png)

Amyloid Beta-42 Simulation
Aptamer Mutation
Optimisation of Electrode Experiments

###### Amyloid Beta-42 Simulation

[Introduction](#overview)

[Cycle 1](#overview)

[Cycle 2](#overview)

Introduction

As not much Literature work has been done into the Simulation of
Amyloid Beta Structures, 1IYT and 5KK3, we couldn’t guess
initially the Simulation time and conditions for the Protein
structures, so we conducted a series of experiments to finally
understand the simulation parameters.

Cycle 1

Design

We obtained the 2-D and 3-D structures of Aptamer, and docked them
with the respective proteins using HADDOCK, wherein we found the
most energetically favorable Clusters on which we are planning to
do MD simulations. We firstly run the simulations on Protein 1IYT,
independently.

![](https://static.igem.wiki/teams/4838/wiki/engineeringsuccess/img1.png)

Build

We generate the input files for MD simulations of the 1IYT
Protein, using CHARMM-GUI, where we set up the MD conditions with
generated Grid Box, and 0.15 M KCl solution, and hence we obtain
the GROMACS input files, in .mdp format, along with the Topology
files.

Test

We initially set the simulation time to be 125 ns. We run the
Energy Minimization Run, NVT, NPT and finally the Molecular
Dynamics run, and hence we obtain the results, through which we
create a MD movie, and finally run the commands for RMSD, RMSF,
H-bond and Radius of Gyration Analysis.

![](https://static.igem.wiki/teams/4838/wiki/engineeringsuccess/img2.jpg)

Learn

After analyzing the results, We understood that Protein stabilizes
in the region of 50 ns - 100 ns, after that protein undergoes
spikes in its RMSD and Gyration radius, hence we could run our MD
Simulation for the time of 100 ns in case of 1IYT which is small
and 50 ns in case of 5KK3, which is large in shape.

Cycle 2

Design

After the previous Cycle, we learned the simulation time for the
proteins. We pick the best clusters for the proteins, run the
GROMACS simulations on it. We pick 50 ns and 100 ns for the docked
structures.

![](https://static.igem.wiki/teams/4838/wiki/engineeringsuccess/img3.png)

Build

We set the simulation time 50 ns and 100 ns. We generate the input
files for MD simulations of the 1IYT Protein, using CHARMM-GUI,
where we set up the MD conditions with generated Grid Box, and
0.15 M KCl + + 0.15 M MgCl2 solution, and hence we obtain the
GROMACS input files, in .mdp format, along with the Topology
files.

Test

We set the simulation parameters and run the Energy Minimization
Run, NVT, NPT and finally the Molecular Dynamics run, and hence we
obtain the results, through which we create a MD movie, and
finally run the commands for RMSD, RMSF, H-bond and Radius of
Gyration Analysis.

![](https://static.igem.wiki/teams/4838/wiki/engineeringsuccess/img4.png)

![](https://static.igem.wiki/teams/4838/wiki/engineeringsuccess/img5.png)

![](https://static.igem.wiki/teams/4838/wiki/engineeringsuccess/img6.png)

Learn

As we can see the graphs above, the Simulation for few structures
is yielding highly unstable data, at 100 ns simulation time, which
indicates we need to study the simulation for a longer time, but
we also learned the simulation condition 0.15 M KCl and MgCl2 is
an ideal as we are able to significantly reduce the RMSD as
compared to first cycle. We need more cycles to fully understand
the stability of conformations that are showing thermodynamic
stability at higher simulation time.

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

References

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6096388/

https://www.mdpi.com/1420-3049/21/4/421

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7689369/

https://www.frontiersin.org/articles/10.3389/fchem.2023.1144347/full

https://pubs.acs.org/doi/pdf/10.1021/ja9719586?src=getftr

https://www.sciencedirect.com/science/article/pii/S0039914011004978

###### Aptamer Mutation

[Introduction](#overview)

[Cycle 1](#overview)

[Cycle 2](#overview)

[References](#overview)

Introduction

As we skimmed through the iGEM directory for the part for
Lactoferrin Aptamer, for our efforts to make a better part, which
shows better affinity for Lactoferrin, as lactoferrin is a protein
that is used in multiple places throughout the world. For this, we
wanted to do Mutations to the aptamer sequence which are in
binding pockets with protein, and analyze the results.

Cycle 1

Design

We pick the Aptamer sequence, for Lactoferrin,
gcaggacaccgtaacacgggcttttgctttatcgtaccctttatgctagattgtcctgc
After analyzing the simulation data for this Aptamer, we make a
few nucleotide substitutions and obtain new simulation data.

![](https://static.igem.wiki/teams/4838/wiki/engineeringsuccess/img8.png)

Build

We set the simulation time for 50 ns. For the Mutated Aptamer
sequence, we obtain the 2-D and 3-D structure of the Aptamer, and
then dock it with Lactoferrin structure using HADDOCK, then we
generate the simulation parameter files using CHARMM-GUI, for
running the GROMACS simulation.

Test

We initially set the simulation time to be 50 ns. We run the
Energy Minimization Run, NVT, NPT and finally the Molecular
Dynamics run, and hence we obtain the results, through which we
create a MD movie, and finally run the commands for RMSD, RMSF,
H-bond and Radius of Gyration Analysis.

![](https://static.igem.wiki/teams/4838/wiki/engineeringsuccess/img9.png)

![](https://static.igem.wiki/teams/4838/wiki/engineeringsuccess/img10.png)

Learn

We learned from the plots that even after Mutation, the RMSD and
Gyration radius values don’t really change much, that means we
have to make more directed mutations in this case, with specific
targets in various pockets. For that we have to make more
simulations and generate more data to improve the Aptamer part.

###### Optimisation of Electrode Experiments

[Introduction](#overview)

[Cycle 1](#overview)

[Cycle 2](#overview)

[Cycle 3](#overview)

[References](#overview)

Introduction

The report to report variation in electrochemical sensing, the
very nascent stage of aptasensor research and the several
different fabrication strategies available makes measuring with
Electrochemical Aptasensors a not so straightforward task. We had
to iterate through multiple cycles to optimize our protocol
design.

Cycle 1

Design

Based on our literature review, a preliminary protocol was
designed based on using a Self-Assembly-Monolayer (SAM) of
Mercaptohexanol (MCH) and Thiolated Aptamer to prevent aptamer
fouling and block the remaining electrode[5].

![](https://static.igem.wiki/teams/4838/wiki/engineeringsuccess/img11.png)

Build

The Aptasensor Fabrication Protocol was followed and MCH was
backfilled as a blocking agent after aptamer immobilization and
incubation overnight. This was then washed with PBS and incubated
again before electrochemical analysis was done. Sensing studies
with analytes (Lactoferrin, 10μL of all concentrations) were also
performed.

Test

Both CV and DPV were performed on fabricated electrodes and bare
SPEs too. CV results didn’t show much variation, and we weren’t
completely satisfied with the DPV results either as we thought we
could optimize for a greater difference between the two currents.
Some electrodes were giving unstable results as well. The current
plots of the analyte sensing studies saturated after two
concentrations.

![](https://static.igem.wiki/teams/4838/wiki/engineeringsuccess/img12.jpg)

Learn

We interpreted the results to indicate possible fouling and
blocking of electrode sites by the aptamer. The unstable
electrodes also probably indicated leaching of aptamers (See Notes
in Wet Lab Results Section) due to re-oxidation of thiol groups.
The saturation of Aptamers with Lactoferrin was possibly due to
the volume of Lactoferrin we used being more than available
aptamers for binding.

Cycle 2

Design

We tried optimizing the protocol for Analyte Sensing Studies by
using lesser concentrations of Lactoferrin on each electrode.

Build

The same immobilization protocol was followed. Sensing studies
were performed using 3μL Lactoferrin on each electrode instead of
10μL.

Test

Successful sensing studies were performed, with distinct DPV plots
in the required range, before reaching saturation current (see
Results page).

![](https://static.igem.wiki/teams/4838/wiki/engineeringsuccess/img13.jpg)

Learn

For successful detection of our biomarker we would have to either
ensure more specific control and studies of sample quantity or
optimize our immobilization protocol to have more available
aptamers for binding. We were also still unsatisfied with the
Aptasensor Fabrication from the first DBTL Cycle.

Cycle 1

Design

We decided to try a second immobilization protocol, this time
co-immobilizing the initial DTT used to reduce the aptamer along
with the aptamer. We had read about SAMs using DTT as well,
proving to be more effective than MCH. [6]

![](https://static.igem.wiki/teams/4838/wiki/engineeringsuccess/img14.png)

Build

The same immobilization protocol was followed except DTT was
co-immobilised with DNA:DTT ratio of 1:10. MCH was backfilled as a
blocking agent after incubation overnight. This was then washed
with PBS and incubated again before electrochemical analysis was
done. This time, sensing studies were also performed subsequently.

Test

DPV was performed on the fabricated electrodes and bare SPEs. The
DPV plots showed a significant increase in current (roughly 72μA).
However, on performing analyte studies (with 3μL), it reached
saturation current at 1μg/mL itself.

Learn

Even though much higher DNA:DTT ratios have been reported, the
available aptamers for binding are clearly not sufficient for
sensing. Perhaps our overall concentration of aptamer solutions
must be optimized. It is possible that DTT was interfering with
our sensing studies and not having the desired effect at all. We
will continue to optimize these protocols until we can develop
reproducible results.

References

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6096388/

https://www.mdpi.com/1420-3049/21/4/421

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7689369/

https://www.frontiersin.org/articles/10.3389/fchem.2023.1144347/full

https://pubs.acs.org/doi/pdf/10.1021/ja9719586?src=getftr

https://www.sciencedirect.com/science/article/pii/S0039914011004978

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

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](http://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)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

---

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

---

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

## Inspirations

---

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

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