iGEM 2022 — Team iiser-pune2-india

{% 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 %} {% block
page\_content %}

![](https://static.igem.wiki/teams/4429/wiki/tittles/notebook.png)

# Monthly Meetings

This is a comprehensive collection of all of the meetings we have ever had, our activities, our successes and our failures. Follow us, 10
undergraduates learning how to navigate the iGEM world, on
our iGEM journey.

Please click on the months to open up into our monthly notebooks!

[APRIL](https://static.igem.wiki/teams/4429/wiki/notebook/april-final-compressed.pdf)

[MAY](https://static.igem.wiki/teams/4429/wiki/notebook/may-final.pdf)

[JUNE](https://static.igem.wiki/teams/4429/wiki/notebook/june-final-compressed-1.pdf)

[JULY](https://static.igem.wiki/teams/4429/wiki/notebook/july-compressed.pdf)

[AUGUST](https://static.igem.wiki/teams/4429/wiki/notebook/august.pdf)

[SEPTEMBER](https://static.igem.wiki/teams/4429/wiki/notebook/september.pdf)

[OCTOBER](https://static.igem.wiki/teams/4429/wiki/notebook/october.pdf)

{% endblock %}

{% extends "layout.html" %} {% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by
going through at least one iteration of the engineering design cycle.{% endblock
%} {% block page\_content %}

![](https://static.igem.wiki/teams/4429/wiki/tittles/engineering.png)

# Determining the Antibody

The existence of four DENV serotypes (DENV 1-4) and the association of
prior DENV infection with an increased risk for severe disease presents
significant challenges to the healthcare infrastructure of subtropical
countries, which have a huge burden of Dengue cases every year. People
who are infected a subsequent time with a different serotype of the
dengue virus may experience “antibody-dependent enhancement” (ADE),
where the dengue virus of the different serotype takes advantage of
pre-existing antibodies from the first infection to increase its
virulence leading to a more severe form of DENV infection. ADE occurs
due to the binding of human antibodies (like IgG) through their Fc
region to immune receptors, and incomplete (non-neutralising) binding to
the pathogen.

**Ideation 1**

IgY, the avian antibody, due to its non-reactivity with human immune
receptors, can prevent ADE by binding specifically to the virus particle
to neutralise it. We planned to switch the constant region of a full
length IgG antibody to IgY. Goose-derived anti-DENV IgY has been shown
to neutralise DENV infection without triggering ADE.

**Problems**

We faced problems with figuring out the mechanism of clearance and the
potential immunogenicity of an antibody from a non-human, non-mammalian
system. We also had multiple questions on the general immunotherapy
techniques we could have used to modify IgY and make it a functional
therapeutic. After thorough research and conversations with experts, we
felt that we weren’t able to answer these questions adequately and
decided to move onto another option.

**Ideation 2**

Another way to prevent ADE is to remove glycans from the DENV-targeting
antibody, producing an aglycosylated IgG molecule. N-linked glycosylated
is necessary for antibodies to bind to effector receptors, so having the
antibody be aglycosylated would abrogate effector functions without
affecting antigen-binding, hence making an effective therapeutic against
Dengue without causing ADE.

**Problems**

The production of full-length antibodies is quite expensive. Bacteria
cannot produce it in high yields, or with the necessary glycosylation.
Yeast can produce the protein, but the differing glycosylation patterns
mean that it is processed differently by the body’s machinery. Mammalian
cell lines can produce antibodies, but are quite difficult to handle and
grow.

**Ideation 3**

We settled on synthesising antibody fragments, specifically scFvs
(single chain fragment variable), which would be a small protein, easy
for bacteria to synthesise, with high affinity and specificity to the
antigen. It would have no means of interacting with immune receptors, so
it would not trigger ADE.

The issue arose that scFvs have quite short half-lives in serum, due to
their size and non-interaction with the FcRn receptor, which is
responsible for preventing the lysis of native antibodies in the body.
We resolved this by adding a short FcRn-binding peptide into the
protein, which mimics the interaction of IgG antibodies with the
receptor.

# Determining the Epitope

**Ideation 1**

The DENV non-structural protein 1 (NS1) is a component of the viral
replication complex and can be found on intracellular membranes as well
as on the cell surface. Endothelial hyperpermeability and vascular leak,
pathogenic hallmarks of severe dengue disease, are directly triggered by
DENV non-structural protein 1 (NS1). As such, anti-NS1 antibodies can
prevent NS1-triggered endothelial dysfunction in vitro and pathogenesis
in vivo.

**Problems**

We discovered that the NS1 protein only tends to accumulate once the
dengue infection has already become quite severe. Additionally, anti-NS1
antibodies tend to cross-react with host proteins. They also target and
destroy infected endothelial tissue, which could potentially lead to the
worsening of the hemorrhagic fever.

**Ideation 2**

We then looked into the Fusion Loop Epitope (FLE), which is highly
conserved across all the serotypes of dengue and is the target for
several known neutralizing antibodies. It is important for the virus’
entry into the cell, so blocking it would effectively erase its
pathogenicity.

**Problems**

The FLE, while conserved, is often hidden on the surface of the virus.
The degree to which it is hidden varies across serotypes and stages of
the virus life-cycle. So while it would be a great target for a
neutralizing antibody, it might not always be accessible by one.

**Ideation 3**

We spoke to Dr Vidya Mangala Prasad and presented our candidates for
epitope. She pointed out the issue with the FLE and suggested that if we
were looking for a conserved epitope that is the target of neutralizing
antibodies, we would be best off with the E-dimer epitope (EDE). EDE is
a quaternary epitope that ticked all the boxes we were interested in. We
looked into the topic ourselves after our conversation with Dr Prasad,
and decided on a specific EDE-targeting antibody - C10.

After deciding our antibody and antigen, our goal now was to clone our
c10-scFv insert DNA into our pET-21b vector. We made competent cells to
transform our positive clone into. We then wanted to optimise the SHuffle
system to produce our protein of interest by standardising certain factors
like expression temperature, concentration of IPTG, and growth (OD) at the
time of induction.

**Cloning**
We started by cloning our c10-scFv insert into our pET-21b vector. We
had already transformed our insert (in a pUCIDT-KAN vector synthesised
by IDT) into a DH5-𝞪 chemically competent cell to amplify it and eluted
50ul of the plasmid using plasmid prep.

[Cycle 1](#!)
[Cycle 2](#!)
[Cycle 3](#!)

---

| Design | Build | Test | Learn |
| --- | --- | --- | --- |
| We wanted to digest both vector and insert plasmid with NdeI and XhoI enzymes, with the final goal of cloning the insert into the vector. | We set up a restriction digest for both the vector and the insert - a 30uL reaction with 1uL of each enzyme, left overnight. The reactions would be run on a 0.8% agarose gel and extracted to conduct the later steps. | When the gel was imaged, we weren’t able to see a defined vector band - there was a lot of background. Insert band was visible but also had a huge smear. | Star activity in vector DNA might have caused multiple fragments in the vector and the smear. We were advised to have a shorter digest period and a larger reaction volume. |

![](https://static.igem.wiki/teams/4429/wiki/engineering/23-7-22-igy-and-pet21b-cigg-rd2-1-first-digestion.jpg)

First restriction digest with pet-21b vector and IgG-IgY-aMBP
insert

| Design | Build | Test | Learn |
| --- | --- | --- | --- |
| We integrated the advice of our mentors from the first round into our experiment plan. | We started with 1000 ng each of vector and insert DNA and set a 50 µL reaction using NEB high fidelity restriction enzymes. We ran the digest for three hours and merged wells in our gel to ensure proper loading of all our DNA. | Vector and insert bands were visible and the DNA was extracted. After setting up ligation and transformation, there were more colonies on the control plate as compared to the 1:3 plate. | It was suggested that our digest had not been complete. We also learned that DNA obtained from alkaline lysis is quite impure, and could possibly have interfered with the efficiency of our digest. Additionally, while it was sufficient this time, quite low amounts of DNA were obtained after extraction. |

![](https://static.igem.wiki/teams/4429/wiki/engineering/engg/tester.png)

Plates after transformation with ligated product, control has more
colonies than 1:3 plate

| Design | Build | Test | Learn |
| --- | --- | --- | --- |
| We used spin columns to isolate plasmids to ensure high purity. We began with 2000 ng of DNA so that we would have sufficient amounts following extraction. We set up a second control for ligation, this one without ligase, to determine how much of the vector had remained undigested. | After determining the total amount of vector plasmid, restriction digest was set again in the manner learned previously. Extracted DNA was set for ligation, with two controls (Vector, Vector+ligase). | There were no colonies seen on the first control plate (Vector). The second control plate (Vector+ligase) had about 30 colonies and there were almost 200 colonies on the 1:3 plate. | Cloning was successful. Positive clones were found through alkaline lysis and agarose electrophoresis. The cloned plasmids were sent for sequencing. |

![](https://static.igem.wiki/teams/4429/wiki/engineering/img-20220927-wa0002.jpg)

Fig: L to R, ligation control, 1:3 plate, digestion control, 1:5
plate

**Making competent SHuffle cells**
In order to express our protein into SHuffle, we needed competent cells
to transform our plasmid into. However, transforming SHuffle K-12 proved
tricky and there were multiple rounds before we were finally able to
successfully make chemically competent SHuffle K-12 cells.

[Cycle 1](#!)
[Cycle 2](#!)
[Cycle 3](#!)

---

| Design | Build | Test | Learn |
| --- | --- | --- | --- |
| Use chemical competency methods to make competent SHuffle cells. | SHuffle K-12 cultures were grown and an ASB buffer (with bivalent cations) along with glycerol was added for ensuring chemical competency. | No colonies were observed after transforming a plasmid into freshly made chemically competent SHuffle cells. Transformation was repeated, to obtain the same results. | We were advised to monitor OD more thoroughly and use freshly prepared ASB buffer; we had re-used the buffer from a previous round of experiments. |

| Design | Build | Test | Learn |
| --- | --- | --- | --- |
| Repeat the previous protocol, with fresh buffer and less lag time between optical density measurements | New ASB buffer was prepared, cultures were kept on ice always and OD readings were taken with a higher frequency. | No colonies were observed after transforming a plasmid | Upon consulting lab members who had previously worked with this strain of SHuffle, we decided to try out the Inoue method. SHuffle K-12 was hypothesised to be incompatible with ordinary chemical competency. |

| Design | Build | Test | Learn |
| --- | --- | --- | --- |
| Research the Inoue method. | Inoue method of chemical competency was applied to SHuffle-K12 cells. Cells were cultured, washed with Inoue buffer, then dried. More buffer was added, along with DMSO. | Colonies were observed after transforming our antibody into ultra-competent SHuffle K-12 cells | SHuffle K-12 was made competent successfully. |

To view the tables, please view on the desktop

**Optimization of antibody production in a prokaryotic system**
We wanted to optimise the production of our protein in our chassis.
After research and conversations with experts, we settled on DOE, or
statistical Design of Experiments. The goal was to find the optimum set
of conditions so the yield of soluble protein was maximised.

[Cycle 1](#!)
[Cycle 2](#!)
[Cycle 3](#!)

---

| Design | Build | Test | Learn |
| --- | --- | --- | --- |
| We identified six factors worth investigating for influence on yield. | We constructed a six-factor classical screening design in JMP. | Found to be unfeasible at the scale and resources - the number of experiments was too high to do. | We needed to find a more efficient way of identifying interactions - a six-factor model was far too laborious. |

| Design | Build | Test | Learn |
| --- | --- | --- | --- |
| We spoke to experts and our mentors, and narrowed the model down to three factors that were deemed the most important. | A response surface design on OD at induction, temperature and IPTG concentration was constructed - yielding 15 experiments. | Due to erroneous execution, we lost a few cultures due to growing beyond the determined value of optical density. These experiments were repeated. | SDS-PAGE gels were run in triplicate, and imaged to measure the amount of the protein in the supernatant. |

| Design | Build | Test | Learn |
| --- | --- | --- | --- |
| ImageJ was used to set up the analysis. The software was standardised using gels run with multiple concentrations of BSA. | The software was set up to analyse the gel images. | The final response outputs were inputted into regression analysis in JMP | Interaction between factors were identified. |

{% 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 %}

![](https://static.igem.wiki/teams/4429/wiki/tittles/experiments.png)

# Cloning

Working Principle:

* A desired sequence of DNA is inserted into a suitable vector, which results in a chimeric or recombinant DNA molecule. The vector
  transports the desired sequence of DNA into the host cell, usually a bacterium.
* Within the host cell, the vector along with the gene it carries, replicates. Then the host cells divide and further replications of the
  vector take place. After multiple divisions, identical host cells are produced, usually called “a colony” or “a clone”
* It consists of several steps, all of which are listed below along with their protocols.

## Restriction digest

Working Principle:

* Restriction Digestion involves fragmenting DNA molecules into smaller pieces with special enzymes called Restriction Endonucleases
  commonly known as Restriction Enzymes (RE).
* These leave behind overhangs (sticky) or blunt ends. depending on the enzyme, in the DNA so that the vector and insert can ligate with
  corresponding ends to make a new plasmid.
* It consists of several steps, all of which are listed below along with their protocols.

[Protocol](https://static.igem.wiki/teams/4429/wiki/experiments/restriction-digest.pdf)

## Gel electrophoresis

Working Principle:

* Gel electrophoresis separates DNA fragments by size in a solid support medium (an agarose gel). DNA samples are pipetted into the sample
  wells. Application of an electric current at the top (anodal, negative) end causes the negatively-charged DNA to migrate (electrophorese)
  towards the bottom (cathodal, positive) end. The rate of migration is proportional to size: smaller fragments move more quickly, and wind
  up at the bottom of the gel.
* DNA is visualised by including in the gel an intercalating dye, Ethidium Bromide. DNA fragments take up the dye as they migrate through
  the gel. Illumination with ultraviolet light causes the intercalated dye to fluoresce with a pale pink colour.
* The digested pieces of DNA are run through the gel alongside a molecular weight ladder and we can easily visualise the parts we want in
  our new plasmid.

[Protocol](https://static.igem.wiki/teams/4429/wiki/experiments/gel-electrophoresis.pdf)

## Gel extraction

Working Principle:

* DNA run on a gel needs to be extracted for further ligation steps.
* The cut gel slice is usually dissolved in buffers, and the DNA is precipitated by ethanol to elute digested DNA fragments.

[Protocol](https://static.igem.wiki/teams/4429/wiki/experiments/gel-extraction.pdf)

## Ligation

Working Principle:

* The cut vector DNA ends are ligated with the corresponding insert DNA ends with the help of ligases.
* After running the reaction, we transform the mixture into competent cells.

[Protocol](https://static.igem.wiki/teams/4429/wiki/experiments/ligation.pdf)

## Transformation

Working Principle:

* Bacterial transformation is a process of horizontal gene transfer by which some bacteria take up foreign genetic material (naked DNA)
  from the environment.
* Not all bacteria are capable of taking up exogenous DNA from their environment. The practical approach to acquire competent cells is to
  make the bacterial cells artificially competent using chemicals or electrical pulses.

[Protocol](https://static.igem.wiki/teams/4429/wiki/experiments/transformation.pdf)

## Confirmation

This is done using alkaline lysis. Alkaline Lysis to separate plasmid DNA from all other cell constituents including genomic DNA.

Working Principle:

* The cell pellets are resuspended in a buffer, usually EDTA or Tris. The cells are lysed using an alkali, like 10 N NaOH and a detergent
  like SDS that denatures most of the cell proteins.
* The cell debris is then precipitated using potassium acetate that helps renature the double stranded plasmid DNA. This is then
  dissolved and the dissolved DNA is eluted out after centrifuging.
* We now need to precipitate the DNA and clean it of the salts it was dissolved in. An ethanol/isopropanol extraction method precipitates
  the final plasmid DNA.
* A miniprep kit works on the same principle.
* Elute in low volumes, typically lower than the instruction manual gives, around 30uL in two rounds of 15uL each. Heat the elution
  buffer or NFW beforehand, and let it soak the membrane for 5-10 minutes before you spin it down.

[Protocol](https://static.igem.wiki/teams/4429/wiki/experiments/confirmation.pdf)

# Bacterial Expression

## Chemical competency

Working Principle:

* The bacterial cells that can take up the foreign DNA from the surroundings by a process called transformation are known as competent
  cells.
* Competent cells have altered cell walls that allow the DNA to easily pass through it. Some cells need to be exposed to some chemical or
  electrical treatments to make them competent. Treatment with calcium ions is the standard method for the preparation of these cells.
  Bacterial cells can also take up DNA through a process called electroporation, which we didn’t use.

[Protocol](https://static.igem.wiki/teams/4429/wiki/experiments/chemical-competency.pdf)

## Streaking/Spreading

Working Principle:

* Bacteria from a culture or a plate need to be spread on an agar plate to form colonies.
* These colonies are then picked and grown in media with or without an antibiotic to make a primary culture.

[Protocol](https://static.igem.wiki/teams/4429/wiki/experiments/streaking-spreading.pdf)

## Primary cultures

Working Principle:

* Bacterial colonies have to be grown in a media to form enough culture that can be further used to express our protein.

## Secondary cultures

Working Principle:

* Secondary cultures are usually inoculated with primary cultures and further induced with a chemical like IPTG to enable the bacteria to
  synthesise our protein of interest.
* Secondary cultures are larger in size and can be scaled up according to the expression level of a certain protein.

[Protocol](https://static.igem.wiki/teams/4429/wiki/experiments/expression-cultures.pdf)

# Sonication

Working Principle:

* The sonication process uses ultrasonic sound waves. During the process, there is a production of thousands of microscopic vacuum bubbles
  in the solution due to applied pressure. The formed bubbles collapse into the solution during the process of cavitation.
* The collapsing of bubbles takes place in the cavitation field leading to the generation of enormous energy as there is a production of
  waves. This results in the disruption of the molecular interactions between the molecules of water. As there is a reduction in the
  molecular interactions, the particles start to separate and allow the mixing process to take place.
* There is a release of energy from the sound waves that result in friction in the solution. Ice cubes are used during and after the
  sonication process to prevent the sample from heating up.
* A buffer that can mimic the environment of the cell and the lysis method are important factors in the cell lysis protocol. Buffers
  commonly include salts, detergent and glycerol.

[Protocol](https://static.igem.wiki/teams/4429/wiki/experiments/sonication.pdf)

# Protein Purification

## Protein A chromatography

Working Principle:

* Protein A chromatography is the most frequently used affinity chromatography method in biomanufacturing. It is the standard technique
  for capturing recombinant monoclonal antibodies, which relies on the reversible and specific binding between the immobilised protein A
  ligand and antibodies.
* Each protein A molecule has five immunoglobulin-binding domains, and each domain can bind proteins from many mammalian species, most
  notably Immunoglobulin G (IgG). The primary binding site is the heavy chain within the Fc region of most IgG. Through such a binding
  between protein A and the Fc region of IgG, antibodies are retained on the column, which later can be eluted in a purified and
  concentrated form.
* The binding strength of protein A to IgG depends upon the source species of the immunoglobulin. The dynamic binding capacity depends
  upon the binding strength and factors such as flow rate during sample application.

[Protocol](https://static.igem.wiki/teams/4429/wiki/experiments/protein-a-chromatography.pdf)

## Ni-NTA chromatography

Working Principle

* The Ni-NTA Purification System is designed for purification of 6xHis-tagged recombinant proteins expressed in bacteria, insect, and
  mammalian cells.
* The system is designed around the high affinity and selectivity of Ni-NTA Agarose for recombinant fusion proteins that are tagged with
  six tandem histidine residues and exhibits high affinity and selectivity for 6xHis-tagged recombinant fusion proteins.
* Proteins can be purified under native, denaturing, or hybrid conditions using the Ni-NTA Agarose. Proteins bound to the resin are
  eluted with a low pH buffer or by competition with imidazole or histidine. The resulting proteins are ready for use in target
  applications.
* Ni-NTA Agarose uses nitrilotriacetic acid (NTA), a tetradentate chelating ligand, in a highly cross-linked 6% agarose matrix. NTA binds
  Ni2+ ions by four coordination sites.

[Protocol](https://static.igem.wiki/teams/4429/wiki/experiments/ni-nta-chromatography.pdf)

# SDS-PAGE

Working Principle:

* SDS PAGE or Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis is a technique used for the separation of proteins based on their
  molecular weight. A charged molecule migrates to the electrode with the opposite sign when placed in an electric field. The separation of
  the charged molecules depends upon the relative mobility of charged species. The smaller molecules migrate faster due to less resistance
  during electrophoresis. The structure and the charge of the proteins also influence the rate of migration.
* SDS is a detergent present in the SDS-PAGE sample buffer. SDS along with some reducing agents function to break the disulphide bonds of
  proteins disrupting the tertiary structure of proteins. It makes the amount of negatively charged protein far exceed its original charge,
  masking the natural charge difference between various protein molecules. Therefore, the mobility of various protein-SDS complexes during
  electrophoresis is no longer affected by the original charge and molecular shape, but only depends on the relative molecular mass.
* Polyacrylamide gel is composed of acrylamide and cross-linking agent N, N'-methylenebisacrylamide under the action of catalysts
  ammonium persulfate (APS) and N, N, N', N'-Tetramethylethylenediamine (TEMED).
* The polyacrylamide gel is usually composed of a stacking gel in the upper layer and a separating gel in the lower layer. The difference
  between the upper and lower gels is the concentration of acrylamide and the pH of Tris-HCl.
* During electrophoresis, an electric field is applied to the gel, and negatively charged proteins migrate across the gel from the
  negative electrode to the positive electrode. The most common electrophoresis buffer consists of Tris and glycine. The pH in the stacking
  gel is 6.8, and only a few glycine molecules dissociate. Therefore, the SDS-treated protein molecules move between the upper glycine
  molecule and the lower Cl- ion. This process compresses the protein sample in the gel into bands that are much thinner than the volume
  initially loaded.
* As the electrophoresis progresses, the protein moves to the separating gel (pH 8.8), where the glycine molecules dissociate. The speed
  of the movement increases and exceeds the protein. In the separating gel, the speed of movement of each protein depends on its molecular
  weight. Proteins with small molecular weights can pass through the pores in the gel easily, while those with large molecular weights have
  more difficulty passing through. After a period of time, proteins reach different distances according to the sizes, achieving the purpose
  of protein separation.

[Protocol](https://static.igem.wiki/teams/4429/wiki/experiments/sds-page.pdf)

# Bradford Assay

Working Principle:

* The Bradford protein assay is used to measure the concentration of total protein in a sample.
* The assay is based on the observation that the absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-250 shifts from
  465 nm to 595 nm when binding to protein occurs. Both hydrophobic and ionic interactions stabilise the anionic form of the dye, causing a
  visible colour change.

[Protocol](https://static.igem.wiki/teams/4429/wiki/experiments/bradford-assay.pdf)

# Western Blot

Working Principle:

* Western Blot refers to the electrophoretic transfer of proteins from sodium dodecyl sulphate polyacrylamide gels to sheets of PVDF
  or nitrocellulose membrane, followed by immunodetection of proteins using antibodies with fluorescent or chemiluminescent detection.
* Bradford Assay can be used to determine protein concentration and thus determine equal volumes of protein to load for binding in a
  Western Blot.

[Protocol](https://static.igem.wiki/teams/4429/wiki/experiments/western-blot.pdf)

# Protein Concentration

Working Principle:

* Proteins are concentrated using a centricon which has a 10kDa membrane, allowing salts to pass through but not proteins.
* Proteins are eluted in an Imidazole buffer which will give readings in a Nanodrop. So, proteins must be dialyzed before taking nanodrop
  readings. Dialysis buffer is the elution buffer without Imidazole.
* After successive washes with the dialysis buffer, most salts pass through the membrane and we get about 200uL of concentrated protein
  from a 2mL sample.

[Protocol](https://static.igem.wiki/teams/4429/wiki/experiments/protein-concentration.pdf)

# Thermal Shift Assay

Working Principle:

* SYPRO Orange dye binds non-specifically to exposed hydrophobic residues in a protein. If the protein is unstable in a given buffer,
  SYPRO will bind to its hydrophobic regions and give out a fluorescence signal at lower temperatures. Getting a signal at a higher
  temperature means that the protein is stable in the given buffer.
* Protein samples are gradually heated to find their melting point.

[Protocol](https://static.igem.wiki/teams/4429/wiki/experiments/thermal-shift-assay.pdf)

# VLP Fusion Assay

Working Principle:

* It involves vesicles concentrated with dye to the extent that the dye is quenched. Dengue virus-like particles are added to these
  vesicles - and the pH is lowered to make the particles fuse with the vesicles.
* As the vesicles grow upon fusion - the dye becomes less concentrated and visible, allowing for a simple colorimetric readout for the
  extent of fusion.

[Protocol](https://static.igem.wiki/teams/4429/wiki/experiments/vlp-fusion-assay.pdf)

# Miscellaneous Protocols/Buffer Recepies

There were some common lab protocols that we followed and are illustrated below. The buffers used in the above protocols are also mentioned.

[Protocols and Recepies](https://static.igem.wiki/teams/4429/wiki/experiments/igem2-web-lab-protocols.pdf)

{% 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 %}

![](https://static.igem.wiki/teams/4429/wiki/tittles/results.png)

While we would have been unable to create a full therapeutic by the end of the cycle, we still made significant progress toward that end. Through our own work, as well as the constant input of experts, stakeholders and more, we were able to achieve the goals we had charted at the beginning of the cycle.
We were successful in modelling our protein and its interactions, and in expressing and assaying the same. We were able to bridge gaps in science communication about Dengue and synthetic biology through extensive efforts at outreach activities and public engagement.

[Integrated Human Practices](#!)
[Dry Lab](#!)
[Wet Lab](#!)
[Education and Communications](#!)

# Integrated Human Practices

**Goals**
A good iGEM project involves a solution that can **realistically combat a real-world problem**. In order for us to ensure this, we had
to
first understand the problem. Our first approach was to attempt to **identify and approach all of our key stakeholders**, and to **collate and
integrate their inputs and suggestions** into every step of our ideation process. We then re-approached some of our stakeholders, presented
our proposal to them and sought their opinions to **further improve** our solution.

**Results**
We successfully managed to design a **human-centered project** by engaging with various stakeholders at every stage of our
project.

## Understanding and identifying the problem

We read and researched as much as we could about the problem - Dengue and ADE - from the news, papers, the WHO website, and from our friends,
families, academicians, and virologists. We also interacted with doctors experienced in treating Dengue, local Municipal bodies, local
vendors, ‘Vasti’ residents, construction workers, and local society members while identifying and understanding the problem better.

## Coming up with a project design

We met and spoke to several IgY experts, recombinant therapy experts, industrialists, experts who have worked extensively with scFvs, and
academicians. We have integrated their advice while coming up with a project design, wet lab, and dry lab plan. As clearly elucidated on
the Integrated Human Practises page, we have made **significant modifications** to our project in terms of our therapeutic, chassis, target
epitope, assays, modelling, and outreach activities we have performed based on the human practices we conducted.

**Future directions**

We hope to **maintain and continue** our relationship with our stakeholders and ensure that they are involved closely with
the future implementation of our project.

Integrating the advice we received into **several rounds of ideation and evaluation**, we designed our project and conducted **extensive
modelling** in Dry Lab, and **experimentation** on the Wet Lab front.

# Dry Lab

## AlphaFold structure predictions

**Goals**
To **predict the structures** of antibodies and antibody fragments as a way to -

1. **Validate** that they are **similar to existing antibody structures** in protein databases
2. To **input the predicted structures** into our **Molecular Dynamics** and **Molecular Docking** simulations

**Results**

1. We installed DeepMind’s AlphaFold 2.0’s entire Algorithm on IISER Pune’s supercomputer ‘PARAM Brahma’ and wrote Slurm batch files to
   execute the commands.
2. We predicted **more than twenty structures** of the antibodies and antibody fragments we designed for our project.
3. We understood that our **FcRn binding peptide**, which has been described as linear in literature, **may not be truly linear** in solution. It
   potentially forms a **pair of β-strands** stabilised by a β-turn even **without a disulfide bridge**.

**Future Directions**

Structures of these proteins can be **experimentally validated** in the future through **X-ray crystallography** or **NMR Spectroscopy**. This will
also verify the hypothesis of the linear FcRn binding peptide folding to form a pair of beta strands.

## Molecular docking simulations with AutoDock Vina

**Goals**

1. To **generate various conformations** of the FcRn binding **peptide complexed to the FcRn receptor**
2. To **find the sensitive residues** associated with FcRnBP binding on the FcRn receptors

**Results**

1. We found that the sensitive residues were very similar to the crystal structures of **another similar FcRn binding peptide** existing in
   literature.
2. AutoDock Vina **unfolded the linear variants** from their secondary structure, making the results seem unreliable.
3. So, for the linear structures, we **generated variants through original crystal structures** which also did not contain disulfide bridges.
4. We **validated our AutoDock Vina results** using Molecular Dynamics simulations.

**Future Directions**

For more reliable characterisation of the NeoFv-FcRn interaction, **mutagenesis studies** should be performed in the lab.

Instead of using the strength of binding to the FcRn as a proxy for the strength of FcRn signalling, **key residues** on FcRn should be
identified that are involved in **downstream signalling**, and further screens should be performed using this.

## Molecular Docking Simulations with HADDOCK 2.4

**Goals**

1. To try to identify the **most suitable position for our FcRn binding peptide** in our antibody fragments (scFv).
2. To **compare the positioning of the peptide** at the N-Terminus, C-terminus and the linker and find the ideal placement for the same.

**Results**

1. HADDOCK Software generated **HADDOCK scores** or **binding energies** for our N-Terminus, C-Terminus and Linker variants.
2. There was **no significant difference** between the results of the HADDOCK values or binding energies.
3. Hence, we could infer that we can place our FcRn-binding peptide in any of these regions for our peptide to work.

**Future Directions**

1. We can use **metadynamics approaches in molecular dynamics simulations** to properly investigate the NeoFv position variants.
2. We can use **surface plasmon resonance** in the lab to identify the actual binding association and dissociation values of our peptide.

## Statistical design of experiments

**Goals**

1. To arrive at the **optimal values** of the factors that **influence the yield** of our protein product.
2. To identify the **interaction between factors** to **optimise the production process** of our NeoFvs.

**Results**
We found out that our scFv **expressed most optimally at 26.5°C**, at a concentration of IPTG of 0.1mM, and OD600 of induction
being 1. While we were able to identify the optimal conditions in our sampled designs, we were not able to profile any statistically
significant interactions, as analysed by regression reports generated by JMP.

![](https://static.igem.wiki/teams/4429/wiki/results/d1.png)

**Future Directions**

1. The future design for modelling a statistically significant DoE model would be more **capital intensive** and will require more definitive
   screening designs for **analysing multiple factors** and arrive at accurate results using fermenters.
2. **Percentage dissolved oxygen** was a very important parameter which we could not analyse due to resource constraints, so further
   experiments which involve examining this factor would help get significant results.
3. Along with percent dissolved oxygen, parameters such as **differences in growth medium, time** and addition of **molecular chaperones** can
   also be included for building a **better statistical model**.

## Molecular dynamic simulations

**Goals**

1. To investigate the **structural stability** of our NeoFv variants.
2. To **estimate the strength of binding** of the FcRn binding peptide to the FcRn receptor.

**Results**

1. The MD trajectories show root mean square deviation of the structure to be **under 2Å**, which is the accepted limit for stability of a
   molecule.
2. The **strength of binding was successfully measured** for the different variants at different pHs. The binding energies were all negative
   - the values are given below.

| Variant | pH | Mean Binding free energy (kcal/mol) | Standard Deviation |
| --- | --- | --- | --- |
| Cyc | 6 | -32.80 | 4.22 |
| CycY12H | 6 | -34.06 | 5.43 |
| Lin | 6 | -37.11 | 6.91 |
| LinY12H | 6 | -32.20 | 3.44 |
| CycY12H | 7.4 | -31.06 | 3.99 |
| LinY12H | 7.4 | -24.00 | 7.51 |

![](https://static.igem.wiki/teams/4429/wiki/results/d2.png)

**Future Directions**
**Metadynamics approaches** should be applied to evaluate stability of folded structures and their complexes. Metadynamics **forces the
system to migrate to the different minima**, thereby helping determine the **free energy landscapes**.

# Wet Lab

## Competency

**Aim**
To **obtain competent SHuffle B and SHuffle K-12 cells** to facilitate uptake of our plasmid.

**Experiment**
The method of **chemical competency** was used to prepare chemically competent SHuffle B and SHuffle K12
cells.

**SHuffle B**

1. The culture was grown and spun down at the appropriate OD600 of 0.5-0.6, based on literature and advice that said that
   cells arrested in the **early log phase** of their cycle yielded the best results.
2. The pellets were **washed with ASB buffer**. More buffer was added to ensure competency; glycerol was added as a cryoprotectant, and the cells were **flash-frozen using
   liquid nitrogen** and **stored at -80C**.

As a test for success, the SHuffle B cells were transformed with the NeoFv plasmid, and plated on LA+Ampicillin plates.

**Results**
**Colonies were found** on the plate after 10-12 hours of incubation at 30C, indicating that the cells were
competent.

![](https://static.igem.wiki/teams/4429/wiki/results/w1.png)

**SHuffle K12**

1. A similar procedure to the one above was performed, but the plates yielded **no colonies** despite repeating the
   procedure multiple times with LA(no antibiotic) plates as **controls** to check for cell death. Colonies on the control plate indicated that SHuffle K-12 was alive, but
   had not become competent.
2. The procedure was repeated with **new ASB buffer** and **less lag time between OD600 measurements**. The cells were **kept
   constantly on ice** to ensure that the failure to produce competent SHuffle K-12 cells was not due to human error. The plates still yielded
   **no colonies**.
3. After a review of literature and discussions with our mentors, the **Inoue method** for producing ultracompetent cells was done.

**Results**
The **plate yielded colonies**, indicating that the transformation was successful, and the cells had become competent.

![](https://static.igem.wiki/teams/4429/wiki/results/w2.png)

## Cloning

**IgY-IgG hybrid insert**

**Aim**
To determine whether our gene of interest, IgY-IgG-aMBP, has been cloned into our pET-21b vector.

**Experiment**

1. A **0.8% agarose gel** was run with the **suspected positive clones** along with the undigested empty vector. The positive clones are expected
   to show an **upward shift of about 2200 bp**.
2. As a further test for confirmation, a **restriction digest with HindIII** was run. HindIII is a **single cutter for the positive clone**, and a
   non-cutter for the empty vector. A 0.8% agarose gel was run with the products of the restriction digest. A positive clone would be
   linearized by HindIII.

**Results**
 We were successful in cloning the gene for the IgY-IgG into our vector. We have confirmed the same by having the **plasmid
sequenced by Barcode Biosciences**.

![](https://static.igem.wiki/teams/4429/wiki/results/w3.png)

Fig: Alkaline lysis check for the primaries made from the 1:3 plate showing a shift of
2.2kB

![](https://static.igem.wiki/teams/4429/wiki/results/w4.png)

Fig: Confirmatory digest by HindIII. Sample 11 showed a linear band, so it was transformed
and sent for sequencing

**scFv insert**

**Aim** To determine whether our gene of interest, scFv, has been cloned into our pET-21b vector.

**Experiment**

1. The scFv-pUCIDT-KAN and pET-21b vector were both digested with Nde1 and Xho1, run on a 0.8% agarose gel, the DNA fragments extracted
   and ligated.
2. To check for positive clones, we performed alkaline lysis on the primaries made from the 1:3 plate. A **0.8% agarose gel** was run with the
   **suspected positive clones** along with the undigested empty vector. The positive clones are **expected to show an upward shift of about 800
   bp**.

**Results**
 We were successful in cloning the gene for the scFv into our vector. We have sent the sample for sequencing.

![](https://static.igem.wiki/teams/4429/wiki/results/w5.png)

Fig: Restriction digest for the vector and insert DNA

![](https://static.igem.wiki/teams/4429/wiki/results/w6.png)

Fig: Alkaline lysis check to select for positive clones

**NeoFv insert**

**Aim** To determine whether our gene of interest, NeoFv, has been cloned into our pET-21b vector.

**Experiment**

1. After a successful run of cloning, a **0.8% agarose gel** was run with the **suspected positive clones** along with the undigested empty
   vector. The positive clones are **expected to show an upward shift of about 800 bp**.
2. As further confirmation, a **restriction digest with NdeI and XhoI** was run. The products of the digest were run on a 0.8%
   agarose gel to see if two bands, of **lengths 800 bp and 5500 bp**, are visible.

**Results**
 We were successful in cloning the gene for the scFv into our vector. We have sent the sample for sequencing.

![](https://static.igem.wiki/teams/4429/wiki/results/w7.png)

Fig: Alkaline lysis check showing multiple positive clones. We decided to go ahead with sample 6, 7, 10, 11 - due to minimal RNA contamination

![](https://static.igem.wiki/teams/4429/wiki/results/w8.png)

Fig: Confirmatory digest for the selected positive clones - we transformed and expressed sample 6

## Ni-NTA purification

**Aim**

To **purify NeoFv, scFv and IgG-IgY-aMBP with Ni-NTA beads** in order to characterise protein interactions and functions.

**Experiment**

1. The culture was **grown at 16℃** overnight (14-16 hours) and spun down at 4C, 5000 rpm, for 15 mins. The pellet was resuspended using Lysis
   buffer and were **sonicated** (at 60% amplitude with a 2 sec ON, 5 sec OFF cycle) until the solution turned slightly brown and translucent.
   The solution was then spun down (12000 rpm for 25 minutes at 4C), and the **supernatant**, which contained our protein, was purified using
   Ni-NTA beads and eluted using **increasing concentrations of Imidazole**.
2. The flow-through, the different elutes, and cracked beads was run on a **12% SDS-PAGE gel** to resolve the different protein bands along with a
   protein ladder that shows 10 bands with 3 reference bands.

**Results**
We tried purifying the proteins that we expressed - IgG-IgY hybrid, scFv and neoFv, and failed multiple times

1. For IgY-IgG antibody, we got **fragmented bands** when we ran a SDS-PAGE. This was possibly be due to working with an **improper protease
   inhibitor**.
2. For scFv, we got bands at a higher size in the elutes with 100mM imidazole. This led us to believe that the elutes did not have
   significant quantities of our protein of interest and that the beads we used might have had an issue with **non-specific pull-down**.
3. For neoFvs, we changed our resin from **agarose to silica**. This may have contributed to its successful purification. A protein band was
   expected at the 29 kDa mark, which we observed in the lane containing the **first elute with 500mM imidazole**.

![](https://static.igem.wiki/teams/4429/wiki/results/w9.png)

Fig: Fragmented bands seen for IgG-IgY purification

![](https://static.igem.wiki/teams/4429/wiki/results/w10.png)

Fig: scFv with bands at lower elutes of Imidazole

![](https://static.igem.wiki/teams/4429/wiki/results/w11.png)

Fig: A 12% SDS-PAGE gel run to resolve the purified NeoFv. Band seen at E1-500 that is
probably the NeoFv showing successful purification

## Protein A purification

**Aim**

To purify IgG-aMBP protein, we needed to do **Protein A chromatography**, as there were no His-tags attached to the sequence.

**Experiment**

1. Cells were sonicated and spun down, the **supernatant was collected** and stored at 4 C.
2. The supernatant was **added to the Protein A beads** and left for binding. Elutes were taken in **0.2mM glycine pH 2.4** after giving washes
   with the lysis buffer.

**Results**
For IgG, we obtained a band at around the **40 kDa mark**, possibly be due to a **truncated antibody**.

![](https://static.igem.wiki/teams/4429/wiki/results/w12.png)

Fig: Fig: IgG purification with band
at the wrong weight mark

## Expression check

**Aim**

For each of the proteins we expressed, we tried to find the **optimum conditions for expression** in each of them, so as to make the downstream steps as efficient as possible.

**Experiment**

1. Six separate cultures each of SHuffle B transformed with aMBP-IgG and IgG-IgY-aMBP. Both were **grown and induced** at an
   OD600 of 0.6 with **different concentrations of IPTG** and allowed to grow overnight.
2. The cultures were then spun down and lysed. The **pellet and
   supernatant** were taken separately for each culture and run on a 12% SDS-PAGE gel to resolve the protein bands.

**Anti-MBP IgG**
**Results**
We obtained **bands of varying intensity** based on the yield of the protein and determined that **induction with a IPTG
concentration of 0.3 mM** was the most conducive for expression.

![](https://static.igem.wiki/teams/4429/wiki/results/w13.png)

Fig: aMBP-IgG cultures induced with
IPTG concentrations ranging from 0.1mM-2mM

**Anti-MBP IgY**
**Results**
We observed **bands of varying intensity** corresponding to the different concentrations of IPTG and determined that a **IPTG
concentration of 0.5mM is ideal** for maximum yield of protein.

![](https://static.igem.wiki/teams/4429/wiki/results/w14.png)

Fig: aMBP-IgY cultures induced with IPTG concentrations ranging from 0.1mM-2mM

## Western Blot

**Aim**

To **verify that the scFv is being expressed** with and without the linker peptide through a **Western blot**.

**Experiment**

1. To ensure that we loaded **equal amounts of protein** in all the wells for the Western blot, we performed a **Bradford Colorimetry assay**.
2. Normalised amounts of proteins were loaded and run on an **SDS-PAGE**. The proteins were expressed under different conditions - scFv expressed
   at 16C, scFv expressed at 25C, scFv expressed at 30C and scFv-FcRnBp (NeoFv) expressed at 16C.
3. The proteins were **transferred to a PVDF membrane** and probed using an **anti-His primary antibody**. The blot was **imaged using
   chemiluminescence**.

**Results**
Bradford Assay showed the concentration of protein present in our sample. We plotted the data obtained to get the line of
best fit.

| Protein conc of standards (ug/mL) | Absorbance measured for standards |
| --- | --- |
| 0 | 0.397 |
| 156 | 0387 |
| 312 | 0.444 |
| 625 | 0.48 |
| 1250 | 0.51 |
| 2500 | 0.68 |
| 5000 | 0.78 |

**Equation of the line of best fit:** y = 0.00008x + 0.412

| Samples | Absorbance measured for samples | Protein conc calculated for samples from the equation of the line of best fit (ug/mL) |
| --- | --- | --- |
| scFv (16 degrees) | 0.803 | 4881.25 |
| scFv (25 degrees) | 1.128 | 8943.75 |
| scFv (30 degrees) | 0.706 | 3668.75 |
| scFv+linker (16 degrees) | 1.2 | 9843.75 |

![](https://static.igem.wiki/teams/4429/wiki/results/w15.png)

**Western blot**

The western blot **confirmed the expression of our proteins** by our chassis. We observed expected bands a little **above the 25kDa mark**, and the
**band for NeoFv was slightly retarded** compared to the scFv, indicating the **presence of the additional peptide linker**. We observed no
non-specific binding in the lane with the NeoFv, while the lanes with the scFv showed non-specific binding.

![](https://static.igem.wiki/teams/4429/wiki/resultsw/resultsw/igem.jpg)

Fig: Antibodies probed with His-antibodies showing expression. First three lanes are loaded with scFv and the last lane is scFv with the
FcRn binding peptide (labelled in this image as 'Linker').

## Thermal Shift Assay with SYPRO Orange:

**Aim**

To test for the **proper folding** of protein and its **melting temperature** produced by SHuffle B

**Experiment**

1. 5000x SYPRO Orange stock solution was added in **5x concentration** to the final sample. The dye was diluted accordingly. Buffers and
   protein were added to a 96-well plate; the dye was added last since it is light sensitive.
2. The well plate was put in an **RT-PCR machine** and a one hour run was started with **increasing temperatures**. The results we obtained are given
   below.

**Results**
We ran **three replicates** for NeoFv along with a control (purely dye without protein). Graphs showed that the protein was
**well-folded** and, averaging the replicates, **started to degrade at about 31.0℃**. This suggests that the protein is **stable in the given buffer**. To compare thermal stabilty of our protein, we would need to run the assay with different buffers.

**Future directions**

1. We must conduct the assay in **multiple buffers** to assay relative stability in them, and understand how the protein might behave in true
   in-vivo systems.
2. We could conduct **mutagenesis experiments** to increase the stability of the protein.

![](https://static.igem.wiki/teams/4429/wiki/results/w17.png)

![](https://static.igem.wiki/teams/4429/wiki/results/w18.png)

The raw data obtained from the Thermal Shift Assay can be found here -

[TSA Data](https://static.igem.wiki/teams/4429/wiki/results/sypro-orange-thermal-shift-assay-results.pdf)

## VLP fusion assay

**Aim**

To test for the **binding and neutralisation** of the EDE epitope with neoFv as a proof of concept for the functionality of our therapeutic.

**Experiment**

1. Vacuoles are **filled with dye** to concentrations so high that the **colour is extinguished**.
2. **Virus-like Particles** are added to the vacuoles and the **pH is lowered** to make the VLPs fuse with the vacuoles. This leads to the dilution
   of the dye, and increase in visible colour.
3. VLPs were incubated with the NeoFv, and were added to the vacuoles, with the expectation that a neutralising interaction between the
   two would **decrease the colorimetric readout**, due to the **NeoFv inhibiting membrane fusion**.

**Results**

1. The assay showed **positive results**, showing **significant
   inhibition of fusion at 5-fold dilution**. The control line at 90% fusion represents the
   experiment conducted with no antibody at all, and the NeoFv at 5X dilution
   brings it down to 33% fusion.
2. The percentage inhibition of fusion decreases from 57% to 5% beginning from 5X to 50000X dilution.
3. We were unable to achieve complete inhibition. Since the assay could
   not be optimised for our antibody in time, we cannot be sure of the reason.

**Future directions**

The next step would be to **standardise the assay**, and understand the details of the parameters that could
influence its results. The protein might not have been at optimum functionality due to inadequate shipment conditions.

![](https://static.igem.wiki/teams/4429/wiki/results/w19.png)

Fig: Graph showing neutralisation
efficiency of our antibody

## Design of Experiments (Wet Lab)

**Aim**

Dry lab and Wet lab worked in tandem to decide on a **set of factors** to evaluate the best growth conditions for our chassis - SHuffle B in
producing NeoFvs.

**Experiment**

**Triplicate SDS-Page gels** were run with varying concentrations of **Bovine Serum Albumin (BSA)** protein to calibrate the gels to estimate the
yield of our protein.

![](https://static.igem.wiki/teams/4429/wiki/results/w20-bsa-calibration.jpg)

Fig: SDS-PAGE set to calibrate our results with BSA

We ran **15 experiments** on three factors:

1. Temperature post-induction: We varied this **between 16C and 37C**. From our literature review, the ideal temperature and its
   interactions was bound to lie between these values.
2. IPTG concentration: Varying from **0.1 to 1mM**
3. Optical Density of the culture at induction: Varying from **0.5 to 1**

**Results**
After measuring the response variable yield, we found the most optimal condition in our sampling to be at **0.1mM IPTG, 26.5°C**
incubation temperature and induction at OD600 of 1.

![](https://static.igem.wiki/teams/4429/wiki/resultsw/doe-1-t3-ladder-c-0-15-0-7-2-3-7-8-9-11-12-13-labelled.jpg)

Fig: One of the triplicate gels for 8 experiments

![](https://static.igem.wiki/teams/4429/wiki/resultsw/doe-2-t1-ladder-c-0-15-0-7-1-4-5-6-10-14-15-results.jpg)

Fig: One of the triplicate gels for the rest of the experiments (7)

**Future Directions:**

1. A **Native PAGE** to gain information about our protein structure in terms of whether **polymerisation is taking place**, sub-unit interactions
   and quaternary structure.
2. A **phagocytosis assay** to determine **Fc effector functionality** of our proteins.
3. A **Transcytosis FcRn binding assay** to determine the **FcRn binding capacity** of our modified protein.

# Education and Communication

## Science fest

**Goals**

To connect with senior secondary students, fellow undergraduates and staff from our institute and around to inform them about dengue.

**Results**

1. We got an **immense response** from students, faculty and staff from the college in this initiative; many **actively participated** in donating
   blood.
2. We had an excellent opportunity to **connect with the masses** and speak with them regarding dengue. We also asked them to participate in
   taking our **‘Let’s Fight Dengue’ Pledge**.
3. We also had the opportunity to interact with the President of the Rotary Club, Pune, **Dr Shobha Rao**. She gave us insights on the health
   issues people face in the rural areas, especially vector borne diseases like Dengue.

**Future directions**

We look forward to **volunteering in more such events**, so as to **disseminate information** about dengue while
giving back to the community.

## School sessions (grades 5th to 10th)

**Goals**

To **connect with school students and teachers** across our town, and inform them about synthetic biology, immunology, and our
project AbDEN.

**Results**

1. We had an amazing opportunity to **interact with young school-going children** and introduce them to the world of SynBio.
2. We also had the chance to **make them aware of the dangers of Dengue** and how they can protect themselves against it.
3. We connected with an **all-girls high school** and discussed the gender gap with the children we met there.

**Future directions**

In the future, we plan to reach out to **parents with girl children** discuss the gender gap in STEM, to figure out
the measures we can take to combat it.

## A session with school science teachers

**Goals**

To **connect with upper primary and secondary grade teachers** across our town, and inform them about synthetic biology,
immunology, and our project AbDEN.

**Results**

1. We were able to discuss **pedagogical differences between school education and college education** and propose ways to **improve the former**.
2. We informed the teachers about the **challenges students face** due to **existing teaching methods** and how they can help alleviate the same.
3. We learnt about the various **issues faced by educators** because of the demands made by school authorities and parents.

**Future directions**

We want to speak with **school administrations and educational authorities** to discuss the pros and cons of
including synthetic biology in greater depth in school curricula, and to **improve the quality of science education** in general.

## Three webinars

**Goals**

To **engage undergraduate students** from institutes around the world through our webinar series.

**Results**

1. We held **two webinars** as part of our Immunotalks series and a **webinar in collaboration with MIT Mahe**.- We had a **great turnover** of students from across the world.
   - The webinar series was very popular, with the attendees asking the speakers a **lot of questions**.
   - We got an amazing opportunity to learn from the speakers - all giants within their fields.

**Future directions**

We plan on making a **regular webinar series** with distinguished members of various biological fields, along with
iGEMers who have worked within that realm, to have **enlightening conversations**.

## Sessions with orphaned children and specially-abled women

**Goals**

To reach out to **socially neglected groups** to learn about their experiences and to understand their perspective of the
problem we plan on addressing

**Results**

1. This venture helped us understand the problems that **different sections of society** face while tackling diseases like dengue.
2. It was a **humbling experience** to interact with people that had overcome hardships like these individuals.
3. We also learned about their **local knowledge on dengue** and the **traditional remedies** that they use.

**Future directions**

We hope to compile a **list of such traditional remedies** from across the country and **evaluate their scientific
accuracy** - to reliably inform people what they can trust and what they cannot.

## Activity book, card game, anti-dengue pamphlet and informative survey

**Goals**

To produce **tangible educational material** in the form of an activity book, a card game, a pamphlet and an informative
survey, all of which can be accessed through our wiki.

**Results**

1. We were able to create an **attractive and interactive activity book**, an **innovative and user-friendly card game**, an anti-dengue pamphlet
   in **seven regional languages**, and a survey designed to **disseminate knowledge** about Dengue translated into **five regional languages**.
2. We took our activity book and game to children from **different communities** across our town and **collected opinions** regarding the same.
3. We **distributed our anti-dengue pamphlets** across the country.
4. We **circulated our survey** across the country and beyond to **gauge the level of awareness** regarding dengue. We also approached several
   people from **varying socio-economic backgrounds**, and had them take our survey.

**Future directions**

We plan on making **further modules of our activity book**, and translate it into regional languages. We also want to
develop a **mobile app version of our card game** so it can have **added features** and is **easier to access** in today's world.

[Integrated Human Practices](#!)
[Dry Lab](#!)
[Wet Lab](#!)
[Education and Communications](#!)

{% endblock %}