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.

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

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.

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.

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.

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.

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.

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.

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.

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.



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.



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



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.



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.





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

TSA Data




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.


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.


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.



Fig: One of the triplicate gels for 8 experiments



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.
2. We had a great turnover of students from across the world.
3. The webinar series was very popular, with the attendees asking the speakers a lot of questions.
4. 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.