Team:IISER-Pune-India/Results
Dry Lab
Designing of inhibitory peptides
The modelling team designed peptide inhibitors against various Plasmodium falciparum proteins that are essential for malarial invasion. More details about each interaction, their modelling methods and discussion can be found in the Model Results subpage.
Here we have summarised the modeling results.
The table gives the parasite-human protein-protein interaction and the designed peptide inhibitors' primary sequence against the corresponding parasite protein. Here we have listed them according to a priority based on the Molecular Dynamics simulation results and their FoldX interaction energy scores.
| Index | Interaction (PDB) | Peptides |
|---|---|---|
| 1 | The DBLb domain of PF11_0521 PfEMP1 interacting with human Intercellular Adhesion Molecule (ICAM) -1 (5MZA) |
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| 2 | Plasmodium falciparum reticulocyte-binding protein homologue 5 (PfRH5) interacting with human basigin (4U0Q) |
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| 3 | The CIDRa domain from MCvar1 PfEMP1 bound to human platelet glycoprotein 4- CD36 (5LGD) |
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| 4 | The CIDRa domain from HB3var03 PfEMP1 binding to endothelial protein C receptor (4V3D) |
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| 5 | Structure of the PfEMP1 IT4var13 DBLbeta domain interacting with ICAM-1 (6S8U) |
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In-silico grafting of peptides on to kalata- B1
The peptide sequences with high priority found for each interaction were grafted into the cyclotide Kalata-B1 using MODELLER and further computational studies were done on these grafted cyclotides to check for their stability.
More details about the protocol and the studies can be found under the Model Results subpage. The MD simulation results for these grafted cyclotides were not promising. The root mean square deviation plots of the simulations showed major conformational changes and the cyclic structure of the cyclotide backbone was lost during simulation. This could be due to the limitations in correctly modeling the grafted cyclotides.
Diagnostics
Through DeLeMa Detect we aimed to create a cost-effective, portable, and easy to use method to accurately diagnose malaria. Images of blood smears captured using a smartphone were tested for the presence of malaria parasites using a Web API making the diagnosis simple and efficient. For attaining the required magnification, a foldscope and a cost-effective centrifuge were used. The malaria detection software works on deep learning based on Convolutional Neural Networks (CNN). We could achieve an accuracy of 95.45% by training and testing on twenty-seven thousand images of blood smears which have been identified by experts to be positive or negative for the presence of malaria parasites. We have also made a User Manual for the detection kit.
Wet Lab
Due to the COVID-19 pandemic, we did not have access to labs until very recently, and thus we don’t have any significant or relevant wet-lab results to report. Our institute was fully closed and non-operational till August 2020, after which students returned in a staggered manner according to a priority order. Obtaining the necessary space and permissions, two members have been able to work in the lab only starting 20th September. Since the institute had been closed since March, we had to learn a lot of lab techniques in a very short period of time. This has been our best attempt to learn and work and compile the same. Below are the results of our cloning that we could achieve in this short time.
Various experiments that were designed to be performed in the lab and their details can be found on the Experiments Page.
The cloning of the Human Platelet Glycoprotein 4 (PDB ID: 5LGD) was achieved in 4 days after receiving the raw synthesised DNA sequences from IDT on 23rd October 2020.
Cloning (Human Protein-Platelet Glycoprotein 4)
This is the human protein component of the 5LGD interaction.
Preparation
Autoclaved tips
Autoclaved water
Prepared and autoclaved LB and LB agar and poured Kanamycin plates
Megaprimer PCR
Enzyme used: PFU DNA Polymerase
Lid: 105℃
| Step | Temperature | Time |
|---|---|---|
| 1 | 95℃ | 10:00 |
| 2 | 95℃ | 0:30 min |
| 3 | 55℃ | 0:30 min |
| 4 | 68℃ | 1:30 min |
| 5 | Repeat Step 2 x30 | |
| 6 | 72℃ | 10:00 min |
Conclusion:This PCR did not give the required results.
Gradient PCR
Enzyme used: PFU DNA Polymerase
Lid: 105℃
| Step | Temperature | Time |
|---|---|---|
| 1 | 95℃ | 10:00 |
| 2 | 95℃ | 0:30 min |
| 3 | 50/65℃ | 0:30 min |
| 4 | 68℃ | 1:30 min |
| 5 | Repeat Step 2 x30 | |
| 6 | 72℃ | 10:00 min |
Conclusion: None of the different annealing temperatures gave the required amplicon(1437bp).
Gradient PCR with
(a) A crowding agent i.e DMSO
Enzyme used: PFU DNA Polymerase
Lid:105℃
| Step | Temperature | Time |
|---|---|---|
| 1 | 95℃ | 10:00 |
| 2 | 95℃ | 0:30 min |
| 3 | 30/45℃ | 0:30 min |
| 4 | 68℃ | 1:30 min |
| 5 | Repeat Step 2 x30 | |
| 6 | 72℃ | 10:00 min |
(b) Lower temperature gradient with crowding agent.
Enzyme used: PFU DNA Polymerase
Lid:105℃
| Step | Temperature | Time |
|---|---|---|
| 1 | 95℃ | 10:00 |
| 2 | 95℃ | 0:30 min |
| 3 | 45/65℃ | 0:30 min |
| 4 | 68℃ | 1:30 min |
| 5 | Repeat Step 2 x30 | |
| 6 | 72℃ | 10:00 min |
(c)An Enzyme change - from Pfu to Paq5000
Enzyme used: PAQ5000 DNA Polymerase
Lid:105℃
| Step | Temperature | Time |
|---|---|---|
| 1 | 95℃ | 10:00 |
| 2 | 95℃ | 0:30 min |
| 3 | 45/65℃ | 0:30 min |
| 4 | 68℃ | 1:30 min |
| 5 | Repeat Step 2 x30 | |
| 6 | 72℃ | 10:00 min |
(a) Failed
(b) Failed
(c) The PCR worked for some of the temperatures (49.5℃, 53.8℃, 57.5℃)
Prep-PCR for gel extraction of Megaprimer(49.5℃, 53.8℃, 57.5℃)
Enzyme used: PAQ5000 DNA Polymerase
Lid:105℃
| Step | Temperature | Time |
|---|---|---|
| 1 | 95℃ | 10:00 |
| 2 | 95℃ | 0:30 min |
| 3 | 49.5,53.8,57.5℃ | 0:30 min |
| 4 | 68℃ | 1:30 min |
| 5 | Repeat Step 2 x30 | |
| 6 | 72℃ | 10:00 min |
Gel Purification
This was done using the NEB Purification Kit
Protocol for the same can be found here.
Obtaining the megaprimer:
Purified megaprimer of the desired length was obtained from the prep PCR (after gel extraction and purification). The concentration of the megaprimer was estimated to be 76.9 ng/uL using the Nanodrop method. A confirmatory gel was run to ascertain the length of the purified megaprimer which gave a positive result.
Restriction-Free PCR using the Megaprimer
Enzyme used: GXL DNA Polymerase
Lid:105℃
| Step | Temperature | Time |
|---|---|---|
| 1 | 95℃ | 10:00 |
| 2 | 95℃ | 0:30 min |
| 3 | 49.5,53.8,57.5℃ | 1:00 min |
| 4 | 68℃ | 8:00 min |
| 5 | Repeat Step 2 x37 | |
| 6 | 72℃ | 10:00 min |
Conclusion - The band in the first column indicates the final cloned plasmid with the gene coding for the Human Platelet Glycoprotein 4 (size - 6652bp). The second column is a NEB 1kb ladder. The third column is a control with an empty plasmid - it’s band indicates that it is in the supercoiled state. With these results, we have successfully cloned the Human Platelet Glycoprotein 4 gene into the pET28-a(+) plasmid vector.