iGEM 2020 — Team IISER-Pune-India

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# Team:IISER-Pune-India/Notebook

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* [Project](/Team%3AIISER-Pune-India/)

  [Description](/Team%3AIISER-Pune-India/Description)
  [Design](/Team%3AIISER-Pune-India/Design)
  [Contribution](/Team%3AIISER-Pune-India/Contribution)
  [Notebook](/Team%3AIISER-Pune-India/Notebook)
  [Results](/Team%3AIISER-Pune-India/Results)
  [Future Implementation](/Team%3AIISER-Pune-India/Implementation)
* [Team](/Team%3AIISER-Pune-India/)

  [Members](/Team%3AIISER-Pune-India/Team)
  [Collaborations](/Team%3AIISER-Pune-India/Collaborations)
  [Attributions](/Team%3AIISER-Pune-India/Attributions)
* [Engineering](/Team%3AIISER-Pune-India/)

  [Engineering Success](/Team%3AIISER-Pune-India/Engineering)
  [Experiments](/Team%3AIISER-Pune-India/Experiments)
  [Measurement](/Team%3AIISER-Pune-India/Measurement)
  [Safety](/Team%3AIISER-Pune-India/Safety)
* [Modelling](/Team%3AIISER-Pune-India/)

  [Overview](/Team%3AIISER-Pune-India/Model)
  [Model Results](/Team%3AIISER-Pune-India/Model_Results)
  [Visualization](/Team%3AIISER-Pune-India/Models_NGL)
* [Diagnostics](/Team%3AIISER-Pune-India/)

  [Motivation](/Team%3AIISER-Pune-India/Motivation)
  [DeleMa Detect](/Team%3AIISER-Pune-India/Software)
* [Human Practices](/Team%3AIISER-Pune-India/)

  [Integrated Human Practices](/Team%3AIISER-Pune-India/Human_Practices)
  [Public Education and Engagement](/Team%3AIISER-Pune-India/Education)
* [Parts](/Team%3AIISER-Pune-India/Parts)
* [Judging Form](https://igem.org/2020_Judging_Form?id=3582)

* [January](#1)
* [February](#2)
* [March](#3)
* [April](#4)
* [May](#5)
* [June](#6)
* [July](#7)
* [August](#8)
* [September](#9)
* [October](#10)
* [Wet-lab Journal](#11)

# January

---

* **2nd January** - We finalised our team members and started brainstorming ideas.
* **18th January** - We had our first HP meeting in which we decided to take part in “Spread The Smile”(STS). It is a program organised by Disha, a social outreach club at IISER Pune.
* **20th-22nd January** - We made a module for STS. It included very basic scientific information along with a few activities to teach children about synthetic biology and antibiotic resistance.
* **30th January** - In the meeting with our PI and Faculty Advisor Dr Sanjeev Galande we discussed various possible project ideas and finalised one.

# February

---

* **7th February** - As a part of the activities that we had planned for the kids the whole team sat together for a few hours to make Tangrams.
* The team performed an extensive literature search for our then project idea regarding biolasers for the entirety of the month.

# March

---

* **23rd March** - In a team meeting all the team members along with PhD mentors decided to change the project idea due to lack of literature and resources available.
* **26th March** - Sponsorship meeting with Delta Faucet Company.
* **30th March** - Sponsorship meeting with Scivic Engineering India Pvt. Ltd.
* **31st March** - The team came up with the new idea of developing a drug against Malaria which later was finalised after a meeting with our PI.

# April

---

[Week - 1](#collapseTwo1)

* **2nd April** - Distribution of work and discussion regarding preparing the iBEC (Indian Biological Engineering Competition) proposal for sponsorship.

[Week - 2](#collapseThree2)

* **10th April** - Literature search for potential interactions to target.

[Week - 3](#collapseFour3)

* **19th April** - We had a HP meeting to discuss future plans and to decide on possible things we could do, which included building on our STS module to modify it for high school students, engaging the public through social media to create awareness about malaria, conducting a survey etc.
* **21st April** - According to their feasibility, the human and parasite protein interactions were shortlisted and different experimental methods were studied.
* **22nd April** - Meeting for deciding the possible experiments to perform.

[Week - 4](#collapseFive4)

* **23nd-25th April** - Designing the three modules for the proposal i.e.

+ **Module 1**: Cloning and biobrick
+ **Module 2**: Expression and purification
+ **Module 3**: Drug efficacy and toxicity check assay

* **25th April** - Confirmation meeting with mentors regarding all three modules.
* **27th April** - Submission of the iBEC proposal after validation from our PI.

# May

---

[Week - 2](#collapseThree12)

* **11th May** - We had a HP meeting to finalize a survey to gauge public awareness levels regarding malaria.
* **12th-16th May** - Making of the HP survey questions and conversion into an appropriate format.
* **13th May** - Collaboration meeting with team Aalto Helsinki. Talked about possible wet lab and drylab collaborations.

[Week - 3](#collapseFour13)

* **17th- 23rd May** - Continued work on the HP survey.
* **20th May** - Collaboration meeting with **[Team USP-Brazil 2019](https://2019.igem.org/Team%3AUSP-Brazil)**. Got us in touch with **[Team UPCH\_Peru 2020](https://2020.igem.org/Team%3AUPCH_Peru) since their team wasn’t going to be participating in this year’s competition.**
* We had our first meeting to decide upon the wiki.
* **21st May** - Tutorial on UCSF Chimera for dry lab members.
* **23rd May** - Collaboration meeting with UPCH Peru. Got us in touch with Dr Dionicia Gamboa, Peruvian parasitologist and associate professor at Institute of Tropical Medicine Alexander von Humboldt, Cayetano Heredia University. Her research focuses on *Plasmodium vivax*, major malaria parasite species in South America. (Here’s the link to her [article](https://www.who.int/news-room/feature-stories/tackling-malaria-hotspots-in-the-amazon).)

[Week - 4](#collapseFive14)

* **25th May** - Collaboration meeting with **[Team Düsseldorf 2020](https://2020.igem.org/Team%3ADuesseldorf)**. Discussed possible wet lab collaboration and the postcard project.
* **26th May** - Collaboration meeting with **[Team Tuebingen 2020](https://2020.igem.org/Team%3ATuebingen)** and got in touch with Dr Velavan from the Institute of Tropical Diseases in Tübingen, Germany.
* **27th May** - Started figuring out the use of Javascript for the wiki.
* **28th May** - Collaboration meeting with **[Team BITSPilani-Goa\_India 2020](https://2020.igem.org/Team%3ABITSPilani-Goa_India)**. Talked about possible collaborations. Helped us get in touch with Dr Shruti Vembar, faculty scientist at Institute of Bioinformatics and Applied Biotechnology (IBAB).

  On the same day we got sponsorship approval from Snapgene.
* **29th May** - Sponsorship: Geneious Prime.
* **31st May** - Meeting with Akashwani station director for a possible radio show.

# June

---

[Week - 1](#collapseTwo21)

* **2nd June** - We formed the diagnostics department.
* **3rd June** - We had a meeting with the HP members to discuss the radio show details.

[Week - 2](#collapseThree22)

* **8th June** - We had a meeting where we discussed work done by past iGEM teams and what methods we could use to build the diagnostics kit.
* **9th-14th June** - Radio show script writing and discussion.
* **10th-14th June** - Tutorials for biobrick building (snapgene, geneious, benchling).
* **15th June** - A rough layout of the landing page was decided.

[Week - 3](#collapseFour23)

* **15th-20th June** - Continuation of tutorials for biobrick building (snapgene, geneious, benchling).
* **15th-18th June** - Radio show script writing and discussion.
* **16th June** - Circulated the HP survey on different social media platforms.
* **21st June** - Started designing biobrick for 5MZA interaction.
* **21st June** - Meeting with Akashwani station director to finalize the radio show script.

[Week - 4](#collapseFive24)

* **22nd-23rd June** - Designing of biobrick for 5MZA interaction.
* **22nd-28th June** - Search for the precursors of host and parasite proteins involved in the interaction and building sample biobricks.
* **23rd June** - Started working on safety form. Made a preliminary draft for all sections.
* **24th June** - Meeting regarding safety issues with mentors.
* **25th June** - Finished with the final draft of the safety form and got it checked from our PhD mentors and PI.
* **26th June** - Submission of the preliminary draft of the safety form.
* **28th June** - The ‘Team Page’ was set up for the wiki.

# July

---

[Week - 1](#collapseTwo31)

* **1st July** - Had a discussion about whether to use a web based or an offline tool for the software to be developed for the diagnostics section of the project.
* **1st-4th July** - Deciding on the cyclotide i.e. choosing between Kalata B1 and MCoTI-II and addressing the issue of haemolytic activity of cyclotides.
* **29th June-5th July** - Inhibitor modelling and Saturated Mutagenesis done for 2-3 molecules. 22 new molecules were shortlisted from a possible list of 46 interactions from PDB.
* **29th June-5th July** - Scoring of inhibitory peptide models and saturated mutagenesis models using FoldX.

[Week - 2](#collapseThree32)

* **6th July** - Collaboration meeting with **[Team IISER\_Berhampur 2020](https://2020.igem.org/Team%3AIISER_Berhampur)**.

  Collaboration meeting with **[Team OhioState 2020](https://2020.igem.org/Team%3AOhioState)**. Possibility of them helping us out with drug disposal methods.
* **9th July** - Collaboration meeting with **[Team MSP-Maastricht 2020](https://2020.igem.org/Team%3AMSP-Maastricht)**, submitted an abstract of our project in Proceedings Journal.
* **6th-12th July** - Scoring of inhibitory peptide models and saturated mutagenesis models using FoldX.
* **5th-9th July**- Choosing the assembling technique.
* Started working on the malaria song.
* Started working on the promotion video.

[Week - 3](#collapseFour33)

* **12th-16th July** - In silico primer designing tutorials.
* **13th July** - MD Simulations for proteins started.
* **14th July** - Had a discussion about the cost of different magnification tools, particularly those that would enable us to take microscopic images using a smartphone camera.

[Week - 4](#collapseFive34)

* **18th-22st July** - Deciding on expression and purification techniques.
* **23rd July** - Recording of the radio show in the Akashwani Radio Station.
* **20th July** - Amended dry lab protocol was given out, obtained PARAM brahma credentials.

[Week - 5](#collapsesix35)

* **27th July** - Airing of the radio show which was broadcasted in Mount Abu, Rajasthan, India.
* **28th July** - Tutorial on how to run GROMACS on the supercomputer cluster by Tejashree Kanitkar, our PhD dry lab mentor and started working on dry lab presentation for the All India iGEM Meet.
* **25th-30th July** - Designing and deciding the experimental details of module 3 i.e. efficacy and toxicity checks.
* **31st July** - Attended the All India iGEM Meet and presented our project in it.

# August

---

[Week - 1](#collapseTwo41)

* **1st August** - Attended the All India iGEM Meet.
* **2nd-6th August** - Addressing the issue of circularization of cyclotides.
* **3rd-9th August** - MD simulations were run on the Param Brahma Cluster.
* **3rd August** - Second meeting with IISER Berhampur to discuss a possible collaboration.
* **3rd August** - Found a cheap and efficient way to take microscopic images using smartphones developed by Dr Manu Prakash for our diagnostic tool.
* **4th August** - Had a meeting with a senior from college, Gulzar to discuss a new homepage idea. This meeting helped us revamp the entire design of the frontpage. Also discussed Protein NGL viewer to have a 3-D structure of a protein on the wiki.
* **5th August** - Collaboration with **[Team Pittsburgh 2020](https://2020.igem.org/Team%3APittsburgh)**. We were invited as a guest on their Podcast.
* **9th August** - Finished training our first CNN (convolutional neural networks) model for diagnostics and obtained an accuracy of 94.5%.
* Illustrations for the website were made and finalized.

[Week - 2](#collapseThree42)

* **8th-14th August** - Grafting the inhibitory peptide sequence in cyclotide and designing the biobrick.
* **9th-15th August** - Script writing and preparation for webinar.
* **10th August** -Sponsorship: Reliance Industries Ltd.
* **10th-16th August** - MD simulations were run on the Param Brahma Cluster.
* **11th August** - Had a meeting to discuss updates and how to move forward with creating a diagnostics kit/tool.
* **13th August** - Collaboration meeting with TAS Taipei. Drylab discussions on image processing and analysis for the diagnostic tool.
* **12th-18th August** - Building biobricks for synthesizing host and parasite protein.
* **16th August** - Hosted a webinar in partnership with IISER Berhampur. It was an hour long webinar meant for students aged between 13-16 years.

  Frontpage of the wiki was finished.

[Week - 3](#collapseFour43)

* **17th-18th August** - Continuation of building biobricks for synthesizing host and parasite protein.
* **17th August** - Had a meeting and discussed additions to the NGL viewer in order to have a download feature for the PDB file of the protein.
* **18th-22nd August** -RFC criteria check.

[Week - 4](#collapseFive44)

* Started with write ups for wiki.
* **29th August** - Meeting with PI, Dr Sanjeev Galande for updates.
* **30th August** - As a part of collaboration with MSP Maastricht, we published a paper in a peer reviewed journal, Proceedings Journal.

# September

---

[Week - 1](#collapseTwo51)

* **2nd-6th September** - Documenting biobricks for 5MZA, 4V3E etc.
* **3rd September** - Meeting with Dr Shanti Kalipatnapu in order to get articles printed in newspapers for public engagement.
* **5th-6th September** - Attended the iGEMeetParis and presented our project.
* **6th September** - Team meeting
* Ordered DNA from IDT and Twist Bioscience.

[Week - 2](#collapseThree52)

* **7th-13th September** - Documenting biobricks for different interactions.
* **10th September** - Safety form was submitted.
* Finalised wiki subpage layout.
* Design team started working on designs and illustrations for protocols.

[Week - 3](#collapseFour53)

* **14th-15th September** - Documenting biobricks for different interactions.
* **19th September** - Therapeutics meet up organised by **[Team KCL\_UK 2020](https://2020.igem.org/Team%3AKCL_UK)**.
* **19th September** - Wet lab experiments began.
* Started uploading pages to the wiki.

[Week - 4](#collapseFive54)

* **20th-26th September** - Project title and abstract finalization.
* **22nd September** - First gradient PCR was run.
* **22nd September** - As a part of collaboration with iGEM IISER Berhampur, CCU iGEM Taiwan, members participated in a survey conducted.
* **23rd September** - Hindi conversion of the promotion video.
* **23rd September** - Second PCR was done.
* **25th September** - Uploaded the promotion video.
* **25th September** - Gel extraction using PCR -2 product.
* **28th-29th September** - Third PCR and Cell transformation.

# October

---

[Week - 1](#collapseTwo61)

* **2nd October** - Team meeting.
* **2nd October** - Colony PCR.
* **3rd October** - Gel electrophoresis, alkaline lysis and second transformation.
* **3rd October** - Received the foldscope.
* **3rd October** - Meeting with Mr. Abhishek Kanyal to discuss the analysis of IC50 assay.
* **1st-4th October** - 4V3E biobrick construction.
* **5th October**-Surveys in 13 different languages were circulated everywhere.
* **5th-9th October** - Making of Diagnostic kit user manual.
* **5th-10th October** - Documentation of Engineering success.
* **7th October** - Shared our promotion video on various social media handles.
* **9th October** - Two team members gave a short interview to Pune Mirror for a newspaper article that was published on 10th October.

[Week - 2](#collapseThree62)

* **10th October** - Presentation script writing.
* **10th-12th October** - Making of Diagnostic kit user manual.
* **11th October** - Shared the malaria song on our social media handles.
* **13th October**-Sent out the first review of the user manual of the diagnostic kit to PHC (Primary Healthcare) workers.
* **14th October** - Finalization of standardisation protocol of IC50 assay.
* **14th-16th October** - Uploading biobricks on registry.

[Week - 3 & 4](#collapseFour63)

* **17th-20th October** - Uploading biobricks on registry.
* **23rd October** -Interview with molecular cloud.
* **23rd October** - We received DNA from IDT.
* **23rd - 26th October:**

1. 1. A gradient PCR and a normal PCR for RF cloning was run.
2. 2. Transformation in e.coli dh5 alpha.
3. 3. Plasmid extraction.
4. 4. Re-transformation in E.coli bl21(de3).
5. 5. Protein expression.

# Wet-lab Journal

---

You can view our wet-lab journal [here](https://static.igem.org/mediawiki/2020/b/bd/T--IISER-Pune-India--Wetlab-journal.pdf).

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:IISER-Pune-India/Engineering

[![](https://static.igem.org/mediawiki/2020/a/a4/T--IISER-Pune-India--wiki-logo-red.png)](https://2020.igem.org/Team%3AIISER-Pune-India)

* [Project](/Team%3AIISER-Pune-India/)

  [Description](/Team%3AIISER-Pune-India/Description)
  [Design](/Team%3AIISER-Pune-India/Design)
  [Contribution](/Team%3AIISER-Pune-India/Contribution)
  [Notebook](/Team%3AIISER-Pune-India/Notebook)
  [Results](/Team%3AIISER-Pune-India/Results)
  [Future Implementation](/Team%3AIISER-Pune-India/Implementation)
* [Team](/Team%3AIISER-Pune-India/)

  [Members](/Team%3AIISER-Pune-India/Team)
  [Collaborations](/Team%3AIISER-Pune-India/Collaborations)
  [Attributions](/Team%3AIISER-Pune-India/Attributions)
* [Engineering](/Team%3AIISER-Pune-India/)

  [Engineering Success](/Team%3AIISER-Pune-India/Engineering)
  [Experiments](/Team%3AIISER-Pune-India/Experiments)
  [Measurement](/Team%3AIISER-Pune-India/Measurement)
  [Safety](/Team%3AIISER-Pune-India/Safety)
* [Modelling](/Team%3AIISER-Pune-India/)

  [Overview](/Team%3AIISER-Pune-India/Model)
  [Model Results](/Team%3AIISER-Pune-India/Model_Results)
  [Visualization](/Team%3AIISER-Pune-India/Models_NGL)
* [Diagnostics](/Team%3AIISER-Pune-India/)

  [Motivation](/Team%3AIISER-Pune-India/Motivation)
  [DeleMa Detect](/Team%3AIISER-Pune-India/Software)
* [Human Practices](/Team%3AIISER-Pune-India/)

  [Integrated Human Practices](/Team%3AIISER-Pune-India/Human_Practices)
  [Public Education and Engagement](/Team%3AIISER-Pune-India/Education)
* [Parts](/Team%3AIISER-Pune-India/Parts)
* [Judging Form](https://igem.org/2020_Judging_Form?id=3582)

* [Overview](#overview)
* [Research](#research)
* [Imagine](#imagine)
* [Design](#d)
* [Experiments](#e)
* [Assay](#a)
* [Learn](#l)
* [Improve](#i)
* [References](#references)

# Overview

---

Illustrated below is the pipeline of experiments that we plan to perform in the lab to prepare the cyclotide drugs and verify their efficacy.

![](https://static.igem.org/mediawiki/2020/b/b4/T--IISER-Pune-India--wet-lab-flowchart.png)

Since we had limited lab access for just a three week period due to the pandemic, we were unable to experimentally validate our system or pursue significant work in the lab. However, keeping in mind the engineering design cycle, we have thoroughly planned and designed all our experiments. We have considered the possible problems we might face through the course of the project and have come up with alternatives (in case of unexpected results) and troubleshooting protocols if faced with any failures in our experiments. To evaluate the outcomes and verify them, we have also made measurement standardization protocols for our Assays.

# Research

---

Our research began with looking up statistics on the prevalence of Malaria and drug resistance of the Malarial parasite. Over the past five decades, *Plasmodium falciparum*, the *Plasmodium* parasite which causes the deadliest form of Malaria in humans, has gained resistance against commonly used drugs like chloroquine, sulfadoxine, quinine and mefloquine.[1]

As of today, Artemisinin is the most potent anti-malarial drug. There is, however, growing evidence of Artemisinin resistance worldwide. Our last line of defence is Artemisinin Combination Therapy (ACT), and it’s failing fast, potentially making malaria untreatable in the years to come.[1]

We were aware that the *Plasmodium* parasite spends a part of its life cycle in RBCs. We thought of blocking potential interactions between RBCs and parasites so as to inhibit them from infecting RBCs. Targeting multiple interactions by creating multiple inhibitors could possibly overcome resistance as parasites begin to develop resistance to a particular drug.

The next question was "What system could we use as a delivery vector for these inhibitors?"

**[Team Heidelberg 2014](https://2014.igem.org/Team%3AHeidelberg)** had worked on the synthesis of cyclotides, which have previously been used as potential therapeutics for HIV and other diseases.[2] Upon further research, we found that cyclotides could be grafted with a peptide of choice to give them more stability.[3]

That’s how we came up with the idea for a cyclotide based drug. Cyclotides act as a scaffold or backbone for inhibitory peptides that target crucial host-pathogen interactions. They are robust, cost-effective, efficient, orally bioavailable and resistant to degradation, making them optimal candidates for drug design.[3]

# Imagine

---

To begin, we decided to study crucial interactions between the parasite and human RBCs proteins that mediated entry and infection, and accordingly design peptide-based inhibitors for said interactions.

One problem we faced was the fact that there are four species of *Plasmodium* that infect humans via mosquitoes.[5] After our interaction with Dr. Velavan (which can be found on our iHP page  [here](https://2020.igem.org/Team%3AIISER-Pune-India/Human_Practices)), we picked *Plasmodium falciparum* to work with, as it causes the deadliest form of Malaria.[5]

The basic idea was to design a short peptide which would act as a competitive inhibitor for a crucial interaction between the parasite and the human RBC proteins. Being small, the inhibitory peptides would cause minimal immunological reactions. This peptide would then be grafted into a cyclotide backbone, which would be used for delivery.

Cyclotides make an optimal drug delivery system as they are more resistant to heat than linear peptides and hence are easier to transport and require minimal cold storage facilities.[6] This stability at elevated temperatures comes in handy particularly in tropical countries that are the most affected, and it allows for easy and safe storage in rural areas where there is insufficient availability of cold storage facilities. Using cyclotides may also allow us to administer the drug orally.

In order to gauge the levels of public awareness about malaria, we decided to conduct a survey, the results of which were used to effectively design our human practices and public engagement strategy. In order to determine the problems faced by public health workers in the treatment and diagnosis of the disease and find ways to ameliorate them, we also contacted doctors, scientists and public health workers to learn more about the challenges faced by them in successfully eradicating Malaria.

* [Design](#d)
* [Experiments](#e)
* [Assay](#a)
* [Learn](#l)
* [Improve](#i)

* [Modelling](#d-m)
* [Biobrick](#d-b)

* [Design](#d-m-d)
* [Build](#d-m-b)
* [Test](#d-m-t)
* [Learn](#d-m-l)
* [Improve](#d-m-i)

We realised that to design a suitable inhibitor, we would require a computational approach, in order to shortlist possible interactions we could target. We contacted Dr Madhusudhan MS of IISER Pune and he generously offered to help us, and along with Tejashree Kanitkar, a PhD student from his lab, we designed the basic layout of our dry lab and modelling experiments.

Our modelling approach has the following steps:

1. Extraction of available data from the Protein Data Bank[7] and further analysis and shortlisting of potential PDB IDs that could be used in our project
2. Identification of the interacting regions of the human protein and the parasite protein.
3. Computational mutagenesis of the interacting region
4. Scoring the mutants and doing some advanced studies for stability in silico

We started with extracting data from the Protein Data Bank using advanced search and a custom web-parser script based on python and found over 400 hits which we narrowed down to 47 interactions.[8] We did literature studies and shortlisted 5 interactions to work with. We performed computational saturation mutagenesis for the identified interacting region using a custom python script built on top of Chimera and obtained mutants.[8] The mutants were scored based on their interacting energy using FoldX.[9] We shortlisted 3 mutant interactions with most negative interaction energy for further studies.

We ran Molecular Dynamics simulations for these inhibitors to study their interaction in silico. We got access to the PARAM BRAHMA facility under the National Supercomputing Mission, Government of India at IISER Pune for our computational needs. We performed the simulations as described in the [Modelling](https://2020.igem.org/Team%3AIISER-Pune-India/Model) page of our project and analysed the results.

We found that some inhibitors did not interact with the parasite protein as expected. One reason may have been the small size which leads to structural instabilities. From the iBEC[10] proposal feedback we also learnt that the PfEMP1 interaction which we had targeted so far is highly variable. We also faced problems in doing homology modelling of cyclic peptides.

From our results, we realised that we needed to search for more non-variable interactions of the plasmodium and the human proteins. We analysed how the distance between the centroids of the interacting proteins evolved over time during the MD simulations and the intermolecular hydrogen bonds between them. As for the homology modelling for cyclic peptides, we contacted Dr David Norman, College of Life Sciences, University of Dundee, for help and he generously provided us with tutorials to build a cyclic peptide using homology MODELLER.[11]

* [Design](#d-b-d)
* [Build](#d-b-b)
* [Test](#d-b-t)
* [Learn](#d-b-l)
* [Improve](#d-b-i)

We conceptualized cyclotides to contain inhibitory peptides in a suitable loop to competitively inhibit the host-parasite interactions of interest. After extensive literature review, we finalised on the cyclotide Kalata B1 which is 29 amino acids long with 6 loops divided by 3 cysteines as a knot. Based on *in-silico* modelling, we decided on the insert sequence for potential competitive inhibition (in loop 6) to graft in pET28-a(+) vector.

We also planned to insert host and parasite proteins into the same vector to express and study their interaction in vitro and the effect of the cyclotide inhibitor on their binding affinity. For tagging the parasite and host proteins we planned to use polyhistidine and Strep tags respectively. This differential tagging is to prevent the host protein from non-specifically interacting with the stationary phase (Ni-NTA) and yielding a false positive interaction with 6xHis-tagged parasite protein immobilized on the stationary phase.

We obtained the cyclotide Kalata B1 precursor (OAK1) and competitive inhibitor sequences from the modelling team. We built 1-3 composite biobricks depending on the potential inhibitors. Codon-optimised inhibitors were grafted into loop 6 of OAK1 and loop 3 was replaced with the Strep tag for purification. We also obtained wild type protein sequences from PDB to build basic Biobrick parts.

**Building the inhibitor construct:**

The sequence of nucleotides from the start to the stop codon was removed and the cyclotide construct containing the inhibitory peptide was inserted into the plasmid. A similar procedure was followed for the host and parasite protein.

Our Biobricks were built on the Benchling platform. Further analysis was done on various softwares including Geneious Prime, Snapgene and Benchling to check for RFC 10 criteria and this was cross checked with our mentors. We tested our Biobricks for the ORFs. The initial codon optimisation was done using the GeneSmart tool of genscript and was tested again with codon optimisation tool in Benchling for better results.

While building Biobricks we encountered various problems. We learned that Biobricks for the split Intein approach needed to be tested for at least 3 loops. Even though we grafted our inhibitors in loop 6, the problem of circularisation still persisted. Another issue that we came across after building the ORF was breaking at both ends of the insert sequence. This problem occurred due to the 7th general nucleotide(N) present at the end of the BSA restriction site i.e (GGTCTCN).

To solve the circularisation issue we took the circularisation construct (BBa\_K1362000) and removed the RFP gene from it. We then grafted the Cyclotide KB 1 sequence into the construct which was subsequently grafted into pET 28-a(+). We made sure that ORFs were on a positive strand. The seventh nucleotide was removed from both strands to complete the ORF.

* [Design](#e-d)
* [Build](#e-b)
* [Test](#e-t)
* [Learn](#e-l)
* [Improve](#e-i)

Designing the experiments was the complete second module of our project. During the synthesis of peptides in the lab we aimed to perform a wide variety of experiments from cell cultures, PCRs, gel electrophoresis, mass spectroscopy, and microscopy to accomplish cloning of the desired sequences, expression and purification of proteins. It would also involve a few experiments to validate the results such as SDS-PAGE.

For cloning we would use Gibson assembly techniques. Once cloning is successful, the next step is the expression of the cloned plasmid into the vector. Protein purification would be done by expressing Strep tag for the Host protein and 6Xhis tag for parasite proteins. The purified protein would then be run over SDS-PAGE to check for its purity.

The required plasmid was obtained from the lab and the gene of interest was ordered from IDT with the primers designed using Gibson. Once all the sequences are received, follow the Gibson assembly steps detailed in the Experiments Section.

Protein gene overexpression would be done in E.coli BL21(DE3) strain using the T7 promoter system. The module 2 of experiments involves Purification by 6xHis tag and Strep tag. 6xHis tag purification is done by preparation of lysates after cytoplasmic expression of His-tag protein under native conditions followed by batch purification under native conditions. Purification of strep tag proteins is done by gravity flow columns.

[Experiments Page](https://2020.igem.org/Team%3AIISER-Pune-India/Experiments)

Cloning would be tested by Single restriction digestion based on length and number of fragments in reaction and PCR amplification using gibson primers and sequencing of the cloned plasmids. The positive control should yield ampicillin resistant colonies when transformed into E.Coli. Transformed E.coli BL21 DE3 culture for incubation and purified protein would be run over SDS-PAGE analysis.

[Experiments Page](https://2020.igem.org/Team%3AIISER-Pune-India/Experiments)

During planning of the experiment design we also came up with the problems we might face while performing the experiments. The protein might not get expressed, it might be degraded or there could be insoluble protein in the column.

General difficulties one might encounter in purification are no, or weak binding of proteins to the strep tag column, contamination of proteins, protein precipitation during purification. We have also listed possible solutions for the above problems.

If we face any problems in Gibson cloning we have considered homologous cloning as an alternative which is very similar to Gibson. For the detailed troubleshooting of the individual sections refer to the respective experiments section on the project page.

[Experiments Page](https://2020.igem.org/Team%3AIISER-Pune-India/Experiments)

* [Build](#a-b)
* [Test](#a-t)
* [Learn](#a-l)
* [Improve](#a-i)

After designing the drug, another essential step in our project would be to test its efficiency in combating the disease. Assay building is a molecular biology procedure for testing or measuring a drug's activity in an organic sample. Various molecular biology experiments and assays can be performed to test, verify, and incorporate the computational predictions. The goal is to select a method that requires the least amount of manipulation and pretreatment of the samples to accommodate substances that intervene with the assay.

In our project, the main focus was to develop experiments for testing the interaction between inhibitory drugs and the binding epitomes of the plasmodium proteins.

The following assays were employed for testing competitive binding of the designer cyclotide:

1. ##### Solid-phase protein interaction assay

   *In vitro* by performing affinity chromatography on a Ni-NTA column with the parasite protein immobilized to it and flowing the host protein through it with and without the cyclotides. The difference between the amount of protein left in the supernatant as compared to what was added indicates the bound fraction. This method assesses specific protein-protein interactions at physiological conditions, utilizes relatively small amounts of protein, is free of protein modification, does not require specialized instrumentation and results in quantitative measurements.
2. ##### In vivo in *Plasmodium falciparum* parasite cultures

   * **Half maximal inhibitory concentration (IC50) assay** is commonly used to measure the potency of a drug in pharmacological research. IC50 is a measure of the potency of a substance in inhibiting any specific biological or biochemical function. This assay is fast, relatively inexpensive with no involvement of radioactive isotopes. Due to high affinity towards DNA ,the assay is very sensitive and IC50 values of known drugs obtained were comparable.
   * **RBC invasion assay** is a flow cytometry-based assay for measuring invasion of RBC by *P. falciparum* merozoites. Although comparatively expensive, the key advantage of flow cytometry is that a very large number of particles can be evaluated in a very short time.

[Experiments Page](https://2020.igem.org/Team%3AIISER-Pune-India/Experiments)

#### Assays Standardization

Standardization aims to ensure that assays of the same analyte in the same samples, done at different places or at different times or both, are readily comparable. Standardization is especially desirable because all external quality assessment surveys have shown that this type of method involves a much greater variability than traditional assays. In our project, we have three assays. The RBC invasion assay is a flow cytometry-based assay for which the general standardization protocol is available in [IGEM’s standard measurement protocols](https://2020.igem.org/Measurement/Protocols). We will contribute the standardization protocols for Solid-phase protein interaction assay and IC50 assay to the same as a part of our project.

Assays can yield unexpected or failing results for a variety of reasons. Even simple assays include multiple steps where mistakes are likely to happen, which significantly increases the difficulty of finding the error sources.

Some potential error sources could include: incorrect, contaminated or expired reagents, equipment error, technician error, and many more. Therefore, resolving the cause of unexpected results is often a time consuming and expensive task in laboratory research. After studying the protocols for each assay in detail, we found some possible errors listed below.

1. **Solid-phase protein interaction assay**

   We need a particular concentration of proteins for proceeding to protocol 3 in this assay. But we might end up getting different concentration values for the bicinchoninic acid (BCA) assay.This has to be changed to the needed concentration for accurate results.
2. **Half maximal inhibitory concentration (IC50) assay**

   Due to handling errors, we may get less growth in the zero drug well than in any well with some drug concentration. This might be due to the addition of more cells in a well or more dye in a well.Also,the solvent we use might contribute to the inhibition effect.Our results may contain some background noise which have to be deducted from readings for more accuracy.
3. **RBC invasion assay**

   Treating infected erythrocytes with α-2-3,6,8– Vibrio cholera neuraminidase trypsin and chymotrypsin can give us unexpected results as these three chemicals can block invasion completely. Because of lack of standardization, there is a chance of FACS not giving promising results.Usage of sorbitol in synchronisation of ring stage in infected erythrocytes can affect reinvasion.

One mode of decreasing assay failure is to ensure equipment errors are removed or at least minimised. This may be done by optimizing the equipment employed before running the assay. Analyzing the possible error sources that can hinder our assay process, we came up with some precautions and troubleshooting techniques that can be adapted to modify these assays.

1. **Solid-phase protein interaction assay**

   Depending on what concentration we get in the BCA assay, we will increase or decrease to attain the concentration we need.
2. **Half maximal inhibitory concentration (IC50) assay**

   To avoid handling errors,this experiment is done in triplets and their average is taken. RPMI and DMSO control are done to check whether the drug solvent is responsible for any inhibition effect or not. DMSO is the drug solvent. If the fluorometer readings of DMSO control and RPMI control are similar then that means that the solvent is not contributing to the inhibition effect.A blank control with no parasites can be performed to deduct the background noise from all readings.To check if the proposed drug acts as an inhibitor we first select a broad drug concentration range and if the IC50 value is obtained then the experiment is performed again with a narrow drug concentration range to get a more accurate value.
3. **RBC invasion assay**

   Treatment of ring stage infected O+ erythrocytes with a solution of α-2-3,6,8– Vibrio cholera neuraminidase, trypsin and chymotrypsin can be done using lower concentrations and the relative effectiveness of different peptide inhibitors can be studied.If FACs fail to give promising results, fluorometry based analysis can be performed using a spectrophotometer, which will be speedier as well. To avoid sorbitol treatment as much as possible, fresh parasite cultures can be revived to grow in a synchronized manner for the initial cycles.

We developed each inhibitor sequence after an intensive computational analysis of each interaction. Although, in the worst scenario, there might come a time when these inhibitors fail. We targeted crucial interactions between the host and parasite protein that would have the greatest possibility of successfully preventing entry and infection of the Plasmodium parasite.

Directed Evolution(DE), a method used in protein engineering that mimics natural selection, can be used as our last expedient if our inhibitors fail to provide results. DE generates random mutations in the gene of interest and can be used as an alternative to rationally designing modified proteins. Site-directed mutagenesis(SDM) is an invitro method used in DE for creating a specific mutation in a known sequence. In our project, we can use this method to select or screen for mutations with the desired property of inhibiting our studied interactions. The simplest and most broadly applicable SDM protocol is the QuickChange Site-Directed Mutagenesis System (QCM) developed by Stratagene, which we plan to use in our project.

[Experiments Page](https://2020.igem.org/Team%3AIISER-Pune-India/Experiments)

# References

---

1: Fairhurst, R. M., & Dondorp, A. M. (2016). Artemisinin-Resistant *Plasmodium falciparum* Malaria. Microbiology Spectrum, 4(3).
DOI: [10.1128/microbiolspec.ei10-0013-2016](https://doi.org/10.1128/microbiolspec.ei10-0013-2016)

2: Gould A, Camarero JA. Cyclotides: Overview and Biotechnological Applications. Chembiochem. 2017;18(14):1350-1363.
DOI: [10.1002/cbic.201700153](https://doi.org/10.1002/cbic.201700153)

3: Gould A, Ji Y, Aboye TL, Camarero JA. Cyclotides, a Novel Ultrastable Polypeptide Scaffold for Drug Discovery. *Curr Pharm Des.* 2011;17(38):4294-4307.
DOI: [10.2174/138161211798999438](https://doi.org/10.2174/138161211798999438)

4: Dondorp, A. M., Nosten, F., Yi, P., Das, D., Phyo, A. P., Tarning, J., Lwin, K. M., Ariey, F., Hanpithakpong, W., Lee, S. J., Ringwald, P., Silamut, K., Imwong, M., Chotivanich, K., Lim, P., Herdman, T., An, S. S., Yeung, S., Singhasivanon, P., … White, N. J. (2009). Artemisinin Resistance in *Plasmodium falciparum* Malaria. *New England Journal of Medicine*, 5, 455–467.
DOI: [10.1056/nejmoa0808859](https://doi.org/10.1056/nejmoa0808859)

5: WHO | Malaria. (n.d.). WHO | World Health Organization. Retrieved October 20, 2020, from [www.who.int/ith/diseases/malaria/en/](https://www.who.int/ith/diseases/malaria/en/)

6: Gould, A., Ji, Y., L. Aboye, T., & A. Camarero, J. (2011). Cyclotides, a Novel Ultrastable Polypeptide Scaffold for Drug Discovery. *Current Pharmaceutical Design*, 38, 4294–4307.
DOI: [10.2174/138161211798999438](https://doi.org/10.2174/138161211798999438)

7: Bank, P. D. (n.d.). *RCSB PDB: Homepage*. Retrieved October 11, 2020, from [www.rcsb.org](https://www.rcsb.org/)

8: igemsoftware2020. (n.d.). *GitHub - igemsoftware2020/IISER-Pune-India: Inhibitor peptides against falciparum Malaria and a Deep learning Web API for Malaria Diagnosis*. GitHub. Retrieved October 20, 2020, from [github.com/igemsoftware2020/IISER-Pune-India](https://github.com/igemsoftware2020/IISER-Pune-India)

9: Schymkowitz, J., Borg, J., Stricher, F., Nys, R., Rousseau, F., & Serrano, L. (2005). The FoldX web server: an online force field. *Nucleic Acids Research, Web Server*, W382–W388.
DOI: [10.1093/nar/gki387](https://doi.org/10.1093/nar/gki387)

10: *Indian Biological Engineering Competition (iBEC) PRE- iGEM Competition* | Department of Biotechnology. (n.d.). Home | Department of Biotechnology. Retrieved October 20, 2020, from [dbtindia.gov.in/schemes-programmes/building-capacities/awards/indian-biological-engineering-competition-ibec-pre](http://dbtindia.gov.in/schemes-programmes/building-capacities/awards/indian-biological-engineering-competition-ibec-pre)

11: Webb, B., & Sali, A. (2014). Comparative Protein Structure Modeling Using MODELLER. *Current Protocols in Bioinformatics*, 1, 5.6.1-5.6.32.
DOI: [10.1002/0471250953.bi0506s47](https://doi.org/10.1002/0471250953.bi0506s47)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:IISER-Pune-India/Experiments

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* [Project](/Team%3AIISER-Pune-India/)

  [Description](/Team%3AIISER-Pune-India/Description)
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  [Contribution](/Team%3AIISER-Pune-India/Contribution)
  [Notebook](/Team%3AIISER-Pune-India/Notebook)
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  [Future Implementation](/Team%3AIISER-Pune-India/Implementation)
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* [Engineering](/Team%3AIISER-Pune-India/)

  [Engineering Success](/Team%3AIISER-Pune-India/Engineering)
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* [Modelling](/Team%3AIISER-Pune-India/)

  [Overview](/Team%3AIISER-Pune-India/Model)
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* [Diagnostics](/Team%3AIISER-Pune-India/)

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* [Judging Form](https://igem.org/2020_Judging_Form?id=3582)

Gibson

RF Cloning

Expression

6xHis-tag

Strep-tag

SDS-PAGE

Ni-NTA

IC50

RBC Invasion Assay

PCRs

Site Directed Mutagenesis

## Gibson's Assembly Protocols

### Supplies

* 15 μL aliquots or a 2X stock of Gibson master mix
* Tubes in both a 96 well holder
* 50 mL tube
* Sterile, nuclease-free water
* Positive control: Positive Control DNA Mix (see below)
* Primer Design using Gibson

### Protocol

1. Use PCR to produce the DNA segments needed for assembling the new construct.
   Generally, it is best to use a high fidelity polymerase, such as Phusion, to amplify the
   Gibson fragments.
2. Confirm the success of each PCR by running 5 μL of the reaction on an agarose gel.
3. Mix 10 ng-100 ng of each of the DNA fragments together (such that their ratios are equimolar)
   into a total volume of 5 μL.
4. If using the 2X Gibson Master Mix from NEB, add 10 μL of total DNA (containing all the
   fragments) to 10 μL of the mix. If using the homemade Gibson mix (refer to the recipe at the
   bottom of this page), add 5 μL of DNA to 15 μL of mix. Be careful with pipetting small
   volumes.
5. Mix well by pipetting.
6. Incubate the reaction at 50°C for 1 hour.
   For each Gibson reaction, perform two transformations.
7. Perform a 1:4 dilution of the Gibson reaction in nuclease-free water, add 4 μL to 50 μL of
   chemically competent cells.
8. Add 4 μL of the Gibson reaction to 50 μL competent cells without dilution.
    (Note: If
   electroporating, dilute the reaction 1:5 in water or PCR purify the reaction prior to
   transformation.)
9. Follow transformation protocol for chemically competent cells.
10. Run an aliquot of the final reaction on a gel to verify the presence of your construct. It
    may be useful to make a 'no incubation' control to determine if the chemistry of the
    reaction happened.

### Expected Results

The positive control should yield ampicillin resistant colonies when transformed into E. Coli.
Depending on the number of fragments in the reaction, different efficiencies will be observed.
Gibson reactions with a higher number of fragments tend to be less efficient, therefore there
will likely be few colonies on the plates.

### Positive Control

Competency of cell was tested by two fragment assembly of pUC 19 plasmid. Positive control mix is obtained by PCR amplification of Fragment 1 and Fragment 2.

Use the following reaction mixture

|  |  |
| --- | --- |
| 32.7 μL | dH2O |
| 10 μL | 5X HF Buffer |
| 1 μL | 10uM dNTP |
| 2.5 μL | 10uM FW primer |
| 2.5 μL | 10uM RV primer |
| 1 μL ~3ng/μL | pUC19 template |
| 0.3 μL | Phusion Polymerase |

Use the following PCR program for both F1 and F2 PCRs

|  |  |
| --- | --- |
| 98°C | 0:30 |
| 58°C | 0:30 |
| 72°C | 0:45 |
| Go to step 2, 25X |  |
| 72°C | 10:00 |

11. After the reaction is completed, add 1 μL of Dpn1 (20,000 U/mL) to each reaction, briefly
    vortex and spin down the reactions, and then incubate in the PCR machine at 37°C for 30
    minutes.
12. To test the effectiveness of Dpn1, construct the above PCR reaction without Phusion
    polymerase, digest and purify it in parallel with the F1 and F2 PCRs, and transform it
    alongside the other reactions.
13. Any ampicillin-resistant transformants produced from transforming this negative reaction
    indicate that the Dpn1 digest was ineffective at digesting the pUC19 template. The solution
    to this is to use fresh Dpn1 in the digest, or to gel extract F1 and F2 instead of using a
    Dpn1 digest and column purification to remove the pUC19 template.
14. Test the success of the PCR by gel electrophoresis and purify the reaction using a PCR
    cleanup kit, and elute with buffer or water without EDTA.
15. Quantify the concentration of F1 and F2 purifications.
16. Combine F1 and F2 PCR products to a final concentration of 2.8ng/μL of each fragment.
17. Dilute with the elution buffer used in the PCR purification if needed. This final mixture is
    the Positive Control DNA Mix.
18. Using 5 μL of this reaction provides approximately 1ng/100bp of each fragment in the Gibson
    Assembly reaction.

### References

1: [Barrick Lab Protocol Manual](https://barricklab.org/twiki/bin/view/Lab/ProtocolsGibsonCloning)

## Outline

* Design complementary hybrid primers.
   These primers should be complementary to both the desired insert and the destination vector.
* ↓
* Perform the first round of PCR.
   This creates a ‘mega-primer’ which comprises the insert sequence flanked by sequences complementary to the destination vector.
* ↓
* Perform the second round of PCR.
   In this the mega-primer initiates replication of the destination plasmid.
   [The new product obtained is an nicked circular plasmid.]
* ↓
* Selectively degrade parental vectors using DpnI.
* ↓
* Transform the product plasmid into competent cells.

## Procedure

### Buffer Compositions

* **Cold lysis buffer:** 50 mM Tris,300 mM NaCl, 20 mM imidazole, pH 8.0
* **Elution buffer:** 50 mM Tris, 600 mM imidazole, pH 7.0

1. PCR
   1. PCR 1 (Mega Primer Formation)
      1. Run for 5 min at 95°C
      2. Repeat 35x:
         1. 30s at 95°C
         2. 30s at annealing temperature (as per gradient PCR)
         3. 1 min/1 kb template at 68°C
      3. Run for 10 min at 72°C
      4. Run PCR products on an agarose gel and purify it using a PCR purification kit.
      5. Dilute the final concentration to 100 ng/μl.
   2. PCR 2 (RF Cloning PCR)
      1. Run for 5 min at 95°C
      2. Repeat 35x:
         1. 30s at 95°C
         2. 30s at the temperature as per the Tm of the primers
         3. 1 min + 1 min/1 kb template at 68°C
      3. Run again for 10 min at 72°C.
      4. Run PCR products on an agarose gel.
      5. Digest the PCR products using REase DPN1 at 37℃ for 2 hrs.
      6. Transform the product into ultracompetent E.coli cells.
2. Protein Expression
   1. Perform test expressions using BL21(DE3) cells induced with 1 mM IPTG in early log phase.
   2. Harvest 10 mL of cells, after 4h, at 37°C and lyse in a 600 μL cold lysis buffer, in the presence of lysozyme and DNaseI.
   3. Lyse cells by sonication and pellet cell debris for 7 min at 14 krpm, at 4°C.
   4. Wash supernatant bound to Ni2+-NTA spin column, 3 times with 600 μL cold lysis buffer and elute it in 200 μL elution buffer.
   5. To discriminate between protein insolubility and absence of expression, use a Ni2+-NTA spin column with the same buffers supplemented with 8 M urea.

## References

1: Bond SR, Naus CC. RF-Cloning.org: an online tool for the design of restriction-free cloning projects. Nucleic Acids Res. 2012 Jul;40(Web Server issue):W209-13. DOI: [10.1093/nar/gks396](https://doi.org/10.1093/nar/gks396). Epub 2012 May 8. PMID: 22570410; PMCID: PMC3394257.

2: van den Ent F, Löwe J. RF cloning: a restriction-free method for inserting target genes into plasmids. J Biochem Biophys Methods. 2006 Apr 30;67(1):67-74. DOI: [10.1016/j.jbbm.2005.12.008](https://doi.org/10.1016/j.jbbm.2005.12.008). Epub 2006 Feb 3. PMID: 16480772.

## **Expression**

### Reagents

| Recommended Buffers/Solutions | Concentration of Ingredients | Notes |
| --- | --- | --- |
| Kanamycin | Stock solution: 50 µg/mL in H2O, sterile filtered | Store in aliquots at -20°C |
| Buffer W | * 100 mM Tris/HCl * 150 mM NaCl * 1 mM EDTA * Adjust pH to 8.0 |  |
| LB Medium | * 10 g/l trypton * 5 g/l yeast extract * 5 g/l NaCl |  |
| Glucose | 20%, sterile filtered |  |
| IPTG | Stock solution (1M): 238 mg/mL in H2O, sterile filtered | Store in aliquots at -20°C |
| 5x SDS-PAGE sample buffer | * 0.250 M Tris/HCl * 25% glycerol * 7.5% SDS * 0.25 mg/mL bromophenolblue * 12.5 % v/v mercaptoethanol * Adjust pH 8.0 |  |

### Equipment

* Culture Plates
* Pipettes
* Glassware
* Microfuge
* Centrifuge

### Protocol

1. Preculture: Inoculate 2 mL of LB medium containing 50 μg/mL Kanamycin with a fresh colony
   harbouring the pET28a expression plasmid and shake overnight (200 rpm) at 37°C.
2. Culture for expression: Inoculate 100 mL of LB medium containing 50 μg/mL Kanamycin with the
   preculture and shake at 37°C.
3. Monitor the optical density at 550 nm (OD550). Cell suspension with an OD550 over 0.3 should
   be diluted with LB medium before measuring.
4. Take a 1 mL sample immediately before induction.
   This sample is the non-induced control; pellet cells (microfuge, 30 seconds) and resuspend
   them in 80 μL Buffer W. Add 20 μL 5x SDS-PAGE sample buffer. Store at -20°C until SDS-PAGE
   analysis. The whole sample must be incubated in an ultrasonic bath for 15 minutes to shear
   the chromosomal DNA into small pieces and should be heated to 70°C for 10 minutes prior to
   SDS-PAGE.
5. When OD550 equals 0.5-0.6, add 50 μL of IPTG stock solution (0.5 mM end concentration).
6. Shake for 3 hours at 200 rpm.
7. Harvest the cells by centrifugation at 4500 x g for 12 minutes (4°C). Add 5 μL 5x SDS-PAGE
   sample buffer to 20 μL supernatant and store at -20°C for SDS-PAGE analysis.

### Troubleshooting

| Problem | Comments and Suggestions |
| --- | --- |
| No or low expression | * Check the culture condition (e.g., IPTG, anhydrotetracycline, antibiotics). * Check vector (sequence, frame). * Check whether the protein is found in the insoluble fraction. Reduction of   temperature during cultivation may solve this problem. (e.g., 16°C, 22°C,   26°C, 30°C) * Use another expression system. Use eukaryotic cells for expression (yeast,   insect or mammalian cells). |
| Protein is degraded | * Use protease deficient E. coli strains. * If degradation occurs during cell lysis, add protease inhibitor. * If the protein is small (<10 kDa), consider adding a terminal carrier   protein. * Lowering the temperature during induction can reduce the problem. * Secretion of the recombinant protein to the periplasmic space can reduce the   problem. |
| Protein is secreted | Remove all signal sequences from the coding region. |
| Inclusion bodies are formed: protein is insoluble | * Reduce expression level by modifying growth and induction conditions, e.g.,   lower culturing temperature (16°C, 22°C, 26°C, 30°C). * Use another expression system (e.g., Tet promoter instead of T7 promoter). |

### References

1. [www.rsc.org/suppdata/cc/c0/c0cc01704c/c0cc01704c.pdf](http://www.rsc.org/suppdata/cc/c0/c0cc01704c/c0cc01704c.pdf)

## Purification of the Proteins using 6xHistidine-tag

![](https://static.igem.org/mediawiki/2020/f/f1/T--IISER-Pune-India--strep-his-comparison.png)

### Reagents

| Buffers/Solution | Concentration of Ingredients |
| --- | --- |
| Ni-NTA lysis buffer | * 20 mM HEPES (pH 7.4) * 150 mM NaCl * 1 mM PMSF |
| Lysozyme |  |
| 5x SDS-PAGE sample buffer | * 0.25 M Tris/HCl * 7.5% SDS * 25% glycerol * 0.25 mg/mL bromophenol blue * 12.5% v/v mercaptoethanol * Adjust pH 8.0 |
| Ni-NTA Lysis Buffer | * 20 mM HEPES (pH 7.4) * 150 mM NaCl * 1mM PMSF |
| Ni-NTA Wash Buffer | * 20 mM HEPES (pH 7.4) * 150 mM NaCl * 20 mM imidazole |
| Ni-NTA Elution Buffer | * 20 mM HEPES (pH 7.4) * 150 mM NaCl * 250 mM imidazole |

### Glasswares and Plastic wares

* Pipettes and tips (including large orifice tubes)
* Vortex mixer.
* Distilled Water
* Bench top shaker
* Pipettes and tips, including large orifice tips for pipetting Antibody Bead.
* 100 mL graduated cylinder and container.

### Protocol

1. Thaw the cell pellet for 15 minutes on ice and resuspend the cells in Ni-NTA Lysis Buffer at
   2-5 mL per gram wet weight (typically, 30 mL is needed for a cell pellet from a litre of
   culture).
2. Sonicate on ice-water mixture (1 on, 3 off, 60% amplitude for 10 mins).
3. Centrifuge lysate at 30,000 x *g* for 30 minutes at 4°C to pellet the cellular
   debris.
4. Add 5 μL 5x SDS-PAGE sample buffer to 20 μL supernatant and store at -20°C for SDS-PAGE
   analysis.
5. Add 1 mL of the 50% Ni-NTA slurry to ~30 mL of cleared lysate (Ni-NTA beads washed and
   pre-equilibrated in Lysis Buffer) and mix gently by shaking (200 rpm on a rotary shaker) at
   4°C for 60 min.
6. Load the lysate/Ni-NTA mixture onto a PD10 column with capped bottom outlet.
7. Remove bottom cap and collect the column flow-through.
8. Wash extensively (~100 mL) with Ni-NTA Wash Buffer; collect wash fractions for SDS-PAGE
   analysis.
9. Elute the protein 4 times with 2 mL each time with Ni-NTA Elution Buffer.
10. Collect the eluate in four tubes and analyze by SDS-PAGE.

### Troubleshooting

#### If the proteins don't bind to Ni-NTA

| Problem | Suggestions |
| --- | --- |
| His tag is not expressed properly | Try moving His Tag to another terminal of protein. Try purification under denaturing conditions instead of native. |
| Degradation of His-tag after expression | Avoid His-tag near the hotspot regions of inserted sequence (Part of protein under process). |
| Change in Binding conditions | Composition and pH of all buffers should be checked prior to use. Dissociation of urea often causes a shift in pH. Proper concentration of imidazole. There should be no chelating agent in the buffer. |

#### If the protein elutes in the Ni-NTA Wash Buffer

| Problem | Suggestions |
| --- | --- |
| Hidden His-tag | Try purification under denaturing conditions. Lower washing stringency. Do not use imidazole in wash buffer. |
| Buffer Composition incorrect | Check the pH and composition of the Ni-NTA buffer. |
| High washing stringency | Increase the pH. Use low concentration of imidazole. |

#### If the protein precipitates during purification

| Problem | Suggestions |
| --- | --- |
| Low temperature | Purify proteins at room temperature |
| Aggregates of protein | Try adding solubilization reagents such as 0.1% Triton X-100 or Tween-20, up to 20 mM β-mercaptoethanol, up to 2 M NaCl, or stabilizing cofactors such as Mg2+. These may be necessary in all buffers to maintain protein solubility. |

#### If the protein does not elute

| Problem | Suggestions |
| --- | --- |
| Precipitation of proteins in column | Elute under denaturing conditions. Perform binding and elution in Batch purification to maintain protein concentrations. |
| Mild elution conditions (Multimer of proteins) | Elute with a pH of imidazole step-gradient to determine the optimal elution conditions. |

#### If the proteins are contaminated or have low purity

| Problem | Suggestions |
| --- | --- |
| Column is large | Use less Ni-NTA resin. |
| Less stringency in binding and washing conditions | Increase the concentration of imidazole in the wash buffer to 20 mM. Use higher salt concentration (500 mM KCl) for wash. |
| Contaminants with tagged proteins | Increase the detergent concentration to avoid nonspecific interactions. Add β-mercaptoethanol to reduce disulfide bonds. (<20mM) |
| Contaminants are truncated forms of the tagged protein | Prevent protein degradation during purification by working at 4°C or by including protease inhibitors. Try purification with both His-tag and Strep-tag (2-step purification) |

#### Discoloration of resin

| Problem | Suggestions |
| --- | --- |
| Less Ni ion concentration | Ensure that there are no chelating compounds (resin color turns white) or reducing agents (resin color turns brown) present in all buffers. |

### References

1: [www.iba-lifesciences.com/](https://www.iba-lifesciences.com/)

2: Hochuli E, Döbeli H, Schacher A, 1987: J. Chromatogr. 411:177-184. New metal chelate adsorbent
selective for proteins and peptide containing neighbouring histidine residues.

---

## Purification of the Proteins using Strep-tag

![](https://static.igem.org/mediawiki/2020/7/7a/T--IISER-Pune-India--strep-tag.png)

### Reagents

| Buffers/Solution | Concentration of Ingredients |
| --- | --- |
| Ni-NTA lysis buffer | * 50 mM NaH2PO4 * 300 mM NaCl * 10 mM imidazol * Adjust pH to 8.0 |
| Buffer W | * 100 mM Tris/HCl, pH 8 * 150 mM NaCl * 1 mM EDTA |
| 5x SDS-PAGE sample buffer | * 0.25 M Tris/HCl * 7.5% SDS * 25% glycerol * 0.25 mg/mL bromophenolblue * 12.5% v/v mercaptoethanol * Adjust pH 8.0 |
| Buffer E   Elution Buffer | * 100 mM Tris/HCl, pH 8.0 * 150 mM NaCl * 1 mM EDTA * 2.5 mM desthiobiotin |

### Glass wares and Plastic wares

* Pipettes and tips (including large orifice tubes)
* Vortex mixer
* Distilled Water
* Shaker
* Pipettes and tips, including large orifice tips for pipetting Antibody Bead
* 100 mL graduated cylinder and container

### Protocol

1. Chill Buffer W at 4°C.
2. Take a 10 µl sample for analysis of the total protein content via SDS PAGE and/or Western
   blotting. 10 µl sample should be thoroughly mixed with 90 µl Buffer W and 25 µl of 5x
   SDS-PAGE sample buffer. Store it at -20 C.
3. Sonicate the residual suspension under ice-cooling.
4. If the lysate is very viscous, add RNase A (10 µg/mL) and DNase I (5 µg/mL) and incubate on
   ice for 10 – 15 min.
5. Centrifuge the suspension at 13,000 rpm for 15 minutes at 4°C.
6. Insoluble cell components are sedimented. If the recombinant protein forms inclusion bodies
   it will be present in the sediment.
7. Carefully transfer the clear supernatant to a clean tube. For the analysis of insoluble part
   of the expressed proteins, dissolve the sediments with 1.25 mL 1x SDS-PAGE sample buffer
   with 1 mL Buffer W.
8. Equilibrate the Strep-Tactin® column with 2 CVs (column bed volumes) Buffer W. Remove the
   first top cap column, then the cap at the outlet of the column. If caps are removed in
   reverse order, the column may run dry. Use a buffer without EDTA for metalloproteins
9. Centrifuge the cleared lysates at 14,000 rpm for 5 min under 4°C. Insoluble aggregates
   formed during storage have to be removed to prevent clogging.
10. Add supernatant of cleared lysates to the column. Volume of lysates should be in range 0.5
    to 10 CVs. Extracts of large volume with recombinant proteins at low concentration may lead
    to the reduced yield.
11. Wash the column 5 times with 1 CV Buffer W, after the cell extract has completely entered
    the column. Collect the eluate in fractions having a size of 1 CV. Apply 2 µl of the first
    washing fraction and 20 µl of each subsequent fraction to an analytical SDS-PAGE.
12. Add 6 times 0.5 CVs Buffer E and collect the eluate in 0.5 CV fractions. 20 µl samples of
    each fraction can be used for SDS-PAGE analysis. Most of the purified Strep-tag® II fusion
    protein usually elutes in the 2nd to 5th fraction. Desthiobiotin and EDTA can be removed, if
    necessary, via dialysis or gel chromatography.

### Troubleshooting

#### If the proteins don't bind to Strep-tag column

| Problem | Suggestions |
| --- | --- |
| pH is not correct | The pH should be > 7.0 |
| Strep-tag is not accessible | Fuse Strep-tag with the other proteins terminus; use other linkers. |
| Strep-tag has been degraded | Check the strep tag is not associated with the portion of proteins which is processed. Avoid purification in discontinuous batch mode. |
| Strep-tag column is inactive | Check activity with HABA. Add 2-3 nmol of avidin monomer per nmol of biotin |

#### If the proteins are contaminated or have low purity

| Problem | Suggestions |
| --- | --- |
| Contaminants are short forms of tagged proteins | Add protease inhibitors after cell lysis. Fuse strep tag with other protein terminus. Check for the presence of the internal translation initiation sites or premature termination sites. |
| Covalent link between contaminants and recombinant proteins via disulphide bond. | All reducing agents to all buffers for cell lysis and chromatography. |

#### Air Bubble in the column

| Problem | Suggestions |
| --- | --- |
| Development of bubbles in the column bed | Keep on working in the cold room. Wash columns immediately with buffers at ambient temperature once the column is removed from cold. |

### References

1: [www.iba-lifesciences.com/](https://www.iba-lifesciences.com/)

2: Schmidt, TGM and Skerra, A, 2007: NATURE PROTOCOLS 2, 1528-1535. The Streptag system for
one-step purification and high-affinity detection or capturing of proteins.

3: Skerra A, Schmidt TGM, 2000: Meth. Enzymol. 326: 271-304. Use of the Strep-tag and
streptavidin for recombinant protein purification and detection.

## SDS-PAGE Protocol

### Equipment

* Erlenmeyer or plastic tubes
* Hypodermic needle attached to the syringe
* Pasteur pipette
* Squirt Bottle
* Micropipette equipped with gel loading tips
* Vertical electrophoresis apparatus (Invitrogen)

### Reagent

| Buffer/Solution | Contents |
| --- | --- |
| 40% Acrylamide (37.5:1) | * Acrylamide 116.8 g * N,N’-Methylene bisacrylamide 3.2 g * DDI H2O to 300 mL * Filter and store in a dark bottle at 4°C |
| 30% Ammonium Persulfate | * Ammonium Persulfate 1.5 g * DDI H2O 5 ml * Store at 4°C |
| RG Buffer--1.5 M   Tris•Cl, pH 8.8 | * DDI H2O 300 mL * Tris–free base 90.75 g * Conc. HCl 8 mL |
| SG Buffer--1.0 M   Tris•Cl, pH 6.8 | * DDI H2O 300 mL * Tris–free base 60.54 g * Conc. HCl 36 mL |
| 4x SDS-PAGE Sample Buffer | * 125 mM Tris•HCl, pH 6.8, 1 M, 5 mL * 20% Glycerol 8 mL * 4% SDS 20% 8 mL * 10% ß-Mercaptoethanol 4 mL * 0.5 mg/mL Bromophenol Blue 20 mg * DDI H2O 15 mL |
| 10x SDS-PAGE Running Buffer | * 30.3 g Tris base * 144.0 g Glycine * 10.0 g SDS * Fill upto 1000 mL with deionized water |
| Destain Solution | * Ethanol 1200 mL * Glacial Acetic Acid 400 mL * DDI H2O 2.4 L |

### Casting the Gel

1. Assemble glass plates and spacers in gel casting apparatus. See BioRad instruction manual.
2. Mix the components for the resolving gel as described in the Mini-Protean II protocol.
3. Pour the resolving gel mixture into the gel plates to a level 2 cm below the top of the shorter plate.
4. Place a layer of DDI H2O over the top of the resolving gel to prevent meniscus formation in the resolving gel.
5. Allow resolving gel to stand 30 min at room temperature.
6. Drain the DDI H2O from top of the resolving gel. Rinse with DDI H2O, drain, and wick any remaining DDI H2O away with a Kimwipe.
7. Mix components for stacking gel.
8. Pour stacking gel solution into gel plates (on top of running gel), so that gel plates are filled. Insert comb to the top of the spacers.
9. Allow gel to stand for at least 1 hr at room temperature, or overnight at 4°C (wrapped in saran wrap).

### Cell Samples

1. Harvest 100 μL of cells at O.D. > 0.6. Decant the supernatant media.
2. Resuspend cells in 20 μL of 2x sample buffer.
3. Incubate tubes in boiling water for 5 min.
4. Centrifuge at 12,000 x g for 30 s.

### Solution Samples

1. Place a volume of protein solution (or 1 μL of standard) into a μ fuge tube, such that there is 5-10 μg of protein in the solution.
2. Add an equal volume of 2x sample buffer (or 10 μL for standards).
3. Incubate tubes in boiling water for 5 min.
4. Centrifuge at 12,000 x g for 30 s.

### Running the Gel

1. Remove comb and assemble cast gel into Mini-Protean II apparatus.
2. Add freshly prepared 1x running buffer (300 mL) to both chambers of the apparatus.
3. Load the prepared samples into the wells of the gel.
4. Run the gel at 100 V until the dye front migrates into the running gel (~15 min), and increase to 200 V until the dye front reaches the bottom of the gel (~45 min).

### Staining and Destaining the Gel

1. Remove the run gel from the apparatus and remove the spacers and glass plates. Place the gel into a small tray.
2. Add ~20 mL staining solution and stain for > 30 min with gentle shaking.
3. Pour off and save the stain.
4. Add ~5 mL destain solution and destain for ~1 min with gentle shaking.
5. Pour off and discard the destain solution. Add ~30 mL of destain solution.
6. Distain with gentle shaking until the gel is visibly destained (> 2 hr).
7. Pour off and discard the destain solution.
8. Rinse with DDI H2O. Add ~30 mL DDI H2O and rinse for 5 min with gentle shaking.
9. Dry the gel on the gel dryer at 60°C for 1 hr with a sheet of Whatman filter paper below the gel and a piece of Seran wrap over the gel.

### % Acrylamide in running gel

| Single Percentage | Separation size range (kDa) |
| --- | --- |
| 5% | 100-250 |
| 7.5% | 40-200 |
| 10% | 30-150 |
| 12% | 20-120 |
| 15% | 10-100 |
| 18% | 6-50 |

| Gradient | Separation size range (kDa) |
| --- | --- |
| 4–15% | 20–250 |
| 4–20% | 10–200 |
| 10–20% | 10–100 |
| 8–16% | 6–70 |

### References

1: The BioRad Readygel Manual.

2: Laemmli, U.K. (1970) Nature 227, 680-685.

3: Adapted from Laemmli, U.K. (1970) Nature 227, 680-685.

## Ni-NTA Protein Binding Assay

### Outline of Experimental Procedure

* Membrane dialysis of His-tagged parasite protein to remove Imidazole used for purification of the protein against 150 mM KCl + 20 mM Hepes buffer
* ↓
* Verify thermal stability of cyclotides **(Protocol 1)**
* ↓
* Perform SDS PAGE to check the purity of proteins of interest
* ↓
* Measure the concentration of the host protein, parasite protein and cyclotide protein via bicinchoninic acid (BCA) assay **(Protocol 2)**
* ↓
* Concentrating protein solutions (if necessary) using centricon membrane filters of 10MWCO for storage
* ↓
* Resuspend the proteins in buffer solution and confirm the concentrations via BCA assay Buffer of 150 mM KCl + 20 mM Hepes
* ↓
* Wash NI-NTA functionalized agarose beads in a buffer and incubate with parasite protein in 96 well plate. Add host protein (control) or both host protein and cyclotide (test) and allow for binding **(Protocol 3)**
* ↓
* Directly or Indirectly measure cyclotide and host protein concentrations to check the effect of presence of cyclotide on host and parasite protein binding **(Protocol 4)**

![](https://static.igem.org/mediawiki/2020/0/03/T--IISER-Pune-India--ni-nta.png)

### Protocol 1

* Add equal volumes of 1 μM BSA, 1 μM taq polymerase and 1 μM cyclotide into two eppendorf tubes.
* Heat the first tube in a water bath (90°C for 10 mins).
* Centrifuge contents of both tubes (30 mins at 20,000 g). Separate the supernatant from the pellet. Run supernatants and pellets of both tubes on SDS-PAGE compared to control lanes of taq polymerase, BSA and cyclotide individually. Presence of cyclotide in the supernatant after heat-denaturation is conclusive proof of the thermostability of the cyclotide.

### Protocol 2: Bicinchoninic Acid Assay[1]

**Reagent A:** sodium bicinchoninate (1.0 g), Na2CO3 (2.0 g), sodium tartrate (dihydrate) (0.16 g), NaOH (0.4 g), NaHCO3 (0.95 g), made up to 100 mL. If necessary, adjust the pH to 11.25 with NaHCO3 or NaOH

**Reagent B:** CuSO4 5H2O (0.4 g) in 10 mL of water (see Note 1).

**Standard working reagent (SWR):** Mix 100 vol of regent A with 2 vol of Reagent B.

* To a 100 μL aqueous sample containing 10–100 μg protein, add 2 mL of SWR. Incubate at 60°C for 30 min. The sensitivity of the assay can be increased by incubating the samples longer. Alternatively, if the color is becoming too dark, heating can be stopped earlier. Take care to treat standard samples similarly.
* Cool the sample to room temperature, then measure the absorbance at 562 nm.
* **Standardization:** A calibration curve can be constructed using dilutions of a stock 1 mg/mL solution of bovine serum albumin (BSA)

### Protocol 3

* Wash 20 µL (bed volume of a 1:1 slurry) Ni2+-NTA functionalized agarose beads with water to get rid of the ethanol in the slurry and then equilibrate beads with 150 mM KCl + 20 mM Hepes (HKS) Buffer
* Add 500 µL of 1 µM of the parasite protein in HKS buffer containing the His tag to each well in the plate with the beads and incubate for 30 minutes at 37°C. Spin the beads after incubation and discard the supernatant containing excess unbound protein.
* Add 500 µL of HKS Buffer to the beads. Mix well and centrifuge again on a table top centrifuge for 1 min. Discard the supernatant. Do the same wash three times.
* For the host + parasite sample:
  + Add 500 µL of 1 µM host protein with the StrepII tag to each well in the plate and incubate at 37°C for 60 minutes.
  + Centrifuge, discard the supernatant, and resuspend the beads in buffer after washing (in buffer) 3 times.
  + Add 20 µL of 2x Laemmli’s buffer to the beads.
  + Load the samples on a SDS PAGE to verify the proteins (Control)
  + Obtain concentration of host protein (see Protocol 4)
* For the host + parasite +cyclotide sample (Method 1):
  + Add between 100 nM to 10µM cyclotides (gradation in concentration)in 500 µL volume to each well in the plate and incubate at 37°C for 60 minutes.
  + Centrifuge, discard the supernatant, and resuspend the pellet in buffer after washing (3x times) to get rid of unbound cyclotide.
  + Add 500 µL of 1 µM of the host protein with the StrepII tag to each well in the plate and spin at 37°C for 60 minutes.
  + Centrifuge, discard the supernatant, and resuspend the pellet in buffer after washing (in buffer) 3 times.
  + Add 20 µL of 2x Laemmli’s buffer to the beads.
  + Perform SDS PAGE for qualitative estimation of inhibition of binding in presence of cyclotide.
  + Do BCA assay of the proteins bound on the beads to get a quantitative estimate of inhibition of binding (see Protocol 4)
* For the host + parasite +cyclotide sample (Method 2):
  + Mix the amount of cyclotide showing most inhibition of binding determined by method 1 along with 500 µL of 1 µM of the host protein to each well of parasite protein-functionalized beads and incubate at 37°C for 60 minutes.
  + Centrifuge, discard the supernatant, and resuspend the pellet in the buffer after washing to get rid of excess unbound cyclotide and host protein in solution.
  + Add 20 µL of 2x Laemmli’s buffer to the beads.
  + Perform SDS PAGE to check if the amount of host protein is lesser than the control (parasite protein on beads+host protein)
  + Obtain concentration of host protein (see Protocol 4)

### Protocol 4

* **Direct measurement of bound protein:**
  + Set up two identical reactions with the parasite protein
  + Add 500 µL 1 µM of the parasite protein with the His tag to both reaction setups and incubate at 37°C for 30 minutes.
  + Centrifuge, discard the supernatant, and resuspend the pellet in buffer after washing in both setups.
  + Add 1% SDS to the beads to denature the protein and get it in solution. Do a BCA assay of this to get an estimate of the amount of parasite protein bound on 20 µL of beads under equilibrium conditions. **Let this concentration be p.**
  + **For host + parasite protein sample:**
    - After allowing the host protein to bind and centrifuging and obtaining the pellet (see Protocol 3), obtain the protein remaining bound to the beads by adding 1% SDS to the beads. Measure concentration of this total protein via BCA. Let this concentration be x.
    - **Concentration of host protein bound h = x - p**
  + **For host + parasite + cyclotide protein sample:**
    - After allowing the host protein and cyclotide to bind and centrifuging and obtaining the pellet (see Protocol 3), obtain the total protein bound to the beads by adding EDTA to a final concentration of 1 mM. Spin to get rid of the beads.
    - Take the supernatant and heat-denature the total protein by subjecting it to a water-bath heated at 90°C for 10 mins. Spin the sample at 20,000g for 30 mins to separate supernatant and pellet. The supernatant will contain the cyclotide. Pellet will contain host protein, parasite protein and beads.
    - Perform BCA on the supernatant. Let this concentration be c.
    - **Concentration of cyclotide = c**
    - Obtain all the protein in the pellet and perform BCA. Let this concentration be x’.
    - **Concentration of host protein h = x’ - p**
* **Indirect measurement of bound protein:**
  + Allow parasite protein to bind to the agarose beads according to protocol 3. Let initial concentration of parasite protein be pi. Centrifuge and perform BCA on the supernatant. Let this concentration be pf.
  + **Concentration of parasite protein bound p = pf - pi**
  + **For host + parasite protein sample:**
    - Perform a BCA on host protein before adding it to the reaction setup. Let this concentration be hi.
    - After allowing the host protein to bind and centrifuging and obtaining the pellet (see Protocol 3), perform a BCA of the supernatant. Let this concentration be hf.
    - **Concentration of host protein bound = hf - hi.**
  + **For host + parasite + cyclotide protein sample:**
    - Perform a BCA on host protein before adding it to the reaction setup. Let this concentration be hi.
    - Perform a BCA on cyclotide before adding it to the reaction setup. Let this concentration be ci.
    - After allowing the host protein and cyclotide to bind and centrifuging and obtaining the supernatant (see Protocol 3), thermally precipitate it. Supernatant will contain unbound cyclotides. Pellet will contain unbound host protein.
    - Perform BCA on the supernatant. Let this concentration be cf.
    - **Concentration of cyclotide = ci - cf**
    - Obtain all the protein in the pellet and perform BCA. Let this concentration be hf.
    - **Concentration of host protein h = hi - hf**

### References

1: Walker, J. M. (2009). *The Protein Protocols Handbook (Springer Protocols Handbooks)* (3rd ed.). New York City, NY: Humana Press.

## IC50 Assay

### SYBR Green I DNA Fluorometry

#### Materials and Reagents

* *P. falciparum* culture
* Sterile 96-well plate
* Varioskan Flash
* SYBR Green I DNA staining dye
  [NB: SYBR Green I is light-sensitive; keep it in the dark as much as possible]

| Buffer | Concentration of Ingredients |
| --- | --- |
| Lysis buffer | * 100 mM Tris-HCl * pH 7.5 * 25 mM EDTA * 0.5% Triton X-100 * 0.05% saponin in PBS |

#### Protocol

![](https://static.igem.org/mediawiki/2020/f/fc/T--IISER-Pune-India--ic50.png)

1. Take 100 µL of RPMI in the wells of 96 well-plate.
2. Choose a concentration range for the experiment.
3. Prepare a 200 µM stock of the drug in RPMI.
4. Perform serial dilution (2-fold)
   1. Take 100 µL of the stock solution and transfer it into a well containing 100 µL RPMI (the drug concentration of the solution is now reduced to 100 µM)
   2. Take 100 µL of solution from well 1 and transfer it into well 2 containing 100 µL RPMI (the drug concentration of the solution is now reduced to 50 µM)
   3. Take 100 µL of solution from well 2 and transfer it into well 3 containing 100 µL RPMI (the drug concentration of the solution is now reduced to 25 µM)
   4. Take 100 µL of solution from well 3 and transfer it into well 4 containing 100 µL RPMI (the drug concentration of the solution is now reduced to 12.5 µM)
   5. Take 100 µL of solution from well 4 and transfer it into well 5 containing 100 µL RPMI (the drug concentration of the solution is now reduced to 6.25 µM)
   6. Take 100 µL of solution from well 5 and transfer it into well 6 containing 100 µL RPMI (the drug concentration of the solution is now reduced to 3.125 µM)
   7. Take 100 µL of solution from well 6 and transfer it into well 7 containing 100 µL RPMI (the drug concentration of the solution is now reduced to 1.5625 µM)
   8. Take 100 µL of solution from well 7 and transfer it into well 8 containing 100 µL RPMI (the drug concentration of the solution is now reduced to 0.78125 µM)
5. Take 100 µL of the culture (RBC + RPMI) with 4% hematocrit value and 2% parasitemia and transfer it in well 1 to 8.
   [NOTE: Now the hematocrit value of the solution is reduced to 2% but the parasitemia remains the same. The drug concentration is also reduced by half.]
6. Controls
   1. Blank (background signal control)
      1. Take 100 µL of RPMI in well 9
      2. Add 100 µL of culture (RBC + RPMI) with 4% hematocrit and no parasites
   2. RPMI control (zero drug control)
      1. Take 100 µL of RPMI in well 10
      2. Add 100 µL of culture (RBC + RPMI) with 4% hematocrit value and 2% parasitemia
   3. DMSO control (to check the effect of solvent)
      1. Take 100 µL of DMSO in well 11
      2. Add 100 µL of culture (RBC + RPMI) with 4% hematocrit value and 2% parasitemia
7. The plate is incubated for 48 hours at 37°C under the gas conditions of 5% CO2 and 95% N2
8. After 48 hours transfer the plate to -80°C to freeze.
9. Prepare a 10X stock of SYBR Green 1 + Lysis Buffer (volume = 2500 µL)
10. Thaw the plate at room temperature (this freezing and thawing will cause cell lysis)
11. Add 22.5 µL of 10X SYBR Green 1 + Lysis buffer to each well
12. Mix and incubate for 45 minutes at room temperature in dark
13. Take fluorometer readings in Thermo Fisher VarioskanFlash
    1. Select the lane
    2. Select fluorometry based assessment
    3. Set up mode to:

       |  |  |
       | --- | --- |
       | Excitation | 495 nm |
       | Emission | 520 nm |
    4. Select type of plate i.e. Flat Bottom/Conical/Round Bottom
    5. Record the fluorescence values
14. Import data into this excel sheet.

     [Download Excel Spreadsheet](https://static.igem.org/mediawiki/2020/8/84/T--IISER-Pune-India--IC50-Assay-Analysis.xlsx)

#### Analysis

1. We can calculate the IC50 values by using an online tool, IC50 calculator.[1]

   The tool uses a four parameter logistic regression model to calculate the value. This model typically resolves as a sigmoid function, or "S"-shaped curve.

   All you have to do is input the concentration and readout values obtained from the excel sheet.
2. We have also written R code which you can use to calculate the IC50 value. For the code to work you have to save the concentration and readout values obtained in the Excel sheet in two columns in a CSV file.[2]

   [Download R code file](https://static.igem.org/mediawiki/2020/c/c2/T--IISER-Pune-India--drc-fit.zip)

#### Troubleshooting

1. To check if the proposed drug acts as an inhibitor we first select a broad drug concentration range and if the IC50 value is obtained then the experiment is performed again with a narrow drug concentration range to get a more accurate value.
2. After the results are obtained if the growth in zero drug concentration is less than the growth in any well with some drug concentration then it is because of handling error. We have either added more cells in a well or more dye in a well. To avoid this the experiment is done in triplets and average is taken.
3. We perform RPMI and DMSO control to check if the drug solvent is responsible for any inhibition effect or not. DMSO is the drug solvent. If the fluorometer readings of DMSO control and RPMI control are similar then that means solvent is not contributing to the inhibition effect.
4. We perform a blank control with no parasites (no DNA in sample) so that we can deduct the background noise from all readings.

#### References

1: "IC50 Calculator | AAT Bioquest." [www.aatbio.com/tools/ic50-calculator](https://www.aatbio.com/tools/ic50-calculator). Accessed 26 Oct. 2020.

2: "Bioassay analysis using R - Journal of Statistical Software." 23 Jan. 2005, [www.jstatsoft.org/v12/i05/paper](https://www.jstatsoft.org/v12/i05/paper). Accessed 14 Oct. 2020.

3: SYBR Green I DNA Fluorometry, [sites.psu.edu/llinaslab/files/2020/05/SYBR\_Green\_protocol.pdf](https://sites.psu.edu/llinaslab/files/2020/05/SYBR_Green_protocol.pdf).

## RBC Invasion Assay

### Materials Required

* **Chemicals**
  + RPMI-1640 media
  + HEPES
  + Sodium bicarbonate
  + Hypoxanthine
  + Albumax II
  + α-2-3,6,8–Vibrio cholera neuraminidase
  + Trypsin
  + Chymotrypsin
  + Phosphate buffered saline(PBS)
  + Bovine serum albumin (BSA)
  + Sodium azide
  + Sorbitol
  + SYBR Green I dye
* **Others**
  + 96-well microtiter plate

![](https://static.igem.org/mediawiki/2020/a/af/T--IISER-Pune-India--rbc-invasion-assay.png)

### Protocol

#### Parasite Culture

* **Solution 1:** 4% hematocrit in RPMI-1640 media supplemented with 25 mM HEPES,
  0.21% sodium bicarbonate, 50 mg/L hypoxanthine, and 0.5% Albumax II
* **Solution 2:** α-2-3,6,8– Vibrio cholera neuraminidase (66.7 mU/mL,
  Calbiochem), trypsin (1 mg/ mL, Sigma), and chymotrypsin (1 mg/mL, Worthington)

1. Treat the *P.falciparum* asexual stages maintained in vitro in human O+ erythrocytes
   with solution 1.
2. Perform invasion assays by mixing enzyme-treated infected donor cells with equivalent cell
   number of RPMI treated erythrocyte control cells, or enzyme-treated erythrocyte negative
   control cells.
3. Treat ring stage infected O+ erythrocytes with solution 2 to prevent reinvasion.
4. Plate parasite duplicates in complete RPMI media at a final parasitemia of between 0.45 and
   1% at 2% hematocrit.
5. Following reinvasion, about 48 hours after plating, assess parasitemia by flow cytometry as
   detailed below.

#### Flow Cytometry

* **Solution 3:** 1 x phosphate buffered saline (PBS) + 0.5% bovine serum albumin
  (BSA) + 0.02% sodium azide

1. Culture two-hundred microliters of sorbitol-synchronized, ring-stage parasites in a 96-well
   microtiter plate at 1% parasitemia.
2. Plate samples in triplicate and incubate at 37℃ until reinvasion.
3. Harvest cultures through centrifugation (1200 rpm, 5 minutes) and wash it twice in 100 μL of
   solution 3.
4. Incubate cells with 75 μL of 1:1000 SYBR Green I (Molecular Probes) for 20 min at 25℃.
5. Wash the cells in solution 3 and resuspend it in PBS.
6. Collect flow cytometry data using FACS ARIA. Carry out initial gating with unstained,
   uninfected erythrocytes to account for erythrocyte autofluorescence. Analyse the data with
   FlowJo 8.8.6 software (Tree Star).

### Precautions

The accuracy with which flow cytometry resolves multiply-infected erythrocyte peaks needs the
culture to be at the ring-stage. As the culture shifts from late rings to early trophozoites the
parasite starts to replicate its DNA. The flow cytometer is unable to distinguish between the
fluorescence emitted by a multiple infected erythrocyte having more than one, ring stage
parasites versus one infected by a single, early trophozoite. The requirement for ring-stage
parasites can easily be met for in vitro laboratory-adapted parasites cultured by
synchronization by sorbitol lysis.

### Troubleshooting

1. In step 3, if treatment of infected erythrocytes with solution 2 is very harsh because of
   the α-2-3,6,8– Vibrio cholera neuraminidase and trypsin and chymotrypsin (all three
   chemicals block reinvasion completely) , lower concentrations can be used and the relative
   effectiveness of different peptide inhibitors can be studied.
2. If FACS does not give promising results because of lack of standardization then fluorometry
   based analysis can be performed using a spectrophotometer, which will be speedier as well.
3. Harsh sorbitol treatment can affect reinvasion. To troubleshoot this, fresh parasite
   cultures can be revived to grow in a synchronized manner for the initial cycles. Sorbitol
   treatment should be avoided as much as possible.

### References

1: Bei, A. K., DeSimone, T. M., Badiane, A. S., Ahouidi, A. D., Dieye, T., Ndiaye, D., …
Duraisingh, M. T. (2010). A flow cytometry-based assay for measuring invasion of red blood cells
byPlasmodium falciparum. American Journal of Hematology, 85(4), 234–237. [doi.org/10.1002/ajh.21642](https://doi.org/10.1002/ajh.21642)

2: Jacobberger JW, Horan PK, Hare JD: Analysis of malaria parasite-infected blood by flow
cytometry. Cytometry 1983, 4:228–237.
 [www.sciencedirect.com/science/article/abs/pii/016511619390140U](https://www.sciencedirect.com/science/article/abs/pii/016511619390140U)

## PCR Protocol

### Cloning

**Enzyme:** *Pfu* DNA polymerase

**Template:** Synthesized Gene

#### Steps

1. **Gradient Amplification to determine optimal annealing temperature**

   Gradient PCR is a modification to a standard PCR procedure that allows to optimize an
   annealing temperature in order to increase the specificity of the amplification process.
   It is usually conducted in a thermal cycler in which different annealing temperatures
   can be set in different parts of the block, maintaining constant denaturing and
   elongation temperatures. Thus, after running a gel with PCR products and comparing the
   pattern of bands, it is possible to empirically determine the optimal annealing
   temperature for a given set of primers. Annealing temperatures between 55°C to 65°C are
   ideal for PCR reaction. Deviation of annealing temperature above or below this range can
   cause non-specific bindings or reaction failure.
2. **Gene amplification at annealing temp determined by gradient PCR**

| Name | 50 μL reaction | 20 μL reaction |
| --- | --- | --- |
| Molecular Grade Water | Make up to 50 | Make up to 20 |
| 10X ThermoPol®️ Buffer | 10 μL | 4 μL |
| dNTPs | 2 μL | 0.8 μL |
| Forward primer | X μL | 2X/5 μL |
| Phusion Polymerase | 1 μL | 0.4 μL |
| Reverse primer | X μL | 2X/5 μL |
| DNA | X μL | 2X/5 μL |

|  | Step | Temp. | Time |
| --- | --- | --- | --- |
| Hold 1 | Initial denaturation | 95°C |  |
| Cycling x30 | Denaturing | 98°C | 10 s |
| Annealing | X°C | 30 s |
| Extension | 72°C | 30 s/kb |
| Hold 1 | Final extension | 72°C | 10 mins |

**Tip: Use 15-30 s/kb for extension. Do not exceed 1 min/kb.**

### Clone Confirmation

**Enzyme:** *Pfu* DNA polymerase

**Template:** pET-28a(+) vector

| Name | 50 μL reaction | 20 μL reaction |
| --- | --- | --- |
| Molecular Grade Water | Make up to 50 | Make up to 20 |
| 10X ThermoPol®️ Buffer | 10 μL | 4 μL |
| dNTPs | 2 μL | 0.8 μL |
| Forward primer (M13) | X μL | 2X/5 μL |
| *Pfu* Polymerase | 1 μL | 0.4 μL |
| Reverse primer (M13) | X μL | 2X/5 μL |
| DNA | X μL | 2X/5 μL |

|  | Step | Temp. | Time |
| --- | --- | --- | --- |
| Hold 1 | Initial denaturation | 95°C |  |
| Cycling x30 | Denaturing | 98°C | 10 s |
| Annealing | 50°C | 30 s |
| Elongation | 72°C | 30 s/kb |
| Hold 1 | Final extension | 72°C | 10 mins |

### Troubleshooting

##### → No PCR product is observed

| Possible Cause | Solution |
| --- | --- |
| A PCR component is missing or degraded | A positive control should always be run to insure components are functioning. A checklist is also recommended when assembling reactions. |
| Too few cycles were performed | Increase the number of cycles (3-5 additional cycles at a time). |
| The annealing temperature is too high | Decrease the annealing temperature in 2-4°C increments |
| The primers are not designed optimally | Confirm the accuracy of the sequence information. If the primers are less than 27 nucleotides long, try to lengthen the primer to 27-33 nucleotides. If the primer has a GC content of less than 45%, try to redesign the primer with a GC content of 45-60%. |
| There is not enough template | After increasing the number of cycles has shown no success, repeat the reaction with a higher concentration of template. |
| The template is of poor quality | Evaluate the template integrity by gel electrophoresis. It may be necessary to repurify the template using methods that minimize shearing and nicking. |
| The denaturation temperature is too high or too low | Optimize the denaturation temperature by increasing or decreasing the temperature in 1°C increments. |
| The denaturation time is too long or too short | Optimize the denaturation time by increasing or decreasing it in 10-second increments. |
| The extension time is too short | Increase the extension time in 2-minute increments, especially for long templates. |
| The reaction does not have enough enzyme | 1.0 µL (2.5 units) is sufficient for most applications. It is recommended that the cycling parameters be optimized before the enzyme concentration is increased. In rare cases, the yields can be improved by increasing the enzyme concentration. However, if the enzyme amount is above 2 µl (5 units), higher background levels may be seen. |
| Mg2+ levels are suboptimal | This is unlikely if the 10X reaction buffer (with MgCl2) is used and the deoxynucleotides do not exceed a concentration of 0.6 mM each (as deoxynucleotide triphosphates can bind Mg2+). Typically, MgCl2 is optimized between 1 to 5 mM. Also, EDTA present in the sample at greater than 5 mM will reduce the effective concentration of magnesium. |
| Deoxynucleotide concentration is too low | This is unlikely if the final concentration of each deoxynucleotide is 0.5 mM. This concentration of dNTPs is suitable for a wide range of applications. If the dNTPs are being prepared in the laboratory, be sure that the final concentration of each deoxynucleotide is 0.5 mM. If the concentration of dNTPs is increased, the Mg2+ concentration will need to be increased proportionately. |
| Target template is complex | In most cases, inherently complex targets are due to unusually high GC content and/or secondary structure. |

##### → There are multiple or smeared products

| Possible Cause | Solution |
| --- | --- |
| The annealing temperature is too low | Increase the annealing temperature in increments of 2-3°C. |
| The primers are not designed optimally | Confirm the accuracy of the sequence information. If the primers are less than 27 nucleotides long, try to lengthen the primers to 27-33 nucleotides. If the primer has a GC content of less than 45%, try to redesign the primers with a GC content of 45-60%. |
| Touchdown PCR may be required | “Touchdown” PCR significantly improves the specificity of many PCR reactions in various applications. Touchdown PCR involves using an annealing/extension temperature that is higher than the TM of the primers during the initial PCR cycles. The annealing/extension temperature is then reduced to the primer TM for the remaining PCR cycles. The change can be performed in a single step or in increments over several cycles. |
| Too many cycles were performed | The nonspecific bands may be eliminated by reducing the number of cycles. |
| There is too much enzyme in the reaction mix | 1 µL (2.5 units) is sufficient for most applications. However, this concentration may be too high for some applications. We recommend optimizing the cycling parameters first as described above, then if necessary incrementally reduce the enzyme concentration to determine the optimal concentration. |
| Magnesium concentration is too high | The MgCl2 concentration should be optimized. Typically, the concentration of MgCl2 is optimal between 1 and 5 mM. If the concentration of the dNTPs is 0.5 mM, it is very unlikely that the magnesium concentration is too high. |
| The template concentration is too high | Reduce the concentration of the template in the PCR reaction. |
| The template concentration is too low | Add additional template in 50 ng increments for genomic DNA or 1-2 ng for viral DNA. |

##### → The yield of specific products is low

| Possible Cause | Solution |
| --- | --- |
| Too few cycles were performed | Increase the cycle number in 3-5 cycle increments. |
| Extension times are too short | Increase the extension times in 2-minute increments. |
| A co-solvent is required | Add dimethyl sulfoxide up to a final concentration of 5%. |
| PCR priming opportunities may be low due to reaction conditions or primer design | Modify the reaction conditions by increasing the denaturation temperature to 95°C, increase extension times in 2-minute increments, increase MgCl2 and dNTP concentrations, etc. Redesign PCR primers. |

### Reference

https://www.sigmaaldrich.com/technical-documents/protocols/biology/long-and-accurate-dna-amplification.html

 [Link](https://www.sigmaaldrich.com/technical-documents/protocols/biology/long-and-accurate-dna-amplification.html)

## Site Directed Mutagenesis Protocol

### Background

This protocol has been adapted from the **[Team Wageningen\_UR 2014](https://2014.igem.org/Team%3AWageningen_UR)** which is based on the Stratagene Quikchange protocol. The primary reaction is illustrated below. Here the product of the reaction is never used as a template. This is a linear amplification technique, unlike standard PCR where an exponential amplification of the product is obtained.

![](https://static.igem.org/mediawiki/2020/e/e9/T--IISER-Pune-India--mutagenesis.png)

### Protocol

* **Oligo Designing**

  The protocol in the QuikChange Manual is followed for oligo-design. It designs a primer with a Tm of at least 78°C and centers the mutation in the middle, which works well for single amino acid changes. An automated website to help do this is available at [bioinformatics.org/primerx](http://bioinformatics.org/primerx). According to the Stratagene protocol, the oligos need to be PAGE purified.
* **Solution**
  + 0.5 µL 2.5 pmoles/µL forward primer
  + 0.5 µL 2.5 pmoles/µL reverse primer
  + 0.25 µL 40 mM dNTP mix (10 mM each)
  + 1 µL 2 ng/µL Template DNA
  + 1.25 µL 10x PfuUltra Buffer (contains Mg2+)
  + 0.25 µL PfuUltra Hotstart (Stratagene)
  + 8.75 µL sterile H2O
* **PCR Program**
  + Run for 5 min at 95°C
  + Repeat 18x
    1. 50s at 95°C
    2. 50s at 60°C
    3. 1 min + 1 min/1 kb template at 68°C
  + Run again for 7 min at 68°C
* Run 2.5 µL of the reaction on a gel, there should be a band corresponding to your product.
* Add 0.25 µL of DpnI to the reaction. Incubate at 37°C for 1 hr.
* Transform the final reaction into competent cells.
* Pick a colony, isolate its plasmid DNA and sequence it to check for the mutation and any PCR introduced errors.

### Precautions and Troubleshooting

1. The digestion with Dpnl is crucial as it only cuts at methylated sites. Since the transformation efficiency of the circular template plasmid is better than the linear PCR product, without the DpnI digest, a large number of colonies would be parental. It implies that the template plasmid cannot come from a methylation deficient strain (e.g., JM101).
2. As the PCR reaction goes around the entire plasmid, we need to minimize the chances of introducing unwanted mutations in your gene and the backbone. So a high fidelity polymerase is also crucial.
3. The concentration of primer versus template decides whether primer-primer or template-primer interaction dominates. If a reaction fails and if a strong primer dimer band is seen, it means that primer-primer annealing is favored over primer-template annealing. To troubleshoot this, the following solutions can be implemented: decreasing the primer concentration, increasing the template concentration, decreasing the annealing temperature, or redesigning primers.
4. In case no product is seen, repeating the protocol with 5% DMSO in the solution mix can help.DMSO disrupts base pairing, facilitating strand separation in GC rich regions of DNA, and reducing the propensity of the DNA to form secondary structure.

### References

1: L Zheng, U Baumann, and JL Reymond. An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucl. Acids Res., 32:e115, 2004.

2: [iGEM Team Wageningen 2014, Standard Protocol for Mutagenesis](https://static.igem.org/mediawiki/2014/b/b5/Wageningen_UR_protocols_Site_Directed_Mutagenesis.pdf)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:IISER-Pune-India/Results

[![](https://static.igem.org/mediawiki/2020/a/a4/T--IISER-Pune-India--wiki-logo-red.png)](https://2020.igem.org/Team%3AIISER-Pune-India)

* [Project](/Team%3AIISER-Pune-India/)

  [Description](/Team%3AIISER-Pune-India/Description)
  [Design](/Team%3AIISER-Pune-India/Design)
  [Contribution](/Team%3AIISER-Pune-India/Contribution)
  [Notebook](/Team%3AIISER-Pune-India/Notebook)
  [Results](/Team%3AIISER-Pune-India/Results)
  [Future Implementation](/Team%3AIISER-Pune-India/Implementation)
* [Team](/Team%3AIISER-Pune-India/)

  [Members](/Team%3AIISER-Pune-India/Team)
  [Collaborations](/Team%3AIISER-Pune-India/Collaborations)
  [Attributions](/Team%3AIISER-Pune-India/Attributions)
* [Engineering](/Team%3AIISER-Pune-India/)

  [Engineering Success](/Team%3AIISER-Pune-India/Engineering)
  [Experiments](/Team%3AIISER-Pune-India/Experiments)
  [Measurement](/Team%3AIISER-Pune-India/Measurement)
  [Safety](/Team%3AIISER-Pune-India/Safety)
* [Modelling](/Team%3AIISER-Pune-India/)

  [Overview](/Team%3AIISER-Pune-India/Model)
  [Model Results](/Team%3AIISER-Pune-India/Model_Results)
  [Visualization](/Team%3AIISER-Pune-India/Models_NGL)
* [Diagnostics](/Team%3AIISER-Pune-India/)

  [Motivation](/Team%3AIISER-Pune-India/Motivation)
  [DeleMa Detect](/Team%3AIISER-Pune-India/Software)
* [Human Practices](/Team%3AIISER-Pune-India/)

  [Integrated Human Practices](/Team%3AIISER-Pune-India/Human_Practices)
  [Public Education and Engagement](/Team%3AIISER-Pune-India/Education)
* [Parts](/Team%3AIISER-Pune-India/Parts)
* [Judging Form](https://igem.org/2020_Judging_Form?id=3582)

* [Dry Lab](#1)
* [Diagnostics](#2)
* [Wet Lab](#3)

# 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](https://2020.igem.org/Team%3AIISER-Pune-India/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) | * ILPRGGIVL * GVPRGGDF |
| 2 | *Plasmodium falciparum* reticulocyte-binding protein homologue 5 (PfRH5) interacting with human basigin (4U0Q) | * NPMPMFDMEY * YYYWMWMF * SRSVPPV |
| 3 | The CIDRa domain from MCvar1 PfEMP1 bound to human platelet glycoprotein 4- CD36 (5LGD) | * NQFVQMILNM * NQFVQMILNF * NQFVQMILNW * NKFWRWMWRM |
| 4 | The CIDRa domain from HB3var03 PfEMP1 binding to endothelial protein C receptor (4V3D) | * QSYLLQFNGLVRLVHQER * YMRDKLMIGYWRLR |
| 5 | Structure of the PfEMP1 IT4var13 DBLbeta domain interacting with ICAM-1 (6S8U) | * TSVQPSKVILP * TSVWPSKVILP * TSVIPSKVILP |

## 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](https://2020.igem.org/Team%3AIISER-Pune-India/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](https://static.igem.org/mediawiki/2020/0/0f/T--IISER-Pune-India--Malaria-Diagnostic-Kit-Manual.pdf) 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](https://2020.igem.org/Team%3AIISER-Pune-India/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

1. Autoclaved tips
2. Autoclaved water
3. Prepared and autoclaved LB and LB agar and poured Kanamycin plates

![](https://static.igem.org/mediawiki/2020/thumb/5/50/T--IISER-Pune-India--wlplates.jpg/320px-T--IISER-Pune-India--wlplates.jpg)

Growth Medium Plates

![](https://static.igem.org/mediawiki/2020/6/60/T--IISER-Pune-India--wlplasmid.png)

Our Vector

## 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 |

![](https://static.igem.org/mediawiki/2020/thumb/a/af/T--IISER-Pune-India--wl1.jpg/439px-T--IISER-Pune-India--wl1.jpg)

From left to right we have 1kb NEB ladder, template (gene coding for Human Protein of 5LGD interaction) ~ 1437 bp

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 |

![](https://static.igem.org/mediawiki/2020/thumb/5/5b/T--IISER-Pune-India--wl2.jpg/484px-T--IISER-Pune-India--wl2.jpg.png)

From left to right 1kb Ladder, columns 2-9 increasing temperature gradient from 50-65℃

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 |

![](https://static.igem.org/mediawiki/2020/e/ec/T--IISER-Pune-India--wl3.png)

### (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 |

![](https://static.igem.org/mediawiki/2020/d/d9/T--IISER-Pune-India--wl4.png)
![](https://static.igem.org/mediawiki/2020/5/54/T--IISER-Pune-India--wl5.png)

### (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 |

![](https://static.igem.org/mediawiki/2020/9/97/T--IISER-Pune-India--wl6.png)

* (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 |

![](https://static.igem.org/mediawiki/2020/a/a1/T--IISER-Pune-India--wl7.png)

Prep-PCR Verification Gel - From left to right, 1kb NEB ladder, Product from 49.5℃ annealing temperature, Product from 53.8℃ annealing temperature, Product from 57.5℃ annealing temperature

## Gel Purification

---

This was done using the NEB Purification Kit

Protocol for the same can be found [here](https://static.igem.org/mediawiki/2020/a/a8/T--IISER-Pune-India--gelpurify.pdf).

## 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.

![](https://static.igem.org/mediawiki/2020/3/3f/T--IISER-Pune-India--wl8.png)

## 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 |

![](https://static.igem.org/mediawiki/2020/thumb/0/0b/T--IISER-Pune-India--wl9.png/393px-T--IISER-Pune-India--wl9.png)

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.