iGEM 2021 — Team IISER_Berhampur

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# Team:IISER Berhampur/Notebook

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[![](https://static.igem.org/mediawiki/2021/2/2f/T--IISER_Berhampur--TeamLogo2021.png)](https://2021.igem.org/Team%3AIISER_Berhampur)

[![](https://static.igem.org/mediawiki/2021/8/85/T--IISER_Berhampur--gold_medal.png)](https://2021.igem.org/Team%3AIISER_Berhampur)

* Project

  + [Description](https://2021.igem.org/Team%3AIISER_Berhampur/Description)
  + [Engineering](https://2021.igem.org/Team%3AIISER_Berhampur/Engineering)
  + [Implementation](https://2021.igem.org/Team%3AIISER_Berhampur/Implementation)
  + [Experiment](https://2021.igem.org/Team%3AIISER_Berhampur/Experiment)
  + [Proof of Concept](https://2021.igem.org/Team%3AIISER_Berhampur/Proof_Of_Concept)
  + [Contribution](https://2021.igem.org/Team%3AIISER_Berhampur/Contribution)
  + [Results](https://2021.igem.org/Team%3AIISER_Berhampur/Results)
* [Modelling](https://2021.igem.org/Team%3AIISER_Berhampur/Model)* Human Practices

    + [Integrated HP](https://2021.igem.org/Team%3AIISER_Berhampur/Human_Practices)
    + [Science & Communication](https://2021.igem.org/Team%3AIISER_Berhampur/Communication)
    + [Notebook](https://2021.igem.org/Team%3AIISER_Berhampur/Notebook)
  * Team

    + [Members](https://2021.igem.org/Team%3AIISER_Berhampur/Team)
    + [Attributions](https://2021.igem.org/Team%3AIISER_Berhampur/Attributions)
    + [Collaboration](https://2021.igem.org/Team%3AIISER_Berhampur/Collaborations)
  * [BioBricks](https://2021.igem.org/Team%3AIISER_Berhampur/Parts)
  * Awards

    + [Medal Criterias](https://2021.igem.org/Team%3AIISER_Berhampur/Medals)
    + [Safety](https://2021.igem.org/Team%3AIISER_Berhampur/Safety)
  * [Entrepreneurship](https://2021.igem.org/Team%3AIISER_Berhampur/Entrepreneurship)

iGEM IISER\_Berhampur 2021

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February - March

April

May

June

July

August

September

October

November

## February - March

Week1

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Week2

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Week3

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Week4

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## April

Week2

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### Friday, 9 April

* Final iGEM 2021 team announced.
* First iGEM meet with iGEM 2021 team.
* Brainstorming for the final project.

### Saturday, ‎10 April

* iGEM meet with PI - Dr. Vinay Bulusu, other advisors and 2020 team.
* Overview and summary of the project; division of team-roles and responsibilities.

### Sunday, 11 April

* iGEM student group meet-Planning for wet lab, HP and modelling teams

Week3

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### Monday, 12 April

* iGEM group meeting - brainstorming session.

### Tuesday, 13 April

* iGEM student group meet 1 (HP) with last year’s HP team.
* iGEM 2020 team discussed their experiments, experience and ideas with 2021 team
* iGEM 2021 team also put forward their ideas

### Wednesday, ‎14 April

* iGEM wet lab group meet 2.
* Cas14, detection techniques.and reporter systems

### Thursday, ‎15 April

* iGEM group meet with advisors
* Wet lab and sponsorship discussion.

Week4

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### Wednesday, 21 April:

* iGEM group meeting - brainstorming session
* iGEM group meeting - brainstorming session

### Friday, 23 April

* iGEM group meeting - brainstorming session

### Sunday, 25 April

* iGEM group meeting - brainstorming session

### Wednesday, 28 April

* iGEM registration complete

## May

Week1

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### Tuesday, 4 May

Started campaigning on twitter for Genscript sponsorship (for the month of May)

### Wednesday, 5 May

HP student team meet-

Collaborations and discussions+ Discussed posters for team and funding, demarcation, logo and team introduction posts.

### Saturday, 8 May

* Team meeting with PI
  Discussed :-
  + Team registration
  + Promega project outline
  + Fine tuning proposal
  + Modelling data collection and wet lab literature review

### Sunday, 9 May

* Posting of demarcation
* Student registration into iGEM website
* Wet lab - started project promega outline and literature survey about strain distribution.
* modelling - epidemiology model lit survey, data collection and presentation work.

Week2

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### Monday, 10 May

* Posting the first tile of the project logo and caption - instagram.
* HP - emails sent to DOSA, CSA and social cell regarding retweets for sponsorship.
* Design team - Team poster in the making.

### Tuesday, 11 May

HP student team meet -

+ Final distribution of sub team tasks (collaboration, social media and interviews, designing, sponsorships, blogs) to team members.
+ Collaboration sub team student meet- Timeline and events scheduled and planned, work divided among team members.
+ Continuing the comic strip activity message sent to iGEM team Costa Rica

### Wednesday, 12 May

* Emails sent to NGOs and private TB organizations for Genscript retweets campaign
* Wet lab student team meet -
  + Literature review for finding the relevant MDR-TB strain, promega sponsorship write up, project outline.
  + Tasks were divided among the team members

### Thursday, 13 May

* 3rd and 4th tile of logo posted on instagram
Modelling student team meet

+ Distribution of disease in age, seasonal variation and proportion of MDR, XDR and normal TB in the population
+ Presentation - math model and data collection.

### Friday, 14 May

* iGEM student team meet
  + Discussed overall on goings-designing and social media.
  + Modelling team presentation.
* Design student team meet -
  + Discussed ideas for intro and team poster layout and design.

### Saturday, 15 May

* Logo posted on twitter, genscript crossed 400 retweets!
* iGEM team meet with PI, advisors and 2020 team :-
  + Math modelling presentation (Om and Ritika)- Explained the SEIRD model, equations and graphs, refining of model - considering background mortality
  + Sponsorships - Genscript twitter campaign, promega write up, snapgene license
  + HP team updated PI with - School event, comic strip event, collaboration with the Moscow team, and ideas for interviews with doctors.
  + Wetlab - ongoing literature review

### Sunday, 16 May

* Wiki student team meet-
  + Division of work among sub team members (CSS and HTML)
  + Bar tab to be designed and kept
  + Current log to be updated on the website
  + Team page to be updated
* Design student team meet - Worked on and finalized intro design poster.
* HP student team meet-Worked on treasure hunt patterns and questions
* Sent the comic strip event email to iGEM HQ.

Week3

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### Monday, 17 May

* Tweet for Genscript 50$ gift card sponsorship tweeted.
* HP student short meet for outreach program - treasure hunt, discussed riddles and answers for the hunt

### Tuesday, 18 May

* HP outreach event (treasure hunt) student team meet- Discussed 11th grade hunt rules and riddle questions.
* Project introduction on twitter - part 1

### Wednesday, 19 May

* Wiki team short meet - discussed google site
* HP treasure hunt - 9th and 10th hunt riddles discussion
* Sponsorships meet with PI - Beckman Coulter grant and discussed some budgets

### Thursday, 20 May

* Wiki student team meet - Notebook update and wiki design (template, theme colours, google site)
* Treasure hunt mock run
* iGEM student team meet with ambassador (Ms. Shruti Sridhar) -
  + Technical and non technical collaborations
  + How to increase sponsorships
  + Contribution for future teams
  + Interviews - more important than surveys.
  + Social work

### Saturday, 22 May

* Poster and form for scrofula (treasure hunt) posted on social media
* Poster for rise of the phoenix (comic strip) posted on twitter
* Awareness Program for School Students (treasure hunt) email sent to CSA who circulated among faculties
* iGEM team meet with PI and advisors
  + Wet lab presentation - lit review on mutation patterns, future plans
  + HP treasure hunt-outreach program presentation
  + Drawbacks were discussed and it was suggested to conduct the events in regional languages.
* Sponsorships, interviews and collaborations -
  + Promega, Beckman Coulter, Merck , Biocon sponsorships
  + International NGOs partnership/collab to spread awareness about TB and get funds
  + Interview with MKCG - Dr Taher
  + International collaboration (comic strip) email to HQ.
* Design student team meet -
  + Team poster design
  + Brochure design and content for crowd funding and sponsorships.
* iGEM student team meet with Vilnius-Lithuania iGEM team -
  + Vilnius team presented their project outline and human practices.
  + Bpr team presented our project and HP activities
  + Discussed collaborations - surveys and interviews with Indian NGOs or doctors working in amoebiasis
  + Future collaborations involving- combined interviews, podcasts, software development using mobile phone (for us)
* Wiki team student meet
  + Discussed main design, social media icon images, description for origin of the project proposal, logo description and design and team 2021 design.

### Sunday, 23 May

* Modelling and wet lab student team meetings
* Discussed - interview questions, podcast

Week4

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### Monday, 24 May

* First Outreach program held - Treasure hunt (The Tale of Scrofula) and interactive presentations for spreading awareness about MDR-TB and TB.
* Certificates for participation and winners to be given by 27th
* Received license for snapgene

### Tuesday, 25 May

* HP student team meet -
  + Treasure hunt in regional language and targeting international students
  + Fundraising - contacting NGOs, Colouring book edition 2 - get funding estimate
  + Emails to international and local schools for funds and awareness.* HP team meet with PI - discussed fundraising and sending emails to NGOs
  * Team Poster posted on twitter

### Wednesday, 26 May

* Team poster posted on instagram
* Genscript campaign (ongoing since announcement) - 4 days left
* iGEM student team meet with IISER Tirupati iGEM team
  + Discussed- each other’s project, wet lab (presentations-both) and modelling.TPT - contraceptive method using bacteria to block sperm entry.+ suggestions from TPT team -
      - Making a disposal system for waste from our experiments
      - Contacting Doctors from Safdarjung hospital- huge TB facility
      - IGIB Delhi - can contact profs for interview
      - Ongoing Human Practises discussion (from our side mainly)
      - General discussions and collaborations ideas with both teams

### Thursday, 27 May

* iGEM student team meet-
  + Giveaway for retweets
  + Created sub team group for managing Interviews
  + Selected All India iGEM Meet (AIIM) representatives Promotion video discussion
* HP (collaboration, social media & interviews sub team) student team meet -
  + Discussed - collaborations, prepared team list
  + promoted comic strip
  + Villinus team requirement list.

### Friday, 28 May

* Genscript twitter campaign

### Saturday, 29 May

* iGEM student team meet with PI and mentors (Wet lab brainstorming) -
  + A paper that used cas14a+guide RNA recombinant plasmids
  + Ordering of relevant plasmids
  + Different approaches by PI and students in using sgRNAs (multiple & WT/mutants) and fluorescence production (detection).
  + Settled on specific mutants
* Promega sponsorship project outline - final draft
* Interview sub team meet

### Sunday, 30 May

* Interview with Dr Shaik Taheruddin (microbiologist MKCG medical hospital) -
  + Presentation of project idea - wet lab and modelling questions
  + Documentation
  + General discussions
* iGEM and project brochure done

## June

Week1

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### Wednesday, 2 June

* iGEM HP student team meet -
  + School event - Small skit
  + Crowdfunding ideas
  + Podcast and promotion video ideas
  + Collaboration ideas
  + Merchandise
  + Personal email to carry out comic strip

### Thursday, 3 June

* Biobrick student team meet with 2020 biobrick team -
  + I Plasmid and Restriction enzymes selection - preferably from the registry.

- Emailed Vilnius team for cas14 plasmid

### Saturday, 5 June

* List of researchers made for the Vilnius team

### Sunday, 6 June

* iGEM team meet with Rochester iGEM team -
  + Exchanged each other’s project ideas
  + Our suggestions - attaching it to a BP/heart monitor etc. device
  + Discussed Modelling aspects
  + Human practices ideas exchange - promoted our comic strip, and invited them to our podcast
  + Interviewees from US and India
* Email sent to Vilnius team of interview contacts on people working with amoebiasis
* Received interview contacts from Lithuania on people working with TB

Week2

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### Monday, 7 June

* iGEM student team meet -
  + Promotion video ideas
  + Podcast ideas
  + Nukkad natak
* Podcast team meet with Niraj -
  + Platforms (for editing and recording) and distribution manuals
  + RSS link for podcasters
  + Mic tips
  + Audio to text conversion
* Wet lab student team meet -
  + Hardware and software - general working
    - Fluorescence detector and report software (app)
    - Main hardware device
  + General working of our system - guide RNAs and target genes

### Tuesday, 8 June

* Podcast student team meet -
  + First 4 episodes of podcast - overview and topics to talk about
  + Ideas for name and background music* MIT\_MAHE’s instagram fun activity participation invitation
  * List of possible interviewees (for all sub teams)

### Wednesday, 9 June

* Vinay sir’s intro post posted on instagram
* iGEM HP student team meet
  + Promotional video - animation, graphs etc, one voice
  + Nukkad Natak - video to be made after NGO replies

### Friday, 11 June

* Wet lab student team meet

### Saturday, 12 June

* Submission of Beckman Coulter sponsorship
* iGEM student team meet - presentation of wet lab project
* iGEM podcast team meet - trailer

### Monday, 14 June

* Promotional video meet
  + First draft of story outline headings
  + Writing content for each heading and subheading
* Podcast student team meet -
  + Script for trailer
  + Work on episode 1
* Project Brochure done

Week3

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### Wednesday, 16 June

* Trailer video done
* Modelling Team meet:
  + Discussed about future modelling plans - DNA amplification optimum time, amount of cas14 required, software app

### Friday, 18 June

* iGEM interview with MDR-TB survivor Radhika Jatpag -
  + Discussed in detail the life of an MDR-TB patient - symptoms and diagnosis, cost of treatment, medication and daily routine, mental health, support groups, spreading awareness and outreach to society (giving out free protein rich food)* iGEM student team meet - discussed progress of work in each sub team, wet lab and human practices

### Saturday, 19 June

* iGEM team meet with PI and advisors -
  + Wet lab work on goings
  + Sponsorships, multipurpose outline, rpob strain
  + Human Practices - doctors and professors to interview, future sponsorships, promotional video
  + Biobricks team meet - safety form biobricks details

### Monday, 21 June

* Podcast trailer poster done

### Tuesday, 22 June

* Modelling team meet with Arun sir - discussed docking and dynamics, future prospects
* Podcast trailer poster posted and Trailer released on youtube and spotify.

### Thursday, 24 June

* iGEM student team meet - Hardware and software (modelling)
* iGEM podcast team meet - voice overs for 3rd episode, script writing and panel discussions

### Friday, 25 June

* Modelling team meet -
  + Decided the official document structure
  + Epidemiological model in more detail - updating equations and parameters
  + Sending emails to epidemiologists for cross checking data
* Submission of preliminary safety form

Week4

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### Saturday, 26 June

* Promotional video meet - decided final script, discussed video design and pattern

### Sunday, 27 June

* iGEM team meet with Korea\_HS team -
  + Each others project through presentations
  + Human practices - outreach and human interaction
  + Possible collaboration topics - Modelling (fluorescence), wet lab (cytotoxicity)
* Rice art collaboration sent to Manipal
* Made official project presentation

### Monday, 28 June

* iGEM student team meet
* iGEM team meet with Sridhar sir - Contact microbiologists rather than doctors, BIRAC for possible funds, make project more viable - Indian project for worldwide application, hardware prototype, developing the prototype for commercial use
* iGEM modelling team meet - current SIRD model - graphs, equations and values, possible interviews, future model prospects
* iGEM design team meet- discussed comic strip

### Tuesday, 29 June

* iGEM HP team meet - plans in motion, collaboration ideas for NGOs, mailing for data and epidemiologists
* iGEM Modelling team meet - future model

### Wednesday, 30 June

* Submission of impact grant
* Human Practises - flyers, apps, donation, targeting younger TB affected generation, human transmission

## July

Week1

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### Thursday, 1 July

* iGEM team meet with IISER Kolkata iGEM team - Both team presented their projects. The detailed discussions are as follows :-
  + Wet lab -
    - CRISPR cas system
    - Colorimetry
    - conducting joint webinars/tutorials on crispr
  + Modelling
    - Data collection
    - Graphs and equations
    - Mathematical modelling approaches
  + Human Practices -
    - Podcasts invitation, joint interviews, awareness programs
    - Collaboration on awareness for a larger audience via brochures, pamphlets etc.
* Dry lab and modelling collaboration

### Friday, 2 July * iGEM student team meet with NGO head Amit Tuteja (Discussion and advice) - + Human Practices- Awareness, ideas to assist people currently diagnosed with TB, direct assistance via kit + Wet Lab- discussed LAMP and strains, cost reduction- market survey + Modelling - Epidemiological survey. * Release of first podcast episode Saturday, 3 July * Russian meet event - Presented our project, casual interactions with other teams. * Modelling team meet- discussed the future diagnostic model * Comic strip post by iGEM costa rica and iGEM Kcl Sunday, 4 July * Comic strip post by igem stony brook, iGEM MUANL, iGEM China

Week2

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### Monday, 5 July

* iGEM team meet with PI -
  + Updates of all sub teams given
  + Discussed possible funds and interviews
  + Discussed wet lab strain and mtb genome safety
* iGEM team meet with iGEM team Athens -
  + Podcast
  + Offer to conduct our wet lab experiments and provide reagents
  + They provided inputs on designing tools
* Rochester, Athens and Vilnius agreed for podcast - work started

### Tuesday, 6 July

* Modelling team meet - Discussed early, late and no detection model, enzyme kinetics - collaboration with wet lab
* iGEM team meet - Discussed wet lab, modelling - collab with wet lab for protocols etc. for enzyme kinetics model
* iGEM Global collaboration team meet - Discussed global meet between teams working with CRISPR worldwide for better wet lab collaboration and ideas for wet lab, modelling and other sub teams

### Thursday, 8 July

* iGEM meet with lafafa for merchandise - Detailed discussion on design
* iGEM project and synthetic biology Informative posts on social media

### Friday, 9 July

* Modelling team meet- discussion on ideas for official documentation

### Sunday, 11 July

* iGEM wet lab team meet - Discussed LAMP and interview questions
* iGEM team meet (human practices) - Discussed Global CRISPR meet, AIIM event - organizers and event, Promotional video final

### Monday, 12 July

* iGEM interview with Professor Rajesh Sudhir Gokhale -
  + Emphasis on cost and funding - phosphothiode cost
  + Product management and marketing strategies
  + Funding companies and assistance in getting us contact for funds
  + Efficiency and specificity of our project
* iGEM modelling team meet

### Tuesday, 13 July

* Interview with Dr Sutirth Dey -
  + Modelling aspects of the project presentation and brief introduction to the project
  + Detailed discussion on the model equations and output graphs
  + Data collection
  + Discussion on hardware device

Week3

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### Wednesday, 14 July

* iGEM team with PI and mentors -
  + Wetlab,- multipurpose document, POC and LAMP, promotional video; suggestions were given, modelling main goal and literature review
* HP team meet
  + Event planning - workshop, teaching synthetic Biology
  + Interview consent forms
  + IHP discussion
  + Sponsorship emails
* Release of podcast episode 2

### Thursday, 15 July

* Unboxing of iGEM 2021 distribution kit at IISER Berhampur.

### Monday, 19 July

* CRISPR meet team emails and slot booking

### Tuesday, 20 July

* Meet with HP team and PI - Discussed fundraising ideas
* Project Management team meet - Discussed objectives and approaches towards cost analysis
* Wet lab meet - Interview questions discussion
* Start of IHP wiki work

### Wednesday, 21 July

* Wet lab team meet - Discussion for interview questions
* AIIM meet - Indian iGEM team meets with briefing about events and schedule

### Thursday, 22 July

* Promotional video updated
* iGEM team meet - Editing of presentation for AIIM event
* Updates on fundraiser
* IHP wiki work
* WAO meet - Making and sending questions to teams

### Friday, 23 July

* AIIM events - participation in other events and WAO hosted by IISER Berhampur
* Mock presentation meet
* IGCC email to iGEMHQ

### Saturday, 24 July

* AIIM project presentation
* IGCC meet - Discussed event rules

### Sunday, 25 July

* AIIM event participation
* Fundraiser website sent for verification
* Emails drafted for IGCC
* Promotional video uploaded on social media

Week4

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### Monday, 26 July

* iGEM meet with PI and advisors - Modelling presentation. strain analysis discussion with Arun sir

### Tuesday, 27July

* Biobrick team meet - Parts construct design discussion
* IGCC team meet - Discussed event particulars, schedule etc.

### Wednesday, 28 July

* Fundraiser website approved and live

### Thursday, 29 July

* Wet lab team meet with PI - Discussed strains, LAMP primers and sgRNA design
* School event workshop meet - Discussed techniques to teach them
* IGCC meet - Discussed schedule

### Saturday, 31 July

* iGEM team meet - Discussed fundraiser and campaigning
* Ignite Life Science foundation grant proposal written

## August

Week1

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### Sunday, 1 August

* IHP website design discussion

### Monday, 2 August

* IGCC -
  + Presentations details email sent to teams
  + Details and photos of speakers received
  + Campaign poster done
* Modelling team meet with PI

### Wednesday, 4 August

* Impact grant winners announced [IISER Berhampur won]

### Thursday, 5 August

* Modelling team meet with PI - Discussed Probable future rise in strains model and Enzyme kinetics

### Friday, 6 August

* Received iGEM Rochester collaboration request

### Saturday, 7 August

* HP update to leader
* Mail sent to Dr. Basudev Maji and Sundaram Acharya to confirm timing.
* Planning for campaign

Week2

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### Monday, 9 August

* IGCC work - sending emails

### Wednesday, 11 August

* IGCC wirk

### Thursday, 12 August

* IGCC - Invite emails to institutions, Syn Bio Quiz invitation for registered teams and in All India igem group

### Saturday, 14 August

* IGCC event day 1
  + Team presentations
  + Quiz - Fun with synbio

### Sunday, 15 August

* IGCC event day 2
  + Team presentations
  + Talks by speakers - Dr Sundaram Acharya and Dr Basudev Mazhi

Week3

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### Wednesday, 18 August

* iGEM impact grant form mail received

### Thursday, 19 August

* NEB reagents arrived
* iGEM team wet lab meet with PI - Wetlab planning
* Won genscript sponsorship

### Friday, 20 August

* iGEM team meet - Discussed about merchandise
* Purchasing Video and audio recording software
* iGEM meet team meet with iGEM IISER Kolkata
  + Discussed children’s story book

### Saturday, 21 August

* iGEM team meet with PI -
  + Genscript grant celebration and discussion
  + Hardware meeting
  + Merchandise with grant money

### Sunday, 22 August

* Meeting with theracues -
  + Discussed project - brief introduction and prototype including specifications
* Wiki team meet - Discussed web page design

Week4

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### Monday, 23 August

* iGEM team meet with PI and Professor Sridhar -
  + Presentation for Theracues
  + Inputs from Sridhar sir - focus on prototype, POC and cost analysis
* iGEM team meet with iGEM NTU -
  + Project presentations by both teams
  + Discussed points of collaboration - Exchanging cas12 sequence, help in Hardware

### Friday, 27 August

* Wet lab - Growing competent cells in LB media, preparing LB broth

### Monday, 30 August

* Safety protocol and authorization orientation
  + Lecture on GLP and introduction to equipments to be used
  + Fixed workstation

### Tuesday, 31 August

* Issued pipettes, pipette tips, pipette stand, 2 markers from UG-PG lab

## September

Week1

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### Wednesday, 1 September

* School event meet - Discussed tutorials

### Thursday, 2 September

* Wet lab
  + Autoclaving of equipment
  + Preparation of SOB media

### Friday, 3 September

* Wet lab
  + Primary and secondary inoculation
  + Inoue buffer preparation

### Saturday, 4 September

* Wet lab
  + Elution of competent cells
  + Storage in liquid nitrogen

### Sunday, 5 September

* Inoculation of BAC clones

Week2

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### Monday, 6 September

* Plasmid isolation of BAC clones
* Synverse roundtable with team Vilnius released - episode 4

### Wednesday, 8 September

* Wet lab - Agarose gel run
* Human Practices -
  + Invitation to participate in problem book collaboration by team Russia
  + Confirmation of inventa collaboration
* iGEM team meet with PI -
  + Wet lab updates and future plan
  + Modelling update - phylogenetic analysis
  + Fundraiser and merchandise
  + Discussion about jamboree meet up
  + Biobricks - plasmid design discussion

### Thursday, 9 September

* Hardware team meet with Dr Satyaprasad P Senanayak -
  + General hardware model and brief introduction to the experiment
  + Availability of laser

### Sunday, 12 September

* iGEM all India team meet with ambassadors - Discussion about Jamboree meetup at Goa
* Duke mini Jamboree - Project presentation
* Registration for Dual safety workshop
* iGEM team meet for Giant Jamboree meet discussion

Week3

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### Monday, 13 September

* Human Practices
  + Merchandise planning
  + Confirmation to send NTU contacts

Week4

### Wednesday, 22 September

* Hardware team meet - Discussed new development in the hardware model

### Thursday, 23 September

* Wet lab
  + Media preparation - LB broth and LB agar

### Friday, 24 September

* Inventa talk
* Media preparation

### Tuesday, 27 September

* Wet lab - Poured agar plates
* Meet with PI -
  + Gold medal criteria
  + Modelling - different models and phylogenetic analysis
  + Protocol for transformation
* Wiki team meet - Content discussion

### Wednesday, 28 September

* Wet lab -
  + Absorbance and fluorescence measurements of serially diluted venus, molar concentration calculations
  + Transformation of plasmids (rpoB and KatG) in *E coli*
* Modelling team meet - Phylogenetic analysis discussion

### Wednesday, 29 September

* Wet lab -
  + LB media and broth preparation
  + Autoclaving media, broth pipette tips and plates

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## October

Week1

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### Saturday, 2 October

* iGEM team meet -
  + Medal criteria discussion
  + Assigning tasks to team members

### Sunday, 3 October

* Hardware meet - Prototype discussion
* Team meet for wiki design - Individual wiki page design and layout

Week2

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### Monday, 11 October

* Wet lab - cas14 and Bac clones primary inoculate
* Parts improvement discussion with Dr Selvi Bharatavikru
* Modelling - Phylogenetic trees

### Tuesday, 12 October

* Wet lab planning & inoculation of cas14a1 plasmid
* Modelling - phylogenetic analysis

### Wednesday, 13 October

* Plasmid isolation
* Transformation of plasmid into BL21DE3

Week3

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* Documentation
* Working on Wiki page

## November

Week2

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### Saturday, 13 November

* We reached Resort Marinha Dourada,Goa for our Giant Jamboree Meet

### Sunday, 14 November

* Result declared at Giant Jamboree and we secured the GOLD medal.

* ![](https://static.igem.org/mediawiki/2021/3/3c/T--IISER_Berhampur--Teamsssss3.png)
* ![](https://static.igem.org/mediawiki/2021/4/4d/T--IISER_Berhampur--SPON42021.png)
* ![](https://static.igem.org/mediawiki/2021/7/74/T--IISER_Berhampur--SPON62021.png)
* ![](https://static.igem.org/mediawiki/2021/e/e2/T--IISER_Berhampur--Teamsssss2.png)
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# Team:IISER Berhampur/Engineering

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* Project

  + [Description](https://2021.igem.org/Team%3AIISER_Berhampur/Description)
  + [Engineering](https://2021.igem.org/Team%3AIISER_Berhampur/Engineering)
  + [Implementation](https://2021.igem.org/Team%3AIISER_Berhampur/Implementation)
  + [Experiment](https://2021.igem.org/Team%3AIISER_Berhampur/Experiment)
  + [Proof of Concept](https://2021.igem.org/Team%3AIISER_Berhampur/Proof_Of_Concept)
  + [Contribution](https://2021.igem.org/Team%3AIISER_Berhampur/Contribution)
  + [Results](https://2021.igem.org/Team%3AIISER_Berhampur/Results)
* [Modelling](https://2021.igem.org/Team%3AIISER_Berhampur/Model)* Human Practices

    + [Integrated HP](https://2021.igem.org/Team%3AIISER_Berhampur/Human_Practices)
    + [Science & Communication](https://2021.igem.org/Team%3AIISER_Berhampur/Communication)
    + [Notebook](https://2021.igem.org/Team%3AIISER_Berhampur/Notebook)
  * Team

    + [Members](https://2021.igem.org/Team%3AIISER_Berhampur/Team)
    + [Attributions](https://2021.igem.org/Team%3AIISER_Berhampur/Attributions)
    + [Collaboration](https://2021.igem.org/Team%3AIISER_Berhampur/Collaborations)
  * [BioBricks](https://2021.igem.org/Team%3AIISER_Berhampur/Parts)
  * Awards

    + [Medal Criterias](https://2021.igem.org/Team%3AIISER_Berhampur/Medals)
    + [Safety](https://2021.igem.org/Team%3AIISER_Berhampur/Safety)
  * [Entrepreneurship](https://2021.igem.org/Team%3AIISER_Berhampur/Entrepreneurship)

Modelling 2021

![Our animated logo is keeping you company until the page has loaded.](https://static.igem.org/mediawiki/2021/4/40/T--IISER_Berhampur--LoadCODEM.gif)

[Skip to main content](#slide1-chapter1)

# Introduction

Tb is caused by a rod-shaped bacteria called *Mycobacterium tuberculosis*. It is a human pathogen responsible for causing tuberculosis (TB) and it's a major health problem in India and all across the world. Antimicrobial resistance has risen as the next big threat to the healthcare system. MDR-TB, a drug-resistant variant of TB is also rising due to inadequate facilities for patients and unaffordable detection. Currently, the culture methods to detect MDR-TB are slow and the molecular detection methods are expensive and require expertise. In low-income countries like India, detection for MDR is unavailable in peripheral regions like in rural areas and is only available in far places from the towns. Economically challenged patients usually can't afford doctors’ advice of proper diagnosis and potentially spread MDR-TB. This unavailability of proper diagnostic tests often limits the diagnosis of patients to TB while being MDR-TB patients, resulting in late diagnosis. All this pointed us towards the need for a rapid, affordable, and accessible diagnostic kit for MDR-TB.

# Research

Following the preliminary study, where we discovered the necessity for a new detection method for MDR-Tb. We began a literature review on the antagonist (MDR-TB) and current detection techniques. We divided the literature survey into 3 main parts. Understanding the disease, prevalence of the disease, and current strategies used for its detection.

1. **Understanding the disease:**
   In this part, we found out that rpoB and katG genes of Mycobacterium tuberculosis encode for ß subunit of RNA polymerase and catalase-peroxidase respectively. Mutations in rpoB confer resistance to rifampicin and are confined to an 81 bp region in the gene known as the rifampicin resistance determining region (RRDR). Similarly, a katG mutation confers resistance to isoniazid. The presence of both these mutations in a single isolate of Mtb will highlight it as an MDR-TB while the presence of either mutation makes it a monoresistant TB. [3]
2. **Prevalence:**
   After an extensive literature review, we found that S315T in katG and S450L in rpoB were found to be the deadliest and most widespread mutations respectively across India. This analysis was performed by reading a variety of publications focusing on MDR-TB mutation patterns in different regions (North and South) and states in which MDR-TB was highly prevalent [4]. We mostly analyzed collected data from these regions in terms of sample size, percentage of resistance, and the recovery rate in patients as well as on more recent publications. Along with studying the mutation patterns, we also analyzed the strains where these experiments were conducted[5]. The most common strain used in the laboratory we found was  *Mycobacterium tuberculosis* H37Rv [6].
3. **Current strategies used for its detection:**
   Studies in this section showed that current detection methods are divided into culture methods, phenotypic methods, and molecular methods. However, the culture methods to detect MDR-TB are slow and take time in weeks, and also have requirements of resources and skilled technicians. On the other hand, molecular detection cuts down on time to three to four hours but these methods are expensive and require expertise.
   This section also included working and applications of different diagnostic kits currently used in medicine. This helped us to come up with a prototype design.[7] [8] [9]

Design

Build

Test

Learn & Improve

Introduction

LAMP

sgRNA and Cas14 complex

Hardware

CODE M Constructs

Proposed Constructs

Building Our BioBricks

Building LAMP Primers

Building sgRNA

Building Hardware Components

Testing LAMP Primers

Test: sgRNA + Cas14

Test: Hardware

Learn Section

Improve Section

References

## Introduction

The goal was clear: To make a cheap, accessible, sensitive, and rapid diagnostic kit for MDR-TB detection. We started by finding out sensitive and cheaper alternatives to the current system and added them as modules in our detection kit.
 The final design of our kit had 3 modules.

1. LAMP amplification
2. sgRNA and Cas14 complex
3. Hardware component.

Each module was designed to fulfill a definite purpose to reach our goal.

## LAMP

Loop-mediated Isothermal Amplification technique (LAMP) is an amplification method that amplifies DNA at a constant temperature. It is a novel nucleic acid amplification method that amplifies DNA with high specificity, efficiency, and rapidity. This technique has been used as a detection method to detect bacterial, parasitic, and viral pathogens. This method of amplification in our kit will replace the general PCR-based amplification. The advantages of LAMP over conventional PCR are (1) its minimal setup (only a water/dry bath is required) and (2) a greater yield of amplified DNA.
LAMP will serve three purposes in our kit:-

1. it will help increase the sensitivity of the kit
2. As it removes the need for a thermal cycle for amplification it will help decrease the cost.
3. Phenol-mediated color change, if amplification happens, will also help to detect the presence of TB.

## sgRNA and Cas14 complex

Cas14a1 belongs to a family of exceptionally compact RNA-guided nucleases which are about the size of 400-700 amino acids. It has a bilobed architecture with REC (recognition) and nuclease (NUC) lobes. The single guide RNA (sgRNA) has a scaffold region and guiding region. The sgRNA is an engineered version of natural guide RNA found in archaea and bacteria, which confers ease and flexibility to use with Cas14a1 nuclease due to its reduced size and enhanced activity. Also, it does not require a PAM (Protospacer Adjacent Motif) sequence for target recognition. Cas14a1 and sgRNA can readily form complexes. We wanted to use a cost-effective and highly sensitive tool for SNP genotyping in the *Mycobacterium tuberculosis* genome. The main reasons for exploiting this tool in project CODE M are:

1. The miniature size of Cas14a1 will help to reduce the cost of production of the kit by decreasing the materials required for its synthesis as compared to other Cas proteins.
2. Its high-fidelity SNP genotyping will make it fit for use in our kit for highly sensitive detection of MDR-TB.
3. sgRNA can be easily customized by engineering the spacer sequence to effectively target complementary sequences from the katG and rpoB genes in the Mtb genome.

## Hardware

A fluorometer is essential for the detection of fluorescence in our kit as the amount of fluorescence produced is very low and cannot be detected by the naked eye. But a fluorometer also requires a computer set up to operate and computer setups are not cheap nor is it accessible to remote locations and because of this in low-income countries like India it is not possible to have a fluorometer at the periphery level. Hardware components in our kit will include mobile phone microscopy. Mobile phone microscopy can be considered as a plausible alternative to a fluorometer as mobile phones are more accessible and easy to handle than computers.
Hardware will serve two purposes in our kit.

1. It will help us make our kit more accessible and easy to handle as it will be a small compact device.
2. It will also help to reduce the cost of our kit as phones are much cheaper than a fluorometer and a computer setup.

For a detailed description of each module go to [proof of concept>>design](https://2021.igem.org/Team%3AIISER_Berhampur/Proof_Of_Concept)

## CODE M Constructs

From literature and countless brainstorming sessions, we finalized upon using Cas14a1 protein for detecting SNPs in the Mtb genome extracted from sputum samples. The miniature size and high-fidelity SNP genotyping made us choose this for our project.
As the Cas14a1 was never used in any iGEM project before, it was never a part of the iGEM Parts Registry. So, our team took the lead to introduce its coding sequence and several other relevant parts as BioBricks in the Parts Registry.

The first challenge that we encountered was the selection of highly specific single-guide RNAs (sgRNAs) to minimize the off-target effects. Secondly, was the presence of a few unwanted restriction sites in our sequences. We worked on these challenges as we designed our biobricks.

## Proposed Constructs

Using various basic parts, we assembled them into the required constructs for our project. Check out the constructs in the table below:

| Part Number | Construct | Part Number | Standard Construct |
| --- | --- | --- | --- |
| BBa\_K3982025 | CODE M Construct C1 | BBa\_K3982026 | CODE M Construct S1 |
| BBa\_K3982030 | CODE M Construct C2 | BBa\_K3982038 | CODE M Construct S2 |
| BBa\_K3982031 | CODE M Construct C3 | BBa\_K3982039 | CODE M Construct S3 |
| BBa\_K3982032 | CODE M Construct C4 | BBa\_K3982040 | CODE M Construct S4 |
| BBa\_K3982033 | CODE M Construct C5 | BBa\_K3982041 | CODE M Construct S5 |

## Building Our BioBricks

1. For our construct C1, we propose to clone Part [BBa\_K3982006](http://parts.igem.org/Part%3ABBa_K3982006) along with Tet repressor (TetR), Tet operator (TetO), overlapping tetR/tetA promoters, RBS and T7Te terminator in pSB1C3 backbone.
2. For each construct C2, C3, C4, C5, we propose to insert each of our four CODE M sgRNA sequences in the [Addgene Plasmid # 15030](https://www.addgene.org/15030/) for targeting wild-type and mutant variants of katG and rpoB genes in Mtb genome, thus building four different constructs. For this, we propose using PCR-based Site-directed mutagenesis (SDM) with mutagenic primers. We plan to use SDM instead of conventional restriction cloning to avoid the generation of any overhangs in our construct and it will allow us for the introduction of specifically designed sgRNA sequences.
3. In addition to the above-mentioned constructs, we propose to build a series of standard constructs S1 - S2 for comparing and testing the efficiency of expression. The standard constructs consist of coding parts from the main constructs along with well-characterized T7 promoters, RBS, and T7Te terminator.
4. We propose to transform all our constructs in E. coli DH5α cells and the expression system to be used will be BL21(DE3) Competent Cells. We choose to use these, as E. coli DH5α cells are versatile and widely used for transformation experiments whereas BL21(DE3) cells have highly efficient transcription machinery.

   Visit the [BioBrick](https://2021.igem.org/Team%3AIISER_Berhampur/Parts) page for more information.

## Building LAMP primers

We first attempted designing our primers manually however manual primer designing proved to be a challenging task. The utilization of software tools was a much more effective method that helped in making the process faster and increasing the likelihood of reaction success. After going through several software, we built our LAMP primers using the NEB LAMP primer Design tool (New England Biolabs) [10] since we found this to be more user-friendly. We also used it as a guide to select our primers. We designed six sets of primers for each gene FIP ( F2 + F1c), BIP (B2 + B1c), F3,B3 and LF and LB ( loop primers).

The primers for both katG and rpoB were chosen according to the below parameters.

![](https://static.igem.org/mediawiki/2021/3/3e/T--IISER_Berhampur--ENG_pic_1.jpg)

The below set of primers are the ones we chose for the rpoB gene sequence. Our project mainly focuses on the S450L mutation pattern represented in green (TCG); however, we optimized our search by selecting primers flanking the 81bp mutation hotspot which also includes the other 3 highlighted codons. These are H445D, D435V, and L431P.

Sequence retrieved from NCBI for strain H37Rv (NCBI Reference Sequence: NC\_000962.3)

**Primer 1:-**

5’Gccggtggaaaccgacgacatcgaccacttcggcaaccgccgcctgcgtacggtcggcgagctgatccaaaaccagatccgggtcggcatgtcgcggatggagcgggtggtccgggagcggatgaccacccaggacgtggaggcgatcaca
ccgcagacgttgatcaacatccggccggtggtcgccgcgatcaaggagttcttcggcaccagccagctgagccaattcatg
gaccagaacaacccgctgtcggggttgacccacaagcgccgactgtcggcgctggggcccggcggtctgtcacgtgagcgtgccgggctggaggtccgcgacgtgcacccgtcgcactacggccggatgtgcccgatcgaaacccct
gaggggcccaacatcggtctgatcggctcgctgtcggtgtacgcgcgggtcaacccgttcgggttcatcga 3’

3’Cggccacctttggctgctgtagctggtgaagccgttggcggcggacgcatgccagccgctcgactaggttttggtctaggcccagccgtacagcgcctacctcgcccacca
ggccctcgcctactggtgggtcctgcacctccgctagtgtggcgtctgcaactagttgtaggccggccaccagcggcgctagttcctcaagaagccgtggtcggtcgactcggttaagtacctggtcttgttgggcgacagccccaactgggtg
ttcgcggctgacagccgcgaccccgggccgccagacagtgcactcgcacggcccgacctccaggcgctgcacgtgggcagcgtgatgccggcctacacgggctagctttggggactcccc
gggttgtagccagactagccgagcgacagccacatgcgcgcccagttgggcaagcccaagtagct 5’

Dimer dG: -2.20 (required oligos are orange)

F3:

CCGCAGACGTTGATCAACAT

B3:

CCCCTCAGGGGTTTCGA

FIP:

CAGCGGGTTGTTCTGGTCCATG GTCGCCGCGATCAAGG

BIP:

CCGGCGGTCTGTCACGTGA AGTGCGACGGGTGCA

F2:GTCGCCGCGATCAAGG

F1c:CAGCGGGTTGTTCTGGTCCATG

B2:AGTGCGACGGGTGCA

B1c:CCGGCGGTCTGTCACGTGA

Dimer dG: -1.66 (required oligos are orange)

LF:

AGCTGGCTGGTGCCGAA

For the same parameters, we obtained multiple sets of primers which we further selected based on a less negative delta G for dimer formation and primer sequences that flanked the target region more uniformly. Unfortunately, there were no loop primers available for the backward reaction.

In a similar manner, primers were also selected for the katG sequence. In katG we mainly focused on the S315T mutation pattern and likewise chose a primer set flanking this mutation. The katG sequence does not have any mutation hotspot region. The parameters considered were similar to those of rpoB primers.

Sequence retrieved from NCBI for strain H37Rv (NCBI Reference Sequence: NC\_000962.3)

**Primer 2:-**

5’GCCGATCTGGTCGGCCCCGAACCCGAGGCTGCTCCGCTGGAGCAGATGGGCTTGGGCTGGAAGAGCTCGT
ATGGCACCGGAACCGGTAAGGACGCGATCACCACCGGCATCGAGGTCGTATGGACGAACACCCCGACGAA
ATGGGACAACAGTTTCCTCGAGATCCTGTACGGCTACGAGTGGGAGCTGACGAAGAGCCCTGCTGGCGCT 3’

Dimer dG: -2.36 (required oligos in orange)

F3:

AGGCTGCTCCGCTGGA

B3:

AGCAGGGCTCTTCGTCAG

FIP:

GGTGATCGCGTCCTTACCGGTGCAGATGGGCTTGGGC

BIP:

ACCGGCATCGAGGTCGTATGGCACTCGTAGCCGTACAGGAT

F2:GCAGATGGGCTTGGGC

F1c:GGTGATCGCGTCCTTACCGGT

B2:CACTCGTAGCCGTACAGGAT

B1c:ACCGGCATCGAGGTCGTATGG

Dimer dG: -1.23 (required oligos in orange)

LF:

GCCATACGAGCTCTTCCA

LB:

CGACGAAATGGGACAACAG

## Building sgRNA

sgRNAs targeting LAMP amplified ssDNA sequences from Mtb will be generated by in vitro transcription using the plasmid vector<https://www.addgene.org/15030/>. The sgRNA sequence containing the crRNA (CRISPR RNA) and trRNA (Tracer RNA) was obtained from Harrington et al. (Harrington et al., 2018) and Kim et al. (Kim et al. 2021)

5’CTTCACTGATAAAGTGGAGAACCGCTTCACCAAAAGCTGTCCCTTAGGGGATTAGAACTTGAGTGAAGGTGGGCTGCTTGCATCAGCCTAATGTCGAGAAGTGCTTTCTT
CGGAAAGTAACCCTCGAAACAAATTCATTTTTCCTCTCCAATTCTGCACAAGAAAGTTGCAGAACCCGAATAGACGAATGAAGGAATGCAACNNNNNNNNNNNNNNNNNNNN3’

The last stretch of N nucleotides is gene and mutation-specific and the corresponding 20 nucleotide sgRNA sequences to be used in the study are shown below:

| S.No | Gene | Mutation | Sequence |
| --- | --- | --- | --- |
| 1 | rpob | Wild Type | CGCCGACTGTCGGCGCTGGG |
| 2 | rpob | S450L | CGCCGACTGTTGGCGCTGGG |
| 3 | katG | Wild Type | GCGATCACCAGCGGCATCGA |
| 4 | katG | S315T | GCGATCACCACCGGCATCGA |

DNA gblocks encoding these sgRNAs with flanking BglII overhangs are assembled and have been ordered from IDT technologies, who are supporting iGEM teams by providing free oligos and DNA fragments. Following receipt of these fragments, they will be cloned in pSP64T plasmid (<https://www.addgene.org/15030/>). The plasmids will then be used for RNA synthesis (sgRNA) by using the SP6 Hiscribe RNA synthesis kit from NEB (New England Biolabs). The in vitro transcribed RNA will be purified and precipitated and used for hybridization.

## Building hardware components

Our hardware component was inspired by the Fozouni et al. (Fozouni et al., 2021) kit for the detection of COVID-19[13]. To detect fluorescence with the highest efficiency. We planned to use a low-powered laser, with a wavelength range between 480 nm to 500 nm, a mirror, an aperture, an interference filter, a sample chamber, and a mobile phone. The laser will act as a light source to illuminate the sample chamber so that fluorescence can be produced. Mirrors and the aperture will help us to adjust the intensity of light and help the light to fall uniformly on the sample chamber. The interference filter will filter out non-essential light (noise) to take up the maximum signal. The sample chamber will be a chip containing space where mutation detection and fluorescence production will happen. The mobile phone will be an actual detector detecting the presence of fluorescence. The mobile phone will take multiple images and calculate the pixels present in the image and then average them out to give an output which is the presence or absence of fluorescence.

## Testing LAMP primers

1. BLAST
2. eLAMP
3. Wetlab experiment with LAMP

# BLAST

In this step, we analyze the accuracy of our NEB-generated primers for this we performed BLAST. Basic Local Alignment Search Tool (BLAST) finds regions of similarity between sequences. It is an algorithm for comparing primary biological sequences like the nucleotides of DNA and/or RNA. We checked the accuracy by measuring the off-target effects in different organisms by performing a BLAST nucleotide search for all 11 primers (rpoB and katG) and limiting the search to 1000 sequences.

# eLAMP

eLAMP is a free PERL script that electronically simulates Loop-mediated isothermal AMPlification(LAMP). It is a fast and inexpensive test of LAMP primer suitability. It helps to electronically simulate amplification ability and the sensitivity of primers. The graphical user interface (GUI) is simple which allows non-specialists users to easily use it, also the command-line interface is suitable for use in pipelines. Results are presented either in a single .csv file or in a GUI panel and they are straightforward to interpret. [11]

Go to experimentation for eLAMP [Experimentation>>eLAMP](https://2021.igem.org/Team%3AIISER_Berhampur/Experiment).

# LAMP sensitivity test (optimization)

The WarmStart Colorimetric LAMP 2X Master Mix is a mixture of Warmstart DNA Polymerase and WarmStart RTx it is a special low-buffer reaction solution having a visible pH indicator for rapid and easy detection of Loop-Mediated Isothermal Amplification (LAMP). Detection of amplification is shown by producing a change in solution color from pink to yellow. This happens due to the production of protons and subsequent drop in pH that occurs due to the extensive DNA polymerase activity in a LAMP reaction.
This system is designed to provide a fast, clear visual detection of amplification. This mix is easy to handle and efficient as it needs only a heated chamber and samples, it also provides readouts of positive amplification in 15–40 minutes which is easy to judge by eyes. [12] WarmStart Colorimetric LAMP 2X Master Mix Typical LAMP Protocol (M1800) was ordered for our kit. But due to COVID restrictions, they didn't arrive on time.

To check the sensitivity of our LAMP assay amount of target DNA will be taken at different concentrations and LAMP assay can be done for each concentration. The presence of phenol-mediated color change will show us that amplification has occurred. In this way, we can find out the minimum amount of Target DNA required for our LAMP assay to function. In our case for proof of principle study, the target DNA is present in the form of BAC clones. So after isolating the BAC clones from the bacteria and measuring the amount of DNA isolated a 1000 fold dilution can be done for the acquired DNA and then LAMP can be performed to check for sensitivity. The minimum concentration of target DNA at which LAMP occurs will help us in the optimization of the LAMP assay.

## Testing sgRNA + Cas14

# BLAST

For preliminary testing to assess the activity of the Cas14a1-sgRNA complex, we ran a BLAST of the spacer sequences. This enables us to find similar or identical sequences in the database for katG and rpoB sequences. A BLAST search was performed with a limit of 1000 organisms to find off-target organisms for both genes. Check the [Results](https://2021.igem.org/Team%3AIISER_Berhampur/Results) page to know more about this.

# Optimization:- (using varying FQ concentration)

As the readout of a perfect hybridization of ssDNA and sgRNA is Cas14a1 mediated trans cleavage of a Fluorescent-Quencher containing DNA probe, we plan to optimize the concentration of DNA probe to get a good signal to noise ratio. We can test sgRNA and Cas14 activity at varying concentrations of FQ pairs. The reaction could be monitored on a plate reader and fluorescence measurements can be taken depending on the fluorophore used. Towards this end, we used a pure recombinant fluorescent protein (Venus) and found that a signal-to-noise threshold of 1000 could be reached at a concentration of 0.4µmoles.
[Go to results section](https://2021.igem.org/Team%3AIISER_Berhampur/Results)

## Testing Hardware

From preliminary analysis, we were able to find out the minimum concentration required for the fluorescence signal of Venus, which was available in our labs at that time. We used a Commercial plate reader (Spectramax ID5) for our analysis. However, for our final design, we will be using Green Fluorescent Protein “EGFP” that shows fluorescence in the 480 nm - 500 nm range (peak at 488 nm). Using the concentration obtained from preliminary analysis, which is 0.4 micro Molar or 40picoMoles giving a signal/noise ratio of 1000, we planned to test it in the proposed model using EGFP. Unfortunately, due to the pandemic, we were unable to access labs and carry out the testing. But we were expecting positive results even with Green Fluorescent Protein “EGFP”, similar to the preliminary analysis done using the YFP. However, if we wouldn't be able to get the desired results, we would have re-done the experiments by either increasing the laser's power while taking necessary precautions or the concentration of the GFP.
[Go to results section](https://2021.igem.org/Team%3AIISER_Berhampur/Results) for YFP venus results

## **Learn Section**

## LAMP

* # 1. Learning from Primers BLAST

   The BLAST result for F3 rpoB, B3 katG, B3 rpoB, LB katG showed minimum off-targets i.e., 174 for F3 rpoB, 164 for B3 katG, 146 for B3 rpoB, and 129 for LB katG. However, off-targets for F3 katG and LF rpoB were substantially higher i.e., 530 and 812 respectively. For *Mycobacterium tuberculosis*, we found that the LAMP primers were specific to our target gene. When we analyzed the off-targets containing other organisms, we found that most of them were not human pathogens. We ruled them out as the likelihood of them being present in a human sputum sample was extremely low. Also, we ruled out some pathogens that were targeted by only 1 LAMP primer, as for the amplification to occur multiple primers are required. The remaining sequences showed an output of “no significant similarity found”.

  We also went deeper into our analysis to find the common human pathogen off-targets across all the BLAST searches. The below table summarizes it.

  | Off-target organism | Pathogenesis | Analysis |
  | --- | --- | --- |
  | *Mycobacterium tuberculosis variant Bovis* | Can cause TB in humans | Part of the tuberculosis complex. Treatment is similar to normal TB. |
  | *Mycobacterium tuberculosis variant microti* | Microti can cause severe pulmonary TB in immunocompetent patients however it mostly harms animals | Part of the tuberculosis complex. Treatment is similar to normal TB. |
  | *Mycobacterium tuberculosis variant africanum* | Can cause TB | Part of the tuberculosis complex. Treatment is similar to normal TB. |
  | *Salmonella enterica* | CODE M sgRNA for targeting wildtype katG gene in *Mycobacterium tuberculosis* | Usually found in the intestine. The chances of being in the sputum sample are negligible. |
  | *Pseudomonas aeruginosa* | Can cause pneumonia | Potential misdiagnosis |
  | *Pseudomonas* | Can cause multiple infections Eg-endophthalmitis, pneumonia, etc. | Potential misdiagnosis |
  | *Chryseobacterium* | Can cause multiple infections Eg - myositis, keratitis, pneumonia, etc. It is also intrinsically multi-drug resistant |. Potential misdiagnosis (rare disease) |
  | *Aspergillus brunneoviolaceus* | Can cause serious illnesses when people with weakened immune systems, underlying lung disease, or asthma inhale their fungal spores. | Potential misdiagnosis if not combined with preliminary testing like X-Ray/CT scan. |

  In this list, the tuberculosis complex group causes pulmonary tuberculosis and has the same treatment regimen as normal TB. For other organisms that can be potentially misdiagnosed as TB by our device can be ruled out in the next step of SNP detection by Cas14. If the cas14 does not recognize the product either as wild type, rifampicin-resistant, or isoniazid-resistant we can send the sample for further testing.
* # 2. Learning from e-LAMP

  As eLAMP electronically simulates the isothermal amplification. The results obtained from eLAMP will tell us if primers made by us will work or not. It will also tell us about the amplification ability of our LAMP primers. The sensitivity of our LAMP primers will also be simulated in the eLAMP. eLAMP also provides the exact number of mismatches in our LAMP primers. However, eLAMP is limited to primers made on LAVA and Primer explorer as our primers were made with NEB LAMP Primer Design Tool it did not show proper results for us.

* # 3. Learning from LAMP sensitivity test(optimization)

  This test when performed will give us the minimum amount of target DNA required for amplification using the LAMP kit. This will help us to optimize the amount of LAMP reagents needed and the amount of bacterial DNA needed from patient samples. To get some more insights into this section we modeled it by making an enzyme kinetics model.
  [Modelling Page](https://2021.igem.org/Team%3AIISER_Berhampur/Model)

## Learning from sg RNA+ Cas14

+ # 1. BLAST

  The BLAST analysis will give an idea of the potential mismatches that might arise as a result of non-specific hybridization. The majority of the BLAST results were Mycobacterium tuberculosis. When we analyzed these off-targets, we found that most of them were not human pathogens. We ruled these out as the likelihood of them being present in a human sputum sample was extremely low. The BLAST search recorded a total of 455 organisms for katG wild-type sgRNA and 250 organisms for rpoB wild-type sgRNA.
  The organisms which could give us a false positive result are listed below

  | Off-target organism | Pathogenesis | Analysis |
  | --- | --- | --- |
  | *Mycobacterium canettii* | Can cause TB in humans | Novel pathogenic taxon of the Mycobacterium tuberculosis complex |
  | *Mycobacterium bovis* | Can cause TB in humans | Part of the tuberculosis complex. Treatment is similar to normal TB. |
  | *Pseudomonas* | Can cause multiple infections Eg-endophthalmitis, pneumonia, etc | Potential misdiagnosis |
  | *Mycobacterium tuberculosis variant africanum* |  | Part of the tuberculosis complex. Treatment is similar to normal TB. |
+ # 2. Optimization:- (using varying FQ concentration)

  This will give us an idea of how much probe concentration to use to detect a particular sequence type in the sample with a good signal-to-noise ratio. This result will also help us to optimize the probe concentrations to their minimum. So that the reaction can be performed at a minimum concentration of reagents.
  [Modelling Page](https://2021.igem.org/Team%3AIISER_Berhampur/Model)
+ # 3. Learning from Hardware:

  Producing desirable results from experiments can help us prove the validity of our prototype. We will then have to focus on improving our working model. Since a lot of manual pre-processing (like vortexing, centrifuge, isolation, and transfer of reagents and products) is required even before the role of hardware comes into play, we thought of proposing an automated hardware device. This device will be handy and only require the user to load the sputum sample to test for TB/MDR-TB.

## Improve Section

# Optimizing Cas14-sgRNAs activity

1. **Potential challenge:** Occurrence of unwanted off-target effects and less efficiency of Cas14a1-sgRNA complex.
2. **Solution:** Though unintended off-target effects are rare for Cas14a1, in case we detect any off-target sites, we can analyze them using targeted deep sequencing. Also, we propose to use varying concentrations of Cas14a1 and sgRNA complex in our experiments to check its efficiency.

# Further improvement in our kit:

1. **Potential challenge:** Detection of other pathogens using this kit.
2. **Solution:** This kit can be repurposed for the detection of other deadly pathogens too. To do this a few modules will need to be altered. Firstly LAMP primers will need to be altered in a way that they amplify the conserved region of the other pathogen. Visible readout of amplification by the phenol-mediated color change will confirm the presence of the pathogen. Furthermore, if resistance to antibiotics happens due to mutation in certain regions, guide RNAs specific to that mutation can also be created, this step can also be done for wild-type sequences for detection.

# Hardware

**Design:** The automated design consists of three chambers - the pre-processing chamber, the lamp chamber, and the detection chamber. The device will be governed by software, and the user will have to set up the time for each step manually. The user will have to insert a test tube consisting of the sputum sample and glass beads into the pre-processing chamber of the proposed model. The test tube will sit on a vortex that will run for a set time and complete the lysis. A suction tube will then suck the sample to another test tube sitting on a centrifuge machine to isolate MTB DNA. After the centrifugation is completed, another suction tube will suck the supernatant from the test tube into the LAMP chamber, leaving behind the pellet. The LAMP chamber is a beaker inside a water bath already loaded with the LAMP primers and LAMP reagents, with a transparent screen on the top to observe what is going on inside.
Once the sample is added to the LAMP Chamber, the water bath starts heating up to a temperature of 65 Degrees Celsius, and the reaction continues for a set amount of time. After the reaction is completed, the device stops. The user will then have to observe a color change from the transparent screen. The color change indicates the presence of TB, and then the user will have to check the detection of MDR-TB strains. For this, the user will turn on a switch, telling the machine to go for MDR-TB detection. The suction tube will then suck the sample in the LAMP chamber and transfer it into the detection chip (detection chamber). If the user observes no change in color, the testing stops. No color change indicates that the sample does not contain TB infection. The detection chip will be loaded with T7 exonuclease, cas14 + guide RNA complex, FQ pairs. After a set amount of time, the laser will be switched on, and the beam will be directed to the chip. The detection of fluorescence will prove the presence of MDR-TB in the given sample.
The components in the chip, the pellet tube, and the sample collection tube are then discarded for the next detection cycle.

 **Test:** The assembled automated hardware design will be made to go through the same preliminary analysis. If the hardware achieves a satisfactory result, it will be sent for clinical trials after proper approvals and conformations, including necessary bio-safety documentation.

# Dealing with unexpected results:

1. **Transformation not working:**
   1. **Potential reasons:-** mistakes in certain steps.
   2. **Solution:-** Maintaining a logbook is very important as it will help to troubleshoot the mistake we made while performing the experiment. After troubleshooting, if we find a mistake in any step we can redo the experiment by taking proper precautions.
2. **eLAMP not working for NEB primers:**
   1. **Potential reasons:-** issues with designing primers.
   2. **Solution:-** This problem can be solved by designing primers on LAVA and Primer explorer as they are supported by eLAMP software. Further eLAMP software can also be modified so that it takes primers made by different methods. The electronically simulated lamp just like online PCR is a very useful tool for researchers and its importance is increasing due to the increased use of LAMP and other isothermal techniques in diagnostic kits.

## References

[1] Kim, D.Y., Lee, J.M., Moon, S.B. et al. Efficient CRISPR editing with a hypercompact Cas12f1 and engineered guide RNAs delivered by adeno-associated virus. Nat Biotechnol (2021).<https://doi.org/10.1038/s41587-021-01009-z>

[2]Harrington, L. B., Burstein, D., Chen, J. S., Paez-Espino, D., Ma, E., Witte, I. P., Cofsky, J. C., Kyrpides, N. C., Banfield, J. F., & Doudna, J. A. (2018). Programmed DNA destruction by miniature CRISPR-Cas14 enzymes. Science (New York, N.Y.), 362(6416), 839–842.<https://doi.org/10.1126/science.aav4294>

[3]Yao C, Zhu T, Li Y, Zhang L, Zhang B, Huang J, Fu W. Detection of rpoB, katG and inhA gene mutations in Mycobacterium tuberculosis clinical isolates from Chongqing as determined by microarray. Clin Microbiol Infect. 2010 Nov;16(11):1639-43.<https://doi.org/10.1111/j.1469-0691.2010.03267.x>

[4]Prasad, P. G., Jasmine, M. S., kanta, K. N. M., Deepthi, K., & Allam, U. S. (2019). Analysis of drug resistance mutations in pulmonary Mycobacterium tuberculosis isolates in the Southern coastal region of Andhra Pradesh, India. The Brazilian Journal of Infectious Diseases. doi:10.1016/j.bjid.2019.07.002<https://www.ingentaconnect.com/content/iuatld/ijtld/2015/00000019/00000004/art00013>

[5]Singhal, R., Arora, J., Sah, G. C., Bhalla, M., Sarin, R., & Prasad Myneedu, V. (2017). Frequency of multi-drug resistance and mutations in Mycobacterium tuberculosis isolates from Punjab state of India. Journal of Epidemiology and Global Health, 7(3), 175–180. doi:10.1016/j.jegh.2017.05.002<https://www.nature.com/articles/s41598-020-74432-y>

[6]Jain, A., Singh, P. K., Chooramani, G., Dixit, P., & Malhotra, H. S. (2016). Drug resistance and associated genetic mutations among patients with suspected MDR-TB in Uttar Pradesh, India. The International Journal of Tuberculosis and Lung Disease, 20(7), 870–875. doi:10.5588/ijtld.15.0874

[7]Chatterjee, S., Jain, Y., & Poonawala, H. (2018). Drug-resistant tuberculosis: Is India ready for the challenge? In BMJ Global Health (Vol. 3, Issue 4).<https://doi.org/10.1136/bmjgh-2018-000971>

[8]Nguyen, T. N. A., Berre, V. A. le, Bañuls, A. L., & Nguyen, T. V. A. (2019). Molecular diagnosis of drug-resistant tuberculosis; A literature review. Frontiers in Microbiology, 10(MAR).<https://doi.org/10.3389/fmicb.2019.00794>

[9]Migliori, G. B., Matteelli, A., Cirillo, D., & Pai, M. (2008). Diagnosis of multidrug-resistant tuberculosis and extensively drug-resistant tuberculosis: Current standards and challenges. In Canadian Journal of Infectious Diseases and Medical Microbiology (Vol. 19, Issue 2).<https://doi.org/10.1155/2008/857901>

[10]Electronic LAMP:<https://lamp.neb.com/#!/>

[11]Electric LAMP: Virtual Loop-Mediated Isothermal AMPlification
Nelson R. Salinas and Damon P. Little
<https://doi.org/10.5402/2012/696758>

[12]Warmstart Colorimetric LAMP 2X Master Mix (DNA & RNA)<https://international.neb.com/products/m1800-warmstart-colorimetric-lamp-2x-master-mix-dna-rna#Product%20Information>

[13] Fozouni et al., 2021, Cell 184, 323–333 January 21, 2021, ª 2020 Elsevier Inc.<https://doi.org/10.1016/j.cell.2020.12.001>

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# Team:IISER Berhampur/Experiments

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# Team:IISER Berhampur/Results

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iGEM IISER\_Berhampur

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[Skip to main content](#slide1-chapter1)

BAC Clones plasmid isolation

Absorbance of purified recombinant venus protein

Fluorescence of purified recombinant venus protein

NCBI Basic Local Alignment Search Tool (BLAST)

Standardization of Plasmids

In-silico Restriction Digests

## Experimental Results

As the global pandemic continues, we couldn’t get hold of our laboratories on time. However, we got access to our labs during the last couple of months before wiki-freeze. Although we ordered the reagents beforehand, we couldn’t get them all on time due to transportation and other issues. With the available reagents, we did some basic and optimization experiments. We have attached below the results of the experiments done so far.

## BAC Clones plasmid isolation

Mycobacterium tuberculosis (a kind gift from Prof. Rajesh Gokhale, National Institute of Immunology, Delhi) was isolated with the help of the NEB Monarch Plasmid isolation kit (New England Biolabs). We did an agarose gel run on those BAC plasmids in a 0.8% agarose gel with 10kb Ladder (1kb minimum) and got the following result. Then we did an in-silico agarose gel run for rpoB and katG genes as a preliminary analysis for PCR to stock the gene fragments (Lane 1 - rpoB, Lane - katG). For the Protocol, refer to the [Experiment](https://2021.igem.org/Team%3AIISER_Berhampur/Experiment) page.

Results are as follows:

![](https://static.igem.org/mediawiki/2021/4/47/T--IISER_Berhampur--RES_pic_1.png)

**BAC clone plasmid isolation**

![](https://static.igem.org/mediawiki/2021/f/f5/T--IISER_Berhampur--gel_image.png)

**In silico Gel Run**

## The absorbance of purified recombinant venus protein :

For the Preliminary analysis of hardware to detect fluorescence, we measured the absorbance of purified Venus protein with emission excitation (YFP: 505 nm, EGFP: 488). It has properties similar to EGFP to perform an optimization experiment for our detection system.

Absorbance of venus :

A = εcl (“Beer-Lambert Law”)
From this equation, we can calculate the concentration present in well 12
c = A/εl
L = 1cm
ε = 92,200M-1L-1 (Excitation coefficient of venus)
A = 1.23 (plate reader)

c = A/εl
c = 13.3μM

![](https://static.igem.org/mediawiki/2021/f/f5/T--IISER_Berhampur--RES_pic_2.jpg)

**Clear plates used for Absorbance**

![](https://static.igem.org/mediawiki/2021/8/8e/T--IISER_Berhampur--RES_pic_3.png)

**Absorbance of venus protein (3D Graph)**

![](https://static.igem.org/mediawiki/2021/b/b5/T--IISER_Berhampur--RES_pic_4.png)

**Absorbance of venus protein**

## Fluorescence of purified recombinant venus protein :

For the chemical kinetics of our experiment modeling, we analyzed the fluorescence emission of venus protein in fluorometry (YFP: 520, EGFP: 509). This helped us in the optimization of EGFP fluorescence emission which we will be using in our detection kit.

The 7th well showed a S/N (Signal to Noise ratio) ≈ 103 during the fluorescence check.
Since the dilution is halved in the next well from the well 12,

The concentration of 7th well = Absorbance / dilution factor = 13.3/25 = 0.4μM

In moles, 0.4μM = 40 picomoles.
Hence, a minimum concentration of 0.4μM of venus protein is required for a fluorescence S/N of ≈ 103.

![](https://static.igem.org/mediawiki/2021/4/44/T--IISER_Berhampur--RES_fluor_3Dgraph.png)

**Fluorescence of venus protein (3D Graph)**

![](https://static.igem.org/mediawiki/2021/8/8d/T--IISER_Berhampur--RES_fluor_reading.png)

**Fluorescence of venus protein**

![](https://static.igem.org/mediawiki/2021/3/3e/T--IISER_Berhampur--RES_graph_1.png)

**Fluorescence spectrum of venus protein**

![](https://static.igem.org/mediawiki/2021/6/67/T--IISER_Berhampur--RES_graph2.png)

**Fluorescence spectrum of venus protein**

## NCBI Basic Local Alignment Search Tool (BLAST)

NCBI BLAST is one of the well-known tools used for nucleotide or protein sequence alignment. In order to make our sgRNAs with minimal off-target effects, the spacer sequences which we had identified from *Mycobacterium tuberculosis* H37Rv (complete genome) from the NCBI database (NCBI Reference Sequence: NC\_000962.3) were run through the NCBI algorithm to assess their specificity. Here, BLAST serves the purpose of finding any sequences which are identical or similar to the spacer region in sgRNA.

Shown below are the results obtained from Multiple Sequence Alignment Viewer (MSA) version 1.20.1 on NCBI BLAST. The top 20 similar alignments for each spacer sequence are shown here. The 20 bases in the spacer sequence are colour coded.

![](https://static.igem.org/mediawiki/2021/9/93/T--IISER_Berhampur--RES_katG_wt.jpg)

**Results for katG (wild type) spacer sequence**

![](https://static.igem.org/mediawiki/2021/7/77/T--IISER_Berhampur--RES_katG_mutant.jpg)

**Results for katG (Mutant) spacer sequence**

![](https://static.igem.org/mediawiki/2021/5/5e/T--IISER_Berhampur--RES_rpoB_wt.jpg)

**Results for rpoB (wild type) spacer sequence**

![](https://static.igem.org/mediawiki/2021/d/df/T--IISER_Berhampur--RES_rpoB_mutant.jpg)

**Results for rpoB (mutant) spacer sequence**

## Standardization of Plasmids

Since we did not have lab access for most of the iGEM 2021 period and our access to lab materials got delayed due to the lockdown restrictions, we planned to optimize our approach for cloning and transformation experiment protocols for our CODE M Constructs using in-silico RE digestion and gel electrophoresis.

pLBH559\_Tet-HisCas14a1Locus is a plasmid that expresses 10xHis-Cas14a1 locus (including crRNA and tracrRNA) under control of a tetracycline-inducible promoter. It has a pTet vector backbone and it consists of a coding region for Chloramphenicol acetyltransferase (CmR) and p15A origin. It was added to addgene by Jennifer Doudna (Addgene plasmid # 112502 ;[http://n2t.net/addgene:112502](http://n2t.net/addgene%3A112502) ; RRID:Addgene\_112502).

## In-silico Restriction Digests

In-silico restriction digests were performed in pLBH559\_Tet-HisCas14a1Locus to identify the restriction sites to be used for excision of 6x-His Tagged Cas14a1 along with TEV protease (which is shown as ORF a in the restriction digestion map given below) for cloning them into pSB1C3 and also, to avoid any illegal restriction sites. We used the NEB cutter tool <http://nc2.neb.com/NEBcutter2/> for this purpose.

Caption: Restriction Enzyme Digestion Map for pLBH559\_Tet-HisCas14a1Locus; for all commercially available NEB enzymes.

![](https://static.igem.org/mediawiki/2021/4/49/T--IISER_Berhampur--RES_digest_adgene.jpg)

**We double digested it with EcoR1 and Pst1 enzymes. We found that EcoR1 and Pst1 sites were present in the ORF a, as shown in the figure below, which is the coding region for 6x-His Tag, Cas14a1, and TEV protease. Also, in another double digestion using Xba1 and Spe1, the sites were not present in ORF a. So, we propose to avoid using EcoR1 and Pst1 restriction enzymes and instead use Xba1 and Spe1 enzymes for cloning protocols that will be using this plasmid. We did a virtual gel run to get an expected gel image to be matched with an experimental gel run. We set a parameters of 1% agarose and 1kb DNA ladder as marker.**

**Caption:** In-silico custom double digests for pLBH559\_Tet-HisCas14a1Locus done using: A) EcoR1 and Pst1, and B) Xba1 and Spe1 restriction enzyme sets.

![](https://static.igem.org/mediawiki/2021/6/63/T--IISER_Berhampur--Res_IDK_1.jpg)

**EcoR1 and Pst1 double digest**

![](https://static.igem.org/mediawiki/2021/c/ce/T--IISER_Berhampur--RES_IDK_2.jpg)

**Xba1 and Spe1 double digest**

**Caption:** Virtual gel runs using the enzyme sets A and B.

![](https://static.igem.org/mediawiki/2021/9/98/T--IISER_Berhampur--RES_ECOR_1.jpg)

**Gel run for EcoR1 and Pst1**

![](https://static.igem.org/mediawiki/2021/d/d6/T--IISER_Berhampur--RES_XBA_1.jpg)

**gel run for Xba1 and Spe1**

* ![](https://static.igem.org/mediawiki/2021/3/3c/T--IISER_Berhampur--Teamsssss3.png)
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* ![](https://static.igem.org/mediawiki/2021/e/e2/T--IISER_Berhampur--Teamsssss2.png)
* ![](https://static.igem.org/mediawiki/2021/6/63/T--IISER_Berhampur--Teamsssss1.png)
* ![](https://static.igem.org/mediawiki/2021/b/bb/T--IISER_Berhampur--SPON32021.png)