iGEM 2023 — Team iiser-mohali

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# Segments

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[Experiments](experiments)

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[Results](results)

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[Parts](parts)

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[Notebook](notebook)

# Notebook

Following is the journal containing information regarding our weekly
activities from the beginning to the end of the cycle:

We ordered our DNA aptamers from IDT as two µmole DNA oligo in
standard desalting conditions. We resuspended the lyophilized aptamer
with 0.1 molar PBS. Then we measured the Cyclic voltammetry and
impedance of the bare Glassy Carbon electrode before modifying it with
GO/PANI solution.

Ideation Stage

* May 3 – May 7, 2023

1. Final Team Formation and Role Assignment.
2. Consultations with Mentors, Advisors, and Instructors for Project Discussion.
3. The Wet Lab team started looking for the literature on DNAzymes.

* May 8 – May 14, 2023

1. Sub-Team Creation for Project Initiatives.
2. Human Practices and Outreach Team Goal Setting.
3. Conversing about overall operational details and anticipations.

* May 15- May 21

1. The fundraising team started their search for sponsors.
2. Team had a meeting for how to do collaboration and started looking for teams which should be
   suitable to collab with as they had similar area of research to us this year, and also last year.
3. The software and dry lab teams initiated the process of defining their initial objectives.

* May 22- May 28, 2023

1. The team initiated conversations regarding the project's name and the creation of a team logo.
2. The wet lab team has initiated the process of generating sequences for the relevant DNAzymes.
3. The human rights team had a meeting to organize various events for local IISER, mohali students.

* May 29- June 4, 2023

1. The team decided to work with a colorblind-friendly palette throughout the season (as IISER mohali
   iGEM team did last year).
2. The team started working survey questionnaire.
3. Dry lab started looking for volunteers to help in aptamer data and their FASTA sequences.

* June 5- June 11, 2023

1. The wet lab team ordered dsDNAzyme sequences and primers for their initial asymmetric PCR.
2. The outreach team started contacting other iGEM team for collaboration.
3. The team had another meeting to make the final decisions on our logo and also deliberated on the
   prospect of commencing the sharing of our work on social media platforms.

* June 12- June 18, 2023

1. The team had a meeting to strategize how we can elevate our project to a nationwide scale in India
   and raise awareness among a larger audience.
2. The outreach team had its first meeting with a potential sponsor.

* June 19- June 25, 2023

1. The team met with Volunteers and explained their work to them.
2. The outreach team decided to take interview of Doctors from various part of covid effect areas of
   India.

* June 26- July 2, 2023

1. The outreach team took an interview with doctor from rajasthan.
2. The wet lab team received the primers and DNAzyme sequences in powdered form, subsequently
   preparing solutions from them.
3. Following this, the wet lab team initiated their first PCR by employing the received primers and
   DNAzyme sequences, ensuring the specificity of the primers.
4. The team had meeting to set future goals of what to be done by the end on July.

* July 3- July 10, 2023

1. The wet lab team again set a symmetric PCR reaction and a set of them were also run on agarose gel
   to make stock for their future experiments.
2. The wet lab team order primers for further asymmetric PCR.
3. The team made a keto funding page.
4. The team has initiated talking with fellow Indian teams concerning the All India iGEM Meet
   scheduled for 2023.

* March 20 – March 26

1. Meeting with Prof. Satyajit Jena, our Primary PI, to discuss
   our project to seek his guidance and support.
2. Meeting with Prof. Ananth Venkatesan to help us with plasma
   ion sputtering of our neural chip.
3. Meeting with Prof. Samarjit Bhattacharyya to seek his
   guidance and support for neural chip development.

* March 27 - April 2

1. Another meeting with Prof. Satyajit Jena, our Primary PI, to
   discuss neural chip development and training process.

* April 3- April 9

1. Meeting with Prof. Ramendra Sunder Dey to discuss the
   project's aptamer aspect and seek his support in developing
   the aptasensor.

* April 10 – April 16

1. In preparation for the final meeting with the institutional
   iGEM committee, we met our mentors and instructors to refine
   our project idea further.

* April 17-April 23

1. Final meeting with the iGEM committee to discuss the chosen
   idea.
2. Our idea got approved to work with, but only if we made a
   small modification to our initial plan.

* April 24- April 30

1. Break due to Midsemester exams.

Getting Started

* May 1 – May 7

1. Formation of the team and assignment of team roles.
2. Meeting with mentors, advisors, and instructors to discuss
   our project idea and initial plans.
3. Discussing general logistics and expectations.
4. Everyone joins sub-teams to begin working on the project.
5. Human Practices and Outreach teams decided their long-term
   goals and the list of professionals and events they will be
   managing the whole season.
6. The Wet Lab team determined the aptamer sequences against
   the respective microbes and an initial protocol for neural
   chip development.

* May 8 – May 14

1. Reaching out to the IISER, Mohali community to see what we
   can do to have a local impact.
2. The hardware and dry lab teams started setting their initial
   goals.

* May 15- May 21

1. The fundraising team started looking for sponsorship.
2. The team started discussions for Team-Logo and the name of
   the Project.
3. We started our discussion for collaboration, and we
   collaborated with Team iGEM Patras to develop a video
   discussing iGEM, Synthetic Biology, Wet Lab and Dry Lab, and
   the Survey on GMO and Syn-Bio.
4. The Wet Lab team started looking for alternatives to
   thiol-modified aptamers and did an extensive literature
   search and discussion with experts to attach unmodified
   aptamers to GO.
5. The dry lab Team started the discussion to perform GROMACS
   simulations of the proteins against our aptamer sequences.

* May 22 – May 28

1. The team decided to work with a colorblind-friendly palette
   throughout the season and decided on our logo.
2. The team started working on our collaborations with iGEM
   Team-Patras and finalized our application for Promega Grant.
3. The team started out working on Outreach and Human practices
   activities.

* May 29 – June 4

1. The team finalized the Logo for the project and submitted
   the survey questionnaire for review to Institutional Ethics
   committee member Anu Sabhlok.
2. The team applied for Beckman Coulter free reagent kits.
3. Team also started having discussions with other Indian Teams
   regarding the All India iGEM Meet 2022.

Summer

* June 5 – June 11

1. The team discussed with Science communicator Ipsa Jain and
   received valuable feedback from her regarding our
   Communication and Education work.
2. The team had another meeting to finalize our Logo and
   discussed starting out sharing our work on social media.
3. We discussed having a potential meeting with Team iGEM
   Berhampur who was also working with Aptamers this year.
4. We also finalized ordering our custom DNA oligo from IDT for
   our Aptamer experiments.

* June 12 – June 18

1. The team had its meeting with iGEM Team IISER Berhampur and
   discussed potential collaborations with them.
2. Wet Lab Co-head met with Anca from iGEM 2021 Team- Rochester
   to gain insights into attaching unmodified aptamers over GO
   and perform the electrochemical discussion method.
3. We started discussions regarding Collaboration with other
   iGEM Teams.
4. The Wet Lab team was notified about an issue we could face
   while using a neural chip to detect the initially chosen
   microbes. The Wet Lab team decided to work with Penicillin
   to sort out the problems that could have arisen.
5. The social media team had a meeting to kick off the season
   over our social media handles officially.
6. Team also decided on our mascot Monsieur Neurapurr.
7. The team met to finalize our application for Impact Grant
   Award 2022.

* June 19 – June 25

1. The team had its initial meeting with Team AFCM Egypt and
   Team Linkoping Sweden and discussed potential collaboration.
2. The team decided on the goals for fulfilling medal criteria
   and planned work accordingly.
3. The team finalized the preliminary safety form for
   submission and met with advisors and mentors over it.
4. The team finalized their plan to host the Syn-Bio camp and
   discussed it with volunteers.

* June 26 – July 2

1. The team met with Volunteers, brainstormed together for the
   conduction of the Syn-Bio camp, and also launched posters
   for registration.
2. The team discussed our participation in AIIM 2022 and got
   two Researchers on board for the Virtual Talk Series.
3. The team again met to discuss the Impact Grant Applications.
4. Syn-Bio Workshop plans finalized.
5. The team had an initial collaboration meeting with High
   School Team ASIJ Tokyo.
6. The marketing and fundraising team started a massive
   campaign to raise funds.
7. The outreach Team collaborated with Team- IISER TVM to
   develop a lab tour video.

* July 3 – July 9

1. Brochures and infographics designed by the design team.
2. The team had review meetings to analyze the work and plan
   for the goals.
3. The human Practices and Wet Lab team met with Dr. Richa
   Sharma and Dr. Raghavarao to discuss some of the
   electrochemical sensing aspects of the aptasensor.

* July 10 – July 16

1. The team met with volunteers to prepare for Syn-Bio
   workshops.
2. The hardware team finalized the design to sputter for the
   neural chip.
3. The marketing and Fundraising team reached out to sponsors
4. The team decided on track selection for the project.
5. The team received the distribution kit.

* July 17 – July 23

1. The neural chip development team placed an order for
   Retinoic acid.
2. Initial designs for the Neural chip got sputtered and tested
   out.
3. The team had a meeting and finalized the presentation for
   AIIM 2022.
4. The design team got done with the 3D rendering of our
   prototype.
5. The team successfully conducted the Syn-Bio workshop for
   high school students.
6. The human Practices team met Prof. Anu Sabhlok to discuss
   the social and ethical aspects of the Project.
7. The team started the survey on GMOs and Syn-bio to
   understand the general public's perspective on GMOs and
   Syn-Bio.

* July 24 – July 30

1. The team attended the AIIM 2022.
2. Aptamers received from IDT.
3. We had a meeting with iGEM Team Montpellier to discuss our
   collaboration.
4. The dry Lab team finalized its goals for docking and figured
   out its work.

* July 31 – Aug 6

1. The team received Impact Grant Award.
2. We had our first meeting with our collaborator iGEM
   Thessaloniki, Greece.
3. The wet lab team met to finalize the protocol and start the
   experiments related to Aptasensors.
4. The team visited Verka Milk Plant, Chandigarh.
5. The Wet Lab team started InterLab Calibration Experiments.
6. The team placed an order for neural cell lines.
7. The team scheduled the meeting with engineers from BLACKROCK
   NEUROTECH.
8. The team successfully concluded its virtual talk series
   event.
9. The team submitted IBSC Application to get BSL-2 clearance
   from Insitute’s BioSafety Committee.
10. The team decided on the script for Project's Promotional
    Video.
11. InterLab study calibration done.

* Aug 7 – Aug 13

1. The team received sponsorship from INDUS PROTECH.
2. The team completed the Inter-Lab study measurements.
3. The team completed shooting clips for Project's Promotional
4. The wet Team successfully modified Glassy Carbon electrodes
   with GO/PANI mixture.
5. The human Practices team met Prof. Indranil Banerjee to
   discuss the project.
6. The human Practices team met Prof. TVR from IISc to discuss
   the sustainability aspect of the project.
7. The comic Strip development team finalized the story to work
   upon.

* Aug 14- Aug 20

1. The human Practices Team visited PBTI, Chandigarh.
2. The Design Team completed the animation for Project's
   Promotional Video.
3. Wiki Team started designing the basic templates for the
   Wiki.
4. The team met Nanjiang-WorldShaper High School students and
   discussed a potential collaboration.
5. Wet Lab Team successfully attached Aptamer over GO/PANI
   modified GC.
6. The team got BSL-2 clearance from Insitute’s BioSafety
   Committee to work with cell lines and S. typhimurium.
7. The human Practices team visited Dr. Archana at PGIMER to
   discuss some of the aspects of the project.
8. The dry Lab team finalized the parameters for running MDS.

* Aug 21- Aug 27

1. The wet Lab team successfully tested aptamer for E.coli.
2. The human Practices and Dry lab team met with the CEO of
   Cortical Labs to discuss some aspects of neural chip
   development.
3. The dry Lab team finalized its plans to develop our
   software-” KAMI.”

Closing the loop

* Aug 28- Sept 3

1. The wet Lab Team placed an order for Penicillium chrysogenum
   and S. typhimurium.
2. The dry Lab Team, under the guidance of our Instructor
   Soumadeep, started the development of the software -” KAMI.”

* Sept 4 – Sept 10

1. The wet Lab Team received N2A and SH-SY5Y cell lines.
2. The human Practices team visited KVK, Mohali.
3. The team received sponsorship of $1250 from Blue Copper
   Technology Pvt. Ltd.

* Sept 11- Sept 17

1. The wet Lab Team started to culture N2A and SH-SY5Y cell
   lines.
2. The team delivered student talk over iGEM and our Project to
   the BS-MS community of IACS, Kolkata.
3. The team finalized the development of “Book on Aptamers”
   with Team Linkoping Sweden and others.

* Sept 18- Sept 24

1. The wet Lab Team received Penicillium chrysogenum and S.
   typhimurium.
2. Wet Lab Team tested aptamers for Penicillin G as well.
3. The wiki team finalized their plans regarding the web design
   and started working on it.
4. Comic strip development finalized the illustrations for the
   comic.
5. The dry Lab team finalized the Docking and almost completed
   it.

* Sept 25 – Oct 1

1. The wet lab team tested the sensitivity of aptamers against
   E. coli and S. typhimrium.
2. The wet lab team started two different cell lines.
3. The dry Lab team performed MDS and finalized modeling work
   related to the project.
4. The outreach team conducted Synthetic Biology based
   demonstration for middle and High school students on the
   occasion of 16th Foundation Day.
5. The wiki Team tested uploading and extensively worked on
   Wiki development.
6. The comic strip development team started collating the
   panels for the comic and did some final editing.
7. Gold medal criteria of long-term partnership achieved and
   discussed with iGEM Team Montpellier.
8. The dry Lab Team wrapped up the development of the
   software-” KAMI” successfully.

* Oct 2 – Oct 8

1. The dry Lab team wrapped up their work.
2. The comic strip got published on our social media handles
   and sent out to high school students we met during Syn-Bio
   Workshop.
3. Boi–Safety video final editing is done and released over our
   YouTube channel and social media pages.
4. The team received a sponsorship offer of $500 from ACS
   Publication.
5. The team extensively documented the work, and Wiki Team
   worked on Wiki development and uploading.

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# Segments

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[Description](description)

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[Engineering](engineering)

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[Contribution](contribution)

# Cycle 1:

Design:

* Initial mining and alignment software for DNAzymes
* Expanding DNAzyme usage beyond the primary focus, in literature, of logical processing capabilities.

Build:

* Built the Strucgen module in PrAtZy software to generate a variety of DNAzymes by mutating sequences and
  selecting the ones with similar secondary structures

Test:

* Characterization of Strucgen module

Learn:

* Additional validation stem is needed to ensure a high level of accuracy in the prediction
* Need to estimate the energy values of of the new structure to ensure the stability of the sequence at a
  temperature of interest.

# Cycle 2:

Design:

* Identified a problem of multiple bands of PCR gel and poor detection of ssDNA due to multiple
  freeze-thaw cycles and contamination from the surroundings

Build:

* Development of a different protocol for easy and effective storage and transport
* Generating a 300bp dsDNA template with the DNAzyme at the terminal to maintain stock using normal PCR
  and extracting DNAzyme using asymmetric PCR

Test:

* Newly built protocols were implemented and tested to see if the multiple bands were avoided and if the
  detection was robust while surviving the freeze-thaw cycles and contamination

Learn:

* Efficient to store DNAzyme as a double-stranded template
* Production of DNAzyme on a requirement basis

# Cycle 3:

Design:

* Increase the RecLoop-RecSeq interaction which was extremely low due to the diluted solution
* Deployment of DNAzyme in such a way the occlusion of one or more sites is avoided

Build:

* Immobilize the DNAzyme on nitrocellulose paper to enable easy visualization, less but concentrated
  DNAzyme and direct exposure to RecSeq-containing solution

Test:

* Verification of the result to avoid occlusion problem
* Market analysis for cost efficiency of nitrocellulose paper-based test vs existing kits

Learn:

* Enables efficient ReqLoop-ReqSeq interactions
* Maintains substrate fluorescence
* About 20-30% cheaper to develop compared to existing methods

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[Notebook](notebook)

We are working on pioneering a novel detection method that leverages enzymes crafted from single-stranded DNA,
specifically DNAzymes. Our goal is to create a platform that can detect DNA and RNA sequences with an
exceptional level of specificity, down to the resolution of single nucleotide changes. This innovative platform
is primarily designed for the accurate identification of unique sequences, especially in the context of pathogen
detection. Additionally, we are exploring its potential for advancing the field by developing organic logic
circuits, showcasing the versatility and broad applicability of this groundbreaking technology.

We have included the methods used by us for conducting these experiments below.

# PCR reaction - The template DNAZymes FBS1, FBS2, and FBS3, initially present in 100mM concentration, were amplified through the symmetric Polymerase Chain Reaction (PCR) process. This amplification was done using specific forward and reverse primers, allowing for the targeted replication of the desired DNA sequence. For three separate DNAzymes a total of 3 primers were used. The CommonSR primer, which serves as the shared reverse primer for all three templates, and two particular forward primers—FBS1S/FBS2S for FBS1 and FBS2, and FBS3S for FBS3 The PCR reaction mixture components included: | | | | --- | --- | | Template | 1μl | | Forward primer | 2.5 μl | | Reverse primer | 2.5 μl | | dNTPs | 1 μl | | Taq polymerase | 0.5 μl | | Taq DNA buffer | 6 μl | | Nuclease free water | 17 μl | | Total | 30 μl | Initial Denaturation | Denaturation | Annealing | IInitial extension | Final extension | Hold || 95° C | 95° C | 55° C | 72° C | 72° C | 4° C | || | | | | | | | --- | --- | --- | --- | --- | --- | | 1 min | 10 sec | 30 sec | 20 sec | 2 min | ∞ | Gel Electrophoresis for confirmation of PCR Product: 1. Measured 1 g of agarose. 2. Agarose powder was mixed with 100 mL 1xTAE in a microwavable flask 3. Microwaved for 1-3 min until the agarose is completely dissolved. 4. Agarose solution was allowed to cool down to about 50 °C (about when you can comfortably keep your hand on the flask), about 5 mins. 5. Ethidium bromide (EtBr) was added to a final concentration of approximately 0.2-0.5 μg/mL . 6. The agarose was poured into a gel tray with the well comb in place. 7. The gel was kept at room temperature for 20-30 mins, until it had completely solidified. Loading Samples and Running an Agarose Gel: 1. Once solidified, the agarose gel was placed into the gel box 2. Fill the gel box with 1xTAE (or TBE) until the gel is covered. 3. 100bp molecular weight ladder was loaded. 4. Samples were loaded into the additional wells of the gel 5. The gel was run at 80-150 V until the dye line was approximately 75-80% of the way down the gel. 6. Turn OFF power, disconnect the electrodes from the power source, and then the gel was carefully removed from the gel box. Visualization of gel 1. BioRad gel viewer used. 2. Gel placed into the viewing platform. 3. Gel drawer closed and the gel manipulation window opened. 4. Alignment of gel done using incandescent light setting into appropriate visualization box. 5. Protocol for EtBr selected. 6. Run protocol. 7. Picture analyzed using inbuilt gel analyzer. The PCR product was visualized on a 2% agarose gel containing ethidium bromide for size confirmation, utilizing a 100bp DNA ladder as a molecular weight marker. PCR product purification : 1. 500μl of binding buffer was added to a MCT containing the PCR product 2. Then it was added to a purple column (Monarch® DNA Cleanup Columns) 3. Centrifuged it for 1 min at 14000 rpm. 4. Passed the flowthrough again to the purple column. 5. Centrifuge it for 1 min at 14000 rpm. 6. Discard the flowthrough 7. 500μl of wash solution was added to the purple column. 8. Centrifuged for 1 min at 14000 rpm. 9. Discard the flowthrough 10. Again 500μl of wash buffer was added to the purple column. 11. Centrifuged for 1 min at 14000 rpm. 12. Flowthrough was discarded. 13. Dry spined the purple column for 3 mins at 14000 rpm 14. Kept the purple column in new MCT 15. 30μl of preheated elution buffer was added in it 16. Kept for 5-10 mins 17. Centrifuged for 3 min at 14000 rpm. 3. Asymmetric PCR After the initial PCR amplification of the stock DNA, an asymmetric PCR was conducted using the purified PCR product. The purpose of this asymmetric PCR was to selectively amplify a specific region of interest, focusing on one DNA strand. In this process, a single primer was used, designed to anneal precisely to the target region of interest. This primer served as the initiation point for DNA synthesis, facilitated by a DNA polymerase enzyme. This led to the preferential amplification of a single DNA strand. The PCR reaction mixture components included: | | | | --- | --- | | Template | 10μl | | Primer | 2.5 μl | | dNTPs | 1 μl | | Taq polymerase | 0.5 μl | | Taq DNA buffer | 6 μl | | Nuclease free water | 10 μl | | Total | 30 μl | Initial Denaturation | Denaturation | Annealing | Initial extension | Final extension | Hold || 95° C | 95° C | 55° C | 72° C | 72° C | 4° C | || | | | | | | | --- | --- | --- | --- | --- | --- | | 1 min | 10 sec | 30 sec | 20 sec | 2 min | ∞ | Loading Samples and Running an Agarose Gel: 1. Once solidified, the agarose gel was placed into the gel box 2. Fill the gel box with 1xTAE (or TBE) until the gel is covered. 3. 100bp molecular weight ladder was loaded. 4. Samples were loaded into the additional wells of the gel 5. The gel was run at 80-150 V until the dye line was approximately 75-80% of the way down the gel. 6. Turn OFF power, disconnect the electrodes from the power source, and then the gel was carefully removed from the gel box. Visualization of gel: 1. BioRad gel viewer used. 2. Gel placed into the viewing platform. 3. Gel drawer closed and the gel manipulation window opened. 4. Alignment of gel done using incandescent light setting into appropriate visualization box. 5. Protocol for EtBr selected. 6. Run protocol. 7. Picture analyzed using inbuilt gel analyzer. The PCR product was visualized on a 2% agarose gel containing ethidium bromide for size confirmation, utilizing a 100bp DNA ladder as a molecular weight marker. GEL extraction: 1. 500μl of binding buffer was added to a MCT containing the PCR product and kept at 65 °C till the gel properly got mixed with the binding buffer 2. Then it was added to a purple column (Monarch® DNA Cleanup Columns) 3. Centrifuged it for 1.5 min at 14000 rpm. 4. Passed the flowthrough again to the purple column. 5. Centrifuge it for 1.5 min at 14000 rpm. 6. Discard the flowthrough 7. 500μl of wash solution was added to the purple column. 8. Centrifuged for 1.5 min at 14000 rpm. 9. Discard the flowthrough 10. Again 500μl of wash buffer was added to the purple column. 11. Centrifuged for 1.5 min at 14000 rpm. 12. Flowthrough was discarded. 13. Dry spined the purple column for 3 mins at 14000 rpm 14. Kept the purple column in new MCT 15. 30μl of preheated elution buffer was added in it 16. Kept for 5-10 mins 17. Centrifuged for 3 min at 14000 rpm. Qubit fluorometer is measurement to get ssDNA conc 1. A mastermix of the QubitTM working solution was prepared. 2. For calibration, the Qubit ssDNA kit needs two standards. 3. Give the tube lids labels. 4. To each tube, aliquot the QubitTM working solution: 5. QubitTM working solution and Sample tubes filled to 180–199ul 6. Each tube must have a final volume of 200 l after the sample and standard have been introduced.. 7. Vortex each tube for 3 to 5 seconds 8. Two minutes of room temperature incubation . 9. "Read standards and samples" should be performed on all tubes. 10. On the QubitTM 3 Fluorometer's Home screen, tap DNA, then choose 1X ssDNA HS as the test type. 11. Close the cover after placing a sample tube within the sample chamber and push the Read tube. 12. Remove the sample tube after the reading is finished (around three seconds). 13. The original sample concentration is shown at the top (in large type), and the dilution concentration is shown at the bottom. 14. Thoroughly document all outcomes and save the Qubit's run file to a memory stick CD spectroscopy 1. The purified DNA samples was diluted to make a solution of 200ul with concentration of 0.2mg/ml 2. The quartz cuvette was filled with the buffer solution used for dilution. This served as a reference or blank. 3. The reference cuvette was inserted into the CD spectrophotometer and performed a baseline correction by measuring the CD signal at around 200-300 nm. 4. Now the quartz cuvette was filled with diluted DNA sample, inserted in the CD spectrophotometer, the spectrum was recorded and the data was analyzed Concentrating DNAzyme 1. Samples pipetted (200μl) in 1.5ml Eppendorf MCT 2. Samples balanced and kept uncovered. 3. Concentrator plus from Eppendorf used at 60 °C using Function V-AQ- Run at setting for 30 minutes uncovered. - 15μl of concentrated DNAzyme obtained - Same protocol, run on dsDNA, primers with same initial concentration. - Take a part of concentrated samples and dilute to 25mM in 200μl - Send all diluted samples for CD spectroscopy to check for quality after concentration. Methylene blue staining: 1. Measured 1 g of agarose. 2. Agarose powder was mixed with 100 mL 1xTAE in a microwavable flask 3. Microwaved for 1-3 min until the agarose is completely dissolved. 4. Agarose solution was allowed to cool down to about 50 °C (about when you can comfortably keep your hand on the flask), about 5 mins. 5. Ethidium bromide (EtBr) was added to a final concentration of approximately 0.2-0.5 μg/mL . 6. The agarose was poured into a gel tray with the well comb in place. 7. The gel was kept at room temperature for 20-30 mins, until it had completely solidified. Sample preparation 1. The 5μl PCR products were mixed with 5μl of methylene blue (used to stain ssDNA) 2. The solution was kept for 30 minutes. Loading Samples and Running an Agarose Gel: 1. Once solidified, the agarose gel was placed into the gel box 2. Fill the gel box with 1xTAE (or TBE) until the gel is covered. 3. 100bp molecular weight ladder was loaded. 4. Methylene blue stained samples were loaded into the additional wells of the gel. 5. The gel was run at 80-150 V until the dye line was approximately 75-80% of the way down the gel. 6. Turn OFF power, disconnect the electrodes from the power source, and then the gel was carefully removed from the gel box. Making DNAzyme action solution 1. Take Nuclease free water (we made a stock solution of 100ml) 2. Add Magnesium chloride powder (20.33g) to the 100ml solution 3. Add Sodium Chloride (5.488g) in the same solution 4. Dissolve and use when setting up DNAzyme-loop reactions Fluorescence Reading 1. Load the sample in the platform 2. Keep the platform to incubate at room temperature for 30mins 3. Switch on the Fluorescence reader, and go to protocols 4. If TamR protocol is present then use the same 5. Otherwise, make a new protocol for tamR with excitation at 550nm and emission at 578nm 6. Load the platform 7. Run the protocol and take a reading 8. For multiple readings of the plate, re-run the protocol again and again at different time intervals to get as many readings as needed

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# Segments

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[Experiments](experiments)

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[Parts](parts)

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[Notebook](notebook)

# Results

Background information

## Microbes Chosen:

Initially, as team we were looking out to develop a device which
can work as one of the best point of care technology for detecting
diseases. We soon realised that the technology we envisioned to
develop can not only detect the microbes commonly responsible for
causing sepsis like diseases but rather can detect any target
molecule against which we can generate a highly specific and
sensitive aptamer.

Our institutional iGEM committee’s head Prof. Anand Bachchawat
also suggested to us that since our technology could detect any
target molecule if an aptamer could be generated against it then
why don’t we develop a “ Proof of Concept” that it could detect
microbes which are more commonly available around us and are
easier to work with.

We considered these aspects and decided to work with E. coli, S.
typhimurium and Pseudomonas aeruginosa. Through the literature
search, we found that these common microbes have already aptamers
developed against them using whole cell-SELEX which are highly
sensitive and specific.We decided to choose the microbes that had
already available aptamer sequences in the literature as we
understood that whole cell-SELEX process takes time to model an
aptamer sequence and given the time frame of the competition we
won’t be able to complete everything we wanted to exhibit if we
are to begin with SELEX.

Our team wanted to make a system that would be better than the
existing fluorescent-based detection methods due to its high false
positive rate. We also wanted to quickly expand to the sample's
multiple, simultaneous, multi-cohort detection. This was not
possible with existing technologies with individual detection
capabilities and data with differing error margins, which are
later processed for a complete analysis of the disease. We solved
all the above issues by implementing the processing system within
the detection system using a neural chip that simultaneously
detects and processes signals. It is also cheap, easy to mass
manufacture, and accessible with minimal electrical equipment (in
the proposed implementation a battery-powered reading box would
suffice). The system would also have an error margin which would
be easy to calculate against accurate world data, unlike data
derived from individual detection, which have errors that cannot
be easily calculated. Neurons are also susceptible to changes in
the electrical fluctuations at the 70-100mV range, thus reducing
the need for high-powered devices like spectrophotometers that
also increase measurement and maintenance costs.

Further, our team member figured out later that the technology we
envisioned developing the device and the neural chip we were
developing for bio-computation could not perform the computation
without dimension reduction. We could not have three inputs and
three outputs if we had to use a neural chip to perform
bio-computation and analyze the electrochemical changes that would
happen upon the binding of microbes to the aptamer. The dimension
reduction property would prove our chip's biocomputation ability
and help reduce stray signals that would be generated and
measured. This is why the chip was planned to have 3input signals
corresponding to three different organisms but only two output
signals. The interpretation of these signals would be based on the
current output of the chip. The chip would present an overall
signal lowering in both outputs when detecting the third organism.
This ensured that the number of outputs and the interpreting
device's involvement were minimized.

Hence, we further, our team member figured out later that the
technology we were envisioning to develop the device and the
neural chip we were developing for bio-computation cannot perform
the computation without the dimension reduction. We cannot have
three inputs and three outputs if we had to use neural chip to
perform bio-computation anad analyse the electrochemical changes
that would happen upon the binding of microbes to the aptamer.
This would act as a biomarker for the fungi Penicillium
chrysogenum.

Thus, we decided to work with E.coli MTCC 443, S. typhimurium MTCC
, Penicillium chrysogenum as our chosen microbes.

Why Aptamers, Neural Chips and Electrochemical sensing?

Aptamers have emerged as a great tool in the recent years in the
development of point of care diagnostics, drug delivery and
therapeutics. Aptamers could be either ss DNA or RNA
oligonucleotide sequences that adapt a secondary structure able to
specifically bind to a target molecule. They are mainly generated
by SELEX (Systematic Evolution of Ligands by Exponential
Enrichment), which is a reiterative process starting with a large
DNA or RNA library, and at each step, only high binding affinity
sequences are separated from the library. One example is Silver
Enhanced Fluorescence-Activated Cell Sorting. In this process, the
target protein or the control/negative proteins (which are used to
ensure specificity of the aptamer) are immobilized on polystyrene
microspheres and added to the large DNA/RNA initial library,
modified with a fluorophore. Silver decahedral nanoparticles
conjugated to a fluorophore are also added, and then these NPs
will interact with the labelled DNA and clump together. Through
fluorescence-associated cell sorting, one can then choose the
cells that had DNA clones that tightly bound to the protein.

We can observe that only aptamers that bind to the cell or protein
of interest are selected from the initial library. This is the
first round, where enrichment occurs. All other rounds are
negative selection, where the protein or cell of interest reacts
with silver nanoparticles, and the non-binding DNA sequences are
directed to the waste container via an electric current. The
collection container sequence is then amplified via PCR, and ssDNA
or RNA aptamers have been generated as a result. They can then be
sequenced via standard sequencing methods. Aptamers have the
advantage that any molecules can be targeted, and there are no
immunizations of animals required. This results in a high affinity
and high specificity receptor that can be selected for and then
synthesized completely in vitro. Moreover, since aptamers are just
a DNA sequence, they can be ordered from DNA synthesis companies.

Fortunately, the sequences we wanted to work with were highly
specific and sensitive with KD value in nM range and were
available under IDT’s sponsorship programme.

Fluorescence detection using Aptamers was initially considered,
but our team soon realized a number of problems with this method.
Aptamer being a small molecule is prone to more molecular motion
than traditional antigen-antibody detection systems. This makes
the fluophore and the quencher interaction more prone to
misfiring. This increases the false positive rate in such systems.
We also considered the problems faced by the use of spectroscopes
and image processing of biological data. Spectroscopes have a
error percentage of 5% at best condition. This makes the data
highly unsuitable for further processing as additional processing
will further add error to the measurement.

Also most spectroscopes in a developing country like ours, do not
have regular maintenance. We were made aware of this issue by food
scientists working in Verka, during our vist. This has made the
food industry trust less on spectroscopic data for bacteria
detection and prefer culturing the bacteria to check for its
presence. This is a time consuming process that is prone to
environmental contamination.

To solve all of these problems we decided to use a property of the
aptamer which is linear, easy to measure, shows large range
changes, scalable and devoid of further processing steps. We hence
decided to use the impedance change property of Aptamers and
interpret out results using a neural chip, which solves all the
above problems. The chip processes the data from the aptamer and
directly outputs a easily quantifiable signal, generated from an
Ensemble of neurons. The neurons are also more sensitive than
traditional current detection systems, firing at 70-100mV range.
The trainability of the network also allowed us to build the
compuational system within the detection system rather than taking
the data separately and processing it. This reduces dependency on
big and expensive machinery and is easily deployable in the field.
The results are instantaneous and can be monitored in real time.
The neural chip itself is reusable, cheap to manufacture and easy
to dispose. It contains no heavy metal contaminants, other than
the wiring of the chip, which itself can be reused to make more
chips. It also requires no maintenance, other than a normal
temperature of 37\*C, and once a supply chain can be established
will be a perfect tool for rapid, cheap and sustainable
multicohort detection.

For E. coli ATCC generic strain 25922 (MTCC 443) , according to
the studies, whole bacterial cell is used as a target and P12-31
is highly specific and sensitive aptamer towards it. Its Kd value
is 87.03± 17.32, Bmax value is 0.56±0.04 and around 10113 no. of
binding sites are available for it. Fluorescently labeled aptamers
label the surface of E. coli cells, as viewed by fluorescent
microscopy. Specificity tests with twelve different bacterial
species showed that one of the aptamers–called P12-31—is highly
specific for E. coli.

Sequence of P12-31 aptamer is given below which is obtained by
Whole-Cell SELEX:

5'CCCTCCGGGGGGGGGGGTCATCGGGATACCTGGTAAGGATACCCTCCGGGGGG
GTCATCGGGATACCTGGTAAGGATA
3'

Structure:

![Image of structure](https://static.igem.wiki/teams/4391/wiki/results-img-one.png)

* Whole bacterial cell is used as a target and ST2P aptamer
  demonstrates the highest affinity for S. typhimurium.
* Its Kd value of this is 6.33±0.58 mM.
* ST2P has highest binding ability with gated fluorescence above
  background values of around 82 %.
* ST2P is highly specific for S. typhimurium and doesn’t binds
  with other bacteria.
* Sequence of ST2P aptamer is obtained from Whole-cell SELEX and
  is as following:

5′ATAGGAGTCACGACGACCAGAAAGTAATGCCCGGTAGTTATTCAAAGATGAGTAGGAA
AAGATATGTGCGTCTACCTCTTGACTAAT3′

Structure of the ST2P Aptamer:

![](https://static.igem.wiki/teams/4391/wiki/results-img-two.png)

Aptamers
Neural Chip

## Results and Conclusions

EIS measurement is a powerful technique for analyzing the
physiochemical changes at the interface (i.e., between the bulk
solution and solid electrode) arising from the formation of insulating
film incurred by the interaction of the analytes with their probing
molecules immobilized on electrode surfaces. Measurement of charge
transfer resistance RcT the impedance increases across the interfacial
layer EIS provides a rapid, sensitive, and nondestructive technique
for detecting various analytes, including biomolecules that would
otherwise be difficult to quantify.

Impedance is usually expressed as a Nyquist plot. A typical Nyquist
plot consists of a semicircle in the high-frequency region
representative of the electron transfer limited process and a straight
line in the low-frequency region associated with the
diffusion-controlled process. In fig:3, The bare GC showed a small
semicircle in the high-frequency region with the calculated RCT value
of 3300Ω. After the electrochemical deposition of GO/PANI, the Nyquist
plot for EIS spectra shows the decrease in RCT value showing the
reduction in the surface resistance, therefore providing a more
effective platform for developing aptasensor.3 The Nyquist plot of the
electrode where the aptamer molecule shows an increase in the RCT
value to 11000, showing the successful assembly of the aptamer layer
on the modified GC. Aptamer acts as a barrier to the anion
electrolyte's restriction [Fe(CN)6]-3/-4 to the electrode surface.4

The Nyquist response shows a vast increase in the RCT value to 40000 Ω
showing the complete coverage of E. coli by the biosensor.

Similar observation can be observed for CV (fig:5) and EIS response
(fig:6) for developing the Penicillin aptasensor. On another GC, a
Change in the RCT from 1400 Ω to 1100 Ω after deposition shows the
GO/PANI layer formation on the GC surface. EIS curves also show the
change in RCT value for penicillin aptasensor and penicillin, which is
seen by the RCT value or the semicircle size of the EIS response. The
RCT values for penicillin aptasensor of around 1500 Ω shows the SAM of
aptamer onto the deposited GO/PANI surface, the RCT value changes to
3600 Ω after dropcasting of penicillin onto modified aptasensor GC.
Hence, the sensitivity of the developed aptasensor towards penicillin
is good.

![](https://static.igem.wiki/teams/4391/wiki/results/rs1.png)

![](https://static.igem.wiki/teams/4391/wiki/results/rs2.png)

![](https://static.igem.wiki/teams/4391/wiki/results/rs3.png)

![](https://static.igem.wiki/teams/4391/wiki/results/rs4.png)

![](https://static.igem.wiki/teams/4391/wiki/results/rs5.png)

![](https://static.igem.wiki/teams/4391/wiki/results/rs6.png)

## Sensitivity measurement:

For the measurement of the sensitivity of the E.Coli
aptasensor for the given E.coli strain, the concentration of the
E.coli was varied in two different ratios in terms of optical density,
i.e., 0.7OD and 0.07OD.

The experiment was performed in
two different GCs with two different E.coli concentrations. The
previous experiments for the development of aptasensor were the same,
and the GCs CV (Figures 7 and 8 ) and EIS (Figures 9 and 10) responses
were recorded.

For GC with E.coli concentration of 0.7OD,
the CV responses were recorded (Figure: 7). The change in CV curves
area with the going from bare GC to the E.coli assembled form was
observed. As seen in figure: 7, the CV responses show a decrease in
the current value with the given potential window. Figure 8 shows the
CV of the 0.07 OD E.coli, showing a similar response as that of 0.7OD
E.coli. Still, the CV curve for the 0.7 OD E.coli assembled aptasensor
is more horizontal than the other concentration of 0.07 OD E.coli.
This shows the current is hindered more in 0.7OD E.coli than the other
concentration ratio.

Nyquist plot for 0.7OD (figure: 9)
and 0.07OD (figure: 10) concentration of E.coli shows the immense RCT
value for 0.7OD concentration than 0.07OD concentration, i.e., 5000 Ω
in former and 13800 Ω in later, this is the inference of the increase
in bacterial assembly on the electrode surface.

![](https://static.igem.wiki/teams/4391/wiki/results/rs7.png)

![](https://static.igem.wiki/teams/4391/wiki/results/rs8.png)

![](https://static.igem.wiki/teams/4391/wiki/results/rs9.png)

![](https://static.igem.wiki/teams/4391/wiki/results/rs10.png)

## Results for the hardware resistance measurements

To ensure that the physical hardware provided as little resistance to
the incoming signals, to the neural chip we use Ti-Au layered input
and output electrodes. Titanium provides structural support to the
electrode and the Gold layer makes the electrode bio-safe and highly
conductive. We however suspected that the electrode may show slight
variability in its resistance, due to oxidation of exposed titanium.
To ensure that the change in resistance was minimal, we kept a record
of the resistance values over 8 days for the input electrodes of a
selected chip, plated without liquid media. We report that the change
in the electrode resistance was minimal, and stabilised over the span
of 8days. This indicates that the sputtering was successful and the
gold layer was preventing oxidation of Titanium underneath.

![](https://static.igem.wiki/teams/4391/wiki/results/nr1.png)

![](https://static.igem.wiki/teams/4391/wiki/results/nr2.png)

Measuring resistance of electrodes

![](https://static.igem.wiki/teams/4391/wiki/results/nr3.png)

Resistance values

## Taking a reading from the chip

We modelled our neural chip to be most sensitive around a current
range of 150mA. A current drop below 100mA would virtually cause the
neurons around the input electrodes to stop firing. We simulated these
conditions using Arduino, where initially all input of the electrodes
were kept at 160mA and then selectively switched off inputs by
lowering their current strength to 50mA.

We used a bifocal fluorescence microscope to image the
neuron-like cells. We used it in bright field mode and captured the
pictures using an attached camera.

![](https://static.igem.wiki/teams/4391/wiki/results/nr4.png)

# References

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