iGEM 2022 — Team iit-roorkee

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Inspirations

---

* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

{% endblock %}

[# CRISPRLY](%7B%7B%20url_for%28%27index%27%29%20%7D%7D)

* Link
* Link
* Link
![]({{ url_for('static', filename = 'assets/img/hamburger-icon.svg') }})

# ENGINEERING

# Engineering

To bring our iGEM project to life, and to fulfil our goal of using biological systems for solving
real-world problems, we had to use the engineering cycle in multiple stages of the project.

![](https://static.igem.wiki/teams/4434/wiki/igem-cycle.jpg)

# The Origin of Project

During the initial literature review and brainstorming phase of our project, we were interested in the role of the human BRCA mutation, which predisposes women to much higher risk of developing breast cancer. Our initial hypothesis was to design a method of using this as a marker for early breast cancer screening. However, on further reading, we discovered the link between HPV and a small percentage of breast cancer cases. HPV grabbed our attention due to it being the primary causative agent for cervical cancer, which has a higher mortality rate and is heavily underdiagnosed due to lack of appropriate screening tools. The identification of this large unsolved problem, that disproportionately affects people of developing countries, with a potential for detection and implementation of preventative measures was the inception of our further efforts.

# Design of the proposed kit

## 1. Biomarker Selection

HPV has over 150 strains. However, only close to 20 strains have the potential to cause cancer. 90% of the sexually active population would test positive for HPV. This makes it essential for us to limit our diagnosis to the detection of only the high-risk strains of HPV in order to convey useful results to the patient.HPV16 being the most carcinogenic. These statistics clearly demonstrate the importance and need to focus on the diagnosis of HPV16 and 18.
Previous attempts at HPV detection in the literature and existing diagnostics target the viral capsid i.e. the L1 capsid protein gene as a biomarker. Literature review to assess the suitability of L1 as a target in high-risk strains suggested that the gene has a high variability, even among HPV 16 sublineages.[1] We performed Multiple Sequence Alignment of the L1 genes of HPV16 and HPV18 and clear variations can be directly observed.

![](https://static.igem.wiki/teams/4434/wiki/msa.png)

A more appropriate biomarker was required.
A recent study demonstrated the importance of the E7 oncogene in cervical cancer progression and how its conservation plays an important role.[2] To confirm this theory, multiple sequence alignment of E7 genes of HPV16 sublineages was performed.HPV16 can be classified into 4 sublineages (A1-4, B1-4, C1-4, D1-4) [3]. All of them have different cervical cancer risks. After reviewing literature, it was found that sublineages A4, C, D2 and D3 of HPV16 are most prevalent in Asia. Thus, we chose to perform a Multiple Sequence Alignment on these 4 sublineages to confirm the conservation of HPV16The results clearly show that E7 of all high risk sublineages of HPV16 is highly conserved and can serve as a suitable biomarker.

MSA of oncogenic sublineages of HPV16:

![](https://static.igem.wiki/teams/4434/wiki/alignment.jpg)

## 2. Choice of detection method

We decided to opt for a cell-free system as the basis for our kit, due to reasons of simplicity and practicality, as our solution is intended for low-resource settings.

Our initial approach to HPV DNA detection was based on the use of a RNA toehold switch complementary to the target gene, coupled with LacZ expression. However, the system required a cell-free expression system for colorimetric readout and was not practical. As an alternative, we chose to focus on CRISPR-Cas based approaches due to their high specificity for user-defined targets. The transcleavage property of Cas 12, led us to choose it as our tool of choice, allowing for a measurable readout triggered by presence of target DNA.

## 3. Primer Design for PCR

Forward and Reverse primers need to be designed for the amplification of our target gene i.e. E7. General set of rules followed for these primers:

1. The GC content of the primers should be greater than 40%.
2. The length of each primer should range anywhere from 18-20 base pairs.
3. The primers should end at either a GG or a CC. This is to ensure a strong bond between the primer and the target. A stronger bonding is ensured because of a triple hydrogen bonding between G and C.

The primers are figured out by taking one strand as it is and one reverse compliment of the same strand. According to this, the sequence of our primers was:

1. E7 Forward Primer (5’ - 3’) - ATGCATGGAGATACACCTAC
2. E7 Reverse Primer(5’ - 3’) - TTATGGTTTCTGAGAACAGATGG

The feasibility of these primers was analysed using IDT’s oligo analyzer tool

## 4. Guide RNA Design

PAM or Protospacer Adjacent Motif is an essential component of any CRISPR system and plays an important role in designing the guide RNAs for the CRISPR reactions. A PAM is a 2-6 nucleotide long sequence that guides the Cas12a enzyme to cut the target DNA. The PAM sequences are different for different Cas enzymes.Cas12 recognizes TTTV as its PAM site (V = A/C/G). This PAM site is recognized on the 5’ to 3’ strand of the target DNA and the guide RNA binds immediately downstream of the PAM but on the opposite strand.

![](https://static.igem.wiki/teams/4434/wiki/cpf1.jpg)

Our first goal was to determine the PAM sites on our target sequence i.e. the E7 gene. The sequence was taken from NCBI. 297 conserved bases of E7 were taken into consideration and our own [PAM finder algorithm](https://pamfinder.herokuapp.com/PAM_finder/) was used to find out three PAM sites on the 5’ to 3’ strand of the E7 gene. The guide RNA is supposed to bind immediately downstream of the PAM sequence on the 5’ - 3’ strand.

According to our literature review, 21 bases was the optimum length for a Cas12 guide RNA. The constant region of a Cas12a guide RNA is 21 bases long hence, making our guide RNA 42 bases in length.

Here are the three PAM sequences highlighted in red on the 5’-3’ strand of E7 :

ATGCATGGAGATACACCTACATTGCATGAATATATGTTAGATTTG**CAACCAGAGACAACTGATCTC**TACT
GTTATGAGCAATTAAATGACAGCTCAGAGGAGGAGGATGAAATAGATGGTCCAGCTGGACAAGCAGAACC
GGACAGAGCCCATTACAATATTGTAACCTTTTG**TTGCAAGTGTGACTCTACGCT**TCGGTTGTGCGTACAA
AGCACACACGTAGACATTCGTACTTTG**GAAGACCTGTTAATGGGCACA**CTAGGAATTGTGTGCCCCATCT
GTTCTCAGAAACCATAA

Therefore, the three guide RNAs sent to be synthesized from IDT are:

     1. CAACCAGAGACAACTGATCTC
     2. TTGCAAGTGTGACTCTACGCT
     3. GAAGACCTGTTAATGGGCACA

# EXPERIMENTAL PROTOCOLS

Initially, the CRISPR/Cas12a reaction protocol was followed according to the standard protocols of the manufacturers. However, multiple unsuccessful experiments led us to alter the protocol to better suit our needs. After discussion with experts and some out of the box thinking, multiple iterations of the protocol were performed by varying reaction parameters and reagent concentrations.  Finally, an optimal combination of reaction time, Cas12a concentration and ssDNA reporter concentration were arrived at that yielded successful results.

We designed the initial protocol, tested it and when it failed, we read more and altered the protocol till fruitful results were obtained. The engineering cycle greatly helped us formulate the correct protocol. For more information, please refer to our Experimental Protocols page.

# MACHINE LEARNING MODEL

This model was intended to tell us about the most accurate guide RNA based on existing CRISPR/Cas12 reactions. It tells us about the indel frequency of CRISPR reaction at the target i.e. 3’ to 5’ strand of the E7 gene. One of the main outputs of CRISPR indel analysis is the indel frequency, which represents the CRISPR editing efficiency. This parameter is used to assess the suitability of crRNA that will give the most efficient results.  Please refer to the [Model page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D) for more details

# COMMUNITY FEEDBACK

We interacted with various experts in the industry and incorporated their valuable insights into the project as much as possible. We also interacted with various doctors and patients to help understand the severity of the problem statement in depth. This helped us in the “Learning” aspect of the engineering cycle on the basis of which, we could formulate new protocols and test them out to achieve the desired results. For more information, please refer to the [Human Practices page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)

# USEFUL TOOLS

To ease out the design cycle, we developed a software tool that could help find the PAM sites in long DNA sequences, and it also gives the guide RNA sequences as the output. This tool helped us in our project, and we hope it also comes of some help to fellow researchers while finding out the PAM sites and designing guide RNAs for their CRISPR reactions. For more information, please refer to the [Web Application page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27software-1%27%29%20%7D%7D) under Drylab.

References:

1. El Aliani, A., El Abid, H., Kassal, Y., Khyatti, M., Attaleb, M., Ennaji, M.M. and El Mzibri, M., 2020. HPV16 L1 diversity and its potential impact on the vaccination-induced immunity. *Gene*, *747*, p.144682.
2. Mirabello, L., Yeager, M., Yu, K., Clifford, G.M., Xiao, Y., Zhu, B., Cullen, M., Boland, J.F., Wentzensen, N., Nelson, C.W. and Raine-Bennett, T., 2017. HPV16 E7 genetic conservation is critical to carcinogenesis. *Cell*, *170*(6), pp.1164-1174.
3. Clifford, G.M., Tenet, V., Georges, D., Alemany, L., Pavón, M.A., Chen, Z., Yeager, M., Cullen, M., Boland, J.F., Bass, S., Steinberg, M., Raine-Bennett, T., Lorey, T., Wentzensen, N., Walker, J., Zuna, R., Schiffman, M. and Mirabello, L. (2019). Human papillomavirus 16 sub-lineage dispersal and cervical cancer risk worldwide: Whole viral genome sequences from 7116 HPV16-positive women. Papillomavirus Research, 7, pp.67–74. doi:10.1016/j.pvr.2019.02.001.

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### People

* [Team](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%20%7D%7D)
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### Project

* [Description](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D)
* [Problem](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27problem%27%29%20%7D%7D)
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### Wet Lab

* [Overview](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27overview-wetlab%27%29%20%7D%7D)
* [Protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D)
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* [Proof of Concept](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27proof-of-concept%27%29%20%7D%7D)

### Dry Lab

* [Model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)
* [Hardware](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27hardware%27%29%20%7D%7D)
* [Software](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27software%27%29%20%7D%7D)

### Human Practices

* [Overview](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27overview-hp%27%29%20%7D%7D)
* [Communication](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27communication%27%29%20%7D%7D)
* [Integrated Human Practices](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)
* [Collaborations](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27collaborations%27%29%20%7D%7D)
* [Sustainable Development Goals](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27sustainable%27%29%20%7D%7D)

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#### Address

The Department of Biosciences and Bioengineering,
Indian Institute of Technology Roorkee,
Roorkee, Uttarakhand
India - 247667

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#### Contact us

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+91 9599219678

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# People

[Team](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%20%7D%7D)[Attributions](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27attributions%27%29%20%7D%7D)[Sponsors](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27sponsors%27%29%20%7D%7D)

# Project

[Description](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D)[Problem](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27problem%27%29%20%7D%7D)[Solution](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27solution%27%29%20%7D%7D)[Implementation](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27implementation%27%29%20%7D%7D)[Engineering](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)[Contribution](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27contribution%27%29%20%7D%7D)

# Human Practices

[Overview](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27overview-hp%27%29%20%7D%7D)[Communication](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27communication%27%29%20%7D%7D)[Integrated Human Practices](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)[Collaborations](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27collaborations%27%29%20%7D%7D)[Sustainable Development Goals](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27sustainable%27%29%20%7D%7D)

# Dry Lab

[Model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)[Hardware](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27hardware%27%29%20%7D%7D)[Software](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27software%27%29%20%7D%7D)

# Wet Lab

[Overview](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27overview-wetlab%27%29%20%7D%7D)[Protocols](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D)[Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)[Safety](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27safety%27%29%20%7D%7D)[Proof of concept](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27proof-of-concept%27%29%20%7D%7D)

[# Judging](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27judging%27%29%20%7D%7D)

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+91 9899725258
+91 9599219678

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igem@iitr.ac.in

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The Department of Biosciences and Bioengineering,
Indian Institute of Technology Roorkee,
Roorkee, Uttarakhand
India - 247667

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{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

{% endblock %}

[# CRISPRLY](%7B%7B%20url_for%28%27index%27%29%20%7D%7D)

* Link
* Link
* Link
![]({{ url_for('static', filename = 'assets/img/hamburger-icon.svg') }})

# Result

# Workflow of the Kit

Click on the following tabs to view the results, observations and inferences of the wetlab experiments conducted -

**CONCENTRATION AND VOLUME CONTROL OF THE REPORTER**

![](https://static.igem.wiki/teams/4434/wiki/modal-arrow.svg)

CONCENTRATION
AND VOLUME CONTROL OF REPORTER

![]({{ url_for('static', filename = 'assets/img/modal-close.svg') }})

A series of concentration and volume control experiments were set up.

OBSERVATIONS FOR VERSION #1
100uL was a lot of fluid for the strip to absorb. The quantity of the solution needed reduction.

* In the first case, a very strong control line developed indicating that the strips worked perfectly fine.
* In the second case, solid control and test ines were seen indicating that the reporter with the FAM and biotin was in order and worked as expected.
* In the last case, a very strong test line and a weak control line was observed. The control line intensity was weak because there was a lot of FAM to which the Anti-FITC antibodies on the conjugate pad could bind to. This conjugate went to the test line where biotin bound to Streptavidin. Due to this, there was very little concentration of the Anti-FITC was left to go to the control line. Hence, the weak intensity.

![](https://static.igem.wiki/teams/4434/wiki/conc1.jpg)

A second set of experiments with reduced concentrations was performed to figure out the optimum concentration for the reaction.

OBSERVATIONS FOR VERSION #2

* The solution was able to flow properly and reached the end of the strip almost instantly. A solid control was observed
* The observed result and speed of flow of the buffer were very similar to the first strip.
* A weak control line was observed but the solution could flow till the end of the strip.
* Weak control line was observed but the solution could not flow till the end indicating that 20uL volume for the ssDNA concentrated reporter is too less mostly because the viscosity of the reporter solution is greater than the buffer that was initially made to flow on the strip with the same volume.
* Extremely weak control line mostly because the total number of moles of FAM available for the Anti-FITC antibodies increased a lot and hardly anything must have been left to go to the control line.

![](https://static.igem.wiki/teams/4434/wiki/conc2.jpg)

![](https://static.igem.wiki/teams/4434/wiki/conc3.jpg)

**PROTOCOL #1**

![](https://static.igem.wiki/teams/4434/wiki/modal-arrow.svg)

PROTOCOL #1

![]({{ url_for('static', filename = 'assets/img/modal-close.svg') }})

The following image shows the result of the 6 reactions that were set up following the first protocol

![](https://static.igem.wiki/teams/4434/wiki/protocol1.png)

OBSERVATION

Both test line and control line were observed in all of the cases. We were expecting only the control line in cases where all of the DNA reporter would be cleaved.

INFERENCE

* CRISPR reactions didnt work properly. We inferred and after literature review, it was realised that the incubation time was less. For all further experiments, the incubation time was decided to be increased to 2.5 hours.
* It was also thought that the enzyme was not enough to cleave all the reporter so the enzyme volume used was also increased for further experiments.
* A possibility was considered that the NEB enzyme was not compatible with the guide RNA ordered from IDT.

**PROTOCOL #2**

![](https://static.igem.wiki/teams/4434/wiki/modal-arrow.svg)

PROTOCOL #2

![]({{ url_for('static', filename = 'assets/img/modal-close.svg') }})

The following image shows the result from the first time the second protocol was followed. This was the first successful experiment.

![](https://static.igem.wiki/teams/4434/wiki/protocol1-2.png)

OBSERVATION

A heavy intensity control line is observed. The intensity of the test line is very weak; almost negligible.

INFERENCE

* All of the reporter molecules were cleaved. Very few remained that gave a very weak test line. This is very close to the desired result.
* After this experiment, the protocol was standardized according to the above mentioned table and the same procedure was followed for all further replicates.

REPLICATE SET #1

The following image shows the result of the 8 reactions that were set up in accordance with the table mentioned along with Replicate#1 in the second protocol.

![](https://static.igem.wiki/teams/4434/wiki/protocol2-2.png)

![](https://static.igem.wiki/teams/4434/wiki/protocol2-3.png)

OBSERVATION

* A higher intensity test line can be seen on strip 1 and 2 due to the higher concentration of reporter used.
* A very low intensity test line is seen when E7 is not added. This is not in accordance with the expected results. Ideally, a very strong test line should have been observed in combination with a control line.
* No test line was observed in the case where reporter was not added (Strip 4)
* A weak intensity test line was observed when the system was incubated for only 1hr 10mins. This could mean that the reaction time was not sufficient for all of the reporter to be cleaved
* When the Cas12 enzyme was omitted from the system, a weak test line and a very solid control line was obtained. Again, this result is not clearly distinguishable from the positive result.
* Extremely weak test line was obtained when guide RNA 2 and 3 were used in the system.

INFERENCE

* This result agrees with the previous successful attempt. However, the negative controls also display a very weak test line making it very difficult for the user to accurately distinguish between a negative and a positive HPV case.
* There is very little reporter in the system which is why the test line in any case is weak.

REPLICATE SET #2

![](https://static.igem.wiki/teams/4434/wiki/protocol2-4.png)

OBSERVATION

Here, the first strip is the positive reaction. The results are very different from the results obtained the first time this protocol was followed. Moreover, there is no significant difference between strip 1 and strip 2. Thus, in this reaction setup, there is no way to distinguish between a positive and negative reaction.

REPLICATE SET #3

Exact same replicate was carried out again with unsatisfactory results

![](https://static.igem.wiki/teams/4434/wiki/protocol2-5.png)

OBSERVATION

Negative and Positive results are almost identical. The protocol needs to be altered yet again.

**PROTOCOL #3**

![](https://static.igem.wiki/teams/4434/wiki/modal-arrow.svg)

PROTOCOL #3

![]({{ url_for('static', filename = 'assets/img/modal-close.svg') }})

![](https://static.igem.wiki/teams/4434/wiki/protocol3-1.png)

OBSERVATION

* All of the reporter molecules were cleaved. Very few remained that gave a very weak test line. This is very close to the desired result. After this experiment, the protocol was standardized according to the above mentioned table and the same procedure was followed for all further replicates.
* However, in order to differentiate between a positive and a negative result, all of the reporter needs to be cleaved.

INFERENCE

* The reporter concentration needs to be minimised in order to make sure that almost all of the reporter gets cleaved as a result of the trans cleavage activity of Cas12.
* In order to ensure that CRISPR reactions work properly, the concentration of E7 was also increased.

DEBUGGING PART #1

![](https://static.igem.wiki/teams/4434/wiki/protocol3-2.png)

OBSERVATION

* The first strip here shows 2nM concentration of the reporter used. The second, third and fourth strip are the results of using 5nM, 10nM and 15nM respectively
* A very weak test line is observed in the case of 2nM concentration of the reporter used. The remaining strips show a good signal at the test line.

INFERENCE

* The second strip shows a good test line and utilises the least reporter concentration. Hence, for protocol 4, the concentration of the DNA reporter was fixed to 5nM.

**PROTOCOL #4**

![](https://static.igem.wiki/teams/4434/wiki/modal-arrow.svg)

PROTOCOL #4

![]({{ url_for('static', filename = 'assets/img/modal-close.svg') }})

Here, the concentration of E7 was increased to almost two times the previous concentration and the concentration of the reporter was reduced to 5nM as a result of Debugging Part#1

![](https://static.igem.wiki/teams/4434/wiki/protocol4.png)

OBSERVATION

* All of the reporter molecules were cleaved. Very few remained that gave a very weak test line. This is very close to the desired result. After this experiment, the protocol was standardized according to the above mentioned table and the same procedure was followed for all further replicates.
* However, in order to differentiate between a positive and a negative result, all of the reporter needs to be cleaved.

INFERENCE

* The reporter concentration needs to be minimised in order to make sure that almost all of the reporter gets cleaved as a result of the trans cleavage activity of Cas12.
* In order to ensure that CRISPR reactions work properly, the concentration of E7 was also increased.

DEBUGGING PART #1

![](https://static.igem.wiki/teams/4434/wiki/protocol3-2.png)

OBSERVATION

* The first strip here shows 2nM concentration of the reporter used. The second, third and fourth strip are the results of using 5nM, 10nM and 15nM respectively
* A very weak test line is observed in the case of 2nM concentration of the reporter used. The remaining strips show a good signal at the test line.

INFERENCE

* The second strip shows a good test line and utilises the least reporter concentration. Hence, for protocol 4, the concentration of the DNA reporter was fixed to 5nM.

**PROTOCOL #5**

![](https://static.igem.wiki/teams/4434/wiki/modal-arrow.svg)

PROTOCOL #5

![]({{ url_for('static', filename = 'assets/img/modal-close.svg') }})

![](https://static.igem.wiki/teams/4434/wiki/protocol5-1.png)

OBSERVATION

* Here, the first two strips display the results of a positive HPV sample adn the last strip shows the results of a negative HPV sample
* There is a clear and significant difference between a negative and a positive sample.

INFERENCE

* The new protocol works perfectly as any reporter that gets cleaved goes to the control line and gives a signal there.

REPLICATE #1

![](https://static.igem.wiki/teams/4434/wiki/protocol5-2.png)

OBSERVATION

* There is a very clear distinction between a negative HPV (Left Strip) and a Positive HPV (Right Strip)

INFERENCE

* The new reagent composition and procedure works well and could be successfully replicated twice.

REPLICATE #2

![](https://static.igem.wiki/teams/4434/wiki/protocol5-3.png)

OBSERVATION

* A clear distinction between a negative HPV and a positive HPV is observed.
* The intensity of the control line remains constant even on changing the guide RNA.

INFERENCE

* The new reagent composition and procedure works well and could be successfully replicated twice.

REPLICATE #3

![](https://static.igem.wiki/teams/4434/wiki/protocol5-4.png)

OBSERVATION

* Successful replicates can be observed in the case of Guide RNA 1 and Guide RNA 2
* Satisfactory results cannot be seen when the incubation time was reduced to one hour.
* There is no significant difference between a negative HPV and a positive HPV when the reaction was incubated at Room Temperature for 2.5 hours.

INFERENCE

* All three guide RNAs work perfectly well and the reaction could be reproduced successfully.
* 1 hour of incubation is not enough to achieve the desired result
* The incubation time needs to be increased to produce successful results at Room Temperature. Thus, for all further replicates, the intubation time was increased to 4 hours.

REPLICATE #2

![](https://static.igem.wiki/teams/4434/wiki/protocol5-5.png)

OBSERVATION

* The fourth replicate of each guide RNA (strips 1 to 3), was successfully carried out thereby, giving us proof of concept. There is a significant difference between positive HPV results and negative HPV results.
* By reducing the E7 concentration, no significant control line could be seen.

INFERENCE

* E7 concentration plays an important role in the development of the control line and upon reduction of the concentration down to 30nM, there is no significant difference between a positive HPV and a negative control.

REPLICATE #2

![](https://static.igem.wiki/teams/4434/wiki/protocol5-6.png)

OBSERVATION

* After 4 hour incubation, strong signals on the test line were observed in the case of all three guide RNAs

INFERENCE

* Once again it was observed that the intensities of the signal did not vary significantly with the choice of guide RNA
* It was confirmed that room temperature incubation of this reaction system requires a greater time to produce successful results.

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Indian Institute of Technology Roorkee,
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