iGEM 2023 — Team montpellier

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## Planning Aedes'n'seek

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## Notebook

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You will find more information about all the wetlab experiments we conducted [here](https://docs.google.com/document/d/1B9BuN7Um1ppcNejkLcTBspgU-ygeBuy9ifpQLvOKzsQ/edit?usp=sharing).

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# Overview

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![](https://static.igem.wiki/teams/4697/wiki/engineering.png)

## **Cycle 1 :** Designing target and guide RNA

# Design

The first design made for the project was in favor of the greater stability of DNA over RNA. Despite working with the latter one, the choice leaned in favor of the longer-lasting stock and virtually limitless quantity of materials offered by amplification through PCR, for as long as enough template was present. For this purpose, the base design that has to be tested was a set of primers fixing one to another in order to reconstruct a complete sequence.

![](https://static.igem.wiki/teams/4697/wiki/seq-guide.png)

**Fig 1:** Designing of the primers for target, LwCas13a guide, and casRX guides

# Build

The first sequences were manually crafted since no software was available at the time. Data was acquired from NCBI after conducting a BLAST on the West Nile Virus (WNV). Additional details can be found in the [Proof of Concept](https://2023.igem.wiki/montpellier/proof%20of%20concept) section
In the [Software section](https://2023.igem.wiki/montpellier/software), further information can be found about the software developed by our team during the project, that facilitates and automatizes guide design. The functionality can be found there, as well as the operational mechanics and the results.

![](https://static.igem.wiki/teams/4697/wiki/blast2.png)

**Fig 2:** Scheme of the process to obtain conserved sequences between variants of WNV. First a BLAST is performed, then a MAFFT alignment, finally the alignment is visualized in Jalview, and 2 conserved sequences > 28 nucleotides were found.

# Test

Two sequences needed to be re-ordered for the reverse primers of LwCas13a (target sequence 2.1 and 2.2) as they proved to be unsuitable for the binding with the forward primer (incorrect orientation of the 28 nucleotides sequence to target). Other than this, all processes ran smoothly, from PCR to transcription.

# Learn

As the initial cycle got completed, the next step can start, involving the preparation of everything concerning CasRx (transformation, expression, purification). However, it is imperative to acknowledge what was learned and make adjustments in the future. For instance, the improvement of the gel electrophoresis, mostly because no ladder was suitable to confirm the weight of the bands except that it is below 100 bp, but also optimizing the conditions by modifying the agarose concentration and type, the buffer composition, the equipment...(see cycle 5)

## **Cycle 2 :** Transformation of CasRx’s plasmid

# Design

The CasRx plasmid was acquired through [Addgene](https://www.addgene.org/153023/).Upon receipt, the plasmid was initially in a strain of *E.Coli* not suited for expression (DH5 alpha strain). Consequently, we needed to transform it according to the [reference material](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587836/).The effective expression strain for the plasmid seemed to be Rosetta2 (DE3), which was already present in the lab.

![](https://static.igem.wiki/teams/4697/wiki/rfxcas13d-plasmid.png)

**Fig 3:** The plasmid contains the coding sequence for RfxCas13d, also known as CasRx. Expression is controlled by the lactose operon and purification can be achieved using the MBP site (Maltose Binding Protein) and a 6-histidine site, situated just before MBP. Post-purification, a TEV site facilitates the removal of these additional sites, leaving only the target protein. The plasmid is designed to ensure integration into an *E.Coli* cell by incorporating a Kanamycin resistance site.

# Build

To transform our plasmid into the new strain, the initial step consists of rendering the cells chemically competent for transformation.

![](https://static.igem.wiki/teams/4697/wiki/transfo.png)

**Fig 4:** Scheme of the miniprep experiment

# Test

We succeeded in making competent cells and transforming the casRX plasmid into Rosetta cells. See the [Results section](https://2023.igem.wiki/montpellier/results#Transformation) for more information.

# Learn

Understanding that the protocol varies a lot depending on the strain, the starting point was the preparation and competency of cells, ending with the transformation itself, leading to the next cycle.

## **Cycle 3 :** Expression and purification of CasRx

# Design

**Production and purification preparation and discussion :** CasRX protein was new to us, so we started by doing expression tests in LB medium to see how it grows. We saw that protein was not well produced, but still decided to try the high scale production in TB medium as it was done in the [article](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587836/) we relied on. In order to avoid losing more time, we started preparing cultures and protocols for our purification. Some small details were slightly changed, for example buffer composition. As cold temperature equipments are heavily used in our host laboratory, we were not able to secure a time slot to perform purification at 4C and decided to try the procedure at room temperature instead.
.

# Test

Refer to the [CasRX expression and purification](https://2023.igem.wiki/montpellier/results#RfxCas13d) result section for more information .

# Learn

First of all, we concluded that our protein is not well produced in *E.coli* cells and there is little to no difference between before and after IPTG induction. Also, the problem might be related to IPTG concentrations, which is why we made after purification expression tests to see the influence of 0.25 mM, 0.5 mM and 1 mM of IPTG. After staining our gel we once again observed no difference between non induced and induced fractions. We therefore tried to quantify it with ImageJ, but it did not give any significant results. We then have been suggested that we should have tried different bacteria strains or even using eukaryotes cells like Saccharomyces. To dig further, we sent our plasmid to sequencing and discovered that there were a few mutations in the ORI region which possibly could have an effect on protein production and result in the experiment failure.

## **Cycle 4 :** Fluorescence detection by plate reader

# Design

In order to validate our system, it is imperative to generate quantifiable data to back-up our claims. Initially, employing an agarose gel electrophoresis was considered, but after reviewing alternative results, no conclusion could have been drawn from it. Consequently, the usage of plate reader detection sounded like a good compromise, considering that it can quantify fluorescence and provide kinetic information.

# Build

First, the Cas will detect the target sequence, thanks to the synthetic guide designed, and it will attract the protein thanks to the stem loop. Once the binding between the target and the guide is done and the Cas detects the complex, it starts cutting everything, firstly the target sequence. After that, every RNA strand in close proximity will be cut, including the string between the fluorescent molecule and the quencher, allowing the separation between those two parts and getting them away from each other, making the fluorescence not quenched, and thus detectable.

![](https://static.igem.wiki/teams/4697/wiki/fluoprinciple.png)

**Fig 4:** Scheme of the miniprep experiment

# Test

The detection results can be found in the [graph 1](https://2023.igem.wiki/montpellier/results#Fluorescence) of the Result section.

# Learn

The initial detection didn’t yield discernible kinetic reactions. In the future, it was considered placing the plate on ice prior to the sample loading to delay the reaction process as much as possible and potentially observe initial reactions kinetics on the plate reader. Additionally, discrepancies were observed in the outcomes of seemingly similar samples, such as those between 1.1 and 1.4, despite only having 2 different nucleotides between each successive sequence. Investigating this interesting difference could offer us valuable insights to help designing new target sequences or even improve our design software tool with new parameters.

## **Cycle 5 :** Optimisation of agarose gel conditions

For this cycle, the purpose of the experiments will be to get better gels through variations of parameters, in order to get an easier time analyzing.

# Design

Better visualization of the agarose gel is important to verify the quality of our materials all along the protocol, from initial amplifications to transcription, even more considering the length of our sample (below 100 bp). Small size samples give rise to new challenges to be visualized on gels.

# Build

In pursuit of optimisation, adjustments were made on all the parameters, here is an exhaustive list:- Varying agarose concentration from 0.8% to 5%
- Switching between buffers (TAE and TBE) as TBE should demonstrate better results as the fragments gets smaller
- Experimenting with different type of agarose (low quality one, [SeaKem](https://www.ugap.fr/achat-public/seakem-le-agarose-125-g_2552246.html) one, we believe NuSieve would have been the best option but we did not have time to order it.
- Testing between 3 different machines
- Altering the voltage setting, between 60V and 150V
- Adjusting the migration time, from 15 minutes to 2h
- Modifying the fluorescent nucleic acid dye (GelRed, SYBR Safe, Diamond nucleic acid dye) and the quantity used

# Test

See [final gel](https://2023.igem.wiki/montpellier/results#Amplification) in the Result section. The gel was prepared using a 2% (m/V ratio) agarose composition with TBE buffer. Electrophoresis was conducted for 45 minutes at a constant voltage of 130V, and the gel was stained with Diamond Nucleic Acid Dye. The sample configuration (from left to right): 25 bp ladder, 100 bp ladder, Negative control, Positive control, Synthetic target sequences 1.1 to 1.4, Synthetic target sequences 2.1 to 2.2, 100 bp ladder, 25 bp ladder.

# Learn

Obtaining a high-quality gel extends beyond parameter optimization. Depending on the choice of materials, a totally different outcome can be seen. For instance, selecting a certain fluorescent nucleic acid dye can affect both the quality of the bands and the noise during the visualization. Likewise, changing the machine necessitates modification of the combs and gel length, which influence the sample behavior under the same voltage. Agarose concentration influences the sample passage through the gel, making a lower concentration more permeable. Additionally, the quality of the agarose powder significantly impact the result consistency during the revelation.

## **Cycle 6 :** Improving detections

# Design

Following the first successful detection, different conditions were tested. The first one was comparing the first and second batch of prepared RNA, while the second part contrasts the difference between raw mass per microliter (ng / µL) and normalized quantity (molecular count) per microliter (µM / µL), with also a change in either the target or the guide concentration (doubling, halving).

# Test

For the differences between old and new RNA batch as well as between ng/µL and µM/µL, check [graph 2 and 3](https://2023.igem.wiki/montpellier/results#Fluorescence) in the Result section. For the differences between guide and target concentration, check graph 4.

# Learn

This phase of the experiment investigated the difference in concentration, giving more insights about the influence on normalizing the experimental settings. It proved that normalizing gives overall better results compared to the concentration analysis, as the molarity acts as a huge factor.

## **Cycle 7 :** Detection using Mosquitoes

# Design

As the detection worked well from a purely in vitro context, the next step aimed to transition into an in vivo setting, using infected mosquitoes. With the support of [MIVEGEC](https://mivegec.fr/fr), the team acquired mosquitoes infected with deactivated viruses, ensuring a safe workplace and not preventing disease development. The goal of the experiment would be to assess if the detection works in an in vivo environment.

# Build

The initial steps involved purifying viral RNA out of the mosquitoes using [Macherey-Nagel’s kit](https://www.mn-net.com/fr/nucleospin-rna-virus-mini-kit-for-viral-rna-from-cell-free-fluids-740956.50), in order to facilitate the detection process. In order to verify if detection could take place in a noisy sample, water was removed from the detection reactions and was fully replaced by mosquito juice. The most stable guide observed in the first rounds of detections, which was selected as “reference sequence”, was used in a detection reaction with purified viral mosquito RNA to see if we could detect RNA coming from a real mosquito. Lastly, we tested a detection reaction with a mix of different guides to explore the feasibility of multiplexing detection possibilities and offer more flexibility to our future automatized system.

# Test

The results of this cycle can be found in [graph 5](https://2023.igem.wiki/montpellier/results#Fluorescence) of the Result section.

# Learn

Thanks to this cycle, the viability of the detection was proved to be possible in an in vivo context, allowing future perspectives. As the detection in the purified viral RNA of the mosquitoes didn’t work, some questions rose up, including the fact that the virus included the sequence or not, as well as the adequacy of the viral charge for effective detection. In the results section, we brainstormed some improvement ideas to understand experiments that did not succeed, but also improve what already works and expand the system, to push its limits further in hopes of achieving the automatized detection system, which could help many.

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Importance of the experiments

Miniprep

Rosetta

Transformation

Glycerol Stock

PCR

Transcription

Fluorescence

RNA Extraction

Expression

Purification

Miniprep

The CasRx plasmid arrived in the DH5 alpha strain of E.Coli, which is suitable for DNA transformation, but not ideal for expression. In order to enhance the efficiency of expression, the solution used was to transfer the plasmid to the Rosetta BL21-DE3 strain, which is built for this task, and also aligns with our reference article  [[1]](https://pubmed.ncbi.nlm.nih.gov/34714054/). After completing the Miniprep procedure, the plasmid will be isolated and thus, the transformation in the appropriate strain can be done, facilitating the expression of the protein.

Competent Rosetta

As the quantity of the needed strain, Rosetta BL21-DE3, runned low in the lab, the iGEM team seized the opportunity to acquire new skills while preparing additional stocks. Those would not only be valuable for the team, but it will also benefit the whole laboratory staff.

Transformation

Following the completion of Miniprep to obtain the plasmid, and the acquisition of competent Rosetta BL21-DE3 strains, the next step is to execute the transformation process, by transferring the plasmid into the previously talked expression strain. This step is crucial to obtain our protein and conduct the desired researches.

Glycerol Stock

With the glycerol stock, our objective is to secure a long-term storage solution for the previously transformed strain. After cultivating and verifying its viability, the next step is to aliquot it into glycerol stocks. This preservation method ensures that, for future experiments, the strain will be available, and thus enhances at the same time the laboratory’s resources and our management of it.

PCR

Considering the experimental design, for both target and guide samples, a PCR step is essential in order to get complete sequences. Not only will it permit the production of testing materials at will, but also it will reduce the necessity to produce any orders in the future, ensuring availability of the genetic substance.

T7

As the subject is synthetic sequences derived from a virus, the WNV, the approach aims to mimic as closely as possible the natural conditions. In order to achieve this, tests involving the conversion of these sequences into RNA were conducted, necessitating in the process a T7 transcription step.

Fluorescence detection (Plate reader)

The primary of the project being the virus detection through fluorescence, with the eventual goal of integrating this into a device equipped with optical measurement tools to detect the presence or not of pathogens. The so-said device would then relay the results to epidemiologists and hospitals, but first, the concept needs to be validated. To do so, in vitro experiments need to confirm the functionality of the detection method.

Viral RNA Extraction

Conducting in vitro experiments represents the majority of the proof of concept part, but the project doesn’t end here. As the ultimate goal involves testing our detection method with mosquitoes, we sought collaboration with MIVEGEC, a research institute on viruses in Montpellier. Through this, we acquired our test subjects for evaluating the functionality, or not, of the detection in an in vivo setting.

Expression of CasRX

Considering the focus of this study being a Cas protein not already available in the lab, the first part of this new experiment would be to express the protein from the transformed cells that previously integrated the ordered plasmid.

Purification of CasRX

Producing the protein is but the beginning of this experiment, the ultimate goal being the utilization of the Cas. Consequently, a significant part of this project consists in actually extracting a purified solution of the protein from the cellular media. To do so, the purification process needs to be done in specialized columns for the Histidine tag and the MBP site, which are present on the target.

## Protocols

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## Miniprep (Extraction of CasRX plasmid)

## Competent Rosetta BL21-DE3

## Transformation of competent BL21-DE3 with CasRX plasmid

## PCR (Polymerase Chain Reaction)

## T7 Transcription

## Fluorescence detection by plate reader

## Viral RNA Extraction

## Expression of CasRX

## Purification of CasRX

#### PCR (Polymerase Chain Reaction) ▼

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#### T7 Transcription ▼

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## What should this page contain?

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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

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* [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)

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[PCR Amplification](#Amplification)
[T7 Transcription](#Transcription)
[RfxCas13d (CasRX) Expression](#RfxCas13d)
[Fluorescence detection](#Fluorescence)

During this project, many results were achieved, including making chemically competent cells, transforming a plasmid in the so-said cells, making de novo sequences through primer amplification, with sequences that are able to proceed a transcription step and even work with enzymes to make fluorescent detections.More details can be found below, including the steps, the results and the discussion of everything that was completed.

## Rosetta Competent

The full protocol can be seen in Experiments in the part [Competent Rosetta BL21-DE3](https://2023.igem.wiki/montpellier/experiments). According to the reference article for the plasmid, it was retrieved from the DH5 alpha and transformed into the Rosetta BL21-DE3 cells. To do so, it was mandatory to prepare some chemically competent cells. Here are the final results of this step:

![](https://static.igem.wiki/teams/4697/wiki/kan.png)

**Fig 1:** Test Transformed Rosetta2 DE3 kanamycin

![](https://static.igem.wiki/teams/4697/wiki/amp.png)

**Fig 3.1:** Test Transformed Rosetta2 DE3 ampicilin

![](https://static.igem.wiki/teams/4697/wiki/cam.png)

**Fig 2:** Test Transformed Rosetta2 DE3 chloramphenicol

![](https://static.igem.wiki/teams/4697/wiki/amp2.png)

**Fig 3.2:** Test Transformed Rosetta2 DE3 ampicilin bis

As neither the cell nor the transformed plasmid (PUC 19) contains the resistance to the antibiotic Kanamycin, results can be seen in the figure above that no colony can be found on the plate.

On the contrary, the strain naturally got embedded in its system the resistance against the Chloramphenicol, leading to the positive control being overwhelmed by bacterias.

To check about the transformation, it is important to look on the plates for the Ampicillin resistance, as it’s the antibiotic that get rid of everything besides what contains our transformed test plasmid (PUC 19), and a few colonies are transformed, and can be seen on the photos, showing that the bacterias are competent, considering that they integrated the plasmid correctly.

## Transformation

Once it was deemed possible to actually transform the newly competent, it is time to go onto the next stage and perform the transformation using CasRx’s plasmid. Again, the protocol can be found here (inserer lien de experiment).

![](https://static.igem.wiki/teams/4697/wiki/transforx.png)

**Fig 4.1:** First Transformation Rosetta CasRx

![](https://static.igem.wiki/teams/4697/wiki/transforx2.png)

**Fig 4.2:** Second Transformation Rosetta CasRx

Those pictures were made on an agarose plate containing both chloramphenicol (for the natural resistance of the Rosetta strains) and kanamycin (for the natural resistance of the CasRx plasmid). Colonies can be seen as the white dots determine the fact that bacteria grew on the media.

We confirmed that the plasmid was properly transformed in the strains using antibiotic resistance and moved on to the next steps of the purification process, namely, investigating the proper expression of the protein.

The transformed cells were safely stored by making a [glycerol stock](https://2023.igem.wiki/montpellier/experiments) and keeping it at -80C for later uses.

## PCR Amplification of target and guide DNA

While part of the team was working on the expression and purification of the Cas, others were in charge of the production and purification of the guides and targets RNA. As they were ordered in the form of DNA, the first step was to amplify the sequences.

The expected bands for each sample is:

- 91 base pairs for all the targets
- 87 base pairs for all the guide of LwCas13a
- 74 base pairs for all the guide of RfxCas13d

The amplification was made using a [PCR Protocol](https://2023.igem.wiki/montpellier/experiments) and the [Thermo Fisher superfi II kit](https://www.thermofisher.com/order/catalog/product/12361250?SID=srch-srp-12361250). The amplification was verified by looking at the DNA band sizes on agarose gel

![](https://static.igem.wiki/teams/4697/wiki/25-bp-ladder.png)

**Fig 5:**  25-bp-ladder

![](https://static.igem.wiki/teams/4697/wiki/100bp.png)

**Fig 6:**  100-bp-ladder

![](https://static.igem.wiki/teams/4697/wiki/pcr-target.png)

**Fig 7:**  Target agarose Gel TBE 0.5 X 130 V 45 min. NC : Negative control, PC : Positive control. The negative control of the target was made using 2 primers that does not hybridize (see Engineering), and the positive control correspond to the FlaA target sequence of Shell'lock project. T1.1-T2.2 are the differents target mentionned in Proof of Concept.

![](https://static.igem.wiki/teams/4697/wiki/25-bp-ladder.png)

**Fig 8:**  25-bp-ladder

![](https://static.igem.wiki/teams/4697/wiki/100bp.png)

**Fig 9:**  100-bp-ladder

![](https://static.igem.wiki/teams/4697/wiki/pcr-guide-new.png)

**Fig 10:**  Guide agarose Gel TBE 0.5 X 130 V 45 min. NC : Negative control, PC : Positive control. The negative control of the target was made without using the polymerase that is used for the amplification step in PCR, and the positive control correspond to the FlaA guide sequence of Shell'lock project. 13a 1.1-2.2 are the differents LwCas13a designed guides, and RX 1.1-2.2 the one for CasRX (see Proof of Concept).

## T7 Transcription of target and guide DNA

After proceeding with the DNA clean up, the next step was the transcription from DNA samples to RNA. It can be quite challenging to work with those as they are degraded easily thanks to all the RNAse present everywhere, which were taken care of using some [RNAse away](https://www.thermofisher.com/order/catalog/product/fr/fr/7003PK), found in Thermo Fisher’s website. After cleaning everything, from the benches to the pipettes, nuclease-free water was needed in order not to contaminate our samples, [special tips](https://www.starlabgroup.com/en/product/300-ul-graduated-tipone-filter-tip-natural-sterile-pf-sl-920412.html?childSku=S1120-9710) were also used.

Once everything is done, the T7 transcription was ready to go using this [protocol](https://2023.igem.wiki/montpellier/experiments) and it was purified using the clean up protocol found at the same place. We used the Nanodrop to verify the presence of DNA and determine its concentration.

![](https://static.igem.wiki/teams/4697/wiki/nanodrop2.png)

**Tab 1:** Tables showing RNA concentration for both the first (old) RNA and the second (new) batch.
 As the Nanodrop isn’t precise enough, every concentration below 50 ng/µL was considered negative.
 Also, the conversion between the result provided (in ng/µL) is also present normalized (in µM).

## RfxCas13d (CasRX) Expression

Daniel J. Brogan et al.'s research [1] served as the foundation for CasRX production. Within the Cas13 enzyme family, RfxCas13d, a member of class-d, distinguishes itself with several notable features:

- - It is 20% smaller than Cas13a-Cas13c variants
- - It does not require a PFS
- - It exhibits enhanced sensitivity

Those properties make it easier to produce in bacteria.

We ordered the high copy plasmid OA-1136J [2] from addgene as agar stab. It was growth in DH5alpha at 37°C.

![](https://static.igem.wiki/teams/4697/wiki/rfxcas13d-plasmid.png)

***Fig 11:** OA-1136J plasmid with CasRX gene, MBP, and 6-his-tag*

First, we used the RfxCas13d Expression protocol mentionned in experiments, but while doing a SDS-page gel, we did not obtained significant results as a band for our protein was not visible. As a matter of fact, it seems that the CasRX protein is insoluble, so we did back the experience this time making an IPTG gradient for the induction as well as taking either the supernatant alone after centrifugation, or the pellet + supernatant.The purification step was not possible a second time, if you are interested by it, you will find the protocol in the experiments section. Below, is detailed the protocol we used for this expression.

Rosetta cells ON were transformed at 37°, using chloramphenicol and kanamycin to filter out other bacteria than our transformed Rosetta.A colony was taken from the petri dish to inoculate 10 ml of LB, and was grow at 37° with the same antibiotics.The preculture was then split in 4 tubes that we named : 1A,1B,1C,1D, by putting 10 ml in each.

Once the OD was around 0.5-0.6, we induced tubes :

- 1A,2A without IPTG
- 1B,2B with 0.25 mM IPTG
- 1C,2C with 0.5 mM IPTG
- 1D,2D with 1 mM IPTG

Afterwards, the culture were grow at 18°C overnight to induce production.Once the induction was one, we run a SDS Page using Bis-Tris 10 % precast gel, and MOPS buffer.

This is the SDS-page gel, we obtain for the RfxCas13d protein

![](https://static.igem.wiki/teams/4697/wiki/prot-ladder.png)

**Fig 12:** Protein Ladder, Bis-Tris 10 % + MOPS

![](https://static.igem.wiki/teams/4697/wiki/gel-prot.png)

**Fig 13:** SDS-page gel Bis-Tris 10 % + MOPS. PL: Protein Ladder, 1: Non iduced supernatant + pellet, 2: Non induced supernatant, 3 : 0.25 mM supernatant + pellet, 4 : 0.25 mM supernatant, 5 : 0.5 mM supernatant + pellet, 6 : 0.5 mM supernatant, 7 : 1 mM supernatant + pellet, 8 : 1mM supernatant

It look like our protein (MW = 155 kDA) was much less present than expected. We did some ImageJ analysis for the intensity of the bands, and the higher one seems to be at 0.25 mM induction which is the concentration recommanded in the CasRX paper. On the other hand the 1mM IPTG induction seems to be lethal for the bacteria as no band are observed. You will find below a table that resume the result of the intensity value for each band.

As the expression did not work the first time, we ask ourselves if the plasmid was mutated, so we did a nanopore sequencing.

![](https://static.igem.wiki/teams/4697/wiki/nanopore-seq.png)

**Fig 14:** Result of the nanopore sequencing on Transformed E.Coli BL21-DE3 with CasRX plasmid

As you noticed there is only 1 red spot on the sequencing which would indicate a mutation in the ORI region, but looking back at addgene sequence of CasRX plasmid there was a nucleotide in this region that was not mentionned. Nevertheless a mutation in the ORI region would not be able to modify CasRX expression pattern according to one of our advisors : Maxime Bello.

## Fluorescence detection by plate reader

![](https://static.igem.wiki/teams/4697/wiki/graph1.png)

**Graph 1:** Fluorescence emission of the different pairs of synthetic targets and guides. Testing all the different guides with their specific synthetic target sequence to see if there is any difference in the fluorescence level of detection between the 2 main sequences, and the
 sub parts of each.

This first graph shows plate reader fluorescence detection for every sample and our positive control (sequence 1.1, green). Interestingly, a difference above 25k units can be seen between sequences 1.4 and 1.1, which are highly similar. This observation indicates that small nucleotide differences in the guides can lead to big detection differences.

While the numbers of each type of nucleotides (A,T,C,G) remains relatively constant for each sequence, a higher fluorescence can be seen for both sequences 2.1 and 2.2, indicating that it might be a better sequence for fluorescence detection.

According to [Konnerman et al. (2019)](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5910255/), Cas13d should have a preference for cutting uracil bases.The results found here were that a higher A and/or G concentration provides more fluorescence (both sequence 2.1 and 2.2 contains more than 10 A and 10 G in their sequences), which means at the same time a higher cutting proficiency. It would be interesting to investigate these results in the future, to check how the nucleotides A, U and G lead to the highest cutting efficiency.

After looking at the overall sequence efficiency and responses, we started looking into the kinetics of the reactions and the overall impact of concentration on the system.

![](https://static.igem.wiki/teams/4697/wiki/graph2.png)

**Graph 2:** Fluorescence detection for old RNA (ng/uL)

![](https://static.igem.wiki/teams/4697/wiki/graph4.png)

**Graph 3:** Fluorescence detection for normalized RNA (uM/uL). Differences in fluorescence emission depending on the batch of RNA (first
 batch in Graph 2 or second batch in Graph 3) and the method used for the concentration calcul (ng / µL or µM / µL respectively in Graph 2
 and Graph 3)

In those graphics, different concentrations and batches of RNA preparation can be seen. The objective was to determine if there are differences between the batches at first, but in a second time, the main goal became to check which is the best method to proceed with our analysis (concentration in ng/µL or normalization in µM).

Unexpected behaviors were observed in both graphs 2 and 3, where the fluorescence signal decreased. (once the probe is cut, it will emit fluorescence and would not be able to bind back to reduce the units emitted). Nonetheless, the sequences 2.1 and 2.2 can be seen as really homogeneous no matter the conditions and the batch, with a fluorescence around 27k RFU (Relative Fluorescence Unit).

Despite observing overall more noise and fewer positive signals using the new RNA batch compared to the old one, we can see that once everything is normalized, either the sequence is working or not, and the results are constant no matter the sequence used.

The normalization process from ng/µL to µM was made by using the concentration obtained after the Nanodrop in ng/µL, and then converting it into g/L (\*10^-9 to go from ng to g, then /10^-6 to go from µL to L). It was then divided with the molar mass of the sequence, leading to mol / L as a unit, or molar M. Last step is to scale this result down to µM by multiplying it with 10^6. By doing so, we reduced by half the quantity of target used (1 µM of target), but the quantity of guides got upped, in order to get the 2:1 ratio between guides and targets present in the [CasRx reference article](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587836/).

Considering that the normalization method is using up to 4 times less materials, and provides better reproductivity and better fluorescence, it will be adopted for all the future experiments, and is also advised to do so for whomever will think about doing a similar project.

In the end, the sequence 1.2 from the new RNA will be used as a reference sequence in further experiments according to the results last shown (RFU around 37k, compared to the 15k in the old RNA), but mostly considering that, paired with those fluorescent data, it also uses less materials compared to the others.

As the difference in fluorescence between the reference and the changed concentrations is noticeable, we concluded that the quantity of target and guides in the mix could be optimized further and provide clearer results. In order to understand our system better, we decided to investigate the impact of concentration variations on total fluorescence. Fluorescence was not observed for all of the tested sequences. It is possible that some of the RNA concentrations were in reality lower than what we measured and not sufficient for Cas detection. Nanodrop is indeed known for not being the most precise way to measure RNA. It could be interesting to measure concentrations again by using the Qbit method to validate this hypothesis.

Also, normalizing the data seems to give overall better results compared to the concentration analysis, considering that the sequences either work really well, or do not give any detection at all. Henceforth, the molarity acts as a huge factor in the fluorescence detection.

![](https://static.igem.wiki/teams/4697/wiki/graph5.png)

**Graph 4:** Differences in fluorescence emission according to the guide concentration or the target concentration.

For this experiment, the goal was to vary either the targets or the guides concentration (twice or halved) to gather more intel on the optimization possibilities of those parameters.

While we expect less fluorescence when scaling down the concentration, we also expect better fluorescence values when increasing the quantity.

On the contrary of the expectations, the results came out as higher fluorescence for all the conditions compared to the reference, no matter if the parameters were doubled or halved.

Those results can be explained because the concentrations used (1 µM of target, 2 µM of guide) might provide different results depending on the sequence used.

In the future, it would be interesing to push the optimisation further by systematically testing different concentration ratios of guides and targets. this would help understanding if the concentration impact is sequence specific or general for Cas13 functions.

![](https://static.igem.wiki/teams/4697/wiki/plateau.png)

**Fig 15:** Python Analysis of slope angle (alpha) and plateau values

As we decided to get more insight on the quantitative aspect of this analysis, we developed a short python script that is able to fit the curves and sort out parameters such as the angle of the slope (alpha), the value of the plateau, and the time it took to reach the plateau.

Thanks to that, it can be seen that amongst the different conditions tested, higher concentrations generally means higher fluorescence (above 16k units of fluorescence in the plateau for both conditions, target and guide) and the slope also as an higher alpha angle (above 15 and above 14.5 relative fluorescent unit per second, RFU / s). When lowering the concentration of guides and targets we can see that, despite getting lower fluorescence values than when the concentrations are doubled, they are still above the reference concentrations, with an alpha unit above by more than 2 points (with the lowest value, corresponding to the half target condition) , but the plateau also gets lower, achieving a whole 3k units of fluorescence difference between the low target and the reference.

Those results mean that more optimisation can be made concerning the optimal concentration of targets and guides, and also that a lower amount of materials is needed to proceed with the detection.

![](https://static.igem.wiki/teams/4697/wiki/graph6.png)

**Graph 5:** Fluorescence emission with the presence of mosquito’s RNA and mosquito juice compared with our reference sequence.

The purpose for this experiment was to test the fluorescence on the reference sequence (1.2, orange curve) and check for the first time whether or not it is possible to detect viruses in in more realistic conditions, with the digested mosquito instead of the water and also the presence of deactivated viral RNA (green curve). After that, tests were made simultaneously with purified viral RNA from the mosquitoes with either the reference guide only, or a mix containing all the guides. The reason behind it was to check if multiplexing was possible, to have a fluorescence signal when all the guides are present in the mix.

The expected results are that both the reference only and the one containing the mosquito juice and viral RNA are working just fine. For the guide’s reference with the viral RNA from mosquitoes, a few fluorescence units are expected, and even less for the condition including the mix of all the guides.

While what was expected got obtained when using the reference guides and targets in both conditions, nothing was detected when no synthetic targets were added to the mix.

As the detection is proved to be possible, the lack of fluorescence can be the results of a couple of parameters. The first one being the fact that the viral charge of our target is too low to be detected, while the second one being that the virus got a mutation on this specific part of his sequence. The third possibility is that our target sequence could have been degraded over time. Last but not least, our sample might have no traces of the target as the experiments did not took place the day the mosquitoes were received and, even though anti-RNAse was added, the viral mRNA in the mosquitoes could have been degraded by the time we used them.

In order to understand if the viral charge was too low, in the future we want to perform RT PCR on the mosquito juice and with that verify if the viruses were present, and at the same time, validate that our guide can bind to the viruses properly. Using this PCR, a sequencing can also be done to verify the sequences of the viruses and if they match our guide properly. Also, checking the state of the RNA throughout our experiments in a bioanalyzer could also give insight on the quality of the RNA. Lastly, if the detection works, it can be interesting to test if the same results can be obtained using isothermal PCR (RPA), as this method would amplify the viral material without requiring a PCR machine and could be done out of a lab, to investigate if whether or not, it could be implemented in an automatic device.

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