iGEM 2022 — Team montpellier

{% extends "layout.html" %}
{% block title %}Implementation{% endblock %}
{% block page\_content %}
{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the
engineering design cycle.{% endblock %}
{% block page\_content %}

Synthetic biology is defined as “a growing discipline that has two subfields. One uses unnatural molecules to
reproduce emergent behaviors from natural biology, with the goal of creating artificial life. The other seeks
interchangeable parts from natural biology to assemble into systems that act unnaturally.” [**(1)**](#index_references)
To successfully develop engineering systems, synthetic biologists follow the Design Build Test Learn Cycle (see
figure 1).

![](https://static.igem.wiki/teams/4473/wiki/engineering-im1.png)

*Figure 1: Design Build Test Learn cycle*

The problem we are trying to solve is the lack of rapide and simple detection of oyster pathogens in water
samples.
To do so, we adapted an existing detection technique called SHERLOCK to our purpose. This is how Shell’lock
born.

In our project Shell’lock, we were able to design guide RNAs specific to target sequences of *Vibrio
Aestuarianus* and RNA probes, successfully detect this pathogen through the recognition of the target
sequence by
the designed guide RNA and finally develop a fluorescent output and paper-based test.

To illustrate our way to reach these successful results, we decided to illustrate all the steps through the
DBTL cycle.

## Cycles

* Cycle 1: design of all the guide RNA
  specific for the pathogen and test the positive control using paper strip

  ### Design

  The first step of this cycle consists in choosing *V.Aestuarianus* genes that will be targeted.
  Bibliography was needed to choose relevant genes, involved in pathogenicity or virulence of the pathogen and that are specific for *V. aestuarianus*. Because we wanted first to perform the test on synthetic sequence before testing it
  with
  true samples, the first step was to design synthetic target sequence as shown in figure 1. The
  second
  step consists in designing guide RNAs that are able to recognize a 28 nucleotides sequence present
  in
  each chosen gene. The design is done using bio informatic tools and literature. For this step, we
  developed a blast program that is able to find matches between designed guide RNAs and other species
  present in water.

  In our idea, we wanted first to test the SHERLOCK reaction using a paper strip. To be able to
  perform
  these experiments, we ordered already purified Cas13a, a plasmid containing this enzyme and a
  FAM-biotin
  probe.

  Finally, we started to design a model for the Cas13a kinetic and a data analysis codepipeline for
  the
  fluorescence experiment we planned to perform.

  ![](https://static.igem.wiki/teams/4473/wiki/engineering-im2.png)

  Figure 2: template of the design part of the first engineering cycle.

  ### Build

  The build step of this first cycle consists [in recombinant expression and purification of LwaCas13a.](https://benchling.com/s/prt-lW0HOn03GkNNXTEjbHtW/edit?m=slm-0rgmYEEICIvMxdb5dbty)

  Moreover, we did [PCR](https://benchling.com/s/prt-gurFJqOXoDNEjhMVgCTn/edit?m=slm-AsElllnLKxP9VMTPAjTz) amplify target and guide sequences, [T7 transcription](https://benchling.com/s/prt-F30n0nBTC7tQFUsoiLTC/edit?m=slm-GnHzOpJoAglWTKlENdHs ) to transcribe them
  into
  RNA.

  Both the purification and the PCR - transcription of target and guide sequences worked. We had all
  the material to perform our first paper-based test using [Milenia Hybrideted](https://www.milenia-biotec.com/en/product/hybridetect/) strip and reagent.

  ![](https://static.igem.wiki/teams/4473/wiki/engineering-im3.png)

  Figure 3: build process of the first cycle

  ### Test

  The first test we did is the positive control using the synthetic sequences described by Kelner et
  al.
  [**(2)**](#index_references). This first test worked but we wanted to optimize the concentration. Meaning, that we
  tested
  several
  concentrations of Cas13a, guide RNA, target sequences and probes. The idea was to optimize the result
  without throwing a lot of reagents.

  ### Learn

  We were able to draw different conclusions from this first cycle. First, the paper-based test
  provides
  visual information about the presence of the pathogen in water but it is subjective and relies only on
  the analysis of the person performing the test. To better evaluate the response given by this test, we
  decided to use a gel imager to expose our readouts to white light and obtain a RGB image of our test.
  Moreover, we found out that the Cas13a already purified protein does not work.
  Finally, at the end of this first cycle all the concentrations were optimized.
* Cycle 2: “quantify” paper-based test
  results and test it under several conditions with the positive control and with specific target / guide
  sequences

  ### Design

  The first step of this second cycle was to develop a way to analyze the paper-strip images obtained
  after white light exposition. We did that using Image J and python (see figure 3).
  Moreover, with this second cycle, we wanted to realize the paper-based test under several
  conditions. We design experiments to monitor the consequence of temperature, water salinity and
  incubation time under the results.
  Finally, during this second cycle we also tried the paper test with specific target and guide
  sequences.

  We did positive and negative control tests again, used the gel imager and the python code to analyze the results obtained.
  This is our first proof of concept: the positive control works with our Cas13a protein.

  ![](https://static.igem.wiki/teams/4473/wiki/engineering-im4.png)

  Figure 4: SHERLOCK detection on synthetic sequence. PC: positive control (synthetic sequence), NC: negative control without the Cas13a.

  Then, we tested the influence of incubation time, water salinity and temperature of the reaction
  (results).
  Concerning the *V.aestuarianus* target and guides, none of them produced positive results.

  ### Learn

  This cycle approved the way we read the paper-based test results and gave us information about
  the
  test robustness under different conditions: 15 minutes is not enough to obtain a positive test,
  the
  reaction does not work at 4°C, the salinity has an influence on the result.
  However, by using a paper-strip to realize the test, we do not have access to the kinetic of the
  reaction. This is why in the third cycle, we decided to develop a fluorescence assay.
  Moreover, we found out that the design of the guide RNA was incorrect, it will be modified in the
  fourth cycle.
* Cycle 3: Fluorescent assay to monitor
  the kinetic of the reaction

  ### Design

  For this new cycle we designed a fluorescent probe and we decided the protocol of the experiments. We also designed the fluorescence data analysis codepipeline.
  To perform the fluorescence experiment, fluorescent probes were ordered.
  As the latter provides the kinetic of the reaction, we also developed a kinetic model.
  Because we read the fluorescence output using a plate reader, we decided to test several experimental conditions at the same time.

  ### Test

  For the first fluorescent experiment, we tried several probes (see figure 4a and 4b) and target (see
  figure 4c and 4d) concentrations. Moreover, we fitted the data with the model and used it to have
  access to the unspecific catalytic constant.

  ![](https://static.igem.wiki/teams/4473/wiki/engineering-im5.png)

  ![](https://static.igem.wiki/teams/4473/wiki/engineering-im6.png)

  Figure 4: A. Kinetic of the reaction with probe dilutions. B. End-point of the reaction with probe dilutions. C. Kinetic of the reaction with target dilutions. D. End-point of the reaction with target dilutions.

  What we learn from this cycle is that the efficiency of the detection technique strongly depends on the probe and target concentration. 2µM of probe and as low as 10nMof target are enough to obtain positive results. Moreover, the model allowed us to have a better understanding of the Cas13a kinetic.
* Cycle 4: paper-test and fluorescence
  assay with new guide sequences

  ### Design

  As we found out an error in the design of the *v.aestuarianus* guide RNAs, we designed and re ordered
  them again.

  ### Build

  We did PCR to amplify the new guide sequences, T7 transcription to transcribe them into RNA.

  ### Test

  We tested both the paper-based test and the fluorescence assay on all the target sequences. Moreover,
  we modified the model to obtain the best one and fitted all the data with it. A remarkable result is
  the one of the detection of the toxR gene (see figure 5).

  ![](https://static.igem.wiki/teams/4473/wiki/fig5-engineering.png)

  Figure 5: Shell’lock is able to detect *V.aestuarianus* gene using both readouts methods
  A. Lateral flow detection of ToxR sequence B. Fluorescent readout of ToxR sequences with decreasing concentration.

  ### Learn

  We have a proof of concept. We are able to detect *vibrio aestuarianus* genes using both fluorescence
  assay and paper-based test.

### Conclusion

***Designing a project from scratch is complex and sometimes slow. Careful design is key, especially when time is limited. The learning part is extremely important to correct our mistakes, better understand our system and optimize it. And repeating the cycle is time consuming but necessary to bring the project to maturity. Here we detailed the process that we followed to achieve success.***

#### References

1. A.Benner S, Sismour AM. Synthetic biology. 2005 Jul; Available from: [10.1038/nrg1637](https://doi.org/10.1038/nrg1637)

2. J.Kellner M, Koob J, S.Gootenberg J, O.Abudayyeh O, Zhang F. SHERLOCK: Nucleic acid detection with CRISPR nucleases. 2020 Mar; Available from:
[10.1038/s41596-019-0210-2](https://doi.org/10.1038/s41596-019-0210-2)

{% endblock %}

{% 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 %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

The end goal of our experimental work was to be able to use the Sherlock technique to detect target genes. For
that we devised four main experiments to arrive at our desired outcome:

* Cas13 production and purification
* Guide and target RNA amplification and transcription
* Fluorescence based Shell’Lock
* Paper-based Shell’Lock

### Cas13 production and purification

Cas13 production was adapted from a protocol published by kellner et al (1)
Cas13, which encompasses four divergent family members (Cas13a–d), is an RNA-guided RNase that produces multiple
cleavage sites in single-stranded areas of an RNA target with specific base preferences(1). An essential part of
our work was to be able to purify this protein. The plasmid pC013 (containing LwCas13a) were shipped by Addgene
in stab culture format (Rosetta).

![](https://static.igem.wiki/teams/4473/wiki/perspective-im2.png)

*Fig1: pC013 plasmid containing the cas13 gene and the his-tag*

1.4 L of cultures were grown to an OD600 ~0.6 in TB-media  [(Cas13a purification protocol)](#faq)
containing 100 µg/mL
of ampicillin and induced with 0,5mM IPTG for 16 hours at 21°C. Next, the culture was harvested by spinning down
at 5000 rpm for 15 minutes at 4°C, resuspended in a lysis buffer (40ml Tris-HCl (pH 8.0, 1M), 200ml NaCl (5M)
and 2ml DTT (1M)), homogenized and sonicated.

The plasmid that we used contained two tags, a SUMO tag, Sumo tag is most frequently used as N-end fusion
sequence in yeast to increase the expression and solubility of the desired recombinant protein and an hist
tag(2),and his-tag.Expressed His-tagged proteins can be purified and detected easily because the string of
histidine residues binds to several types of immobilized metal ions, including nickel, cobalt and copper, under
specific buffer conditions (3). we decided to purify the cas13 using the hist tag without cleaving the SUMO tag.
And so we developed our own protocol for this purification. [( Cas13a Purification protocol)
.](#faq)

We purified this protein first using a histidin trap.
In brief we loaded 10mL of our lysate on the histidin-trap column that was pre-equilibrated with a buffer (20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.4) We then eluted the protein with a buffer containing Imidazole. We applied several concentrations in a stepwise manner . Our column is equipped with a spectrophotometer to follow UV absorption of eluted proteins. We collected the fractions containing our protein and we ran an SDS-PAGE to check the purity and size of our protein. The next step was to run a dialysis to get rid of the Imidazole. We finally performed a size exclusion chromatography to get rid of remaining contaminants.

![](https://static.igem.wiki/teams/4473/wiki/perspective-im6.png)

*Fig 2: the first lane displays a sample of lysed bacteria before his-tag purification. The second lane displays the flow-through form the His-Trap. Lanes 3 to 5 show the different elution fractions. There is a clear band at 140 kDa , which is the mass of our protein.*

We collected the fraction from our SEC and ran an SDS-PAGE to view our protein.

![](https://static.igem.wiki/teams/4473/wiki/perspective-im4.png)

*Fig 3: the first lane displays a sample before injection into the SEc (BI). Lanes 3 to 5 show the different elution fractions. There is a clear band at 140 kDa correlating with the mass of our protein.*

The last step was concentrating our protein and achieving a concentration of 2 mg/mL of protein. Then our
protein was stored at -80°C.
In the end to validate the enzymatic activity of our protein we relied on aSHERLOCK test that we performed with a synthetic RNA target that was used in the paper by Kellner et al (1). We also performed a negative control that does not contain the enzyme. Thus we confirmed that our enzyme is active (Fig.4).

![](https://static.igem.wiki/teams/4473/wiki/perspective-im5new.png)

*Fig 4:lateral flow assay graph showing a positive detection compared to a negative control not containing the enzyme.*

## Protocols

* Cas13a Purification Protocol

#### References

1. Kellner, Max J., et al. "SHERLOCK: nucleic acid detection with CRISPR nucleases." Nature protocols 14.10 (2019): 2986-3012 [https://doi.org/10.1038/s41596-019-0210-2](https://www.nature.com/articles/s41596-019-0210-2)

[2. https://www.sinobiological.com/resource/protein-review/sumo-tag-purification#:~:text=Sumo%20tag%20is%20most%20frequently,amino%20acid%20sequence%20of%20ubiquitin.](https://doi.org/10.1038/s41596-019-0210-2)

 [3.https://www.thermofisher.com/fr/fr/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/his-tagged-proteins-production-purification.html](https://www.thermofisher.com/fr/fr/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/his-tagged-proteins-production-purification.html)

{% endblock %}