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Team:EPFL/Detection

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Detecting
Pathogenic DNA

Abstract

What to do with our amplified bacterial DNA? And how to make a signal for humans to understand?

An underlying problem in synthetic biology remains the limited number of parts for constructing genetic circuits and difficulties that arise when integrating multiple components into a large, complex synthetic network. Unlike electronic circuit elements, which can be insulated from each other, biological components can interact with one another in the complex cellular environment and suffer from unwanted interactions.

We solved this problem by using newly designed toehold switches with low system crosstalk to regulate the expression of catechol 2,3-dioxygenase (CDO). Since we want our diagnostic to be field-deployable, we're using a colorimetric signal (CDO) instead of a fluorescent one (GFP) because it's easier to detect. In the end, the presence of pathogenic DNA will produce a yellow color in our OnePot PURE cell-free system that can be visually detected in under 2 hours.

Principle

A toehold switch is a small strand of RNA that regulates the expression of a downstream gene. It is composed of four main parts:

- Trigger binding region
- Ribosome binding site
- Start codon
- Downstream gene

Detailed toehold sensor design schematic and putative detection mechanism, Green et al., 2014


The hairpin structure sequesters the Ribosome Binding Site (RBS) and start codon making it impossible for the ribosome to bind to the mRNA and transcribe the protein. A second strand of mRNA, complementary to the Switch region needs to be present to activate the toehold. It will bind to the switch region and linearize the hairpin structure, freeing the RBS for the ribosomes to bind.

For our project, we created 3 different toehold switches to detect each target:
- Flavescence Dorée
- Bois Noir
- The endogenous plant DNA used as a control

The generation of a colorimetric signal is the final result of the test, as it will inform the person running it which diseases are present in the leaf. To have a clear signal we decided to induce a colour change instead of expressing a fluorescent protein such as GFP. Fluorescence can be difficult to observe out of the lab relative to colour changes. To do so, we chose to express the enzyme catechol-2,3-dioxygenase (CDO). It cleaves the substrate catechol into a colourful product, which can be observed as a change of colour from transparent to yellow, visible by the naked eye.

Chemical CDO colorimetric reaction, M. Verosloff et al. 2019


Our choice to use CDO as a signalling protein was motivated by its usage in the literature for a similar application to our project. Also since it is a small protein of about 300 amino acids, it requires less energy from the cell-free transcription and translation system, which increases its efficiency.

Before settling for CDO, we also considered using another signalling protein, β-galactosidase (β-gal). β-gal is quite a large protein, with a little more than 1000 amino acids, as well as being a tetramer. This makes it more difficult to express into a cell-free system as it requires a lot of energy. One way to solve this problem is to use alpha complementation: the gene coding for β-gal (LacZ) can be separated into two parts, a small one lacZalpha and a large one LacZomega.

We had the idea to express and purify LacZomega and to add it to the OnePot PURE proteins, this would result to a OnePot Pure system containing the omega part of β-gal, which is not functional on its own. To be able to create a signal, LacZalpha needs to be expressed into this cell free system. It will complement the omega part and create a functional β-gal protein which can cleave a substrate to create a color. This method is called split β-gal as the enzyme is split into two of its subunits.

In the end, we did not use β-gal because the OnePot PURE system is known to be contaminated with β-gal, which is problematic if we want to create an accurate and precise test.

Methods

Toehold design

We designed our first generation of toeholds based on the Green et al. 2014 paper. As the trigger mRNA will be the amplicon from our RPA product (approx. 70 bp), the trigger binding region is designed to contain the complimentary nucleotides. As described in the paper, one of the optimal sizes of the trigger binding region is 36 nts. Based on that number, we designed a library of approximately 35 toeholds for each disease and control. In order to keep the hairpin structure stable when it is deactivated, we calculated its Normalized Ensemble Defect (NED, The average percentage of nucleotides that are incorrectly paired at equilibrium relative to the specified secondary structure, evaluated over the Boltzmann-weighted ensemble of (unpseudoknotted) secondary structures (0% is best, 100% is worst)) using the NUPACK software suite. All the toeholds were then evaluated using the following equation provided by the paper and ranked accordingly (0 is best, 100 is worst):

where ϕ is the design score for the sensor at location i of the mRNA.
- lmRNA is the local single-strandedness (calculated as NED) of the mRNA at the sensor binding site.
- ltoehold is the local single-strandedness of the toehold of the sensor.
- nsensor is the NED of the sensor.
- The score weight factors used were β1 = 5, β2 = 4, and β3 = 3.
Only the top 4 toeholds were selected and carried on to the next step.

Toehold assembly

In order to monitor the kinetics of our detection using toehold regulation, we chose sfGFP as the reporter gene and therefore connected our toeholds to an sfGFP sequence. We ordered forward primers for sfGFP, each containing a toehold as overhang and a common reverse primer. To assemble it we simply performed a PCR using the iGEM distributed sfGFP plasmid as template. You can check the protocol here.
To check if the products matched our desired sequence, we performed a gel electrophoresis followed by Sanger sequencing to confirm the results.

Functionality test

We have a higher sfGFP expression rate than our reference set result, but we still got a low leakage at the OFF state.
The functionality test was then performed in both OnePot PURE and the NEB commercial PURExpress system, using ssDNA ordered from IDT as triggers to limit undesired factors (RNase contamination, DNA transcription, etc...). For each test, we ran a 5 μl reaction in a 384-well-plate, then recorded the GFP absorbance in a microplate reader at 37°C for over 2 hours. You can check the protocol here.

CDO expression

CDO was expressed in OnePot PURE with 1mM of catechol and incubated at 37°C. You can check our protocol here.
To be sure that the change of color was the result of the cleavage of catechol by CDO we made two controls. The first was to express CDO into OnePot PURE without catechol, the second was to add catechol without CDO.

Results

A functional toehold riboregulator has two main characteristics:
1) High or distinguishable protein expression in its ON state
2) Zero or very little protein leakage in its OFF state
At the end of our experiment, we managed to create a sfGFP toehold system, containing three toeholds with low crosstalk which prove our concept of using toehold switches to genetically distinguish between Flavescence Dorée and Bois Noir.


Difference between two triggers we've used

In our project, we've used two kind of triggers. For the ssDNA trigger, there is no need of T7 polymerase transcription, therefore eliminate some uncertainties. But in the real case, we will use dsDNA as trigger, thus it is essential to compare their expression rates (BN 2.1 Toehold):

The error bar of the expression rate overlaps, no significant difference is detected.


Detection limits

In order to test the limit of detection of our toehold, we've run a test for toehold expression in different concentration of trigger DNA, by theory in the detectable range the difference should be bigger when the concentration of trigger increases:

There is a detectable difference from 100nM, and it grows when the concentration goes up, which suits our theory.


Colorimetric signal

A yellow color can be seen after 30 minutes of incubation, and it becomes brighter one hour after the start of the reaction. There are no colors in both controls, this proves that the color is indeed created by the reaction of CDO with catechol and not by self-oxidation of catechol.

Conclusion

In the end, we designed and constructed novel low-crosstalk toehold switches to detect our three targets: Flavescence Dorée, Bois Noir, and the endogenous grapevine. The trigger for these will be the product from our previous RPA reaction. We also demonstrated, as a proof-of-concept, that our toehold for Bois Noir produced a clear expression in our OnePot PURE system with minimal leakage, thus ensuring its usage as a reliable riboregulator of CDO.

We decided to use a colorimetric signal as it produces an easier readout. The enzyme CDO can be used to generate this signal. With catechol as a substrate, it creates a bright yellow color after one hour of incubation. We also considered using other signalling systems, such as β-gal and split β-gal, but due to the limitations of these systems with our cell free system, we chose to focus on CDO.

References

Toehold Switches: De-Novo-Designed Regulators of Gene Expression, Green et al., 2014 [link to paper]

NUPCK online DNA structure modelling [link to website]

BioBitsTM Explorer: A modular synthetic biology education kit, Huang et al., 2018 [link to paper]