Difference between revisions of "Team:Munich/Design"

We started our iGEM journey with brainstorming sessions resulting in many great ideas. The best of these ideas were framed into written project proposals by teams of two to five students and, after a constructive peer-review process, presented to the whole team. The final two ideas were subjected to SWAT-analysis, after which we settled on CascAID, our CRISPR-based diagnostic device for point-of-care testing.

Our project has both an experimental and an applied side and we quickly realized that our construct was quite complex, consisting of many pieces that had to work together. We therefore split our project into parts that could be investigated seperately. First, the infectious pathogens had to be lysed and their target sequences amplified to yield an amount of target RNA that is detectable by Cas13a. Then, Cas13a had to be purified and characterized in the lab. Next, we had to figure out how to make the signal visible to the user. Finally, everything had to be framed into a portable and user-friendly format, by designing appropriate hardware.

We therefore formed sub-teams who focused their work on these modules. A high degree of modularization requires a high level of cooperativity between the sub-teams, but also provides an increased flexibility in design and better possibilities for customization. Another advantage is that many ideas can be developed and tested in parallel, which speeds up the develpment process.

RNA Extraction and Amplification

The goal of this module is to obtain a detectable amount of target RNA from a sample of E. coli cells, which we used as a dummy target. The extraction method should be distributive, efficient and not require the use of hazardous chemicals. Therefore, the first step was to test common lysis techniques differing in the required equipment and use of chemicals. We compared their efficiencies by agarose gel electrophoresis.

The standard approach for RNA extraction, Guanidinium-Phenol-Chloroform extraction, using chaotropic salts as a lytic agent, requires purification of the lysis product and involves use of toxic chemicals. Another commonly used lysis process: incubation at high temperature with detergents such as SDS, gave good results, but again, requires separation of SDS from the RNA afterwards. For RNA purification purposes we investigated silica based procedures, as a less harmful solution. However, a point-of-care device has to be robust and reliable. In this regard, a simple design is considered superior to complex ones. We therefore abandoned chemical lysis methods that require purification and chose a combination of heat lysis followed by isothermal PCR.

Recombinase Polymerase Amplification (RPA), as an isothermal alternative to PCR, is conducted at 37°C without the need for thermocycling and therefore reduces the requirements and costs of the accompanying hardware significantly. Since Cas13a targets RNA rather than DNA, we coupled RPA to in-vitro transcription (TX). Since both reactions take place at same temperature of 37 °C, this can be done in a one-pot reaction. To render the RPA-TX distributable, we lyophilized the enzymes on filter paper, which, when sealed in a tight container, stabilizes the assay for storage.

Cas13a, Purification and Characterization

We chose Cas13a as it, as RNA-guided RNAse, could practically be optimised to detect any sequence of interest, this way allowing customization of device for any pathogen of interest. Also it is highly specific and sensitive, which enables detection even when sequence of interest is only present in traces in sample.

Our plan was to investigate proteins of the Cas13a family from different organisms - Leptotrichia buccalis (Lbu), Leptotrichia shahii (Lsh) and Leptotrichia wadei (Lwa) - and choose the one that performs best. Lbu and Lsh were available at AddGene, while we cloned and codon optimised Lwa ourselves. We therefore used two different E. coli strains for protein expression. We used BL21 Star for optimised plasmid and for not optimised ones, we chose Rosetta strain which is a BL21 derivate that provides tRNAs for codons that are usually not present in E. coli. In order to prevent the large Cas-protein to form inclusion bodies we expressed it together with SUMO (Small Ubiquitin-like Modifier) or MBA (Multiple Banded Antigen) proteins, which force Cas13a to stay in solution. We opted for Ni-NTA purification followed by size exclusion, with the His-tag attached to SUMO/MBA. To determine the purity of protein and see if there are differences between investigated proteins we used SDS-PAGE.

For characterisation of Cas13a-Proteins we used the RNaseAlert assay, a cleavage assay in which Cas13a is activated through the presence of appropriate crRNA and target RNA. We transcribed target RNA in vitro to accelerate the process and then screened for optimum conditions. Using this assay for different target sequences from Noro Virus, Hepatitis C Virus and Bacillus subtilis we investigated the specificity of the Cas13a activity.

Gold nanoparticles: Other than in the other two colorimetric readouts, aeBlue and Intein-Extein, the only protein involved in the gold nanoparticle (AuNP)-readout is Cas13a, like in our RNase Alert readout. This reduces the necessary fine tuning of the biochemical circuit to a minimum, favoring high robustness of the readout. Due to the phenomenon of Localized Surface Plasmon Resonance, AuNPs appear in a distinct color, ranging from intense red to blue, black and colorless. This property depends on particle size, shape, the immediate environment, and -most critical for our purpose- aggregation state.

In our project we use AuNPs with a diameter of roughly 10 nm, giving them a bright red color in solution. Their small size and therefore high surface-to-volume ratio makes them ideal for functionalization with thiolated compounds, forming covalent Au-S bonds. The first step of our concept is to use these properties to functionalize AuNPs with either 5’- or 3’- thiolated DNA and, through addition of linker- RNA which hybridizes with both thiolated DNA strands, form aggregates, changing the color from red to blue. The design of the linker-RNA includes an uracil-rich, single-stranded segment between the DNA-complementary termini, making it prone to Cas13a-mediated promiscuous cleavage.

It has been shown that, for purely DNA-based hybridization, AuNP aggregates can be spotted on filter paper, dried and severed by addition of a nuclease-containing solution, visible through diffusion of red AuNPs on the paper. Thus, the second part of our concept is to spot RNA-linked AuNPs on paper, dry them alongside the Cas13a mixture and detect specific target RNAs and resulting Cas13a activity with a simple change from blue to red.

Hardware and Paper Fluidics

We developed an open source fluorescence detector as a portable solution for fluorescence based readout. With the rise of 3D printing and microcontroller-based DIY electronics, the open source philosophy has spread from software engineering into the world of hardware. By providing detailed documentations and CAD-drawings of our hardware, we want to enable other engineers and hobbyists to improve our designs, democratize access to the system and empower the user to self-educate about the working principle of our hardware.

We chose to perform our detection reaction on filter paper, which enables us to freeze-dry the mixture for long-time stability, and use very small reagent volumes per test. To enable transport of the sample-containing fluid to the areas containing the detection mixture, we used a technique called paper-fluidics. Thereto, we hacked our regular office printer, to pattern the paper with hydrophobic wax. Defined areas were left uncoated in order to form hydrophilic channels that promote fluid transport by capillary forces. This technique enabled us to rapidly prototype and test different channel geometries for optimal flow, with very short iteration cycles of about one hour.

After first tests, we recognized that bleached cellulose paper limits the resolution of fluorescence readout due to its auto-fluorescence. This led us to integrate glass microfiber filter paper into the paper strip in the respective detection areas, which shows little to no auto-fluorescence. Further, we used adhesive tape to seal the paper strip to avoid RNase contamination and keep the glass microfiber filter paper in place.

In a similar way, we prototyped our sample preparation module, consisting of a fluid control unit, a heating system and a fluidic chip. For the fluidic chip we used 3D-printed masters, rather than photo-lithography, to achieve faster prototyping cycles to optimise our design, while being open tolater miniaturization. The heating system is controlled by a microcontroller, and for the fluid control unit we chose to build valves driven with the pressure from a bike tire to direct flow without the need of carrying pressurized air. The design of our hardware components allows their integration in a single portable device.