iGEM 2024 — Team lethbridgehs

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## Wet lab

The lab book for our project can be found [here](https://static.igem.wiki/teams/5092/wetlab-notebook-summer-24.pdf).

## Hardware

Steps for creating our heating device can be found [here](https://2024.igem.wiki/lethbridgehs/hardware).

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## First Iteration

To facilitate the in-field testing, we are engineering a portable unit to provide the BRD test results. Vets or farmers would take a deep nasopharyngeal swab of the cattle. Nasal swabs would be added to a tube with a prepared extraction buffer solution. This tube would then be heated to 100℃ for 10 minutes. The sample would then be transferred to the detection tubes. Each detection tube contains a different set of primers for pathogen detection. The RPA reaction will be allowed to start upon the addition of the prepared sample DNA mixture, then incubated at 37℃ for 30 minutes. Finally, the amplified DNA will be detected through a simple colour change due to hydroxynaphthol blue (HNB).

The first iteration of the device was one rectangular box design that had the extraction and detection steps occurring in close proximity. A 12V adaptor powered the heating element used for the detection and extraction steps, and a separate temperature controller was used to regulate heat appliances. Similar to the current device, there is one slot where a microcentrifuge tube will be placed for the extraction portion of the amplification. Next to this slot are five smaller slots for microcentrifuge tubes containing the primers required for RPA to occur. The fifth microcentrifuge tube is a control. We proposed a clear acrylic lining to enclose the device's elements. This decision was made solely because of the aesthetic properties of acrylic materials.

![Device1](https://static.igem.wiki/teams/5092/engineering1.png)

## Second Iteration

![Device2](https://static.igem.wiki/teams/5092/engineering2.png)

We revised the device's design after speaking to Amino Labs and participating in the 2024 TFC competition. Our second iteration is our minimal viable product using items obtained from Amazon. We worked with U of L tech services to drill holes in two aluminum blocks to create heating elements for the microcentrifuge tubes. Dr Chad Povey from the University of Lethbridge physics department helped with putting together the correct circuit for our first prototype. Our initial test was successful; the heating element did heat the aluminum holder. While our prototype does indeed achieve our goal of heating the tubes to the correct temperature, we wanted to make the design more streamlined and easy to use. It was quite heavy and bulky, so we worked on scaling down the current design. A Raspberry Pi was proposed to control the heating cycles needed for RPA to occur.

![Device3](https://static.igem.wiki/teams/5092/engineering3.png)
![Device4](https://static.igem.wiki/teams/5092/engineering4.png)

## Current Iteration

![Device5](https://static.igem.wiki/teams/5092/engineering5.png)

In our current iteration, we envision two separate heating elements enclosed in one rectangular block made of chitosan. Chitosan is an autoclavable, biodegradable polymer that can withstand temperatures up to 200℃. It is renewable and economically viable. Previously, we proposed an acrylic lining to shelter the device's components. After further discussion, we realized this would not work due to the low melting point of chitosan and its environmental dangers. The current device contains the same microcentrifuge placement as the first design, with the addition of an LCD 1602 screen and six buttons for various uses. An Arduino controls all the device's functions, unlike the previous design which used a temperature controller and a proposed Raspberry Pi. The heating elements/portions of the device are entirely separate from the rest of the internals. The heating blocks will now be insulated to prevent heat damage to other device parts. While the colour change of HNB can be easily observed with the naked eye, we anticipate creating a colour sensor that will be incorporated into our hand-held device. The sensor would have a colour detection threshold; when reached, it will send a message to the user’s phone.

![Device5](https://static.igem.wiki/teams/5092/device3.png)
Please contact us for our downloadable design!
More information on the electronic components can be found on our [hardware page](https://2024.igem.wiki/lethbridgehs/hardware).

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## Design

Since the aim of our project was to create an easy-to-use on-site test for BRD, we didn’t want to use traditional DNA amplification methods like PCR. Conventional PCR requires expensive and relatively large thermocyclers. Isothermal methods, like LAMP or RPA, don’t require this equipment.

We chose to use RPA or recombinase polymerase amplification as our detection method. This method is easier to use than other isothermal techniques, since it only requires two primers (Daher et al., 2016). Simply, RPA components are combined with target DNA. Recombinase proteins bind to the primers and help to scan the DNA for complementary sequences (Tan et al., 2022). Primers anneal to the target DNA and single-stranded DNA binding proteins then stabilize the displaced DNA strand, preventing the ejection of the annealed primer. DNA polymerase can then extend the primer using the target DNA as a template. No temperature changes are required and the whole process can take place at temperatures ranging from 20 to 42°C. RPA is an affordable, simple, and fast method for DNA detection (Sumit Jangra et al., 2021; Magrina Lobato & O'Sullivan, 2018).

![RPA1](https://static.igem.wiki/teams/5092/rpa-rxn1.png)
![RPA2](https://static.igem.wiki/teams/5092/rpa-rxn2.png)

For our first proof-of-concept experiments, we utilized the TwistDx (Cambridge, UK) TwistAmp Liquid Basic kit. This kit provides all the RPA components in easy to use liquid form and costs $300 CAD for 100 reactions.

The amplified DNA from RPA reactions are usually detected using agarose gel electrophoresis. However, we wanted to investigate the use of hydroxynaphthol blue (HNB) as a colourimetric detection method. This chemical is a metal-sensing dye that changes colour in the presence (or absence) of magnesium ions. During DNA amplification, DNA synthesis generates inorganic pyrophosphate (PPi) as a byproduct. PPi in turns binds to Mg2+ ions and precipitates out of solution as Mg2P2O7. As magnesium is pulled out of solution, it can no longer interact with HNB and the colour changes from violet to sky blue. However, it is important that the correct form of HNB is used. Both the disodium and trisodium salts are available for purchase, but it is only the trisodium version that can under the correct colour change.

![HNBmech](https://static.igem.wiki/teams/5092/hnbmech.png)

In practice, Bo-Find would require vets or farmers to take a deep nasopharyngeal swab of the cattle. DNA from the collected samples is traditionally isolated using a commercial kit, such as DNeasy Blood & Tissue Kit or QIAmp PowerFecal DNA Kit, both from Qiagen. However, we proposed that may be unnecessary and a simple heat treatment step could be used instead. The swab would be placed in a microfuge tube with a PBS buffer. The tube would then be heated to 100°C for 10 minutes. A defined volume would then be transferred to the prepared detection tubes. Each detection tube would contain the RPA components and a different set of primers for pathogen detection or control reactions. Our Bo-Find device would then incubate the tubes at 37°C for 30 minutes and a colour change due to DNA synthesis would occur in any positive samples.

![device_overview](https://static.igem.wiki/teams/5092/device-overview.png)

While superior in theory, the kit’s design will need to be tested and improved for practical use. The device’s current design allows for testing of only one cow at a time. This can make Bo-Find unfeasibly slow if large-scale detection is required, so we plan to find a way to improve the design to allow for faster testing of multiple animals at once. To overcome these potential problems we can consider developing a larger testing device that will be able to hold more rows on test, thus allowing for less waste while still testing the same amount of cattle. In feedlots cattle are usually divided into multiple pens. In these pens they are in close proximity to each other. Because of this, they develop very similar microbiomes within around 14 days from initial arrival at the feedlot. Consequently, only one to three cows would need to be tested per pen, depending on the size of the pen. This would allow for use of less tests, creating less product waste and less time for farmers or researchers to test each cow.

## Proof-of-Concept

Since we cannot use actual BRD pathogens in the lab, we used readily available E. coli lab strains containing plasmids that coded for GFP and asPink. Typically cell lysis methods include mechanical or chemical disruption of the cell membrane. Our first goal was to establish that we could detect DNA from these cells using a simple boiling step. DNA was detected using both PCR and RPA to validate our approach. Next, these strains were also used to validate DNA detection by HNB.

![POC](https://static.igem.wiki/teams/5092/pocmech.png)

## References

Daher, R. K., Stewart, G., Boissinot, M., & Bergeron, M. (2016). Recombinase Polymerase Amplification for Diagnostic Applications - PMC. *Clinical Chemistry, 26*(7), 947-958. Retrieved April 22, 2024, from <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7108464/>

Magrina Lobato, I., & O'Sullivan, C. K. (2018). Recombinase polymerase amplification: Basics, applications and recent advances. *Trends in Analytical Chemistry, 98*, 19-35.

Tan, M., Liao, C., Liang, L., Yi, X., Zhou, Z., & Wei, G. (2022). Recent advances in recombinase polymerase amplification: Principle, advantages, disadvantages and applications. *Frontiers in Cellular and Infection Microbiology, 12*.

Sumit Jangra, P., Baranwal, V. K., Dietzgen, R. G., & Ghosh, A. (2021). A rapid field‑based assay using recombinase polymerase amplification for identification of *Thrips palmi*, a vector of tospoviruses. *Journal of Pest Science, 94*, 219-229.

## Protocols

All experimental protocols can be found [here](https://static.igem.wiki/teams/5092/igemhs-protocols2024.pdf).

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## Getting the work done!

Most of our lab work was completed by Mercy Awosoga this summer as part of the High School Youth Researcher Summer program at the U of L. This is a six week intensive research program for students in grade 11. They are paired with a lab at the U of L to work on a research project during the summer. Mercy was supervised by our Faculty Advisor Dr Laura Keffer-Wilkes. She was able to complete a lot of wet lab work as well as a lot of the hardware engineering. Many thanks to Mercy for all her hard work!

![MercyLab](https://static.igem.wiki/teams/5092/mercylab.png)

## RPA vs PCR Detection of Purified Plasmid

We confirmed that the primers we designed for RPA work as intended using purified plasmid DNA. The primers should produce products of 427 bp and 585 bp, respectively from the asPink and GFP DNA templates. (Figure 1).

![PlasmidMaps](https://static.igem.wiki/teams/5092/plasmidmaps.png)

Figure 1. Plasmid maps of pSB1C3-asPink (left) and pSB1C3-GFP (right) used as template DNA in our proof-of-concept reactions. Primer annealing regions are shown in purple.

We were successful in producing the right size product for both asPink and GFP templates using RPA but did not amplify asPink using PCR. Overall, our primers were successfully used in RPA with a purified plasmid template (Figure 2).

![PCRvsRPAgel](https://static.igem.wiki/teams/5092/rpavspcr.png)

Figure 2. Analysis of PCR and RPA proof-of-concept reactions using 1% agarose gel.

## DNA Extraction using Boiling Technique

The two proof-of-concept cell strains carrying pSB1C3-GFP and pSB1C3-asPink were grown in LB overnight. Cells were collected by centrifugation and then resuspended in fresh LB. Into a new tube containing PBS, 1 OD of each cell strain was added. This was repeated three times for a total of four sample tubes each containing an equal number of cell strains. The tubes were then subjected to heating at 100C for 0, 2, 10, or 20 minutes. A 100 uL sample was removed from the boiled tubes and plated on LB plates and grown overnight (figure 3). No growth was seen on plates with samples that had been boiled for 10 or 20 minutes but some growth was observed at the 2 minute time point allowing for quantification of the different cell strains, which was approximately 50:50. A lawn of bacterial growth was observed on the 0 minute boiling time point, which was expected.

![GrowthPlates](https://static.igem.wiki/teams/5092/growthplates.png)

Figure 3. An aliquot of the boiled cell solution was plated on LB and grown overnight. Growth was only observed on the no boil and 2 min boil plates.

Next, the boiled solutions were used as the DNA template for RPA reactions. This would demonstrate that enough DNA is present following the boiling step for DNA detection in the field. We were able to detect the GFP plasmid even with no boiling, indicating that perhaps during our preparation steps some of the cells are bursting without the need to boil. We were also able to detect purified templates as low as 2.5 ng.

![GFPboilRPA](https://static.igem.wiki/teams/5092/gfpgelboil.png)

Figure 4. The pSB1C3-GFP plasmid was able to be detected in all samples, even at 0 min boiled.

Unfortunately, we had more trouble being able to detect the asPink plasmid in the boiled cell samples (below). We have to do some troubleshooting for this detection step.

![asPinkboilRPA](https://static.igem.wiki/teams/5092/aspinkboil.png)

Figure 5. The pSB1C3-asPink plasmid was only detected in the 10 and 20 minute boiled samples. No DNA amplification was observed for any of the BRD pathogen DNA.

We also need to troubleshoot detection of the BRD pathogen DNA. Dr Tim McAllister and Cheyenne Conrad, both from the Lethbridge Agriculture and Agri-Food Canada Research Station, provided us with purified DNA from the four main BRD pathogens, Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis. This allowed us to test our RPA pipeline but still remain safe. Unfortunately, we were unable to detect the DNA using our primers and RPA components (figure 5 lanes 6 - 9). Using a higher percentage agarose gel may help with resolution for detection.

## Hardware and HNB Testing

We were able to test our hardware prototype in the lab. It was able to heat the RPA reactions to 37℃ (figure 6)

![Hardwareinlab](https://static.igem.wiki/teams/5092/hardwareinlab.png)

Figure 6. Bo-Find prototype heating RPA samples.

Interestingly, when we used HNB as our detection method, we observed a colour change despite not being able to detect a band on the gel for the asPink sample (figure 7).

![HNBvsRPAgel](https://static.igem.wiki/teams/5092/hnbvsgel.png)

Figure 7. Analysis of RPA proof-of-concept reactions using 1% agarose gel (right). HNB was also used to detect DNA amplification (left).

## Summary

We were able to successfully use RPA to detect GFP and asPink plasmids from purified DNA but more troubleshooting is needed for confirmation and accuracy. DNA extraction via boiling demonstrated that DNA could be detected in as little as 2 minutes, meaning no elaborate extraction method is needed in the field. BRD pathogen DNA detection was unsuccessful with current RPA components, therefore more fine tuning needs to be completed before field deployment, Despite the correct colour change being observed with HNB, no band on the gel was observed indicating a need for further optimization. The hardware prototype effectively maintained the RPA reaction temperature at 37°C.

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