Contribution

Parts

Our team added primers used in recombinase polymerase amplification reactions. They include primers for detecting the four main bovine respiratory disease pathogens, as well as our proof-of-concept E. coli strains. Additionally, since we plan to use RPA, we attempted to produce our own components in-house instead of purchasing to cut down on production costs.

Name	Type	Description	Source
BBa_K5092000	Primer	Histophilus somni Hs_0116 forward	GenBank: CP000947.1 Conrad et al 2020
BBa_K5092001	Primer	Histophilus somni Hs_0116 reverse	GenBank: CP000947.1 Conrad et al 2020
BBa_K5092002	Primer	Mannheimia haemolytica NmaA forward	GenBank: NC_021082.1 Conrad et al 2020
BBa_K5092003	Primer	Mannheimia haemolytica NmaA reverse	GenBank: NC_021082.1 Conrad et al 2020
BBa_K5092004	Primer	Mycoplasma bovis UvrC forward	GenBank: AF003959.1 Conrad et al 2020
BBa_K5092005	Primer	Mycoplasma bovis UvrC reverse	GenBank: AF003959.1 Conrad et al 2020
BBa_K5092006	Primer	Pasteurella Multocida Kmt1 forward	GenBank: FJ986389.1 Conrad et al 2020
BBa_K5092007	Primer	Pasteurella Multocida Kmt1 reverse	GenBank: FJ986389.1 Conrad et al 2020
BBa_K5092008	Primer	E. coli DH5ɑ AsPink forward	BBa_K1033933
BBa_K5092009	Primer	E. coli DH5ɑ AsPink reverse	BBa_K1033933
BBa_K5092010	Primer	E. coli DH5ɑ GFP forward	BBa_I20270
BBa_K5092011	Primer	E. coli DH5ɑ GFP reverse	BBa_I20270
BBa_K5092012	Coding	UvsX-H6	BBa_J435367 Patchsung et al CRISPR J. 2022 Nov 11. doi: 10.1089/crispr.2022.0048.
BBa_K5092013	Coding	Bsu polymerase	Patchsung et al CRISPR J. 2022 Nov 11. doi: 10.1089/crispr.2022.0048.
BBa_K5092014	Coding	UvsY	BBa_J435357 Patchsung et al CRISPR J. 2022 Nov 11. doi: 10.1089/crispr.2022.0048.
BBa_K5092015	Coding	gp32	BBa_J435388 Patchsung et al CRISPR J. 2022 Nov 11. doi: 10.1089/crispr.2022.0048.

Conrad, C. C., Daher, R. K., Stanford, K., Amoako, K. K., Boissinot, M., Bergeron, M. G., Alexander, T., Cook, S., Ralston, B., Zaheer, R., Niu, Y. D., & McAllister, T. (2020). A sensitive and accurate recombinase polymerase amplification assay for detection of the primary bacterial pathogens causing bovine respiratory disease. Frontiers in Veterinary Science, 7, 208. https://doi.org/10.3389/fvets.2020.00208

Patchsung, M., Homchan, A., Aphicho, K., Suraritdechachai, S., Wanitchanon, T., Pattama, A., Sappakhaw, K., Meesawat, P., Wongsatit, T., Athipanyasilp, A., Jantarug, K., Athipanyasilp, N., Buahom, J., Visanpattanasin, S., Niljianskul, N., Chaiyen, P., Tinikul, R., Wichukchinda, N., Mahasirimongkol, S., ... Uttamapinant, C. (2023). A multiplexed Cas13-based assay with point-of-care attributes for simultaneous COVID-19 diagnosis and variant surveillance. CRISPR Journal, 6(2), 99–115. https://doi.org/10.1089/crispr.2022.0048

DNA Detection using HNB

Hydroxynapthol blue (HNB) has been envisioned as a useful colourimetric tool for DNA amplification detection. However, it is important to note that there are two preparations of HNB available - disodium salt and trisodium salt. The two versions are not interchangeable and do not function in the same manner to detect DNA. Future iGEM teams should be careful when using this chemical for DNA detection.

Be aware!

The disodium salt does not change colour. When dissolved the disodium salt is already a brilliant blue colour. It does not change colour during DNA synthesis making this form of HNB useless for DNA detection.

The trisodium salt is opaque and purple when dissolved. This form of HNB will change from purple to blue when magnesium is precipitated during DNA synthesis.

Unfortunately, obtaining the trisodium salt can prove to be rather tricky. It is especially misleading when researching and papers that have used HNB as a DNA detection mechanism did not discuss or give details on how they prepared the HNB solution.

Therefore, we implore future iGEM teams to be cautious when utilizing HNB as a DNA detection method for LAMP or RPA as it may not be the right formulation.

Hardware

We have designed a 3D printed handheld device that can perform the necessary heating steps to carry out a recombinase polymerase amplification (RPA) reaction. Our hardware will significantly reduce the amount of time it takes to perform an RPA reaction by having an all encompassing tool, especially for detecting bovine respiratory disease (BRD) pathogens. The device utilizes an Arduino system, meaning the specific temperatures can be adjusted if needed for different primers. Because of this specificity, future iGEM teams can use this heating device for their own RPA reactions. More information about our design can be found on our engineering and hardware pages.

Overall, we hope that this design is a helpful contribution to future iGEM teams and the industry.

Laboratory Protocols

This year, we have developed protocols to efficiently prepare samples to perform RPA reactions. The traditional preparation method for DNA collected from nasopharyngeal swabs of cattle is using commercial DNA extraction kits. Based on a study from Priti, Jangra et al. (2021), we believe this step to be unnecessary and a heating step will suffice for sample preparation. To find more information about our specific protocols, please visit our wet laboratory page (link).

References

Priti, Jangra, S., Baranwal, V.K. et al. A rapid field-based assay using recombinase polymerase amplification for identification of Thrips palmi, a vector of tospoviruses. J Pest Sci 94, 219–229 (2021). https://doi.org/10.1007/s10340-020-01284-w

Community Outreach

Our team has participated in events throughout the year to facilitate community outreach and public reducation.

Tech Futures Challenge competition
Farmers market
Destination Summer Camps
Arts Showcase
Fleetwood-Bawden Grade 1 class visits

High School iGEM Symposium

We organized an informal symposium to learn more about other high school iGEM projects. We invited Outaouais, IEA and Khan Lab School to participate. Ms Emily Hicks, co-founder of FREDSense and former iGEMer to give the keynote address and former iGEM participants Justin Vigar, Emily Hagens, and Jessica Semmelrock were asked to be judges. This was a great way to learn from our peers and get to know each other before we all travel to Paris for the Jamboree.