Contribution to RNA Mango Ⅱ Biosensor
          Previous iGEM teams have characterized RNA mango (BBa_K3001024), however this construct contained an unused sn34 RNA sequence that was unnecessary for the function of RNA Mango. Therefore, we removed the sn34 RNA sequence and created the part BBa_K4351010 of RNA Mango Ⅱ. With the modeling of aptamers and wet lab fluorescence spectrophotometer biosensor tests by our team this year, further modeling of RNA mango and characteristics with adjacent biosensors has also been documented. For our system, we needed to test the fluorescence of RNA mango along with our glucose and insulin aptamers for our proof of concept (https://2022.igem.wiki/lethbridge-hs/proof-of-concept.html). Our results provided updated information on RNA mango in different RNA biosensor systems such as the ability to act as a downstream reporter. RNA mango alone has well-researched fluorogenic properties with its ligand of thiazole orange, but its use alongside other RNA aptamers is less documented. The results of measuring endpoint fluorescence levels of RNA mango in the presence and absence of trigger molecules showed that our aptamers for glucose and scrambled sequence bound to glucose. Only certain conditions seemed to allow our glucose aptamer to achieve its purpose. The results page under the project of our wiki explains the condition in full, but these results were obtained by our application and refashioning of RNA mango.

          Furthermore, the real-time transcription rate of RNA mango was observed through a Quanta Master 60 fluorescence spectrophotometer. By recording fluorescence intensity when the template was added, then every 10 minutes for an hour we detected the increase, maximum, and leveling off of the intensity. The graphed intensity over time and wavelength can be found in our results and lab notebook. Documenting this information for RNA mango provides useful data for future experiments involving this aptamer.

          As previously mentioned, our team did the modeling of our aptamers. Secondary and tertiary structures of each aptamer with and without RNA mango were recorded. Computer software was used to predict the conformation of these RNA strands, and by adding RNA mango, our team contributed to additional records of the predicted structure of RNA mango with other biosensors. Again, RNA mango’s relationship and interaction with other aptamers such as our glucose aptamers advance community knowledge of this specific aptamer.


 
Self-amplifying messenger RNA (sa-mRNA)
         This is the first iGEM project to utilize sa-mRNA sequences. This technology was reported as early as 1997 for use in gene delivery (Herweijer and Wolff, 1997). In 2019 there was a boom in sa-mRNA being researched for vaccination. Dia-Beatable builds on these concepts by using it to create a protein replacement therapy. Specifically, a treatment for diabetes. Only a few labs are currently publishing on advancements in sa-mRNA technology for protein replacement - the most influential for our team being the labs of Dr. Niek Sanders at UGhent and Dr. Ron Weiss at MIT.

          Our interview with Dr. Blakney revealed that there is a lot of potential for development in the sa-mRNA realm of research (see interviews under human practices). There are still new self-amplifying sequences to be found and optimized for safety, bioavailability, and immunogenicity; VEEV sequences are used for convenience sake, they are not necessarily the best sequences available. There are new control mechanisms to build for both transcription and translation. There are also delivery mechanisms and applications to be explored. 

          We hope that by introducing sa-mRNA to iGEM, teams will now be able to use and improve upon these self-amplifying RNA parts in the future. We hope that we have also included enough information about dual use research on concern to ensure future teams navigate this topic with the utmost respect for safety.


High School iGEM in North America
          We started a podcast aimed at high school students interested in being part of an iGEM team. High school teams are still rare in North America. We are currently the only Canadian High School team. It would be great to see more participation!


References 
[1] Herweijer, H.; Wolff, J. A. Self-amplifying vectors for gene delivery. Adv. Drug Delivery Rev. 1997, 27 (1), 5– 16,  DOI: 10.1016/S0169-409X(97)00018-5