Team:Lethbridge HS/Parts
CADAR Parts Overview
For this project, we are serving the main purpose of detecting and targeting specific pathogens in the hope to enable physicians to improve the accuracy of their diagnosis and assist more people in their recovery from bacterial infection.
The types of parts included in our project are the Cas13a variants, GFP and Mango crRNA, and purification tags.
Our Cas13a parts Lba, Lbu, Lwa, and Lsh are abbreviations of the organisms from which they were obtained:
- Lachnospiraceae bacterium
- Leptotrichia buccalis
- Leptotrichia wadei
- Leptotrichia shahii
Our crRNA parts were designed based off of Munich 2017’s designs for their parts. We chose to target our RNA Mango II aptamer for our in vitro assays and GFP for our in vivo assays. Please see visit our Demonstrate page to see our final results.
Parts Collection
In our assay, the RNA Mango II RNA aptamer was into a native stem loop of the snoRNA snR30. Our target sequence on our crRNA was designed to target the aptamer sequence itself. While the snR30 RNA Mango II component is not compatible with the BioBrick, the RNA Mango II aptamer itself is. The aptamer should function the same in other RNA molecules as long as it is designed to be within a stem loop. This makes this part collection suitable for future iGEM teams to use for testing the activity of any RNA cleaving enzyme.
Basic Part
All of our new parts can be found on the basic parts page.
Composite Parts
Our CRISPR Cas13a construct contains a T7 promoter, and a medium/strong ribosomal binding site which is required for translation. Following these are our tags required for purification which is then followed by our pure cas13a gene. Finally, we have our terminator.
After a T7 promoter our crRNA construct contains a direct repeat stem-loop (DRSL) which is an RNA structure that is recognized by the Cas13a. Attached to this is an RNA sequence identical to a gene found in the pathogen this helps the Cas13a identify and cut specific sequences. Finally, there is a double terminator.
snR30-RNA Mango II is a fluorescent-tagged RNA with a vibrant orange color due to the incorporation of the dye thiazole orange. This construct begins with a T7 promoter, and then the coding sequence for snR30 with the RNA Mango II aptamer integrated within it. This construct was gifted to us and the lab utilizes run-off transcription.
We are utilizing GFP and RFP parts that are already in the parts registry. Both are under a constitutive promoter. Directly after this, there is a ribosomal binding site, followed by the coding sequence. Finally, there is a single terminator. Here is an alignment of the two DNA sequences that aided us in being able to determine which region of GFP we should target with our crRNA that would not permit cleavage of RFP.
Improved Parts
The parts Lsh and Lwa Cas13a proteins have additional tags for purification, and will be used in in vitro experiments. Additionally, these parts were added in a fully inducible and expressible format with all necessary components for overexpression and purification in one construct. We improved these parts to further diversify the way that other teams can purify the proteins to suit their own projects.