This part collection is generated for plant pathogen detection. It houses our toehold switch library including toehold switches capable of detecting a large number of different plant pathogens. The toehold switches can be assembled into a sensor plasmid by combining them with a promoter, a protein coding sequence and a terminator.The assembled sensor construct can be used for detecting the presence of the target pathogen in a sample. The part collection also contains fourteen ready-made sensor plasmids that can be used in the detection of barley yellow dwarf virus (BYDV). The toehold switches are exchangeable between the sensor plasmids and this collection serves as a demonstration of the global impact of CropFold.
Being a fairly new detection method, toehold switches are not yet that widely used. Especially for plant pathogen detection, only a few toehold switch-based methods exist and, for the pathogens we are using, there is nothing similar to our toehold switches in the iGEM registry. Therefore, we believe that our toehold switch collection is novel, important, and useful for development of plant disease detection in the future.
Plant infections can rarely be detected visually in the early phases of the plant-pathogen interaction, which necessitates the creation of powerful detection kits. We wanted to create a universal cell-free detection system for plant pathogens. The detection uses toehold switch-based sensing of nucleic acids of pathogenic origin. Toehold switches are engineered 5’ UTR modules of mRNA that regulate the translation of a downstream protein coding sequence. The toehold switch allows the translation to occur only in the presence of a specific trigger sequence, which in our system is an RNA sequence either found in or derived from a pathogen’s genome.
Toehold switches need to be assembled into sensor plasmids to create a functional product. As the toehold is the basis of detection in the system, the system can be tailored to target a specific pathogen by exchanging the toehold switch. Our modular assembly, leveraging the benefits of Golden Gate assembly, allows for easy modification of the sensor to facilitate universal pathogen detection. To showcase the global impact of the project, we created a toehold switch library including toehold switches for various relevant plant pathogens (see more on design). These parts make up our Part Collection.
Our part collection contains 73 parts, 14 of which are ready toehold sensor plasmids for detecting BYDV and other 59 are toehold switches designed to detect 12 different pathogens. The collection is divided into smaller subcollections, each containing multiple toehold switch designs for the detection of one target pathogen. The toehold switches were designed using our design algorithm and ranked based on our model (see dry lab and model). The best-ranking toehold switches were selected for this collection to provide an easy selection of the best designs. The scores predicted by our model, as well as visualizations of the minimum free energy structures of these toehold switches, are provided for easy estimation of their performance.
We included multiple designs for each pathogen, as our model cannot predict the performances of the toehold switches with absolute certainty, which, in turn, necessitates experimental verification of functionality. Our part collection contains multiple designs for barley yellow dwarf virus (BYDV) as we used it to demonstrate the viability of the detection system. The designs BBa_K4207002-BBa_K4207007 were designed by us at the beginning of the project, whereas parts BBa_K4207008-BBa_K4207013 were designed by our partner team TAU (more about the partnership can be read on our partnership page). Parts BBa_K4207061-BBa_K4207074 include ready-made sensor plasmids for BYDV detection that were assembled in our lab. As we refined our design algorithms during the year, all other toehold switches were designed using an optimized version of the algorithm and therefore are expected to exhibit exceeding performance compared to earlier iterations. The collection contains toehold switches for 12 pathogens including BYDV. These are listed below in Table 1.
(Barley Yellow Dwarf Virus)
|A70 by TrigGate||BBa_K4207012|
|A92 by TrigGate||BBa_K4207013|
|A95 by TrigGate||BBa_K4207014|
|B39 by TrigGate||BBa_K4207015|
|B46 by TrigGate||BBa_K4207016|
|B69 by TrigGate||BBa_K4207017|
(Cucumber Green Mottle Mosaic Virus)
(Papaya Mosaic Virus)
(Potato Virus Y)
(Tomato Brown Rugose Fruit Virus)
(Tomato Chlorosis Virus)
(Wheat Dwarf Virus)
(Pepper Mild Mottle Virus)
(Plum Pox Virus D)
(Soybean Dwarf Virus)
(Sweet Potato Leaf Curl Virus)
(Tobacco Mosaic Virus)