Team:Lethbridge/Project Production

• iGEM
• RNAiCare
• Project
  • Description
  • Design
  • Results
  • Production
  • Judging
• Parts
  • Basic Parts
  • Composite Parts
  • Measurement
• Practices
  • Risks
  • Stakeholders
  • Current Problems
• Notebook
  • July
  • August
  • September
• Team
  • Members
  • Collaborations
  • Attributions
  • Sponsors

Project Production

dsRNA Production:

Our strategy for dsRNA production is a multi-part approach. The construct is expressed dicistronically, with a His-tagged MS2 coat-protein expressed initially. Additionally, we employ the use of a hammerhead ribozyme fused to a theophylline aptamer, termed an aptazyme (Win, M.N., Smolke C.D. 2007). Upon addition of theophylline, the aptazyme undergoes a conformational change, resulting in cleavage at a specifically prescribed nucleotide. This theophylline aptazyme is placed just upstream of an MS2 coat-protein binding site. This allows us to purify only full-length RNA once it has been transcribed, as those transcripts not harbouring the binding domain will not be effectively purified. His-tagged MS2 coat-protein is then free to bind the newly transcribed binding domain. Using affinity chromatography, purification of the MS2 coat-protein and the bound RNA is possible. Upon addition of theophylline, cleavage and liberation of a single stranded RNA occurs, allowing for purification of a single strand of highly pure RNA.

By using two complementary RNA-generating sequences within the aptazyme construct, we are able to generate double stranded RNA for use in pest control simply by annealing the two resultant strands produced by our purification strategy.

Given our chassis and ability to over-express His-tagged MS2 coat-protein, our purification strategy is poised to purify large amounts of highly specific RNA. The scalability of this platform lies in the ability of individuals to design novel pesticides for any target organism, having only requisite knowledge of the genome. New dsRNA-based pesticides will be employed cheaply and specifically without costly design and massive amounts of resources currently utilized in the development of novel pesticides. Pesticides represent a multi-billion dollar industry worldwide, and with the scalability of this synthetic biology mode of production, this project represents a readily commercializable method of producing large quantities of highly specific pesticides applicable to a wide array of pest species.

For large scale production, our group will likely use fermenters for sufficient growth. However, as demonstrated, the concentrations of dsRNA required for gene silencing are sufficiently low that the large-scale production need not necessitate massive amounts of resources.