Difference between revisions of "Team:Lethbridge/RNAi"
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| − | <p class="grey-text text-lighten-4"> | + | <p class="grey-text text-lighten-4">As a part of the design of our project, we looked over the ethical implications of our study. We looked into a variety of different farming methods and uses of pesticides. We examined off-target and non-target effects by ensuring the target sequences we selected were compared against the entire database of sequenced genomes using the NCBI BLAST program. We also spoke with various experts and held a panel discussion regarding the efficacy of our project from the lab to the field. </p> |
| + | <p class="grey-text text-lighten-4">We also contacted major small molecule pesticide distributors to estimate the current cost analysis. Finally, we collaborated with the Canadian Food Inspection Agency (CFIA) and Health Canada to further explore the ethical implications of our project. | ||
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Revision as of 01:55, 20 October 2016
RNAi
Background
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 Herpes Delta Virus Ribozyme (HDVR), which will be evolved through SELEX in order to generate a thermozyme (Win, M.N., Smolke C.D. 2007). Upon an increase in temperature (22 °C to 37°C), the thermozyme undergoes a conformational change, resulting in cleavage at a specifically prescribed nucleotide. This thermozyme 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 a temperature increase, 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 thermozyme 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.
Human Practices
As a part of the design of our project, we looked over the ethical implications of our study. We looked into a variety of different farming methods and uses of pesticides. We examined off-target and non-target effects by ensuring the target sequences we selected were compared against the entire database of sequenced genomes using the NCBI BLAST program. We also spoke with various experts and held a panel discussion regarding the efficacy of our project from the lab to the field.
We also contacted major small molecule pesticide distributors to estimate the current cost analysis. Finally, we collaborated with the Canadian Food Inspection Agency (CFIA) and Health Canada to further explore the ethical implications of our project.
Wet Lab
HDV Ribozyme Library for Selection of Thermozymes General Overview Oligo Assembly Make HDV Library parts Anneal oRAP-X (oRAP-N9 to oRAP-N12) to oRAP-9 Use T4 DNA polymerase to extend oligos Make Promoter and MS2 SL parts Anneal oRAP-8 and oRAP-7 Use T4 DNA polymerase to extend oligos Overlap Extension PCR to join HDV Library and promoter MS2 SL parts Add equimolar amounts of the two extension products to PCR Perform 10 cycles of PCR to extend products (use Phusion) Check on gel (may need to gel extract) SELEX Procedure In vitro transcribe library (degrade DNA) Bind RNA to MS2 beads (Ni-NTA) at room temp (21 deg C) Wash beads several time to remove cleaved products Raise temperature to 37 deg C incubate for 10 min Remove eluate from tube Reverse transcribe using oRAP-6 during RT step PCR amplify RT products using oRAP-5 (fwd) and oRAP-6 (rev) Use overlap extension PCR to add back promoter and MS2 SL with RT-PCR product and Promoter and MS2 SL oligo extension product. Repeat as necessary. PCR amplify positive sequences for cloning Use oRAP-10 and oRAP-11 to PCR amplify successful RT-PCR products Perform few cycles (~20 and no more) Use small amount of template (10 ng per PCR in a 50 uL volume) Clone into pJET and/or pSB1C3 for submission to iGEM parts repository (only the shitty ones). Detailed Experimental Flow
