Difference between revisions of "Team:Lethbridge/RNAi"
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| − | + | In order to generate a Herpes Delta Virus thermozyme1, we first constructed a library of variants to be utilized in systematic evolution of ligands by exponential enrichment (SELEX). To generate our library we incorporated random nucleotides into the U1A-RBD stem loop with either N9, N10, N11 or N12 random nucleotides for a total of 1.7 X 108 potential combinations. All of these variants were then assembled into the HDVR through oligo assembly followed by overlap extension PCR. Upstream of the HDVR there is a MS2 binding domain and a T7 promoter with an XbaI cut site at the 3’ end and a SpeI cut site at the 5’ end. A second module was used in order to generate an MS2 variant that will no longer dimerize and assembly into a viral capsid (BBa_K2109108). This was accomplished by using a characterized MS2 variant (V291-d1FG)2 with an N-terminal HIS tag, controlled through a T7 promoter and strong RBS followed by a double terminator (B0015). The MS2 will be overexpressed and used to saturate a Nickel Sepharose column so that the Thermozyme library can be applied to select for positive mutants. Two HDVR controls were generated (BBa_K2109109, BBa_K2109110) the first is a U1A mutant which was used as a negative control. The second HDVR mutant U1A-C75T is a negative control that can be induced to cleave in the presence of imidazole1. te (10 ng per PCR in a 50 uL volume) | |
| + | Clone into pJET and/or pSB1C3 for submission to iGEM parts repository.</p> | ||
| − | + | <p>References: </p><p> | |
| − | + | 1. Ke, A., Zhou, K., Ding, F., Jamie, H.D., Doudna, J.A. (2004). A Conformational Switch Controls Hepatitis Delta Virus Ribozyme Catalysis. RNA, 13: 1384-1389.</p> | |
| − | + | 2. Batey, R.T., Kieft, J.S. (2007). Improved Native Affinity Purification of RNA. RNA, 429: 201-205.</p> | |
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Revision as of 02:29, 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
In order to generate a Herpes Delta Virus thermozyme1, we first constructed a library of variants to be utilized in systematic evolution of ligands by exponential enrichment (SELEX). To generate our library we incorporated random nucleotides into the U1A-RBD stem loop with either N9, N10, N11 or N12 random nucleotides for a total of 1.7 X 108 potential combinations. All of these variants were then assembled into the HDVR through oligo assembly followed by overlap extension PCR. Upstream of the HDVR there is a MS2 binding domain and a T7 promoter with an XbaI cut site at the 3’ end and a SpeI cut site at the 5’ end. A second module was used in order to generate an MS2 variant that will no longer dimerize and assembly into a viral capsid (BBa_K2109108). This was accomplished by using a characterized MS2 variant (V291-d1FG)2 with an N-terminal HIS tag, controlled through a T7 promoter and strong RBS followed by a double terminator (B0015). The MS2 will be overexpressed and used to saturate a Nickel Sepharose column so that the Thermozyme library can be applied to select for positive mutants. Two HDVR controls were generated (BBa_K2109109, BBa_K2109110) the first is a U1A mutant which was used as a negative control. The second HDVR mutant U1A-C75T is a negative control that can be induced to cleave in the presence of imidazole1. te (10 ng per PCR in a 50 uL volume) Clone into pJET and/or pSB1C3 for submission to iGEM parts repository.
References:
1. Ke, A., Zhou, K., Ding, F., Jamie, H.D., Doudna, J.A. (2004). A Conformational Switch Controls Hepatitis Delta Virus Ribozyme Catalysis. RNA, 13: 1384-1389.
2. Batey, R.T., Kieft, J.S. (2007). Improved Native Affinity Purification of RNA. RNA, 429: 201-205.
