Difference between revisions of "Team:Lethbridge/Project Production"

 
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                             <li><a href="https://2015.igem.org/Team:Lethbridge/Notebook_September">September</a></li>
 
                             <li><a href="https://2015.igem.org/Team:Lethbridge/Notebook_September">September</a></li>
 
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                        <a href="https://2015.igem.org/Team:Lethbridge/Software">Software</a>
 
 
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                     <h2>dsRNA Production:</h2>
 
                     <h2>dsRNA Production:</h2>
  
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                    <img src=“Uleth15_constructschematic.jpg”>
 
  
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                    <p>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, and 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.</p>
 
  
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                     <p>Uleth15_purificationstrategy.jpg</p>
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                     <p>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.</p>
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                    <img src="https://static.igem.org/mediawiki/2015/6/63/Uleth_RNAPureStrat.png" width=50%" height="50%">
  
 
                     <p>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.</p>
 
                     <p>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.</p>
  
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                    <h2>RNA Interference:</h2>
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                     <p>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.</p>
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                     <p>RNA interference is a gene regulatory phenomenon employed by eukaryotic organisms in which double stranded RNA (dsRNA) is employed to down-regulate expression of a given gene. In this process, a cellular enzyme called Dicer cleaves double stranded RNA to generate small interfering RNA (siRNA). Once cleaved, one strand of this siRNA is incorporated into the RNA Induced Silencing Complex (RISC)/</p>
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                    <img src=“Uleth15_RISCloaded.jpg”>
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                    <p>This strand of RNA is then able to base-pair to a complementary region of cellular mRNA, and the argonaute activity of RISC cleaves the mRNA. This targets the mRNA for further nuclease breakdown within the cellular environment, ensuring it will not be used as a translation template.</p>
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                    <img src=“Uleth15_RNAiInPractice.jpg”>
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                    <p>Dicer cuts essentially randomly within a long double stranded RNA, resulting in a wide variety of siRNA species generated. For specific silencing of a given mRNA, this situation is not ideal. We chose to circumvent this issue by generating highly specific siRNAs capable of silencing only one mRNA within the cell.</p>
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                    <h2>dsRNA Target Desgin:</h2>
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                    <p>We chose to target methylenetetrahydrofolate reductase 1 (MET1) as a study by Frandsen et al. showed that knockout mutants of these gene generate a phenotypic mutant that is easily recognizable. In wild type fusarium you see a red pigment produced in the Fusarium and in the MET 12 and MET 13 knockout mutants you see a loss of this red pigment. Five dsRNA targets were picked for this gene one just down stream of the start codon of the gene (Target Sequence (TS) 1), two upstream of the stop codon (TS 2 and 3) and two in the 5' untranslated region (TS 4 and 5).</p>
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                    <img src=“Uleth15_MET1.JPG”>
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                    <p>Some design features that were taken into account when designing dsRNA targets 1 through 5 were Core GC content varying from 36% to 50% among the 5 sequences. The core Gc content variation can affect how difficult it is for the RNA induced Silencing Complex (RISC) to dissociate the two RNA strands.</p>
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                    <img src=“Uleth15_dsRNA1.jpg”>
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                    <p>Asymmetrical End stability differences varying from 3.48 to -2.29, this value represents GC content differences in the two ends of the dsRNA. We selected two RNA targets with a postive value (TS 5 and 2), two RNA targets with a negative value (TS 3 and 1)  and one with a value close to 0 (TS 4). We varied the asymmetrical end stability differences as it is currently unclear in the literature as to how this value will affect the selection of the guide RNA strand.</p>
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                    <img src=“Uleth15_dsRNA2.jpg”>
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                     <p>The length of dsRNA was also varied between 26 to 29 base pairs however the majority of the targets were kept at 27 base pairs in length. As this closely mimics a DICER by product and therefore should not be cleaved further by DICER within the cell. Our final design consideration was to analyze our selected sequences in order to make an initial estimate of potential off target effects. If you would like to learn more about this analysis please visit the Policy and Practices portion of our wiki.</p>
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                     <p>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.</p>
  
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                    <img src=“Uleth15_TS1.jpg>
 
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                    <img src=“Uleth15_TS2.jpg>
 
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                    <img src=“Uleth15_TS3.jpg>
 
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                    <img src=“Uleth15_TS4.jpg>
 
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                    <img src=“Uleth15_TS5.jpg>
 
  
 
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Latest revision as of 00:34, 19 September 2015

iGEM

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.