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| - | ===Small RNA-regulated system=== | + | ===Device=== |
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| | ====Introduction==== | | ====Introduction==== |
| - | <p>Base pairing offers a powerful way for one RNA to control the activity of another. Both prokaryotes and eukaryotes have many cases which a single-stranded RNA base pairs with a complementary region of an mRNA. As a result, it prevents expression of the mRNA.</p>
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| - | <p>'''RNA interference (RNAi)''', also called post transcriptional gene silencing, is a process in which RNA molecules inhibit gene expression by destroying specific mRNA molecules. In 2006, Andrew Fire and Craig C.Mello shared the Nobel Prize in Physiology or Medicine because of their study on RNA interference<sup>11</sup>. This powerful gene silencing tool in eukaryotes has been used in many research. As their counterparts in bacteria, '''small non-coding RNAs (sRNAs)''' are important regulatory roles.</p>
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| - | <p>Small RNAs (sRNAs) have become increasingly significant in playing the role of bacterial gene regulation. Most sRNAs interact with the targeted mRNAs by imperfect base pairing, reducing the translation efficiency and recruiting chaperones such as Hfq for translation termination. The sRNAs would bind to the target with its hairpin-like structure which wound by some of it's own sequences, and the chaperons would stuck between the hairpins in order to protect the sRNA-mRNA combination from degrading. In the end, the sRNAs regulate gene expression by forestalling translation. Since this regulated-system has already been employed in vivo, it is necessary to design an artificial sRNA that targets specifically to a desired genes, so that we can prevent the sRNA from affecting other undesired genes.</p>
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| - | <p>Unlike the regulated-system popular used in iGEM projects (e.g. P<sub>tet</sub> & ''tetR'', P<sub>lux</sub> & ''LuxR'', etc.), sRNA regulated-system seems '''more efficient'''. Because the inhibition works under RNA level, which means the energy wasted in ''E. coli'' is less than other system (producing proteins to regulate).</p>
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| - | Figure 8 depicts how the small RNA chaperone, Hfq, associates with the small RNA and represses a target mRNA.<sup>12</sup>
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| - | [[File:NCTU_sRNA_Introduction.png|center|600px|Figure 8. <br>Hfq and a small RNA may sequester the ribosome-binding site of a target mRNA, thus blocking binding of the 30S and 50S ribosomal subunits and repressing translation.]]
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| | ====Mechanism==== | | ====Mechanism==== |
| - | <p>How do small RNAs regulate gene expression?
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| - | Figure 9 shows the mechanism of repressing one target mRNA.
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| - | The small RNA has three stem-loop double stranded RNA structures,
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| - | and the loop which is closest to the 3’ terminus is complementary
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| - | to the sequence preceding the initiation codon of the target mRNA.
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| - | Base pairing between small RNA and the target mRNA prevents the ribosome
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| - | from binding to the initiation codon, so the translation would be repressed.<sup>13</sup><sup>,14</sup></p>
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| - | [[File:NCTU FORMOSA WEB WIKI sRNA inhibition.JPG|center|600px|Figure 9. <br>sRNA-Hfq complex specifically binds on target gene, forming a blockade of ribosome binding. Therefore, the translation is inhibited.]]
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| | ====Design==== | | ====Design==== |
| - | The following is the process of our idea in designing the sRNA.
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| - | [[File:NCTU_sRNA_Design.png|900px|center|Figure 10. Design process.]]
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| - | We have designed two artificial sRNAs, each modified from different paper. The sRNA-1 is based on afsRNA ARlacZ1, which is created by Hongmarn Park and his colleagues.<sup>15</sup> ''SibC'' is a small RNA sequence which is already found in ''E.coli''. Hongmarn Park and his colleagues changed few base pairs of ''SibC'' to make ARlacZ1, purposed to make the secondary structure more stable. After that, they tried to change 9 different locations of the target-recognition sequence to test the effect on gene silencing. Our design is to modify from the RNA sequence which has the best efficiency : ARlacZ7. In accordance with afsRNA ARlacZ7, we changed the target-recognition sequence to finally complete our sRNA-1.
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| - | [[File:Nctu_formosa_srna_design.png|600px|center|Figure 11. Secondary structure models of SibC and afsRNA ARlacZ1. The replaced bases in ARlacZ1 are boxed. From ''Hongmarn Park, Geunu Bak, Sun Chang Kim & Younghoon Lee.(2013). "Exploring sRNA-mediated gene silencing mechanisms using artificial small RNAs derived from a natural RNA scaffold in Escherichia coli". Nucleic Acids Research,Vol. 41, No. 6, 3787-3804 DOI:10.1093'']]
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| - | The other sRNA sequence we produced is called sRNA-2. It is employed in this project selected from a library of artificial sRNA that was constructed by fusing a randomized antisense domain of Spot42, the scaffold that is known for recruiting the RNA chaperons. The sRNA we picked contains a consensus sequence, 5’-CCCUC-3’, which can base pair with the SD sequence due to complementary binding. This sRNA, as expected, effectively regulates gene expression by reducing translation efficiency and recruiting Hfq. In addition, this sRNA shows high specificity against its targeted gene, ''OmpF'', as it doesn't hold significant activity against other genes from ''E. coli'' genome (Sharma and others, 2011).
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| - | [[File:Nctu_srna2design.png#file|center|1000px|Figure 12. Artificial sRNA based on the Spot42 sRNA scaffold (yellow box). The bases (red) is
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| - | target-recognition region from Vandana Sharma, Asami Yamamura & Yohei Yokobayashi. (2011) . "Engineering Artificial Small RNAs for Conditional Gene Silencing in E. coli". ACS Synthetic Biology.]]
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| - | The sRNA picked is competent, but we hoped that it can target any desired gene. Therefore, we designed a RBS by employing the sRNA targeting region from ''OmpF'' and making the AUG codon sufficiently apart from the SD sequence for ribosome binding. By adding this RBS to the upstream of any desired gene, the gene can be regulated by sRNA.
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| - | [[File:NCTU_sRNA2.jpg|center|400 px|Figure 13. The secondary structure of the sRNA-2 we designed]]
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