Abstract
Knockdown or silencing of a specific gene presents a powerful strategy for elucidating gene function in a variety of organisms. To date, efficient silencing methods have been established in eukaryotes, but not bacteria. In this chapter, an efficient and versatile gene silencing method using artificial small RNA (afsRNA) is described. For this purpose, target-recognizing sequences were introduced in specially designed RNA scaffolds to exist as single-stranded stretches in afsRNA. The translation initiation region of target genes was used as the sequence for afsRNA recognition, based on the theory that this site is usually highly accessible to ribosomes, and therefore, possibly, afsRNA.
Two genes transcribed as monocistrons were tested with our protocol. Both genes were effectively silenced by their cognate afsRNAs.
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Acknowledgments
This study was supported by the Intelligent Synthetic Biology Center of Global Frontier Project (2012M3A6A8054837) and the National Research Foundation of Korea (NRF) Grant (2010-0029167, 2011-0020322) by the Korean Government (MSIP), and the KRIBB initiative programs.
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Bak, G., Choi, J.S., Kim, W., Suk, S., Lee, Y. (2015). An Effective Method for Specific Gene Silencing in Escherichia coli Using Artificial Small RNA. In: Ponchon, L. (eds) RNA Scaffolds. Methods in Molecular Biology, vol 1316. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2730-2_17
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DOI: https://doi.org/10.1007/978-1-4939-2730-2_17
Publisher Name: Humana Press, New York, NY
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