In Vivo Screening of Artificial Small RNAs for Silencing Endogenous Genes in Escherichia coli

  • Vandana Sharma
  • Yohei Yokobayashi
Part of the Methods in Molecular Biology book series (MIMB, volume 1073)


Bacterial noncoding small RNAs (sRNAs) modulate expression of numerous genes through antisense interactions with mRNAs. This chapter describes an in vivo screening strategy to engineer artificial sRNAs that can posttranscriptionally regulate desired endogenous genes in Escherichia coli. Artificial sRNA libraries are constructed by randomizing the antisense domain of natural sRNAs and screened for gene silencing activity using a cotransformed reporter vector. These small synthetic riboregulators can be used in synthetic gene circuits to control cell functions by directly targeting endogenous genes.

Key words

Noncoding RNA Small RNA Gene regulation Gene silencing RNA engineering Translational regulation 


  1. 1.
    Mizuno T, Chou MY, Inouye M (1984) A unique mechanism regulating gene expression: translational inhibition by a complementary RNA transcript (micRNA). Proc Natl Acad Sci U S A 81:1966–1970CrossRefGoogle Scholar
  2. 2.
    Gottesman S, Storz G (2011) Bacterial small RNA regulators: versatile roles and rapidly evolving variations. Cold Spring Harbor Perspect Biol. doi: 10.1101/cshperspect.a003798 Google Scholar
  3. 3.
    Kawamoto H, Koide Y, Morita T, Aiba H (2006) Basepairing requirement for RNA silencing by a bacterial small RNA and acceleration of duplex formation by Hfq. Mol Microbiol 61:1013–1022CrossRefGoogle Scholar
  4. 4.
    Schmidt M, Zheng P, Delihas N (1995) Secondary structures of Escherichia coli antisense micF RNA, the 50-end of the target ompF mRNA, and the RNA/RNA duplex. Biochemistry 34:3621–3631CrossRefGoogle Scholar
  5. 5.
    Møller T, Franch T, Udesen C et al (2002) Spot 42 RNA mediates discoordinate expression of the E. coli galactose operon. Genes Dev 16:1696–1706CrossRefGoogle Scholar
  6. 6.
    Sharma V, Yamamura A, Yokobayashi Y (2012) Engineering artificial small RNAs for conditional gene silencing in Escherichia coli. ACS Synth Biol 1:6–13CrossRefGoogle Scholar
  7. 7.
    Smith C, Heyne S, Richter AS et al (2010) Freiburg RNA tools: a web server integrating INTARNA, EXPARNA and LOCARNA. Nucleic Acids Res 38:W373–W377CrossRefGoogle Scholar
  8. 8.
    Pedelacq JD, Cabantous S, Tran T et al (2006) Engineering and characterization of a superfolder green fluorescent protein. Nat Biotechnol 24:79–88CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2013

Authors and Affiliations

  • Vandana Sharma
    • 1
  • Yohei Yokobayashi
    • 1
  1. 1.Department of Biomedical EngineeringUniversity of CaliforniaDavisUSA

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