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In Vivo and In Vitro Characterization of σ70 Constitutive Promoters by Real-Time PCR and Fluorescent Measurements

  • James Chappell
  • Paul Freemont
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1073)

Abstract

The characterization of DNA regulatory elements such as ribosome binding sites and transcriptional promoters is a fundamental aim of synthetic biology. Characterization of such DNA regulatory elements by monitoring the synthesis of fluorescent proteins is a commonly used technique to resolve the relative or absolute strengths. These measurements can be used in combination with mathematical models and computer simulation to rapidly assess performance of DNA regulatory elements both in isolation and in combination, to assist predictable and efficient engineering of complex novel biological devices and systems.

Here we describe the construction and relative characterization of Escherichia coli (E. coli) σ70 transcriptional promoters by monitoring the synthesis of green fluorescent protein (GFP) both in vivo in E. coli and in vitro in a E. coli cell-free transcription and translation reaction.

Key words

Synthetic biology σ70 transcriptional promoters DNA regulatory elements characterization Relative promoter units Cell-free transcription and translation reaction green fluorescent protein and real-time PCR 

References

  1. 1.
    Endy D (2005) Foundations for engineering biology. Nature 438:449–453CrossRefGoogle Scholar
  2. 2.
    Arkin A (2008) Setting the standard in synthetic biology. Nat Biotechnol 26:771–774CrossRefGoogle Scholar
  3. 3.
    Martin L, Che A, Endy D (2009) Gemini, a bifunctional enzymatic and fluorescent reporter of gene expression. PLoS One 4:e7569CrossRefGoogle Scholar
  4. 4.
    Canton B, Labno A, Endy D (2008) Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26:787–793CrossRefGoogle Scholar
  5. 5.
    Kelly JR, Rubin AJ et al (2009) Measuring the activity of BioBrick promoters using an in vivo reference standard. J Biol Eng 3:4CrossRefGoogle Scholar
  6. 6.
    Goldman SR, Ebright RH, Nickels BE (2009) Direct detection of abortive RNA transcripts in vivo. Science 324:927–928CrossRefGoogle Scholar
  7. 7.
    Patwardhan RP, Lee C et al (2009) High-resolution analysis of DNA regulatory elements by synthetic saturation mutagenesis. Nat Biotechnol 27:1173–1175CrossRefGoogle Scholar
  8. 8.
    Chappell J, Jensen K, Freemont PS (2013) Validation of an entirely in vitro approach for rapid prototyping of DNA regulatory elements for synthetic biology. Nucleic Acids Res 41:3471–3483CrossRefGoogle Scholar
  9. 9.
    Gulati S, Rouilly V et al (2009) Opportunities for microfluidic technologies in synthetic biology. J R Soc Interface 6(Suppl 4):S493–S506CrossRefGoogle Scholar
  10. 10.
    Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3:1101–1108CrossRefGoogle Scholar
  11. 11.
    Kim TW, Keum JW et al (2006) Simple procedures for the construction of a robust and cost-effective cell-free protein synthesis system. J Biotechnol 126:554–561CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2013

Authors and Affiliations

  • James Chappell
    • 1
  • Paul Freemont
    • 1
  1. 1.Centre for Synthetic Biology and InnovationImperial College LondonLondonUK

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