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Ribozyme-based insulator parts buffer synthetic circuits from genetic context.

Chunbo Lou1, Brynne Stanton, Ying-Ja Chen

  • 1Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

Nature Biotechnology
|October 5, 2012
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Summary

Synthetic biology circuits can be reliably connected using insulator parts, like ribozymes, to buffer sequence effects. This enables predictable gene expression control and the construction of complex synthetic genetic programs.

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Area of Science:

  • Synthetic biology
  • Genetic engineering
  • Molecular biology

Background:

  • Synthetic genetic programs rely on layered transcriptional circuits for temporal control of gene expression.
  • Connecting these circuits involves linking output promoters to input promoters, which can introduce sequence-dependent context effects.
  • These context effects can alter the input-output response (transfer function) of the circuits, hindering predictable behavior.

Purpose of the Study:

  • To identify and characterize sequence elements that buffer context effects between connected synthetic gene circuits.
  • To improve the reliability and predictability of layered synthetic genetic programs.
  • To develop a method for reliably permuting circuits for diverse genetic program construction.

Main Methods:

  • Screening a library of putative 'insulator parts' in Escherichia coli.
  • Evaluating the effect of insulators on the transfer functions of synthetic gene circuits.
  • Utilizing ribozymes that cleave the 5' untranslated region (5'-UTR) of mRNA as effective insulators.
  • Developing a mathematical model to predict the behavior of layered circuits incorporating insulators.

Main Results:

  • Ribozymes acting as insulators were identified as effective in buffering sequence context effects.
  • Insulators generated quantitatively identical transfer functions, independent of the input promoter sequence.
  • The behavior of layered synthetic gene circuits connected by insulators could be accurately predicted using a mathematical model.

Conclusions:

  • Insulator parts, particularly ribozymes, are critical for reliable connection of synthetic gene circuits.
  • The use of insulators ensures predictable circuit behavior, irrespective of promoter identity.
  • Insulators are essential for reliably permuting circuits to construct complex synthetic genetic programs.