Quantifying and Modeling Compositional Context Effects on Synthetic Biocircuits in E. coli

Enoch Yeungâ??1 , Andrew Ng2 , Jongmin Kim3 , Zachary Z. Sun4 , Mustafa Khammash5 , and Richard M. Murray1,4*

1 Computing and Mathematical Sciences, California Institute of Technology 2 Bioengineering & University of Washington at St. Louis, 3 Wyss Institute, Harvard University 4 Biology and Biological Engineering, California Institute of Technology 5 Biosystems Science and Engineering, ETH-Zurich

â?? To whom correspondence should be addressed. E-mail: eyeung@caltech.edu

In the past two decades, our ability to rapidly de- sign and implement novel synthetic biocircuits has increased dramatically. Nonetheless, our power to assemble these biocircuits into large-scale genetic programs to implement useful functions is limited by the lack of modularity, or robustness, in existing biocircuit modules. In general, there are three main sources of failure in engineering more complex bio- circuits: compositional context, host context, and environmental context 1 . In this work we discuss how compositional context, i.e. the spatial arrange- ment, spacing, and orientation of biological parts, can affect gene expression in a biocircuit. We in- troduce a simple biocircuit comprised of two genes - each expressing an mRNA aptamer (mSpinach 2 and MG aptamer 3 ) and show that by varying the rela- tive orientation of the two genes, we achieve sig- nificantly different gene expression. We show that by increasing the relative spacing between genes, we can decrease the differences in gene expression, effectively insulating against compositional context effects. We then discuss how compositional context introduces intermediate supercoiling between genes and derive a novel mathematical modeling frame- work that recapitulates the trends seen in our ex- perimental data. We perform time-lapse single cell microscopy experiments to show that by modulating compositional context, it is possible to affect the am- plitude and duration of burstiness in gene transcrip- tion. Our experimental and modeling results cor- roborate recent work 4 , showing that burstiness is a function of supercoiling state and more importantly, that compositional context can be used to modulate the properties of pulsatile gene expression. Addi- tionally, we demonstrate that compositional context effects can be used to enforce a more fundamen- tal form of (positive and negative) feedback, with time-scales much faster than those achievable by protein-mediated feedback. We show that the de- sign choice of gene orientation and spacing can be used to implement simple logic such as XOR logic and dual positive feedback. Finally, we demonstrate
our findings using variants of the genetic toggle switch 5 , showing how compositional context design choices can be used to improve latching of the tog- gle switch.

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