Design and Engineering of Pattern Formation in Gene Expression in Escherichia coli
Justin Hsia
EECS Department, University of California, Berkeley
Technical Report No. UCB/EECS-2016-32
May 1, 2016
http://www2.eecs.berkeley.edu/Pubs/TechRpts/2016/EECS-2016-32.pdf
Synthetic biology aims to develop new biological systems and devices, from the modification of existing pathways to the construction of entirely new genetic circuits. The role of the engineer in synthetic biology is to apply engineering principles to the design and analysis of proposed systems. Building biological systems de novo are the best way to demonstrate our successes on this front and so far has yielded biological devices such as synthetic promoters, toggle switches, oscillators, and logic gates. Here we aim to push the current boundaries of synthetic biology and study the principles behind engineering pattern formation in ensembles of E. coli cells. Motivated by the study of morphogenesis, we hope to develop synthetic systems with the high-minded goals of one day engineering molecular differentiation or even multicellularity. Pattern formation will be a critical part of these goals and brings an additional focus on cell-to-cell communication and signaling molecules. Here we examine two different communication mechanisms, quorum sensing and contact-based signaling, and see what types of patterns we can achieve. Using quorum sensing, we focus on diffusion-driven instability (Turing patterning), where a homogeneous steady state of an ensemble of cells is destabilized in the presence of diffusion. This is made possible by the conflicting interactions of the internal dynamics of the cells and the normalizing effect of diffusion between them. The work in this area thus far has centered around activator-inhibitor network theory and to date has yet to yield a biological experimental demonstration. Here we analyze the Turing mechanism and propose a new network which we call a “quenched oscillator” system and demonstrate its ability to produce diffusion-driven instability. We then propose a synthetic implementation and present work towards a partial implementation. In the process, we use zinc finger proteins (ZFPs) and small RNAs (sRNAs) to construct new synthetic inverters to put together in a ring oscillator for use in a quenched oscillator system. Interest in contact-based signaling has risen recently with the discovery of a contact-dependent inhibition (CDI) system in E. coli. While a synthetic contact-mediated communication channel has not yet been achieved, its realization will provide a huge boost in engineering possibilities, particularly for multicellular applications. Here we develop an analytical framework based on graph theory for analyzing lateral inhibition networks, a category that CDI falls under, for the existence and stability of equitable patterns. Without an actual CDI system to use, we develop what we call a “compartmental lateral inhibition” system using diffusible molecules and engineered communication channels to simulate contact-mediated signaling for verification of our patterning analysis. The current state of our synthetic implementation is presented, highlighting experimental setup details that may prove useful for future applications in engineered multicellular ensembles.
Advisors: Murat Arcak
BibTeX citation:
@phdthesis{Hsia:EECS-2016-32,
Author= {Hsia, Justin},
Title= {Design and Engineering of Pattern Formation in Gene Expression in Escherichia coli},
School= {EECS Department, University of California, Berkeley},
Year= {2016},
Month= {May},
Url= {http://www2.eecs.berkeley.edu/Pubs/TechRpts/2016/EECS-2016-32.html},
Number= {UCB/EECS-2016-32},
Abstract= { Synthetic biology aims to develop new biological systems and devices, from the modification of existing pathways to the construction of entirely new genetic circuits. The role of the engineer in synthetic biology is to apply engineering principles to the design and analysis of proposed systems. Building biological systems de novo are the best way to demonstrate our successes on this front and so far has yielded biological devices such as synthetic promoters, toggle switches, oscillators, and logic gates. Here we aim to push the current boundaries of synthetic biology and study the principles behind engineering pattern formation in ensembles of E. coli cells. Motivated by the study of morphogenesis, we hope to develop synthetic systems with the high-minded goals of one day engineering molecular differentiation or even multicellularity. Pattern formation will be a critical part of these goals and brings an additional focus on cell-to-cell communication and signaling molecules. Here we examine two different communication mechanisms, quorum sensing and contact-based signaling, and see what types of patterns we can achieve.
Using quorum sensing, we focus on diffusion-driven instability (Turing patterning), where a homogeneous steady state of an ensemble of cells is destabilized in the presence of diffusion. This is made possible by the conflicting interactions of the internal dynamics of the cells and the normalizing effect of diffusion between them. The work in this area thus far has centered around activator-inhibitor network theory and to date has yet to yield a biological experimental demonstration. Here we analyze the Turing mechanism and propose a new network which we call a “quenched oscillator” system and demonstrate its ability to produce diffusion-driven instability. We then propose a synthetic implementation and present work towards a partial implementation. In the process, we use zinc finger proteins (ZFPs) and small RNAs (sRNAs) to construct new synthetic inverters to put together in a ring oscillator for use in a quenched oscillator system.
Interest in contact-based signaling has risen recently with the discovery of a contact-dependent inhibition (CDI) system in E. coli. While a synthetic contact-mediated communication channel has not yet been achieved, its realization will provide a huge boost in engineering possibilities, particularly for multicellular applications. Here we develop an analytical framework based on graph theory for analyzing lateral inhibition networks, a category that CDI falls under, for the existence and stability of equitable patterns. Without an actual CDI system to use, we develop what we call a “compartmental lateral inhibition” system using diffusible molecules and engineered communication channels to simulate contact-mediated signaling for verification of our patterning analysis. The current state of our synthetic implementation is presented, highlighting experimental setup details that may prove useful for future applications in engineered multicellular ensembles.},
}
EndNote citation:
%0 Thesis %A Hsia, Justin %T Design and Engineering of Pattern Formation in Gene Expression in Escherichia coli %I EECS Department, University of California, Berkeley %D 2016 %8 May 1 %@ UCB/EECS-2016-32 %U http://www2.eecs.berkeley.edu/Pubs/TechRpts/2016/EECS-2016-32.html %F Hsia:EECS-2016-32