Enabling Rapid Protein and Circuit Engineering with High-Throughput Microfluidic Devices

We developed several methods and microfluidic approaches to enable the rapid engineering and quantitative characterization of proteins and genetic circuits in vitro. Protein engineering remains challenging, primarily due to the cumbersome experimental processes currently required for expressing, purifying and quantitatively characterizing proteins. We developed a novel solid-phase gene synthesis approach allowing the combinatoric assembly of protein variants. We directly coupled our gene synthesis method with high-throughput on-chip protein expression and characterization, allowing us to drastically reduce the time required for a complete design-test cycle. We applied our novel protein-engineering platform to the generation of hundreds of Zn-finger transcription factors and the precise quantitation of the binding specificity of each variant.

In order to streamline genetic network engineering we have recently demonstrated that genetic networks can be implemented and characterized in vitro using a microfluidic chemostat. This chemostat approach allows complex networks such as oscillators to be run in vitro. We have previously shown that several biologically relevant regulatory mechanisms can be implemented on this in vitro platform and we built and characterized the first in vitro genetic oscillator. Over the last year we focused on testing whether we can functionally implement genetic networks that are known to work in vivo in order to assess whether genetic networks can be designed or modified in vitro and consequently transferred in vivo. We have successfully implemented the Elowitz repressilator in vitro and performed a comprehensive analysis of the system that would be difficult to achieve in vivo. We additionally designed and implemented novel 3 node oscillators, and the first 5 node oscillators in vitro and we will present data on how these systems behave in vivo.