Methods to determine the evolutionary stability of engineered biological function

Summary

Evolution poses unique challenges to engineering in living organisms. The successful implementation and development of a sustainable bioeconomy will require biological solutions that reliably perform at promised specifications. Methods to design evolutionary stable constructs or to increase the evolutionary stability of engineered organisms are critical for building robust engineered biological systems that can be used for industrial, medical, and environmental applications.

Description

Advances in engineering biology have greatly expanded our ability to add new and useful functions to living cells. Engineering stable functions has remained a significant challenge, with many different approaches developed to increase the stability, or decrease the variability, of a function over time. Often these approaches require significant investments of time and resources and the outcomes of such efforts are not predictable.  Generalizable methods to design stable constructs or to increase the stability of the host cell would greatly facilitate engineering evolutionarily stable biological function.

The Cellular Engineering Group is exploring factors that impact the evolutionary stability of engineered functions in living cells, and in ways to characterize the instability of the function. To this end, we have examined the activity of a function, fluorescence, in several different strains of E. coli and in several different synthetic constructs. Using flow cytometry to monitor the activity of the function at the single cell level revealed the fitness of the host cell was the primary determinant in the stability of the function over time. We also demonstrated that increasing the fitness of a cell by adapting it to the intended environment greatly improves the stability of a function over evolutionary timeframes. Our results are published here. NIST is interested in other methods to characterize and quantify the stability of engineered functions in living cells, and in developing ways to improve evolutionary stability.

Adaptation increases the stability of an engineered function. Poorly fit E. coli strains have high variance of an engineered function over 250 generations. By first adapting a strain, fitness increases and the variability of the function decreases.

Stability of a function is strain dependent. Poorly fit E. coli strains have high variance of an engineered function over 250 generations. After adaptation the fitness increased and the variability of the function decreased (compare strain DH5α to the adapted strain eDH5α and strain NEB Turbo to the adapted strain eTurbo).