In vivo continuous directed evolution

Abstract

The development and application of methods for the laboratory evolution of biomolecules has rapidly progressed over the last few decades. Advancements in continuous microbe culturing and selection design have facilitated the development of new technologies that enable the continuous directed evolution of proteins and nucleic acids. These technologies have the potential to support the extremely rapid evolution of biomolecules with tailor-made functional properties. Continuous evolution methods must support all of the key steps of laboratory evolution - translation of genes into gene products, selection or screening, replication of genes encoding the most fit gene products, and mutation of surviving genes - in a self-sustaining manner that requires little or no researcher intervention. Continuous laboratory evolution has been historically used to study problems including antibiotic resistance, organismal adaptation, phylogenetic reconstruction, and host-pathogen interactions, with more recent applications focusing on the rapid generation of proteins and nucleic acids with useful, tailor-made properties. The advent of increasingly general methods for continuous directed evolution should enable researchers to address increasingly complex questions and to access biomolecules with more novel or even unprecedented properties.

Figure 1. Schematic representation of directed evolution

All complete directed evolution methods must provide four major components: translation, selection or screening, replication, and mutation. Historical examples of each of these components are listed. Techniques that are particularly amenable to in vivo continuous evolution are shown in green.

Figure 2. Auxostats and chemostats

Fresh culture medium is pumped into a pre-sterilized container containing the microorganism of interest. Medium with cells is pumped out of the culture container. The rates of medium input and output are held constant in a chemostat system, and the growth rate is regulated by the composition of the medium. In an auxostat system, the medium flow rates are dynamically regulated by a controller in response to measurements made in the growing culture, which correspond directly (turbidity meter) or indirectly (other sensors) to the culture density. In schemes for viral continuous evolution, the auxostat or chemostat culture is pumped into a new vessel where the virus of interest is supplied (cellstat). Both cultures can be supplemented with additives supplied through a dedicated inlet. Selection stringency can be regulated by varying flow rates, changing temperature, or adding compounds to the auxostat, chemostat or cellstat. The depleted medium, cells and virus are pumped to a waste container.

Figure 3. The GM3 cultivation device

A growing culture in the first vessel is supplemented with a defined ratio of relaxing and stressing medium. This ratio is regulated by a controller connected to a turbidity meter measuring cell density. After a defined growth time, the whole culture is transferred to the second vessel to limit selection escape through biofilm formation. Following this transfer, the first vessel is sterilized with 5 M NaOH and washed extensively to limit contamination. After another defined growth period in the second vessel, the growing culture is transferred back to the first vessel, and the cycle is repeated. The selection stringency is modulated through the ratio of the relaxing and stressing media, ultimately reaching up to 100% stressing medium. The depleted medium and cells are pumped to a waste container.

Figure 4. Growth chamber of the Evolugator

a) The growing culture is shown on the right, and is completely contained within a tube clamped at regular intervals to limit unintentional mixing. The density of the culture is measured with a turbidity meter connected to a controller that dynamically regulates the in-flux of new medium. Air bubbles are included at regular intervals to increase the oxygenation level of the medium. b) Upon reaching high density, the right clamp closes, the tubing moves along the track, and the next clamp opens allowing for contact between the growing culture and the fresh medium. c) The fresh medium mixes with the growing culture through diffusion, and the culture resumes growing. d) After sufficient time, the culture again reaches high density. The middle clamp then closes, the tubing shifts on the track once again, the next clamp opens and the new medium is allowed in. This cycle is then continued for the duration of the experiment.