Adaptive Laboratory Evolution (ALE) is a powerful tool to obtain production strains with beneficial mutations. Microbes are cultured for an extended time allowing natural selection to enrich mutant strains with improved fitness (e.g, feedstock or product tolerance, increased substrate utilization, etc.). Since evolutionary processes are highly stochastic, more replicates are needed if the ALE experiment is meant to identify distinct molecular mechanisms associated with a trait of interest. The number of replicates can be easily increased when switching from flasks to 96-well plates. We have shown the use of aqueous microfluidic droplets for ALE (dALE) to generate millions of cultures in parallel. Hundreds of isolates are characterized via an automated high throughput (HTP) method for growth and productivity. HTP characterization can be accomplished with the use of cell-based biosensors or by HTP mass spectrometry (HTP-MS). 

Adaptive Laboratory Evolution

Sorting with flow cytometry using a biosensor

Flow cytometry is a powerful technology to rapidly sort a population based on phenotypes. These phenotypes can be natural, such as size and granularity of the cells or engineered such as fluorescence response from a biosensor as a proxy for metabolic efficiency or productivity.

As a Build infrastructure, flow cytometry and fluorescence-activated cell sorting (FACS) can be applied to sort microbial populations solely based on fluorescence. This step is performed in conjunction with an adaptive laboratory evolution (ALE) or droplet adaptive laboratory evolution (dALE) to build a diverse population with altered phenotypes.

Droplet Adaptive Laboratory Evolution (dALE) 

Screening with biosensors

Cell-based sensors are used for the initial characterization of individual clones. The production cells are cultivated in 96-well plates and after cell density normalization the growth rate is monitored. Samples at late-exponential and stationary phase are mixed with the sensor cells and isolates selected for more rigorous characterization using the HTP-MS technology.

High-throughput mass-spectrometry-based (HTP-MS) method

The Agile BioFoundry developed a high-throughput mass-spectrometry-based method for the monitoring of bioproduct concentration from broth without sample derivatization. Traditional LC-MS/MS applications are used to separate and identify components from complex mixtures, such as microbial growth media. High sensitivity methods are available to measure hundreds of molecules with a wide range of abundance. The obtained metabolomic profile is invaluable to identify potential bottlenecks in engineered systems. On the other end of the scale, simple product titer measurements suffice to provide rapid feed-back for closed-loop experiments. A direct inject method can be used to measure the absorbance of samples and monitor 4 ions with a mass detector capable of measuring 96 samples in less than 2 hours (Figure 1). This technique has been used to provide product titers (muconate and adipate) for thousands of samples. The rapid sample processing via automated liquid handlers, coupled with fast analysis and report generation enables in-line analytics (1 sample/min), steering sampling frequencies and providing complete production profiles.

We are developing a system with even higher throughput via the measurement of analytes from liquid plugs and microfluidic droplets (1 sample/sec).