Mass-Activated Droplet Sorting for the Selection of Lysine-Producing Escherichia coli.

Synthetic biology relies on engineering cells to have desirable properties, such as the production of select chemicals. A bottleneck in engineering methods is often the need to screen and sort variant libraries for potential activity. Droplet microfluidics is a method for high-throughput sample preparation and analysis which has the potential to improve the engineering of cells, but a limitation has been the reliance on fluorescent analysis. Here, we show the ability to select cell variants grown in 20 nL droplets at 0.5 samples/s using mass-activated droplet sorting (MADS), a method for selecting droplets based on the signal intensity measured by electrospray ionization mass spectrometry (ESI-MS). Escherichia coli variants producing lysine were used to evaluate the applicability of MADS for synthetic biology. E. coli were shown to be effectively grown in droplets, and the lysine produced by these cells was detectable using ESI-MS. Sorting of lysine-producing cells based on the MS signal was shown, yielding 96-98% purity for high-producing variants in the selected pool. Using this technique, cells were recovered after screening, enabling downstream validation via phenotyping. The presented method is translatable to whole-cell engineering for biocatalyst production.

Advances in coupling droplet microfluidics to mass spectrometry.

Droplet microfluidics enables development of workflows with low sample consumption and high throughput. Fluorescence-based assays are most used with droplet microfluidics; however, the requirement of a fluorescent reporter restricts applicability of this approach. The coupling of droplets to mass spectrometry (MS) has enabled selective assays on complex mixtures to broaden the analyte scope. Droplet microfluidics has been interfaced to MS via electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). The works reviewed herein outline the development of this nascent field as well as initial exploration of its application in biotechnology and bioanalysis, including synthetic biology, reaction development, and in vivo sensing.