Riboswitches are RNA regulatory elements that bind specific ligands to control gene expression. Because of their modular composition, where a ligand-sensing aptamer domain is combined with an expression platform, riboswitches offer unique tools for synthetic biology applications. Here we took a mutational approach to determine functionally important nucleotide residues in the thiamine pyrophosphate (TPP) riboswitch in the THI4 gene of the model alga Chlamydomonas reinhardtii, allowing us to carry out aptamer swap using THIC aptamers from Chlamydomonas and Arabidopsis thaliana. These chimeric riboswitches displayed a distinct specificity and dynamic range of responses to different ligands. Our studies demonstrate ease of assembly as 5′UTR DNA parts, predictability of output, and utility for controlled production of a high-value compound in Chlamydomonas. The simplicity of riboswitch incorporation in current design platforms will facilitate the generation of genetic circuits to advance synthetic biology and metabolic engineering of microalgae.

Novel and improved biocatalysts are increasingly sourced from libraries via experimental screening. The success of such campaigns is crucially dependent on the number of candidates tested. Water-in-oil emulsion droplets can replace the classical test tube, to provide in vitro compartments as an alternative screening format, containing genotype and phenotype and enabling a readout of function. The scale-down to micrometer droplet diameters and picoliter volumes brings about a >10 7 -fold volume reduction compared to 96-well-plate screening. Droplets made in automated microfluidic devices can be integrated into modular workflows to set up multistep screening protocols involving various detection modes to sort >10 7 variants a day with kHz frequencies. The repertoire of assays available for droplet screening covers all seven enzyme commission (EC) number classes, setting the stage for widespread use of droplet microfluidics in everyday biochemical experiments. We review the practicalities of adapting droplet screening for enzyme discovery and for detailed kinetic characterization. These new ways of working will not just accelerate discovery experiments currently limited by screening capacity but profoundly change the paradigms we can probe. By interfacing the results of ultrahigh-throughput droplet screening with next-generation sequencing and deep learning, strategies for directed evolution can be implemented, examined, and evaluated. CONTENTSAA 11.6.2. Combining High-Throughput Selections with High-Throughput Analysis AB 12. Conclusions

Droplet microfluidics is a valuable method to “beat the odds” in high throughput screening campaigns such as directed evolution, where valuable hits are infrequent and large library sizes are required. Absorbance-based sorting expands the range of enzyme families that can be subjected to droplet screening by expanding possible assays beyond fluorescence detection. However, absorbance-activated droplet sorting (AADS) is currently ∼10-fold slower than typical fluorescence-activated droplet sorting (FADS), meaning that, in comparison, a larger portion of sequence space is inaccessible due to throughput constraints. Here we improve AADS to reach kHz sorting speeds in an order of magnitude increase over previous designs, with close-to-ideal sorting accuracy. This is achieved by a combination of (i) the use of refractive index matching oil that improves signal quality by removal of side scattering (increasing the sensitivity of absorbance measurements); (ii) a sorting algorithm capable of sorting at this increased frequency with an Arduino Due; and (iii) a chip design that transmits product detection better into sorting decisions without false positives, namely a single-layered inlet to space droplets further apart and injections of “bias oil” providing a fluidic barrier preventing droplets from entering the incorrect sorting channel. The updated ultra-high-throughput absorbance-activated droplet sorter increases the effective sensitivity of absorbance measurements through better signal quality at a speed that matches the more established fluorescence-activated sorting devices.

Droplet microfluidics allows one to address the ever-increasing demand to screen large libraries of biological samples. Absorbance spectroscopy complements the established fluorescence detection by alternative target identification and providing quantifiable...

Droplet microfluidics is a valuable method to "beat the odds" in high throughput screening campaigns such as directed evolution, where valuable hits are infrequent and large library sizes are required. Absorbance-based sorting expands the landscape of range of enzyme families that can be subjected to droplet screening by expanding possible assays beyond fluorescence detection. However, absorbance activated droplet sorting (AADS) is currently ~10-fold slower than typical fluorescence activated droplet sorting (FADS), meaning that, in comparison, a larger portion of sequence space is inaccessible due to throughput constraints. Here we improve AADS to reach kHz sorting speeds in an order of magnitude increase over previous designs, with close-to-ideal sorting accuracy. This is achieved by a combination of (i) the use of refractive index matching oil that improves signal quality by removal of side scattering (increasing the sensitivity of absorbance measurements); (ii) a sorting algorithm capable of reaching 4 kHz with an Arduino Due; and (iii) a chip design that transmits product detection better into sorting decisions without false positives, namely a single-layered inlet to space droplets further apart and injections of "bias oil" providing a fluidic barrier preventing droplets from entering the incorrect sorting channel. The updated ultrahigh-throughput absorbance activated droplet sorter (UHT-AADS) increases the effective sensitivity of absorbance measurements through better signal quality at a speed that matches the more established fluorescence-activated sorting devices.