Alleviating Cell Lysate-Induced Inhibition to Enable RT-PCR from Single Cells in Picoliter-Volume Double Emulsion Droplets.

Microfluidic droplet assays enable single-cell polymerase chain reaction (PCR) and sequencing analyses at unprecedented scales, with most methods encapsulating cells within nanoliter-sized single emulsion droplets (water-in-oil). Encapsulating cells within picoliter double emulsion (DE) (water-in-oil-in-water) allows sorting droplets with commercially available fluorescence-activated cell sorter (FACS) machines, making it possible to isolate single cells based on phenotypes of interest for downstream analyses. However, sorting DE droplets with standard cytometers requires small droplets that can pass FACS nozzles. This poses challenges for molecular biology, as prior reports suggest that reverse transcription (RT) and PCR amplification cannot proceed efficiently at volumes below 1 nL due to cell lysate-induced inhibition. To overcome this limitation, we used a plate-based RT-PCR assay designed to mimic reactions in picoliter droplets to systematically quantify and ameliorate the inhibition. We find that RT-PCR is blocked by lysate-induced cleavage of nucleic acid probes and primers, which can be efficiently alleviated through heat lysis. We further show that the magnitude of inhibition depends on the cell type, but that RT-PCR can proceed in low-picoscale reaction volumes for most mouse and human cell lines tested. Finally, we demonstrate one-step RT-PCR from single cells in 20 pL DE droplets with fluorescence quantifiable via FACS. These results open up new avenues for improving picoscale droplet RT-PCR reactions and expanding microfluidic droplet-based single-cell analysis technologies.

Constraints on the expansion of paralogous protein families.

Duplication and divergence is a major mechanism by which new proteins and functions emerge in biology. Consequently, most organisms, in all domains of life, have genomes that encode large paralogous families of proteins. For recently duplicated pathways to acquire different, independent functions, the two paralogs must acquire mutations that effectively insulate them from one another. For instance, paralogous signaling proteins must acquire mutations that endow them with different interaction specificities such that they can participate in different signaling pathways without disruptive cross-talk. Although duplicated genes undoubtedly shape each other's evolution as they diverge and attain new functions, it is less clear how other paralogs impact or constrain gene duplication. Does the establishment of a new pathway by duplication and divergence require the system-wide optimization of all paralogs? The answer has profound implications for molecular evolution and our ability to engineer biological systems. Here, we discuss models, experiments, and approaches for tackling this question, and for understanding how new proteins and pathways are born.

Engineering orthogonal signalling pathways reveals the sparse occupancy of sequence space.

Gene duplication is a common and powerful mechanism by which cells create new signalling pathways1,2, but recently duplicated proteins typically must become insulated from each other and from other paralogues to prevent unwanted crosstalk3. A similar challenge arises when new sensors or synthetic signalling pathways are engineered within cells or transferred between genomes. How easily new pathways can be introduced into cells depends on the density and distribution of paralogous pathways in the sequence space that is defined by their specificity-determining residues4,5. Here we directly investigate how crowded this sequence space is, by generating novel two-component signalling proteins in Escherichia coli using cell sorting coupled to deep sequencing to analyse large libraries designed on the basis of coevolutionary patterns. We produce 58 insulated pathways comprising functional kinase-substrate pairs that have different specificities than their parent proteins, and demonstrate that several of these new pairs are orthogonal to all 27 paralogous pathways in E. coli. Additionally, from the kinase-substrate pairs generated, we identify sets consisting of six pairs that are mutually orthogonal to each other, which considerably increases the two-component signalling capacity of E. coli. These results indicate that sequence space is not densely occupied. The relative sparsity of paralogues in sequence space suggests that new insulated pathways can arise easily during evolution, or be designed de novo. We demonstrate the latter by engineering a signalling pathway in E. coli that responds to a plant cytokinin, without crosstalk to extant pathways. Our work also demonstrates how coevolution-guided mutagenesis and the mapping of sequence space can be used to design large sets of orthogonal protein-protein interactions.

Permanent genetic memory with >1-byte capacity.

Genetic memory enables the recording of information in the DNA of living cells. Memory can record a transient environmental signal or cell state that is then recalled at a later time. Permanent memory is implemented using irreversible recombinases that invert the orientation of a unit of DNA, corresponding to the [0,1] state of a bit. To expand the memory capacity, we have applied bioinformatics to identify 34 phage integrases (and their cognate attB and attP recognition sites), from which we build 11 memory switches that are perfectly orthogonal to each other and the FimE and HbiF bacterial invertases. Using these switches, a memory array is constructed in Escherichia coli that can record 1.375 bytes of information. It is demonstrated that the recombinases can be layered and used to permanently record the transient state of a transcriptional logic gate.