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Abstract Precise control of gene expression is critical for biological research and biotechnology. However, transient plasmid transfections in mammalian cells produce a wide distribution of copy numbers per cell, and consequently, high expression heterogeneity. Here, we report plasmid-based synthetic circuits – Equalizers – that buffer copy-number variation at the single-cell level. Equalizers couple a transcriptional negative feedback loop with post-transcriptional incoherent feedforward control. Computational modeling suggests that the combination of these two topologies enables Equalizers to operate over a wide range of plasmid copy numbers. We demonstrate experimentally that Equalizers outperform other gene dosage compensation topologies and produce as low cell-to-cell variation as chromosomally integrated genes. We also show that episome-encoded Equalizers enable the rapid generation of extrachromosomal cell lines with stable and uniform expression. Overall, Equalizers are simple and versatile devices for homogeneous gene expression and can facilitate the engineering of synthetic circuits that function reliably in every cell. 

For color vision, retinal circuits separate information about intensity and wavelength. In vertebrates that use the full complement of four “ancestral” cone types, the nature and implementation of this computation remain poorly understood. Here, we establish the complete circuit architecture of outer retinal circuits underlying color processing in larval zebrafish. We find that the synaptic outputs of red and green cones efficiently rotate the encoding of natural daylight in a principal components analysis–like manner to yield primary achromatic and spectrally opponent axes, respectively. Blue cones are tuned to capture most remaining variance when opposed to green cones, while UV cone present a UV achromatic axis for prey capture. We note that fruitflies use essentially the same strategy. Therefore, rotating color space into primary achromatic and chromatic axes at the eye’s first synapse may thus be a fundamental principle of color vision when using more than two spectrally well-separated photoreceptor types. 

Unraveling the genetic and epigenetic determinants of phenotypes is critical for understanding and re-engineering biology and would benefit from improved methods to separate cells based on phenotypes. Here, we report SPOTlight, a versatile high-throughput technique to isolate individual yeast or human cells with unique spatiotemporal profiles from heterogeneous populations. SPOTlight relies on imaging visual phenotypes by microscopy, precise optical tagging of single target cells, and retrieval of tagged cells by fluorescence-activated cell sorting. To illustrate SPOTlight’s ability to screen cells based on temporal properties, we chose to develop a photostable yellow fluorescent protein for extended imaging experiments. We screened 3 million cells expressing mutagenesis libraries and identified a bright new variant, mGold, that is the most photostable yellow fluorescent protein reported to date. We anticipate that the versatility of SPOTlight will facilitate its deployment to decipher the rules of life, understand diseases, and engineer new molecules and cells. 

Monitoring voltage dynamics in defined neurons deep in the brain is critical for unraveling the function of neuronal circuits but is challenging due to the limited performance of existing tools. In particular, while genetically encoded voltage indicators have shown promise for optical detection of voltage transients, many indicators exhibit low sensitivity when imaged under two-photon illumination. Previous studies thus fell short of visualizing voltage dynamics in individual neurons in single trials. Here, we report ASAP2s, a novel voltage indicator with improved sensitivity. By imaging ASAP2s using random-access multi-photon microscopy, we demonstrate robust single-trial detection of action potentials in organotypic slice cultures. We also show that ASAP2s enables two-photon imaging of graded potentials in organotypic slice cultures and in Drosophila. These results demonstrate that the combination of ASAP2s and fast two-photon imaging methods enables detection of neural electrical activity with subcellular spatial resolution and millisecond-timescale precision.