Optical pooled screening (OPS) holds great potential for observing cells with different genetic modifications in one experiment. By tagging the cells with unique genetic barcodes, we can link each DNA alteration to an observed change in the physiology or dynamic properties of the cells. Until now, the method has been of limited use in bacteria because the barcodes were tricky to introduce into the bacteria’s chromosomal DNA. In this study, we have figured out how to do just that in the frequently used model organism E. coli. As a proof-of-principle, we use the method to measure how long it takes for 81 different red fluorescent proteins to mature, e.i. to start emitting fluorescence upon excitation inside the cells. Protein maturation is an important property of fluorescent proteins when these are to be used to study dynamic processes in living cells.

Figure 1: Phenotyping followed by in situ genotyping. (A.) Example of fluorescence images of cells at three different timepoints after switching to chloramphenicol-containing medium. Time point zero indicates swap to medium with chloramphenicol. (B.) Cartoon showing major steps of in situ genotyping. (C.) Fluorescence images of seven rounds of genotyping for the cell traps shown in A. (D.) Quantified fluorescence density throughout the experiment for one example lineage for the traps highlighted in A together with the decoded genotypes from C. None of the cells in Trap 2 passed the filtering criteria on the cell fluorescence and are thus not shown in the plot.

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