Two years ago, we first introduced the DuMPLING technique in Molecular Systems Biology. The method is used to analyze dynamic phenotypes in large genetic libraries and subsequently recover the underlying genotype; it was conceived in response to the lack of efficient screening tools for complex phenotypes in large cell libraries.

This time we have tested the performance of DuMPLING to investigate the bacterial cell cycle regulation. From a plasmid library, we express sgRNA for 235 different genes together with a barcode that can be used to recover the genotype. The E. coli cells transformed with the plasmid pool are phenotyped individually in a microfluidic chip allowing only one single clone to propagate in each position. When the phenotype has been determined, the barcodes are interrogated by sequential rounds of FISH probe hybridization.

The results are encouraging. The DuMPLING accurately identifies most of the genes that are previously associated with cell cycle regulation, and we can also resolve the particular effect of these genes on specific parameters such as initiation and division-size as well as the variability in these traits. The next step is to expand the library to include all the genes in the E. Coli genome. In this way, we hope to identify the missing regulatory elements and arrive at a model that can satisfactory reproduce the observed precision of replication initiation.

The complete article is available in Nature Methods.

Instructions for how to download the microscopy images and analysis output associated with the DuMPLING experiments are found at IDR. Use idr0065 as User. The data and analysis output resides in the 20190930-ftp folder.