Characterizing a library of cells at single molecule sensitivity
Michel Lawson, Daniel Camsund, Jimmy Larsson, Özden Baltekin, David Fange, Johan Elf
Department of Cell and Molecular Biology, Science for Life Laboratory, Uppsala University, Sweden
We have developed a method to perform sensitive, time-lapse imaging at the single cell level for many different strains simultaneously.
The method has three components. The first is library generation. We have created a library of plasmids, each with a constitutively expressed sgRNA and a uniquely associated barcode RNA (driven by an inducible and orthogonal expression system). We transform these plasmids into Escherichia coli with chromosomally expressed dCas9 to allow for knockdown of the sgRNA-targeted gene.
The second component is phenotyping. We load the library of strains into a microfluidic device, where we can observe growth of isogenic microcolonies of every strain in the library over many generations. The chip is mounted on a microscope where we can track cells through many divisions, as well as count and localize single molecules and quantify any other phenotype discernable via microscopy.
The third and final step is to genetically identify each strain. The chip design allows for observing thousands of strains in one experiment, however the loading is random. We have developed a multiple round oligo-paint based approach to make an encoding between two fluorescently labeled primers (Cy3 and Cy5) and the unique barcode RNAs. In every round a probe in one of the fluorescent markers hybridizes to each barcode, thus providing a binary readout. After successive rounds, the cells in each trap have an associated binary word that uniquely identifies the strain. The encoding from fluorescent primers to barcode RNA sequence is achieved via template oligos, which are amplified by hybridization-round specific primers from an oligo pool.
As a proof of principle, we implemented a library of three strains with LacY-Ypet as the readout.
Credits: None available.
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