Bacterial chromosome conformation and cell-free gene expression in synthetic 2D compartments.

The E. coli genome is encoded on a contiguous ~4.6 Mb-long DNA molecule, compacted inside a micron-cubed cell. When reconstituted in vitro, chromosomes expand in a bulk that is challenging for probing single-chromosome DNA transactions and conformational changes. Here, we report transplanting E. coli chromosomes into 2D semi-open microfluidic compartments, enabling exchange of conditions, stretching by electric field, mapping DNA-bound proteins, and cell-free transcription-translation at steady-state. We find transplanted chromosomes emerge as intact, compacted, blob-like structures, decorated with native proteins from the donor cell. The blobs include clusters of condensin proteins and exclude sparse bright ribosome foci, whereas RNA polymerases uniformly decorate the chromosome. Introducing a transcription-translation system, we measure genome-average transcription rates and image the birth of individual proteins from a reporter gene on the chromosome. Our data suggest a dilute regime without translational amplification or multiple synthesis events per gene. The removal of native proteins reveals a conformation transition from expanded to compacted state upon increased molecular crowding. Interestingly, transcription has a swelling effect, pushing the compaction transition to higher crowding levels. Our work opens a window into genome-scale DNA transactions outside a cell and helps tackle the bottom-up assembly of autonomous artificial cells.