Bacterial cells organize their genomes into a compact hierarchical structure called the nucleoid. Studying the nucleoid in cells faces challenges because of the cellular complexity while in vitro assays have difficulty in handling the fragile megabase-scale DNA biopolymers that make up bacterial genomes. Here, we introduce a method that overcomes these limitations as we develop and use a microfluidic device for the sequential extraction, purification, and analysis of bacterial nucleoids in individual microchambers. Our approach avoids any transfer or pipetting of the fragile megabase-size genomes and thereby prevents their fragmentation. We show how the microfluidic system can be used to extract and analyze single chromosomes from B. subtilis cells. Upon on-chip lysis, the bacterial genome expands in size and DNA-binding proteins are flushed away. Subsequently, exogeneous proteins can be added to the trapped DNA via diffusion. We envision that integrated microfluidic platforms will become an essential tool for the bottom-up assembly of complex biomolecular systems such as artificial chromosomes.@en

Protocells containing enzyme-driven biomolecular circuits that can process and exchange information offer a promising approach for mimicking cellular features and developing molecular information platforms. Here, we employ synthetic transcriptional circuits together with CRISPR/Cas-based DNA processing inside semipermeable protein-polymer microcompartments. We first establish a transcriptional protocell that can be activated by external DNA strands and produce functional RNA aptamers. Subsequently, we engineer a transcriptional module to generate RNA strands functioning as diffusive signals that can be sensed by neighboring protocells and trigger the activation of internalized DNA probes or localization of Cas nucleases. Our results highlight the opportunities to combine CRISPR/Cas machinery and DNA nanotechnology for protocellular communication and provide a step towards the development of protocells capable of distributed molecular information processing.

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