A method for targeting a specified segment of DNA to a bacterial microorganelle

• Published in: Nucleic acids research 2022-10, Vol.50 (19), p.e113-e113
• Main Authors: Otoničar, Jan, Hostnik, Maja, Grundner, Maja, Kostanjšek, Rok, Gredar, Tajda, Garvas, Maja, Arsov, Zoran, Podlesek, Zdravko, Gostinčar, Cene, Jakše, Jernej, Busby, Stephen J W, Butala, Matej
• Format: Article
• Language: English
• Subjects: Methods Online
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkac714

Encapsulation of a selected DNA molecule in a cell has important implications for bionanotechnology. Non-viral proteins that can be used as nucleic acid containers include proteinaceous subcellular bacterial microcompartments (MCPs) that self-assemble into a selectively permeable protein shell containing an enzymatic core. Here, we adapted a propanediol utilization (Pdu) MCP into a synthetic protein cage to package a specified DNA segment in vivo, thereby enabling subsequent affinity purification. To this end, we engineered the LacI transcription repressor to be routed, together with target DNA, into the lumen of a Strep-tagged Pdu shell. Sequencing of extracted DNA from the affinity-isolated MCPs shows that our strategy results in packaging of a DNA segment carrying multiple LacI binding sites, but not the flanking regions. Furthermore, we used LacI to drive the encapsulation of a DNA segment containing operators for LacI and for a second transcription factor.