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

Otoničar, Jan (author), Hostnik, Maja (author), Grundner, Maja (author), Kostanjšek, Rok (author), Gredar, Tajda (author), Garvas, Maja (author), Arsov, Zoran (author), Podlesek, Zdravko (author), Gostinčar, Cene (author), Jakše, Jernej (author), Busby, Steve J. W. (author), Butala, Matej (author)

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.

• Language: English
• Keywords: DNA, physical characterisation, biochemical characterisation
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: BF - Biotechnical Faculty
• Publication status: Published
• Publication version: Version of Record
• Year: 2022
• Number of pages: 12 str.
• Numbering: Vol. 50, no. 19, art. e113
• PID: 20.500.12556/RUL-143717
• UDC: 577
• ISSN on article: 0305-1048
• DOI: 10.1093/nar/gkac714
• COBISS.SI-ID: 120251907
• Publication date in RUL: 10.01.2023

Record is a part of a journal

• Title: Nucleic acids research
• Shortened title: Nucleic acids res.
• Publisher: Oxford University Press, Nucleic Acids Research
• ISSN: 0305-1048
• COBISS.SI-ID: 3269647

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.

Secondary language

• Language: Slovenian
• Keywords: bakterijski mikrorazdelek Pdu, Twin-Strep-tag, represor LacI, molekularna biologija, biokemija