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

Jan Otoničar; Maja Hostnik; Maja Grundner; Rok Kostanjšek; Tajda Gredar; Maja Garvas; Zoran Arsov; Zdravko Podlesek; Cene Gostinčar; Jernej Jakše; Stephen J W Busby; Matej Butala

doi:10.1093/nar/gkac714

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

Jan Otoničar, Maja Hostnik, Maja Grundner, Rok Kostanjšek, Tajda Gredar, Maja Garvas, Zoran Arsov, Zdravko Podlesek, Cene Gostinčar, Jernej Jakše, Stephen J W Busby, Matej Butala^*

^*Corresponding author for this work

Biosciences

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Abstract

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.

Original language	English
Article number	e113
Number of pages	12
Journal	Nucleic Acids Research
Volume	50
Issue number	19
Early online date	27 Aug 2022
DOIs	https://doi.org/10.1093/nar/gkac714
Publication status	Published - 28 Oct 2022

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10.1093/nar/gkac714Licence: Creative Commons: Attribution (CC BY)

OtoničarJ2022methodFinal published version, 4.91 MBLicence: Creative Commons: Attribution (CC BY)

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title = "A method for targeting a specified segment of DNA to a bacterial microorganelle",

abstract = "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.",

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AU - Otoničar, Jan

AU - Hostnik, Maja

AU - Grundner, Maja

AU - Kostanjšek, Rok

AU - Gredar, Tajda

AU - Garvas, Maja

AU - Arsov, Zoran

AU - Podlesek, Zdravko

AU - Gostinčar, Cene

AU - Jakše, Jernej

AU - Busby, Stephen J W

AU - Butala, Matej

PY - 2022/10/28

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N2 - 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.

AB - 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.

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DO - 10.1093/nar/gkac714

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JO - Nucleic Acids Research

JF - Nucleic Acids Research

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M1 - e113

ER -

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

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