- Title
- A mini-ISY100 transposon delivery system effective in gamma proteobacteria
- Creator
- Conte, Emanuele; Mende, Linda; Grainge, Ian; Colloms, Sean D.
- Relation
- Frontiers in Microbiology Vol. 10, no. 280
- Publisher Link
- http://dx.doi.org/10.3389/fmicb.2019.00280
- Publisher
- Frontiers Research Foundation
- Resource Type
- journal article
- Date
- 2019
- Description
- Transposons are invaluable biological tools for the genetic manipulation of microorganisms. ISY100 from Synechocystis, sp. PCC6803 is a member of the Tc1/mariner/IS630 superfamily, and is characterized by high transposition efficiency and a strong preference for TA target sequences. In this paper, we describe the design and application of a mini-ISY100 suicide vector for the in vivo creation of stable random transposon insertion libraries. The system was successfully applied in seven species belonging to four different orders of γ proteobacteria. In all cases, delivery using conjugation consistently showed the highest transposition efficiency compared to chemical transformation or electroporation. We determined the frequency of transposon insertions in all the species and proved the utility of the system by identifying genes involved in colony coloration in Shewanella oneidensis. The ease and the efficiency of the protocol developed here allow the creation of complete knock-out libraries in an extensive range of host microorganisms in less than a week with no requirement for preparatory modification.
- Subject
- ISY100; transposon mutagenesis; knock-out library; ϕC31 integrase; cytochrome c
- Identifier
- http://hdl.handle.net/1959.13/1400706
- Identifier
- uon:34802
- Identifier
- ISSN:1664-302X
- Rights
- Copyright © 2019 Conte, Mende, Grainge and Colloms. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
- Language
- eng
- Full Text
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