A Mini-ISY100 Transposon Delivery System Effective in γ Proteobacteria
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...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2019-02-01
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| Series: | Frontiers in Microbiology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/article/10.3389/fmicb.2019.00280/full |
| _version_ | 1818219159099539456 |
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| author | Emanuele Conte Linda Mende Ian Grainge Sean D. Colloms |
| author_facet | Emanuele Conte Linda Mende Ian Grainge Sean D. Colloms |
| author_sort | Emanuele Conte |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-12T07:35:13Z |
| format | Article |
| id | doaj.art-7ae7ada67cdd451f969023aa60e3b77b |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-12-12T07:35:13Z |
| publishDate | 2019-02-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-7ae7ada67cdd451f969023aa60e3b77b2022-12-22T00:32:56ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2019-02-011010.3389/fmicb.2019.00280439032A Mini-ISY100 Transposon Delivery System Effective in γ ProteobacteriaEmanuele Conte0Linda Mende1Ian Grainge2Sean D. Colloms3Institute of Molecular Cell and Systems Biology, University of Glasgow, Glasgow, United KingdomSchool of Environmental and Life Sciences, University of Newcastle, Newcastle, NSW, AustraliaSchool of Environmental and Life Sciences, University of Newcastle, Newcastle, NSW, AustraliaInstitute of Molecular Cell and Systems Biology, University of Glasgow, Glasgow, United KingdomTransposons 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.https://www.frontiersin.org/article/10.3389/fmicb.2019.00280/fullISY100transposon mutagenesisknock-out libraryΦC31 integrasecytochrome c |
| spellingShingle | Emanuele Conte Linda Mende Ian Grainge Sean D. Colloms A Mini-ISY100 Transposon Delivery System Effective in γ Proteobacteria Frontiers in Microbiology ISY100 transposon mutagenesis knock-out library ΦC31 integrase cytochrome c |
| title | A Mini-ISY100 Transposon Delivery System Effective in γ Proteobacteria |
| title_full | A Mini-ISY100 Transposon Delivery System Effective in γ Proteobacteria |
| title_fullStr | A Mini-ISY100 Transposon Delivery System Effective in γ Proteobacteria |
| title_full_unstemmed | A Mini-ISY100 Transposon Delivery System Effective in γ Proteobacteria |
| title_short | A Mini-ISY100 Transposon Delivery System Effective in γ Proteobacteria |
| title_sort | mini isy100 transposon delivery system effective in γ proteobacteria |
| topic | ISY100 transposon mutagenesis knock-out library ΦC31 integrase cytochrome c |
| url | https://www.frontiersin.org/article/10.3389/fmicb.2019.00280/full |
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