Rescue of mutant fitness defects using in vitro reconstituted designer transposons in Mycoplasma mycoides
With only hundreds of genes contained within their genomes, mycoplasmas have become model organisms for precise understanding of cellular processes, as well as platform organisms for predictable engineering of microbial functions for mission-critical applications. Despite the availability of whole g...
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doaj-69d7b23778964ea4b1212bb9022b44802020-11-24T23:01:12ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2014-07-01510.3389/fmicb.2014.00369105142Rescue of mutant fitness defects using in vitro reconstituted designer transposons in Mycoplasma mycoidesBogumil Jacek Karas0Kim eWise1Lijie eSun2J. Craig Venter3John eGlass4Clyde eHutchison5Hamilton eSmith6Yo eSuzuki7J. Craig Venter InstituteJ. Craig Venter InstituteJ. Craig Venter InstituteJ. Craig Venter InstituteJ. Craig Venter InstituteJ. Craig Venter InstituteJ. Craig Venter InstituteJ. Craig Venter InstituteWith only hundreds of genes contained within their genomes, mycoplasmas have become model organisms for precise understanding of cellular processes, as well as platform organisms for predictable engineering of microbial functions for mission-critical applications. Despite the availability of whole genome writing in Mycoplasma mycoides, some traditional methods for genetic engineering are underdeveloped in mycoplasmas. Here we demonstrate two facile transposon-mediated approaches for introducing genes into the synthetic cell based on M. mycoides. The marker-less approach involves preparing a fragment containing only a small genomic region of interest with flanking transposase-binding sites, followed by in vitro transposase loading and introduction into the cells. The marker-driven approach involves cloning an open reading frame (ORF) of interest into a vector containing a marker for mycoplasma transformation, as well as sites for transposase loading and random genomic integration. An innovative feature of this construct is to use a single promoter to express the transformation marker and the introduced ORF. The marker-driven approach can be conveniently applied to any exogenous or synthetic gene without any information on the effect of the gene on the strain, whereas the marker-less approach requires that the fragment has a recognizable effect. Using the marker-less method, we found that a region containing the nusG gene rescues a slow growth phenotype of a strain containing a larger deletion encompassing this gene. Using the marker-driven approach, we better defined this finding, thereby establishing that nusG is required for a normal growth rate in synthetic M. mycoides. These methods are suitable for complementation tests to identify genes responsible for assorted functions lacking in deletion mutants. These approaches are also expected to facilitate rapid testing of various natural and engineered genes or gene clusters from numerous sources in M. mycoides.http://journal.frontiersin.org/Journal/10.3389/fmicb.2014.00369/fullcomplementationminimal cellsynthetic celltransposomenusG |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Bogumil Jacek Karas Kim eWise Lijie eSun J. Craig Venter John eGlass Clyde eHutchison Hamilton eSmith Yo eSuzuki |
spellingShingle |
Bogumil Jacek Karas Kim eWise Lijie eSun J. Craig Venter John eGlass Clyde eHutchison Hamilton eSmith Yo eSuzuki Rescue of mutant fitness defects using in vitro reconstituted designer transposons in Mycoplasma mycoides Frontiers in Microbiology complementation minimal cell synthetic cell transposome nusG |
author_facet |
Bogumil Jacek Karas Kim eWise Lijie eSun J. Craig Venter John eGlass Clyde eHutchison Hamilton eSmith Yo eSuzuki |
author_sort |
Bogumil Jacek Karas |
title |
Rescue of mutant fitness defects using in vitro reconstituted designer transposons in Mycoplasma mycoides |
title_short |
Rescue of mutant fitness defects using in vitro reconstituted designer transposons in Mycoplasma mycoides |
title_full |
Rescue of mutant fitness defects using in vitro reconstituted designer transposons in Mycoplasma mycoides |
title_fullStr |
Rescue of mutant fitness defects using in vitro reconstituted designer transposons in Mycoplasma mycoides |
title_full_unstemmed |
Rescue of mutant fitness defects using in vitro reconstituted designer transposons in Mycoplasma mycoides |
title_sort |
rescue of mutant fitness defects using in vitro reconstituted designer transposons in mycoplasma mycoides |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Microbiology |
issn |
1664-302X |
publishDate |
2014-07-01 |
description |
With only hundreds of genes contained within their genomes, mycoplasmas have become model organisms for precise understanding of cellular processes, as well as platform organisms for predictable engineering of microbial functions for mission-critical applications. Despite the availability of whole genome writing in Mycoplasma mycoides, some traditional methods for genetic engineering are underdeveloped in mycoplasmas. Here we demonstrate two facile transposon-mediated approaches for introducing genes into the synthetic cell based on M. mycoides. The marker-less approach involves preparing a fragment containing only a small genomic region of interest with flanking transposase-binding sites, followed by in vitro transposase loading and introduction into the cells. The marker-driven approach involves cloning an open reading frame (ORF) of interest into a vector containing a marker for mycoplasma transformation, as well as sites for transposase loading and random genomic integration. An innovative feature of this construct is to use a single promoter to express the transformation marker and the introduced ORF. The marker-driven approach can be conveniently applied to any exogenous or synthetic gene without any information on the effect of the gene on the strain, whereas the marker-less approach requires that the fragment has a recognizable effect. Using the marker-less method, we found that a region containing the nusG gene rescues a slow growth phenotype of a strain containing a larger deletion encompassing this gene. Using the marker-driven approach, we better defined this finding, thereby establishing that nusG is required for a normal growth rate in synthetic M. mycoides. These methods are suitable for complementation tests to identify genes responsible for assorted functions lacking in deletion mutants. These approaches are also expected to facilitate rapid testing of various natural and engineered genes or gene clusters from numerous sources in M. mycoides. |
topic |
complementation minimal cell synthetic cell transposome nusG |
url |
http://journal.frontiersin.org/Journal/10.3389/fmicb.2014.00369/full |
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