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Team:UPO-Sevilla/Foundational Advances/MiniTn7/Experimental Results/attTn7 target site
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<h3>Characterization of the "portable" attTn7</h3> | <h3>Characterization of the "portable" attTn7</h3> | ||
| - | <p>For preliminary characterization of the "portable" attTn7, we attempted to demonstrate transposition into the plasmid-borne target. The miniTn7BB-Gm transposon was transferred into E. coli DH5α bearing pSB1C3-attTn7 by co-electrotransformation with the helper plasmid pTNS2. Transformants bearing transposon insertions were selected on LB plates supplemented with gentamycin and chloramphenicol. To identify clones bearing the transposon insertion at the plasmid-borne target, colony PCR was performed using prefix and Tn7R primers. | + | <p>For preliminary characterization of the "portable" attTn7, we attempted to demonstrate transposition into the plasmid-borne target. The miniTn7BB-Gm transposon was transferred into E. coli DH5α bearing pSB1C3-attTn7 by co-electrotransformation with the helper plasmid pTNS2. Transformants bearing transposon insertions were selected on LB plates supplemented with gentamycin and chloramphenicol. To identify clones bearing the transposon insertion at the plasmid-borne target, colony PCR was performed using prefix and Tn7R primers. Unfortunately, the results of this assay are too preliminary to be shown.</p> |
Revision as of 17:22, 21 September 2011
A portable attTn7 target site
Single-target transposition to the bacterial genome is dependent on the presence of a conserved sequence, encompasing the 3' end of the glmS gene, encoding glucosamine synthetase, and part the intergenic region immediately downstream, designated the attTn7 site. This region is highly conserved (Milewski, 2002), and Tn7 transposition has been demonstrated in over 20 bacterial species (Craig, 1996). However, the lack of a suitable target may be the limiting factor in some of the organisms in which Tn7 transposition does not occur, including a variety of bacteria, but also archea and eukaryotes. The possibility of inserting a functional attTn7 in the genomes of these organisms may enable site-specific Tn7 transposition for at least some of them, thus expanding the host range of this transposon and its derived tools, such as the miniTn7 BioBrick toolkit.
Construction of a "portable" attTn7
As a first step toward this goal, we undertook the cloning of the functional E. coli attTn7 into a plasmid vector, and the demonstration that this "portable" plasmid-borne attTn7 can be recognized as a Tn7 transposition target by our miniTn7BB minitransposons. The attTn7 was PCR-amplified from the chromosome of the E. coli K12 strain MC4100 with primers bearing prefix and suffix restriction sites, cleaved and ligated into pSB1C3. The construct was verified by sequencing.
Characterization of the "portable" attTn7
For preliminary characterization of the "portable" attTn7, we attempted to demonstrate transposition into the plasmid-borne target. The miniTn7BB-Gm transposon was transferred into E. coli DH5α bearing pSB1C3-attTn7 by co-electrotransformation with the helper plasmid pTNS2. Transformants bearing transposon insertions were selected on LB plates supplemented with gentamycin and chloramphenicol. To identify clones bearing the transposon insertion at the plasmid-borne target, colony PCR was performed using prefix and Tn7R primers. Unfortunately, the results of this assay are too preliminary to be shown.
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