Team:UPO-Sevilla/Foundational Advances/MiniTn7/Experimental Results/attTn7 target site

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                                    <li class="current"><a href="/Team:UPO-Sevilla/Foundational_Advances/MiniTn7/Experimental_Results/attTn7_target_site" style="white-space: nowrap; float: left;">attTn7 target site</a><ul></ul></li>
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                            <h1>A portable attTn7 target site</h1>
                            
<p>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 (<a href="http://www.sciencedirect.com/science/article/pii/S0167483802003187" target="_blank">Milewski, 2002</a>), and Tn7 transposition has been demonstrated in over 20 bacterial species (<a href="http://www.ncbi.nlm.nih.gov/pubmed/8556868" target="_blank">Craig, 1996</a>). 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.</p>

     <h3>Construction of a "portable" attTn7</h3>
<p>As a first step toward this goal, we undertook the cloning of the functional <i>E. coli</i> attTn7 (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K510022" target="_blank">BBa_K510022</a>) 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 <i>E. coli</i> K12 strain MC4100 with primers bearing prefix and suffix restriction sites, cleaved and ligated into pSB1C3. The construct was <strong>verified by sequencing</strong>. </p>

     <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 <i>E. coli</i> DH5&alpha; bearing pSB1C3-attTn7 by tetraparental mating using the helper plasmid pTNS2 to provide the Tn7 transposition functions. Transconjugants 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,  transconjugant colonies were pooled, plasmid DNA was extracted from the pool and used to transform <i>E. coli</i>. Transformants were selected on LB plates containing chloramphenicol and scored for gentamycin resistance (Figure 7). 97 out of 100 colonies had become simultaneously resistant to both antibiotics, strongly suggesting that the miniTn7BB-Gm was inserted in the chloramphenicol resistance plasmid, and transferred along with its target. Restriction analysis confirmed that indeed this was the case, and that the transposon was inserted within the att-Tn7 target site at the pSB1C3-attTn7 plasmid (Figure 8). </p>

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<img width="600px" src="https://static.igem.org/mediawiki/2011/f/f4/AttTn7-miniTn7-600px.png" alt="Transformants bearing miniTn7 insertions att protable attTn7"/> </div>

<p class caption><strong>Figure 7. Patches of <i>E. coli</i> transformed with plasmid DNA from pooled colonies obtained after insertion of pUC18Sfi-miniTn7BB-Gm into the genome of <I>E.coli</i> DH5&alpha; bearing pSC1C3-attTn7.</strong> Open squares indicate patches showing sensitivity to gentamycin , that is, <strong>not bearing</strong> the miniTn7 transposon insertion.</p>

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<img width="600px" src="https://static.igem.org/mediawiki/2011/8/8f/UPOSevilla-attTn7-miniTn7_digestion.jpg" alt="pSB1C3-attTn7 with miniTn7BB-Gm insertions digested with EcoRI"/> </div>

<p class caption><strong>Figure 8. Agarose gel of restriction analysis products of eight different samples of pSB1C3-attTn7 with miniTn7BB-Gm module inserted witin it.</strong> Whether the insertion occurs site-specifically in the insertion site of the portable attTn7 is checked by the presence of 2497bp and 1485bp DNA fragments. These restriction products can be easily find in the first six digestion products. The next two looks to be right too, but impurities in their minipreps may make the digestions difficult to understand. We can also see a partial digestion in the second lane.</p>


     <h3>Construction of a miniTn7 transposon bearing the "portable" attTn7</h3>
<p></strong>	This module allows the integration of BioBricks into bacterial chromosomes (using prefix or suffix restriction sites that flank the attTn7), while regenerating an intact Tn7 attachment site for further genetic manipulation. Thus, the miniTn7BB-Gm-attTn7  allow further integration of BioBrick parts in the chromosome of a strain that already has a copy of mimni Tn7 inserted.</p>

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