Team:UPO-Sevilla/Foundational Advances/MiniTn7/Experimental Results/Characterization

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                                    <li><a href="/Team:UPO-Sevilla/Foundational_Advances/MiniTn7/Overview" style="white-space: nowrap; float: left;">MiniTn7</a><ul></ul></li>
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                            <h1>Characterization of miniTn7BB-Gm function</h1>
                            
<p>In order to characterize the function of the miniTn7BB-Gm minitransposon, we performed a set of experiments aimed to (i) determine whether miniTn7BB-Gm <strong>can transpose efficientely</strong> in an enterobacterial host (<i>E. coli</i> DH5&alpha;) and a non-enterobacterial host (<i>P. putida</i> KT2440), (ii) measure the <strong>transposition frequency</strong> when miniTn7BB-Gm was delivered by electroporation, chemical transformation and mating, (iii) determine whether <strong>insertion occurs site-specifically</strong> at the chromosomal attTn7 site, and (iv) determine whether flipase expression in trans can <strong>excise the drug resistance marker</strong> in the chromosomal copy of miniTn7BB-Gm.</p>

      <h3>Transposition of miniTn7BB-Gm in <i>P. putida</i></h3>

<p>In order to demonstrate miniTn7BB-Gm transposition into the <i>P. putida</i> KT2440 chromosome, pUC18Sfi-miniTn7BB-Gm was transferred to this host by two different means: (i) <strong>co-electroporation</strong> with the helper plasmid pTNS2 [pTNS2 is not replicative in <i>P. putida</i>, but transiently produces the Tn7 transposition machinery (<a href="http://www.nature.com/nmeth/journal/v2/n6/abs/nmeth765.html" target="_blank">Choi et al., 2005</a>)], and (ii) <strong>tetraparental mating</strong>, using DH5&alpha;/pUC18Sfi-miniTn7BB-Gm as the donor, DH5&alpha;/pRK2013 as helper to provide the Tra functions, DH5&alpha;/pTNS2 to provide the Tn7 transposase and <i>P. putida</i> KT2440 as the recipient. <i>P. putida</i> clones bearing miniTn7BB-Gm insertions were selected on LB plates supplemented with Gm (and chloramphenicol when mating was used). Viable counts were also performed in both experiments, and <strong>plasmid transformation efficiency</strong> was determined using the gentamycin resistant replicative plasmid pBBR1mcs-5 in the electroporation experiment. The results are shown in Table 1.</p>


 <div class="center">
                            <table class="parameters" cellpadding=0 cellspacing=0>
                                    <tr>
                                           <th>&nbsp;</th>
                                           <th>Transformation efficiency<sup>a</sup></th>
                                           <th>Transposition efficiency<sup>b</sup></th>
                                           <th>Transposition frequency<sup>c</sup></th>
                                    </tr>
                                    <tr>
                                           <th>Mating</th>
                                           <td>NA</td>
                                           <td>NA</td>
                                           <td>1 x 10<sup>-4</sup></td>
                                    </tr>
                                    <tr>
                                           <th>Electroporation</th>
                                           <td>6 x 10<sup>9</sup></td>
                                           <td>7 x 10<sup>1</sup></td>
                                           <td>NA</td>
                                    </tr>
                            </table>
                        </div>

<p><strong>Table 1. Characterization of miniTn7BB-Gm transposition in <i>P. putida</i> KT2440</strong>. <sup>a</sup>Transformant (gentamycin resistant) cfu x &mu;g<sup-1> pBBR1-mcs5. <sup>b</sup>cfu bearing a miniTn7BB-Gm insertion (gentamycin resistant) x &mu;g<sup-1> pUC18Sfi-miniTn7BB-Gm. <sup>c</sup>Recipient cfu bearing a miniTn7BB-Gm insertion (gentamycin and chloramphenicol resistant) x viable recipient (chloramphenicol resistant) cfu<sup-1>.</p>

<p>The results clearly show that the gentamycin-resistance marker was acquired by <i>P. putida</i> KT2440 by both electrotransformation and conjugation, suggesting that the transposon was transferred to this non-enterobacterial recipient. In the <strong>electroporation experiment</strong>, the efficiency was low, with only 70 potential insertions per &mu;g plasmid DNA, despite the fact that the strain had become very competent as shown by the high transformation efficiency achieved with a replicative plasmid. On the other hand, transposition frequency in the <strong>mating experiment</strong> was quite high, suggesting that conjugation may be a more efficient means to transfer the miniTn7BB-Gm transposon to <i>P. putida</i>. Similar results were obtained with the miniTn7BB-Gm transposon borne in the commercial plasmid pMA (Mr. Gene). Interestingly, conjugative transfer of pUC18 or pMA has not been described, and a transfer origin is not documented for any of these vectors. Similar frequencies and efficiencies have been obtained with other miniTn7 delivery plasmids, such as those of the pBK-miniTn7 series (<a href="http://www.sciencedirect.com/science/article/pii/S0167701201002469" target="_blank">Koch et al., 2001</a>) (Fernando Govantes, personal communication).</p>

<p><strong>Site-specificity of the miniTn7-Gm insertions</strong> in <i>P. putida</i> KT2440 obtained was determined by PCR amplification using a primer annealing at the 3' end of glmS and a primer annealing at the Tn7R end. The occurence of a 164 bp product indicates successful site-specific integration at attTn7, while absence of this product suggests non-specific insertion elsewhere. 12 candidates each from the electroporation and mating experiments were tested by colony PCR as indicated (Figure 3). All candidates from the mating experiment and 10 out of 12 from the electroporation experiment displayed a band of the expected size, indicating that miniTn7BB-Gm efficiently inserts at the chromosomal attTn7 site of <i>P. putida</i>. </p>

<div class="center">
<img width="600px" src="https://static.igem.org/mediawiki/2011/c/c9/Geles.jpg" alt="NAME"/> </div>

<p class caption><strong>Figure 3. Agarose gels showing amplification from the chromosomal copies of miniTn7BB-Gm in candidates obtained in mating </strong>(top)<strong> or electroporation </strong>(bottom)<strong> experiments</strong>. Open arrowheads indicate the location of the correct 164 bp PCR products.</p>

<p>Finally, we tested whether the gentamycin resistance cassette in the miniTn7BB-Gm transposon can be excised by <strong>site-specific recombination</strong> performed by the flipase enzyme at the flanking FRT sites. To achieve this, we transferred the pFLP2 plasmid, encoding the yeast flipase, by electroporation into one of the <i>P. putida</i> clones bearing a miniTn7BB-Gm transposon insertion. Upon selection of the transformants on LB-carbenicillin plates, clones were patched onto LB-gentamycin and LB plates. 6 out of 100 candidates failed to grow on the LB-gentamycin plates, indicating the successful loss of the Gm-resistance cassette in the transposon. The low efficiency of excision may be improved by prolonged incubation on medium not containing gentamycin. Alternetively, the introduction of point mutations at the FRT elements to remove the XbaI restriction site may have resulted in decreased recognition of the target by the site-specific recombinase. Nevertheless, our results show that excision occurs and can be detected in a simple, effortless fashion (Figure 4). </p>


<div class="center">
<img width="600px" src="https://static.igem.org/mediawiki/2011/2/27/FRT_action.png" alt="NAME"/> </div>

<p class caption><strong>Figure 4. Plates showing excision of the gentamycin resistance marker in <i>P. putida</i> KT2440 bearing miniTn7BB-Gm.</strong> Gentamycin-sensitive patches are boxed on both LB+gentamycin (left) and LB (right) plates.</p>
</br>
                <h3>Transposition of miniTn7BB-Gm in <i>E. coli</i></h3>

<p>In order to demonstrate miniTn7BB-Gm transposition into the <i>E. coli</i> DH5&alpha; chromosome, pUC18R6KT-miniTn7BB-Gm was transferred to this host by <strong>co-transformation</strong> with the helper plasmid pTNS2 of <strong>chemically competent cells</strong> [pTNS2 is not replicative in DH5&alpha;, as it harbors a R6K replication origin, but transiently produces the Tn7 transposition machinery (<a href="http://www.nature.com/nmeth/journal/v2/n6/abs/nmeth765.html" target="_blank">Choi et al. (2005)</a>)]. <i>E. coli</i> clones bearing miniTn7BB-Gm insertions were selected on LB plates supplemented with Gm. Viable counts were also performed, and <strong>plasmid transformation efficiency</strong> was determined using the gentamycin resistant replicative plasmid pBBR1mcs-5. The results are summarized in Table 2.</p>

<div class="center">
                            <table class="parameters" cellpadding=0 cellspacing=0>
                                    <tr>
                                           <th>&nbsp;</th>
                                           <th>Transformation efficiency<sup>a</sup></th>
                                           <th>Transposition efficiency<sup>b</sup></th>
                                    </tr>
                                    <tr>
                                           <th>Heat-shock transformation</th>
                                           <td>1 x 10<sup>8</td>
                                           <td>4 x 10<sup>2</td>
                                    </tr>
                            </table>
                        </div>


<p class caption><strong>Table 2. Characterization of miniTn7BB-Gm transposition in <i>E. coli</i> DH5&alpha;</strong>. <sup>a</sup>Transformant (gentamycin resistant) cfu x &mu;g-1 pBBR1mcs5. <sup>b</sup>cfu bearing a miniTn7BB-Gm insertion (gentamycin resistant) x &mu;g-1 pUC18R6KT-miniTn7BB-Gm.</p> 

<p>The results indicate that the gentamycin-resistant marker was acquired by the transformed <i>E. coli</i> DH5&alpha; cells, strongly suggesting that the transposon can be tranferred to an enterobacterial host by means of <strong>transformation of chemically competent cells</strong>. The efficiency with which transposon insertion candidates was obtained was somewhat (6-fold) higher than that obtained with <i>P. putida</i>, despite the fact that competence was clearly (6-fold) lower in the <i>E. coli</i> strain. This suggests that transformation is likely a better suited method for transposon delivery in <i>E. coli</i> than in <i>P. putida</i>. </p>

<p><strong>Site-specificity of the miniTn7-Gm insertions</strong> obtained in <i>E. coli</i> DH5&alpha; was determined by PCR amplification as described above for </i>P. putida</i>. 11 out of 12 candidates tested in this fashion displayed the expected 164 bp band (data not shown), indicating that miniTn7BB-Gm efficiently inserts at the chromosomal attTn7 site of <i>E. coli</i>. </p>

           <h3>Conclusions</i></h3>

<p>Taken together, the characterization of the miniTn7BB-Gm minitransposon indicates that this element is <strong>able to transpose efficiently</strong> in both an enterobacterial (<i>E. coli</i>), and a non-enterobacterial (<i>P. putida</i>) host, consistent with the previously described wide host-range of Tn7. The transposon can be delivered by means of <strong>electrotransformation, heat-shock transformation and conjugal mating</strong> from three different delivery vectors. We have also shown that most transposition events in both hosts occur in a <strong>site-specific fashion</strong>, at the known attTn7 sites present in their chromosomes. Finally, we have shown that the gentamycin resistance can be excised by <strong>flipase-dependent site-specific recombination</strong> at least in one of the hosts (<i>P. putida</i>), leaving an unmarked strain that may be suitable for applications in which the use of drug resistance markers is not acceptable.</p>


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