Difference between revisions of "Team:UNebraska-Lincoln/Results"

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{{UNebraska-Lincoln}}
 
{{UNebraska-Lincoln}}
 
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<div class="column full_size">
 
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<h1>Results</h1>
 
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<p>Here you can describe the results of your project and your future plans. </p>
 
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</div>
 
  
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.body-content{
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position: relative;
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<h3>What should this page contain?</h3>
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<ul>
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padding-top: 100px;
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<li> Clearly and objectively describe the results of your work.</li>
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display: flex;
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<li> Future plans for the project. </li>
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<li> Considerations for replicating the experiments. </li>
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<h3>Describe what your results mean </h3>
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<ul>
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        text-align: center;
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<li> Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for. </li>
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<li> Show data, but remember <b>all measurement and characterization data must also be on the part's Main Page on the Registry.</b> Otherwise these data will not be in consideration for any medals or part awards! </li>
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<li> Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project. </li>
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</ul>
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</div>
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<h3> Project Achievements </h3>
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margin-bottom: 2.25rem;
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}
  
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<p>You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.</p>
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.footer h4 {
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margin-bottom: 1rem;
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<ul>
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.footer .list-unstyled .fas {
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<li>A list of linked bullet points of the successful results during your project</li>
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color: #00bfd8;
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<li>A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.</li>
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<div class="column third_size" >
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.footer .fa-stack .fa-stack-2x {
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<div class="highlight decoration_A_full">
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color: #00bfd8;
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<h3>Inspiration</h3>
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transition: all 0.2s ease;
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<p>See how other teams presented their results.</p>
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    padding-left:5px ;
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<ul>
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    padding-right: 5px;
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<li><a href="https://2014.igem.org/Team:TU_Darmstadt/Results/Pathway">2014 TU Darmstadt </a></li>
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}
−
<li><a href="https://2014.igem.org/Team:Imperial/Results">2014 Imperial </a></li>
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<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Results">2014 Paris Bettencourt </a></li>
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</ul>
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</div>
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</div>
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                                            <div class="collapse navbar-collapse" id="myNavbar">
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                                                <li><a href="#section1">Results</a></li>
 +
                                                <li><a href="#section2">Sensing</a></li>
 +
                                                <li><a href="#section3">Motility</a></li>
 +
                                                <li><a href="#section4">Killing</a></li>
 +
                                              </ul>
 +
                                            </div>
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                                          </div>
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                            <br/>
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                            <br/>
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                            <br/>
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                            <!-- card start -->
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                            <div class="row">
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                                <div id="section1" class="col-xs-12">
 +
                                    <h1>Results</h1>
 +
                                    <p>Below are the results of the characterization of the three mechanisms that make up our system: sensing, motility, and killing.</p>
 +
                                </div>
 +
                            </div>
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                            <!-- card end-->
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                            <br/>
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                            <br/>
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                            <br/>
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                            <!-- card start -->
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                            <div class="row">
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                                <div id="section2" class="col-xs-12">
 +
                                    <h1>Sensing</h1>
 +
                                   
 +
                                        <p>
 +
                                                <strong>Accessory gene regulator<em> (agr) </em>Sensing</strong>
 +
                                                </p>
 +
                                                <p>
 +
                                                <strong><span style="text-decoration:underline;">Purpose: </span></strong>
 +
                                                </p>
 +
                                                <p>
 +
                                                We constructed a new BioBrick for the <em>agr</em> system, BBa_K3191101, that includes a sequence modification of an existing <em>agr</em> BioBrick: BBa_K1022100. We first conducted experiments to find if BBa_K3191101 function is improved when compared to BBa_K1022100.
 +
                                                </p>
 +
                                                <p>
 +
                                                The native <em>agr</em> system contains SarA, which is a transcriptional regulator believed to activate P2, increasing transcription in the <em>agr</em> system. To determine if SarA improves P2 transcription, we conducted a second set of experiments adding SarA to BBa_K3191101. The new construct, BBa_K3191102, contains the same sequence as BBa_K3191101 with the addition of the SarA gene.
 +
                                                </p>
 +
                                                <p>
 +
                                                <strong>Results:</strong>
 +
                                                </p>
 +
                                                <img height=auto width=100% src="https://static.igem.org/mediawiki/2019/8/8f/T--UNebraska-Lincoln--iagr_data_1.png" alt="alt_text" title="image_tooltip">
 +
                                               
 +
                                                <p>
 +
                                                Figure 1: Fluorescence values for BBa_K206000, BBa_K1022100, and BBa_K3191101 under varying concentrations of AIP and arabinose using the standard characterization protocol.
 +
 +
                                               
 +
                                                <br/>
 +
                                                <br/>
 +
                                                </p>
 +
                                                <img height=auto  width=100% src="https://static.igem.org/mediawiki/2019/b/b2/T--UNebraska-Lincoln--iagr_data_2.png" width="" alt="alt_text" title="image_tooltip">
 +
                                               
 +
                                                <p>
 +
                                                Figure 2: P-values for fluorescence data of BBa_K206000, BBa_K1022100, and BBa_K3191101 under varying concentrations of AIP and arabinose using the standard characterization protocol.
 +
                                                </p>
 +
 +
                                                <br/>
 +
                                                <br/>
 +
                                                <br/>
 +
                                                <br/>
 +
                                                <p>
 +
                                               
 +
                                               
 +
                                               
 +
                                               
 +
                                                <img height=auto width=100% src="https://static.igem.org/mediawiki/2019/3/3e/T--UNebraska-Lincoln--iagr_data_3.png" width="" alt="alt_text" title="image_tooltip">
 +
                                               
 +
                                                </p>
 +
                                                <p>
 +
                                                Figure 3: Fluorescence values for BBa_K206000, BBa_K1022100, BBa_K3191101, and BBa_K3191102 using the modified sensing characterization protocol
 +
                                                </p>
 +
 +
                                                <br/>
 +
                                                <br/>
 +
                                                <img height=auto  width=100% src="https://static.igem.org/mediawiki/2019/4/40/T--UNebraska-Lincoln--iagr_data_4.png" width="" alt="alt_text" title="image_tooltip">
 +
                                               
 +
                                                <p>
 +
                                                Figure 4: P-values of fluorescence values for BBa_K206000, BBa_K1022100, BBa_K3191101, and BBa_K3191102 using the modified sensing characterization protocol
 +
                                                </p>
 +
                                                <p>
 +
                                                        <br/>
 +
                                                        <br/>
 +
                                                <strong>Discussion:</strong>
 +
                                                </p>
 +
                                                <p>
 +
                                                Our characterization data displays the difference in fluorescence values between BBa_K206000, BBa_K1022100, BBa_K3191101, and BBa_K3191102. This data was analyzed with a two-tail t-test of unequal variances with a significance level of 0.05 to draw conclusions of significance. Based on these values, fluorescence for BBa_K1022100 and BBa_K3191101 tend to exceed the control. This demonstrates that BBa_K1022100 and BBa_K3191101 both function properly.
 +
                                                </p>
 +
                                                <p>
 +
                                                Figure 1 demonstrates fluorescence values under the standard characterization procedure, with figure 2 listing p-values. When comparing total fluorescence, BBa_K1022100 tends to yield significantly greater fluorescence values than BBa_K3191101 at hour 8, as supported by a p-values of 0.002 for the AIP trial. This suggests the modified sequences, while more complete, do not successfully increase GFP production under the original characterization procedures. The addition of SarA also does not appear to provide a significantly greater fluorescence under the influence of AIP. This is seen in a comparison of BBa_K3191101 and BBa_K3191102 at hour 3 in figure 4 (p-value = 0.902).
 +
                                                </p>
 +
                                                <p>
 +
                                                    Experimental results display two trends when AIP or arabinose are added to solution. First, fluorescence values notably decrease when arabinose is induced. In figure 1, 0.2% arabinose is added to the samples in wells B1-B9 and C7-D3 correspond to hour 0, while wells E1-E9 and F7-G3 correspond to hour 8. Other wells do not possess arabinose and display a much greater fluorescence value. This shows that fluorescence values are consistently lower for cells in the presence of arabinose. We hypothesize that overproduction of AgrC and AgrA proteins in the presence of arabinose may have a detrimental effect on the cells, preventing growth and fluorescence.
 +
                                                </p>
 +
                                                <p>
 +
                                                    Secondly, the introduction of AIP appears to have a greater effect on BBa_K3191101 and BBa_K3191102 than on BBa_K1022100 under the modified characterization conditions. The fluorescence values for these conditions are seen in figure 3, and the corresponding p-values are listed in figure 4. In these conditions, a Tris-EDTA solution is used to attempt to increase AIP intake by the cells. BBa_K1022100 does not experience significantly greater fluorescence than the control at hour 3 with AIP induction (p-value = 0.111). In comparison, BBa_K3191101 and BBa_K3191102 have significantly greater fluorescence at hour 3 with AIP induction. This is demonstrated with p-values of 0.010 for BBa_K3191101 and 0.002 for  BBa_K3191102. This suggests that AIP induction does not significantly increase fluorescence in BBa_K1022100, while it does increase in BBa_K3191101 and BBa_K3191102.
 +
                                                </p>
 +
                                                <p>
 +
                                                The increase in fluorescence may be explained by the modified experimental procedure. If the presence of Tris-EDTA successfully increases AIP uptake, then an increase in AIP uptake only improves fluorescence in BBa_K3191101 and BBa_K3191102. This suggests that our parts are truly induced by AIP, while BBa_K1022100 is not.
 +
                                                </p>
 +
                                </div>
 +
                            </div>
 +
                            <!-- card end-->
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 +
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 +
                            <div class="row">
 +
                                <div id="section3" class="col-xs-12">
 +
                                    <h1>CheZ Motility</h1>
 +
 +
<p>
 +
        Purpose: CheZ directed motility allows for the controlled movement of cells in response to a chemical stimulus.  Overexpression of CheZ has been noted to constrict overall movement, therefore our system utilizes a degron sequence, YbaQ, to control this phenomenon. Degron sequences tag a protein for degradation, preventing CheZ overexpression in our system. The CheZ-YbaQ sequence is placed under the control of a P2 promoter controlled by the <em>agr</em> system as a means to encourage movement towards AIP.
 +
        </p>
 +
        <p>
 +
        After characterization, we concluded our degron was successful, however overall motility failed.
 +
        </p>
 +
        <p>
 +
        The strain UU2685 <em>E. coli</em> cells without the transformed CheZ system exhibited the expected lack of motility. The cells transformed with our CheZ parts, pBAD+CheZ and pBAD+CheZ+YbaQ displayed significant motility, even without the induction of arabinose.
 +
        </p>
 +
        <p>
 +
        The pBAD+CheZ showed consistent diffusion motility for both 0% and 0.02% induction. A two-tail t-test of unequal variances with a significance level of 0.05 displayed a p-value of 0.092, indicating a lack of significance in the area of diffusion. This contradicts the hypothesis of pBAD+CheZ relying on arabinose induction for motility. The reasoning behind this lack of significance is inconclusive, but may be a result of arabinose contamination from an unaccounted source such as the cell media. An additional possibility is our overestimation of promoter regulation. The pBAD+CheZ induced with 0.2% arabinose displayed motility significantly different (p-value of 0.005) from the combined motility of uninduced pBAD+CheZ and pBAD+CheZ induced with 0.02% arabinose. It was concluded this was likely a result of the greater concentration of arabinose providing the cells with more sugar to consume over time, permitting a greater displacement.
 +
        </p>
 +
        <p>
 +
                <img height=auto  width=100% src="https://static.igem.org/mediawiki/2019/0/0d/T--UNebraska-Lincoln--pbadchez021.jpg" width="" alt="alt_text" title="image_tooltip">
 +
                <p><strong>Fig1.</strong>  (pBAD+CheZ 0.2%)</p>
 +
                <br/>
 +
                <br/>
 +
                <img height=auto  width=100% src="https://static.igem.org/mediawiki/2019/9/9a/T--UNebraska-Lincoln--pbadchez0021.jpg" width="" alt="alt_text" title="image_tooltip">
 +
                <p><strong>Fig2.</strong>  (pBAD+CheZ 0.02%)                    </p> 
 +
                <br/>
 +
                <br/>
 +
                <img height=auto  width=100% src="https://static.igem.org/mediawiki/2019/2/2b/T--UNebraska-Lincoln--pbadchez01.jpg" width="" alt="alt_text" title="image_tooltip">
 +
                <p><strong>Fig3.</strong>  (pBAD+CheZ 0%)p>
 +
                        <br/>
 +
                        <br/>
 +
        <table>
 +
          <tr>
 +
          <td>
 +
          </td>
 +
          <td>
 +
          </td>
 +
          </tr>
 +
        </table>
 +
       
 +
       
 +
        <p>
 +
        The pBAD+CheZ+YbaQ induced with 0.02% arabinose produced an area of diffusion comparable to that of pBAD+CheZ+YbaQ when induced with 0% arabinose. A two-tail t-test of unequal variances with a significance level of 0.05 displayed a p-value of 0.978, indicating a lack of significant difference. The lack of significance can be attributed to similar conditions discussed in the pBAD+CheZ analysis when the same trend was observed. The pBAD+CheZ+YbaQ induced with 0.2% arabinose was significantly more motile than the motilities of pBAD+CheZ+YbaQ with no induction and 0.02% induction combined. This was maintained by a p-value of 0.0039 generated by a t-test similar to the one mentioned above.
 +
        </p>
 +
        <p>
 +
 +
                <img height=auto  width=100% src="https://static.igem.org/mediawiki/2019/a/af/T--UNebraska-Lincoln--pbadchezybaq002.jpg" width="" alt="alt_text" title="image_tooltip">
 +
                <p><strong>Fig4.</strong>  (pBAD+CheZ+YbaQ 0.2%) </p>
 +
                <br/>
 +
                <br/>
 +
                <img height=auto  width=100% src="https://static.igem.org/mediawiki/2019/9/9a/T--UNebraska-Lincoln--pbadchez0021.jpg" width="" alt="alt_text" title="image_tooltip">
 +
                <p><strong>Fig5.</strong>  (pBAD+CheZ+YbaQ 0.02%)              </p> 
 +
                <br/>
 +
                <br/>
 +
                <img height=auto  width=100% src="https://static.igem.org/mediawiki/2019/2/2b/T--UNebraska-Lincoln--pbadchez01.jpg" width="" alt="alt_text" title="image_tooltip">
 +
                <p><strong>Fig6.</strong> (pBAD+CheZ+YbaQ 0%)
 +
                </p>
 +
                        <br/>
 +
                        <br/>
 +
 +
        <p>
 +
        The motility observed for the pBAD+CheZ construct with no arabinose induction was registered as a false positive; the lack of arabinose should have yielded no motility. This implies the presence of background CheZ. However, the pBAD+CheZ+YbaQ displayed significantly less motility (two-tail t-test p-value of 0.02) indicating a decrease in the CheZ protein with the presence of the degron. We have concluded this indicates the degron is successful in degrading excess CheZ to allow for the efficient function of chemotaxis. The inhibition of excess CheZ is imperative to the success of the motility mechanism because it ensures that MRSA is precisely targeted as intended. Under optimal conditions (an induction of 0.2% arabinose), pBAD+CheZ showed no significant difference (p-value of 0.766) in motility compared to pBAD+CheZ+YbaQ.
 +
        </p>
 +
        <p>
 +
        While there was significant diffusion of the CheZ parts as detailed above, there were no indicators of successful directionality. The cells proliferated in a circular region that contested any form of directed motility.
 +
        </p>
 +
        <p>
 +
        P2+CheZ+YbaQ was never successfully sequenced and therefore never characterized. It remains unknown how the P2 promoter would have reacted in the context of our sensing system.
 +
        </p>
 +
        <br/>
 +
                <br/>
 +
                <img height=auto  width=100% src="https://static.igem.org/mediawiki/2019/b/be/T--UNebraska-Lincoln--motilty.jpeg" width="" alt="alt_text" title="image_tooltip">
 +
                <p><strong>Fig7.</strong> Motility Assays
 +
                </p>
 +
                       
 +
                                </div>
 +
                            </div>
 +
                            <!-- card end-->
 +
 +
                            <br/>
 +
                            <br/>
 +
                            <br/>
 +
                            <!-- card start -->
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                            <div class="row">
 +
                                <div id="section4" class="col-xs-12">
 +
                                    <h1>Killing</h1>
 +
                                </div>
 +
                            </div>
 +
                            <!-- card end-->
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                            <h4>Address</h4>
 +
                            <p>University of Nebraska-Lincoln iGEM Team
 +
                            <br />634 Hamilton Hall
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                            <br />Lincoln, NE 68588-0304</p>
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Revision as of 02:35, 22 October 2019




Results

Below are the results of the characterization of the three mechanisms that make up our system: sensing, motility, and killing.




Sensing

Accessory gene regulator (agr) Sensing

Purpose:

We constructed a new BioBrick for the agr system, BBa_K3191101, that includes a sequence modification of an existing agr BioBrick: BBa_K1022100. We first conducted experiments to find if BBa_K3191101 function is improved when compared to BBa_K1022100.

The native agr system contains SarA, which is a transcriptional regulator believed to activate P2, increasing transcription in the agr system. To determine if SarA improves P2 transcription, we conducted a second set of experiments adding SarA to BBa_K3191101. The new construct, BBa_K3191102, contains the same sequence as BBa_K3191101 with the addition of the SarA gene.

Results:

alt_text

Figure 1: Fluorescence values for BBa_K206000, BBa_K1022100, and BBa_K3191101 under varying concentrations of AIP and arabinose using the standard characterization protocol.

alt_text

Figure 2: P-values for fluorescence data of BBa_K206000, BBa_K1022100, and BBa_K3191101 under varying concentrations of AIP and arabinose using the standard characterization protocol.





alt_text

Figure 3: Fluorescence values for BBa_K206000, BBa_K1022100, BBa_K3191101, and BBa_K3191102 using the modified sensing characterization protocol



alt_text

Figure 4: P-values of fluorescence values for BBa_K206000, BBa_K1022100, BBa_K3191101, and BBa_K3191102 using the modified sensing characterization protocol



Discussion:

Our characterization data displays the difference in fluorescence values between BBa_K206000, BBa_K1022100, BBa_K3191101, and BBa_K3191102. This data was analyzed with a two-tail t-test of unequal variances with a significance level of 0.05 to draw conclusions of significance. Based on these values, fluorescence for BBa_K1022100 and BBa_K3191101 tend to exceed the control. This demonstrates that BBa_K1022100 and BBa_K3191101 both function properly.

Figure 1 demonstrates fluorescence values under the standard characterization procedure, with figure 2 listing p-values. When comparing total fluorescence, BBa_K1022100 tends to yield significantly greater fluorescence values than BBa_K3191101 at hour 8, as supported by a p-values of 0.002 for the AIP trial. This suggests the modified sequences, while more complete, do not successfully increase GFP production under the original characterization procedures. The addition of SarA also does not appear to provide a significantly greater fluorescence under the influence of AIP. This is seen in a comparison of BBa_K3191101 and BBa_K3191102 at hour 3 in figure 4 (p-value = 0.902).

Experimental results display two trends when AIP or arabinose are added to solution. First, fluorescence values notably decrease when arabinose is induced. In figure 1, 0.2% arabinose is added to the samples in wells B1-B9 and C7-D3 correspond to hour 0, while wells E1-E9 and F7-G3 correspond to hour 8. Other wells do not possess arabinose and display a much greater fluorescence value. This shows that fluorescence values are consistently lower for cells in the presence of arabinose. We hypothesize that overproduction of AgrC and AgrA proteins in the presence of arabinose may have a detrimental effect on the cells, preventing growth and fluorescence.

Secondly, the introduction of AIP appears to have a greater effect on BBa_K3191101 and BBa_K3191102 than on BBa_K1022100 under the modified characterization conditions. The fluorescence values for these conditions are seen in figure 3, and the corresponding p-values are listed in figure 4. In these conditions, a Tris-EDTA solution is used to attempt to increase AIP intake by the cells. BBa_K1022100 does not experience significantly greater fluorescence than the control at hour 3 with AIP induction (p-value = 0.111). In comparison, BBa_K3191101 and BBa_K3191102 have significantly greater fluorescence at hour 3 with AIP induction. This is demonstrated with p-values of 0.010 for BBa_K3191101 and 0.002 for BBa_K3191102. This suggests that AIP induction does not significantly increase fluorescence in BBa_K1022100, while it does increase in BBa_K3191101 and BBa_K3191102.

The increase in fluorescence may be explained by the modified experimental procedure. If the presence of Tris-EDTA successfully increases AIP uptake, then an increase in AIP uptake only improves fluorescence in BBa_K3191101 and BBa_K3191102. This suggests that our parts are truly induced by AIP, while BBa_K1022100 is not.




CheZ Motility

Purpose: CheZ directed motility allows for the controlled movement of cells in response to a chemical stimulus. Overexpression of CheZ has been noted to constrict overall movement, therefore our system utilizes a degron sequence, YbaQ, to control this phenomenon. Degron sequences tag a protein for degradation, preventing CheZ overexpression in our system. The CheZ-YbaQ sequence is placed under the control of a P2 promoter controlled by the agr system as a means to encourage movement towards AIP.

After characterization, we concluded our degron was successful, however overall motility failed.

The strain UU2685 E. coli cells without the transformed CheZ system exhibited the expected lack of motility. The cells transformed with our CheZ parts, pBAD+CheZ and pBAD+CheZ+YbaQ displayed significant motility, even without the induction of arabinose.

The pBAD+CheZ showed consistent diffusion motility for both 0% and 0.02% induction. A two-tail t-test of unequal variances with a significance level of 0.05 displayed a p-value of 0.092, indicating a lack of significance in the area of diffusion. This contradicts the hypothesis of pBAD+CheZ relying on arabinose induction for motility. The reasoning behind this lack of significance is inconclusive, but may be a result of arabinose contamination from an unaccounted source such as the cell media. An additional possibility is our overestimation of promoter regulation. The pBAD+CheZ induced with 0.2% arabinose displayed motility significantly different (p-value of 0.005) from the combined motility of uninduced pBAD+CheZ and pBAD+CheZ induced with 0.02% arabinose. It was concluded this was likely a result of the greater concentration of arabinose providing the cells with more sugar to consume over time, permitting a greater displacement.

alt_text

Fig1. (pBAD+CheZ 0.2%)



alt_text

Fig2. (pBAD+CheZ 0.02%)



alt_text

Fig3. (pBAD+CheZ 0%)p>

The pBAD+CheZ+YbaQ induced with 0.02% arabinose produced an area of diffusion comparable to that of pBAD+CheZ+YbaQ when induced with 0% arabinose. A two-tail t-test of unequal variances with a significance level of 0.05 displayed a p-value of 0.978, indicating a lack of significant difference. The lack of significance can be attributed to similar conditions discussed in the pBAD+CheZ analysis when the same trend was observed. The pBAD+CheZ+YbaQ induced with 0.2% arabinose was significantly more motile than the motilities of pBAD+CheZ+YbaQ with no induction and 0.02% induction combined. This was maintained by a p-value of 0.0039 generated by a t-test similar to the one mentioned above.

alt_text

Fig4. (pBAD+CheZ+YbaQ 0.2%)



alt_text

Fig5. (pBAD+CheZ+YbaQ 0.02%)



alt_text

Fig6. (pBAD+CheZ+YbaQ 0%)



The motility observed for the pBAD+CheZ construct with no arabinose induction was registered as a false positive; the lack of arabinose should have yielded no motility. This implies the presence of background CheZ. However, the pBAD+CheZ+YbaQ displayed significantly less motility (two-tail t-test p-value of 0.02) indicating a decrease in the CheZ protein with the presence of the degron. We have concluded this indicates the degron is successful in degrading excess CheZ to allow for the efficient function of chemotaxis. The inhibition of excess CheZ is imperative to the success of the motility mechanism because it ensures that MRSA is precisely targeted as intended. Under optimal conditions (an induction of 0.2% arabinose), pBAD+CheZ showed no significant difference (p-value of 0.766) in motility compared to pBAD+CheZ+YbaQ.

While there was significant diffusion of the CheZ parts as detailed above, there were no indicators of successful directionality. The cells proliferated in a circular region that contested any form of directed motility.

P2+CheZ+YbaQ was never successfully sequenced and therefore never characterized. It remains unknown how the P2 promoter would have reacted in the context of our sensing system.



alt_text

Fig7. Motility Assays




Killing

Address

University of Nebraska-Lincoln iGEM Team
634 Hamilton Hall
Lincoln, NE 68588-0304

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