http://2010.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=250&target=Lepavgom&year=&month=2010.igem.org - User contributions [en]2024-03-29T02:11:34ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:UPO-Sevilla/Project/Assays/ProtocolsTeam:UPO-Sevilla/Project/Assays/Protocols2010-10-27T20:54:29Z<p>Lepavgom: </p>
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<h1>Protocols</h1><br />
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<th colspan="2">ASSAY</th><br />
<th>Download</th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr><br />
<td class="headRow" rowspan="2">QUALITATIVE</td><br />
<td colspan="2">Soft Agar Plates</td><br />
<td><a href="https://static.igem.org/mediawiki/2010/8/87/BacterialCrowdingSoftAgarPlates.pdf"><img src="https://static.igem.org/mediawiki/2010/7/7a/BacterialCrowdingDownload.jpeg" alt="Bacterial Crowding Protocol Download"></a></td><br />
</tr><br />
<tr><br />
<td colspan="2">Capillary Assay using microscope</td><br />
<td><a href="https://static.igem.org/mediawiki/2010/5/5d/BacterialCrowdingCapillaryAssayUsingMicroscope.pdf"><img src="https://static.igem.org/mediawiki/2010/7/7a/BacterialCrowdingDownload.jpeg" alt="Bacterial Crowding Protocol Download"></a></td><br />
</tr><br />
<tr><br />
<td class="headRow" rowspan="7">QUANTITATIVE</td><br />
<td class="headRow">Bacterial dilution recipient or Chemotaxis Chambers</td><br />
<td class="headRow"> Attractant or Buffer recipient or Capillaries</td><br />
<td class="headRow">&nbsp;</td><br />
</tr><br />
<tr><br />
<td>tip chambers</td><br />
<td>syringe’s needles (more or less thin)</td><br />
<td rowspan="3"><a href="https://static.igem.org/mediawiki/2010/c/ce/BacterialCrowdingCapillaryAssayTipChambers.pdf"><img src="https://static.igem.org/mediawiki/2010/7/7a/BacterialCrowdingDownload.jpeg" alt="Bacterial Crowding Protocol Download"></a></td><br />
</tr><br />
<tr><br />
<td>needle’s cups or heated sealed 100ml tips</td><br />
<td>Needles</td><br />
</tr><br />
<tr><br />
<td>tip chambers</td><br />
<td>10 &#956;l micropipette’s tips</td><br />
</tr><br />
<tr><br />
<td>96-well PVC microplates</td><br />
<td>1 &#956;l capillary pipettes</td><br />
<td><a href="https://static.igem.org/mediawiki/2010/4/4b/BacterialCrowdingCapillaryAssayUsing96.pdf"><img src="https://static.igem.org/mediawiki/2010/7/7a/BacterialCrowdingDownload.jpeg" alt="Bacterial Crowding Protocol Download"></a></td><br />
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<tr><br />
<td>drop between microscope slide and cover slip</td><br />
<td>1 &#956;l capillary pipettes</td><br />
<td><a href="https://static.igem.org/mediawiki/2010/5/5d/BacterialCrowdingCapillaryAssayUsingMicroscope.pdf"><img src="https://static.igem.org/mediawiki/2010/7/7a/BacterialCrowdingDownload.jpeg" alt="Bacterial Crowding Protocol Download"></a></td><br />
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<tr><br />
<td>Flow-chamber</td><br />
<td>1 &#956;l capillary pipettes</td><br />
<td><a href="https://static.igem.org/mediawiki/2010/a/af/BacterialCrowdingCapillaryAssayUsingFlowChambers.pdf"><img src="https://static.igem.org/mediawiki/2010/7/7a/BacterialCrowdingDownload.jpeg" alt="Bacterial Crowding Protocol Download"></a></td><br />
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</div></div>Lepavgomhttp://2010.igem.org/Team:UPO-Sevilla/Project/Assays/ProtocolsTeam:UPO-Sevilla/Project/Assays/Protocols2010-10-27T20:52:56Z<p>Lepavgom: </p>
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<td class="headRow" rowspan="2">QUALITATIVE</td><br />
<td colspan="2">Soft Agar Plates</td><br />
<td><a href="https://static.igem.org/mediawiki/2010/8/87/BacterialCrowdingSoftAgarPlates.pdf"><img src="https://static.igem.org/mediawiki/2010/7/7a/BacterialCrowdingDownload.jpeg" alt="Bacterial Crowding Protocol Download"></a></td><br />
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<td colspan="2">Capillary Assay using microscope</td><br />
<td><a href="https://static.igem.org/mediawiki/2010/5/5d/BacterialCrowdingCapillaryAssayUsingMicroscope.pdf"><img src="https://static.igem.org/mediawiki/2010/7/7a/BacterialCrowdingDownload.jpeg" alt="Bacterial Crowding Protocol Download"></a></td><br />
</tr><br />
<tr><br />
<td class="headRow" rowspan="7">QUANTITATIVE</td><br />
<td class="headRow">Bacterial dilution recipient or Chemotaxis Chambers</td><br />
<td class="headRow"> Attractant or Buffer recipient or Capillaries</td><br />
<td class="headRow">&nbsp;</td><br />
</tr><br />
<tr><br />
<td>tip chambers</td><br />
<td>syringe’s needles (more or less thin)</td><br />
<td rowspan="3"><a href="https://static.igem.org/mediawiki/2010/c/ce/BacterialCrowdingCapillaryAssayTipChambers.pdf"><img src="https://static.igem.org/mediawiki/2010/7/7a/BacterialCrowdingDownload.jpeg" alt="Bacterial Crowding Protocol Download"></a></td><br />
</tr><br />
<tr><br />
<td>needle’s cups or heated sealed 100ml tips</td><br />
<td>Needles</td><br />
</tr><br />
<tr><br />
<td>tip chambers</td><br />
<td>10 &#956;l micropipette’s tips</td><br />
</tr><br />
<tr><br />
<td>96-well PVC microplates</td><br />
<td>1 &#956;l capillary pipettes</td><br />
<td><a href="https://static.igem.org/mediawiki/2010/4/4b/BacterialCrowdingCapillaryAssayUsing96.pdf"><img src="https://static.igem.org/mediawiki/2010/7/7a/BacterialCrowdingDownload.jpeg" alt="Bacterial Crowding Protocol Download"></a></td><br />
</tr><br />
<tr><br />
<td>drop between microscope slide and cover slip</td><br />
<td>1 &#956;l capillary pipettes</td><br />
<td><a href=""><img src="https://static.igem.org/mediawiki/2010/7/7a/BacterialCrowdingDownload.jpeg" alt="Bacterial Crowding Protocol Download"></a></td><br />
</tr><br />
<tr><br />
<td>Flow-chamber</td><br />
<td>1 &#956;l capillary pipettes</td><br />
<td><a href="https://static.igem.org/mediawiki/2010/a/af/BacterialCrowdingCapillaryAssayUsingFlowChambers.pdf"><img src="https://static.igem.org/mediawiki/2010/7/7a/BacterialCrowdingDownload.jpeg" alt="Bacterial Crowding Protocol Download"></a></td><br />
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</div></div>Lepavgomhttp://2010.igem.org/Team:UPO-Sevilla/Project/AssaysTeam:UPO-Sevilla/Project/Assays2010-10-27T20:52:31Z<p>Lepavgom: </p>
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<h1>Introduction</h1><br />
<br />
<p><strong>Motility</strong> is one of the most readily demonstrated bacterial characters, and <strong>chemotaxis</strong> is one of the most studied bacterial behaviors. Motile organisms are attracted by certain chemicals and repelled by others (positive and negative chemotaxis). Quantification of chemotactic motion is necessary to identify chemoeffectors and to determine the structure of bacterial communities.</p><br />
<br />
<p>Current methods of quantifying chemotaxis use chemotactic bacteria such as <i>Escherichia Coli</i>, which is assayed by measuring the number of organisms attracted into a capillary tube containing a chemoattractant.</p><br />
<br />
<p>UPO-Sevilla team has carried out different experimental prototypes which try to have under control all the parameters involved in chemotaxis. The goal of the group was to design different assays that allow us to study this effect in both point of views, <strong>qualitative</strong> and <strong>quantitative</strong>. Also we had to test induction of sensing systems and chemoattractant production. Anyway, these behaviours are highly related with the chemotaxis process in Bacterial Crowding project. This is why we wanted to measure chemoattractant production by counting the range of attracted bacteria by using chemotaxis assays. The induction of sensing systems could be tested by using GFP measures when its promoter is <i>PfecA</i> or <i>PprhJ</i>; or also due to the levels of chemoattractant production. </p><br />
<br />
<h1>Qualitative Assays</h1><br />
<br />
<h2>Agar Soft Plates</h2><br />
<br />
<p>Our qualitative assays were made in <strong>soft agar plates</strong> thanks to the protocols we received from Ph.D Parkinson (University of Utah).</p><br />
<br />
<p>This kind of plates allows bacteria to swim through the agar freely and to show their chemotactic capacities. A colony, inserted in soft agar plate, starts to grow while running out of the environmental sources. For this reason chemotactic bacteria move to places where the sources are not limited. That phenomenon produces a number of <strong>halos</strong> which are spread within the plate and increase in volume as the sources are lowered. The number of halos give us information about the number of chemoattractants that are running out.</p><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/3/36/BacterialCrowdingSoftAgarPlate.png" alt="Soft Agar Plate Assay"/><br />
<p class="caption"><i><strong>Fig 1. Soft agar plate assay.</strong> Different <i>E. coli</i> strains are shown. Every strain carries out a mutation that affects its chemotactic response, but the wild type. In the wt you can observe two halos produced by chemotactic responses to aspartate (bigger halo) and glutamate.</i></p><br />
<br />
<br />
<p>The assay protocol is simple; once the soft agar plates are prepared, a colony is inserted in the agar. Let it grow at 30ºC. The soft agar is a delicate element, so it is important to be careful when moving the plates.</p><br />
<br />
<p>In those plates it could appear different concentric circles which represent chemotaxis to certain attractants. For instance, when two amino acids, which act as chemoattractants, are running out from the medium, two circles will appear. The inner one will show the amino acids limit with low chemotactic response; while the outer one will mean that the amino acid which causes a higher response is running down.</p><br />
<br />
<h2>Optical and Fluorescence Microscopy</h2><br />
<br />
<p>The <strong>microscopy techniques</strong> allow us to see the development of the assay <i>in situ</i> without waiting. In this part we will see how we can carry out an experiment that we could see under the microscope.</p><br />
<br />
<div class="imgLeft"><br />
<img class="ileft" src="https://static.igem.org/mediawiki/2010/4/4e/BacterialCrowdingCapillaryAssay.png" alt="Capillary assay look under a microscope" /><br />
<p class="caption"><i><strong>Fig 2.</strong> Preparation of capillary assay to look under a microscope.</i></p><br />
</div><br />
<br />
<p>Two capillaries are put over a microscope slide which will hold up a cover slip. Then we insert the bacterial dilution between the slide and the cover slip. Two new capillaries are inserted between the slide and the cover slip, inside of the bacterial dilution. One of those capillaries would contain a chemoattractant while the other one would be the control. Under a microscope we could see the difference between both capillaries and we would definitely be able to observe if there is chemotaxis toward this chemoattractant.</p><br />
<br />
<p>Apart from that, we could detect fluorescence emitted by a fluorophore which should be presented in bacteria by using a fluorescent microscope.</p><br />
<br />
<p>This assay can become quantitative too if we spread on agar plates the content of capillaries and count the number of colonies that grew there. Also we could measure the fluorescence inside each capillary, taking to this way other quantitative results.</p><br />
<br />
<div class="clear"></div><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/6/6f/BacterialCrowdingCapillaryAssayMicroscope.png" alt="Capillary assay Pictures"/><br />
<p class="caption"><i><strong>Fig 3.</strong>Results of a capillary assay using microscope techniques. We can see that the chemotatic response toward aspartate is increasing as time passes by. Also there are major differences between the control without aspartate and the control with aspartate.</i></p><br />
<br />
<br />
<h1>Quantitative Assays</h1><br />
<br />
<h2>Capillary assays</h2><br />
<br />
<p>The capillary assays are the most useful to quantify chemotaxis. This team has performed different kinds of capillary assays.</p><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/7/73/BacterialCrowdingCapillaryPictures.png" alt="Capillary assay of E. coli chemotaxis toward aspartate"/><br />
<p class="caption"><i><strong>Fig 4.</strong> On the left, a capillary assay with 96-well PVC microplates and 1&#956;l capillary pipettes; capillaries are inserted through a 2% agarose gel in order to hold it. On the right, a capillary assay using needles and a tip chamber. Both of them were being incubated at 30ºC when pictures was taken.</i></p><br />
<br />
<ul><br />
<li><p><strong>Foundamental Points</strong></p><br />
<p>When a capillary with chemoattractant is put in a bacterial dilution a concentration gradient of chemoatractant is developed according to the <a href="http://en.wikipedia.org/wiki/Fick's_laws_of_diffusion" target="_blank">Fick law</a>. This gradient would be sensed by bacteria that are going from low to high concentration places, thus we should have someThis gradient is sensed by bacteria and they start going from low to high concentration places. This is how some of these bacteria finish into the capillary and so it is possible to demonstrate that capillaries with chemoattractant attract more bacteria than another without any substance (the control), just buffer. The control has to continue the same treatment than the other capillaries. In the same way it could be tested repellent’s efficacy by showing that in its capillary there are less bacteria than in the control.</p><br />
<br />
<div class="table"><br />
<table class="tableBio" cellspacing="0" cellpadding="0"><br />
<thead><br />
<tr><br />
<th>Bacterial dilution recipient or <strong>Chemotaxis Chambers</strong></th><br />
<th>Attractant or Buffer recipient or <strong>Capillaries</strong></th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr><br />
<td>tip chambers</td><br />
<td>syringe’s needles (more or less thin)</td><br />
</tr><br />
<tr><br />
<td>needle’s cups or heated closed 100ml tips</td><br />
<td>Needles</td><br />
</tr><br />
<tr><br />
<td>tip chambers</td><br />
<td>10&#956;l micropipette’s tips</td><br />
</tr><br />
<tr><br />
<td>96-well PVC microplates</td><br />
<td>1&#956;l capillary pipettes</td><br />
</tr><br />
<tr><br />
<td>drop between microscope slide and cover slip</td><br />
<td>1&#956;l capillary pipettes</td><br />
</tr><br />
<tr><br />
<td>Flow-chamber</td><br />
<td>1&#956;l capillary pipettes</td><br />
</tr><br />
</tbody><br />
</table><br />
</div><br />
<br />
<br />
<p class="caption"><i><strong>Table 1</strong>. Several ways to permorm chemotaxis assays we have carried out.</i></p> <br />
<br />
<p>This team has performed those assays on different scales, using differents chemotactic chambers where the bacterial dilution was put inside and with differents capillaries. Some types of these variants are presented in the Table 1. The attractant concentration in the capillary depends on the substance itself, but for aspartate the optimum is reached at 10mM.</p> <br />
<br />
<div class="imgLeft"><br />
<img class="ileft" src="https://static.igem.org/mediawiki/2010/e/ea/BacterialCrowdingFlowChamberAssay.png" alt="Flow-Chamber Capillary Assay" /><br />
<p class="caption"><i><strong>Fig 5. Flow-chamber Capillary Assay</strong>. This picture is shown a three channel flow-chamber, which acts as chemotaxis chamber, with 1&#956;l capillaries fixed in each end of one channel. At that time it was observed with a fluorescence microscope.</i></p><br />
</div><br />
<br />
<br />
<p>One assay that deserves to be explain is the <strong>Multi-capillary assay</strong>. It uses 96-well PVC microplates as chemotaxis chamber and 1μl glass capillaries. You can see a picture of it in figure 4, at the left. We designed this chemotaxis assay when we were looking for a good way to perform numerous and simultaneous capillary assays. The assembly of the assay required to make holes in a microplate lid to allow glass capillaries to go through it. The lid is put upside down over the microplate and it is filled with 2% agarose gel. We also used parafilm to avoid that the gel drip through the lid holes while it was still humid. The function of the gel is to hold capillaries, in its dry way. In orther to fill capillaries we closed one of its ends by heating and heated the glass; then it was introduce in a attractant or buffer suspension and automatically it is filled. It is important to introduce the capillaries in the lid and gel by its closed end to keep the sterility on the other end. Then it is only necessary to put the open ends of capillaries inside the wells of the microplate. Previously these wells should have been filled with a bacterial dilution. At this point the assay continues in the same way than others: 1h incubation, clean outside of capillaries with bidistilled water, dilution of the capillaries content and spread in agar plates.</p><br />
<br />
<p>We got our best results in the last kind of capillary assay showed in the table. It uses <strong>flow-chambers</strong> as chemotaxis chambers and 1&#956;l capillary pipettes as capillaries. This method allows us to quantify bacterial density in two different ways, visualization of flow-chambers by fluorescent microscope techniques and spreading bacteria inside capillaries in LB plates . Actually, we think that this assay leads to less errors than others due to some reasons: when we put a capillary into the flow-chamber the fixing is perfect and the capillary can not move, the exposed part of the capillay inside the chamber is minimal so bacteria can not attach to the outside of the capillary making errors in dilutions, also it is a small device easy to work with.</p><br />
<br />
</li><br />
<br />
<li><p><strong>Protocol</strong></p><br />
<br />
<div class="imgRight"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/1/11/BacterialCrowdingCapillaryRepresentation.png" alt="Representation of capillary assay in a tip chambers." /><br />
<p class="caption"><i><strong>Fig 6.</strong>Representation of capillary assay in a tip chambers.</i></p><br />
</div><br />
<br />
<p>The assays can start in two different ways: setting up inocula from the strains we are going to work with into triptone broth or in minimal medium, in a low shaking at 30ºC overnight. High shaking might cause the loss of flagella, and also the production of flagella would not be possible in rich medium since bacteria would not need it. </p><br />
<br />
<p>The following day the inocula should be diluted in the same medium a hundred of times and wait for the growing phase to be the appropriate. For <i>Escherichia Coli</i> it would be necessary to wait for the exponential middle phase, since it is in this phase when flagella develop the flagellar motor. For <i>Pseudomonas</i> instead it would de better to wait for the late exponential phase, as the flagellum is developed later in this organism.</p><br />
<br />
<p>Once the culture is ready, it must be changed to an appropriate medium for mobility and chemotaxis. For that, it is necessary to wash the culture twice with chemotaxis buffer by centrifuging at low speed, since flagella may be lost if culture is treated abruptly.</p><br />
<br />
<p>When the culture is in the right medium, number of bacteria should be adjusted roughly 10<sup>7</sup> fcu/ml. This dilution has to be distributed in chemotaxis chambers where our capillaries will be introduced. The volume of capillaries has not been fully established and usually we used as standard volume 100 &#956;l of diluted chemoattractant in chemotaxis buffer. It is important not to forget controls. Negative controls for chemotaxis assays are filled with chemotaxis buffer. Then chambers and capillaries have to be incubated at 30º during 60 minutes. After that, we have to quantify bacterial popularion contained into capillaries. In order to achieve that we could do it either with dilution and spread in plates or analyzing the fluorescence, supposing that bacteria have any kind of fluorophore. </p><br />
</li><br />
<br />
<li><p><strong>Advices</strong></p><br />
<p>One of the elements we bear in mind is the chemotaxis buffer: chemotaxis medium contains potassium phosphate buffer (pH 7), ethylenediaminetetraacetate (<strong>EDTA</strong>) and <strong>glycerol</strong> (energy source). Glycerol is only necessary in long incubations; meanwhile in short incubations the typical sources of bacteria are enough to maintain the chemotactic machinery. It is all-important to underline that the chemotaxis medium must be free of any other substance which may have chemotactic effects, since this could disturb the results. This is one of the reasons why the carbon source is not glucose, as you may expect. Another important detail to bear in mind is EDTA, this chelation causes the precipitation of magnesium which may dull the movement of bacteria and the flagellar machinery. It would be complicated to be successful in the chemotaxis assays without this chelation. Incubation of bacteria must be carried at 30ºC since it helps motility. Shaking must be low because flagella can be lost in high shaking.</p><br />
<br />
<p>It is crucial to be careful when <strong>choosing the strains</strong> to be used in the chemotaxis assays, since it may not have motility. Strains used in laboratories have normally no motility, as at that point they have usually suffer different screening process in benefit of immobile bacteria. A bacterium which has no motility will not have to invest any source in motility or chemotaxis; this would encourage the creation of a bigger colony and more eye-catching than usual, so Scientifics would be probably leaded to select one of this kind. This problem happened to us and we were trying to attract a non mobile strain toward different attractants until we saw the light.</p><br />
</li><br />
</ul><br />
<br />
<h2>Buridan’s Donkey</h2><br />
<br />
<p>At the beginning of the summer we wanted to test bacterial chemotaxis by using a three-channel device based on <strong>flow-chamber biofilm</strong>. It would be able to produce a linear gradient within narrow tubes that connect the chambers. The linear chemical gradient would be generated by diffusion of the chemoattractant through a dialysis membrane located on the limit of the chamber. This membrane also makes impossible the movement of the chemoattractant-producing bacteria through the tube.</p><br />
<br />
<p>The first assay would involve only chemoattractants, and the second producing bacterias. As result, it would be expected that the movement of the cells in the center chamber was directed to the chamber containing chemoattractant-producing bacterias. It is necessary to clear up that the chemoattractant production would be activated solely by the contact of bacteria with plant cell walls that reside in the same chamber. Bacteria have to “decide” between going toward control empty chamber or going toward chamber with chemoattractant-producing bacteria.</p><br />
<br />
<p>This device could provide some advantages in chemotaxis studies: rapid and easy implementation, parallel and simultaneous test, visual proofs, different assays possibilities. Also some experimental conditions could be changed easily: concentration of bacterial population, chambers distances, bacterial cultures, chemoattractans. Despite its advantages, it maybe requires more than an hour of incubation.</p><br />
<br />
<p>Although we had not enough time to perform this assay, an explaining diagram of this device is provided below.</p><br />
<br />
<h3>Measuring the performance of the chemotaxis circuits (Buridan's donkey assay principle)</h3><br />
<br />
<img class="centerBig" src="https://static.igem.org/mediawiki/2010/a/a8/BacterialCrowdingBuridanDonkey.png" alt="Measuring the performance of the chemotaxis circuits (Buridan's donkey assay principle)"/><br />
<br />
<h3>Buridan's donkey assays with three-channel flow cells</h3><br />
<br />
<img class="centerBig" src="https://static.igem.org/mediawiki/2010/3/38/BacterialCrowdingBuridanDonkeyThree.png" alt="Buridan's donkey assays with three-channel flow cells"/><br />
<br />
<p>Special acknowledgements to Ph.D Parkinson (chemotaxis researcher in University of Utah) who gave us some advices, handed us over some protocols of him, even mobile and mutant <i>E. Coli</i> strains.</p><br />
<br />
<h1>References</h1><br />
<br />
<ul><br />
<li>J. Adler (1972) A Method for Measuring Chemotaxis and Use of the Method to Determine Optimum Conditions for Chemotaxis by Escherichia coli. - Journal of General Microbiology ( I 973), 74, 77-91</li><br />
<br />
<li>Guocheng Han and Joseph J. Cooney (1993) A modified capillary assay for chemotaxis - Journal of Industrial Microbiology, 12 (1993) 396—398</li><br />
<br />
<li>Hanbin Mao, Paul S. Cremer, and Michael D. Manson (2003) A sensitive, versatile microfluidic assay for bacterial chemotaxis - PNAS MICROBIOLOGY vol. 100 no. 9 5449–5454.</li><br />
<br />
<li>Russell Bainer, Heungwon Park, Philippe Cluzel (2003) A high-throughput capillary assay for bacterial chemotaxis - Journal of Microbiological Methods 55 (2003) 315– 319.</li><br />
</ul><br />
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<td class="headRow" rowspan="2">QUALITATIVE</td><br />
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<td><a href="https://static.igem.org/mediawiki/2010/8/87/BacterialCrowdingSoftAgarPlates.pdf"><img src="https://static.igem.org/mediawiki/2010/7/7a/BacterialCrowdingDownload.jpeg" alt="Bacterial Crowding Protocol Download"></a></td><br />
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<td><a href="https://static.igem.org/mediawiki/2010/5/5d/BacterialCrowdingCapillaryAssayUsingMicroscope.pdf"><img src="https://static.igem.org/mediawiki/2010/7/7a/BacterialCrowdingDownload.jpeg" alt="Bacterial Crowding Protocol Download"></a></td><br />
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<td class="headRow" rowspan="7">QUANTITATIVE</td><br />
<td class="headRow">Bacterial dilution recipient or Chemotaxis Chambers</td><br />
<td class="headRow"> Attractant or Buffer recipient or Capillaries</td><br />
<td class="headRow">&nbsp;</td><br />
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<td>tip chambers</td><br />
<td>syringe’s needles (more or less thin)</td><br />
<td rowspan="3"><a href="https://static.igem.org/mediawiki/2010/c/ce/BacterialCrowdingCapillaryAssayTipChambers.pdf"><img src="https://static.igem.org/mediawiki/2010/7/7a/BacterialCrowdingDownload.jpeg" alt="Bacterial Crowding Protocol Download"></a></td><br />
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<td>needle’s cups or heated sealed 100ml tips</td><br />
<td>Needles</td><br />
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<tr><br />
<td>tip chambers</td><br />
<td>10 &#956;l micropipette’s tips</td><br />
</tr><br />
<tr><br />
<td>96-well PVC microplates</td><br />
<td>1 &#956;l capillary pipettes</td><br />
<td><a href="https://static.igem.org/mediawiki/2010/4/4b/BacterialCrowdingCapillaryAssayUsing96.pdf"><img src="https://static.igem.org/mediawiki/2010/7/7a/BacterialCrowdingDownload.jpeg" alt="Bacterial Crowding Protocol Download"></a></td><br />
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<td class="headRow">&nbsp;</td><br />
</tr><br />
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</tr><br />
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<td>10 &#956;l micropipette’s tips</td><br />
</tr><br />
<tr><br />
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<td>1 &#956;l capillary pipettes</td><br />
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<h1>Introduction</h1><br />
<br />
<p><strong>Motility</strong> is one of the most readily demonstrated bacterial characters, and <strong>chemotaxis</strong> is one of the most studied bacterial behaviors. Motile organisms are attracted by certain chemicals and repelled by others (positive and negative chemotaxis). Quantification of chemotactic motion is necessary to identify chemoeffectors and to determine the structure of bacterial communities.</p><br />
<br />
<p>Current methods of quantifying chemotaxis use chemotactic bacteria such as <i>Escherichia Coli</i>, which is assayed by measuring the number of organisms attracted into a capillary tube containing a chemoattractant.</p><br />
<br />
<p>UPO-Sevilla team has carried out different experimental prototypes which try to have under control all the parameters involved in chemotaxis. The goal of the group was to design different assays that allow us to study this effect in both point of views, <strong>qualitative</strong> and <strong>quantitative</strong>. Also we had to test induction of sensing systems and chemoattractant production. Anyway, these behaviours are highly related with the chemotaxis process in Bacterial Crowding project. This is why we wanted to measure chemoattractant production by counting the range of attracted bacteria by using chemotaxis assays. The induction of sensing systems could be tested by using GFP measures when its promoter is <i>PfecA</i> or <i>PprhJ</i>; or also due to the levels of chemoattractant production. </p><br />
<br />
<h1>Qualitative Assays</h1><br />
<br />
<h2>Agar Soft Plates</h2><br />
<br />
<p>Our qualitative assays were made in <strong>soft agar plates</strong> thanks to the protocols we received from Ph.D Parkinson (University of Utah).</p><br />
<br />
<p>This kind of plates allows bacteria to swim through the agar freely and to show their chemotactic capacities. A colony, inserted in soft agar plate, starts to grow while running out of the environmental sources. For this reason chemotactic bacteria move to places where the sources are not limited. That phenomenon produces a number of <strong>halos</strong> which are spread within the plate and increase in volume as the sources are lowered. The number of halos give us information about the number of chemoattractants that are running out.</p><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/3/36/BacterialCrowdingSoftAgarPlate.png" alt="Soft Agar Plate Assay"/><br />
<p class="caption"><i><strong>Fig 1. Soft agar plate assay.</strong> Different <i>E. coli</i> strains are shown. Every strain carries out a mutation that affects its chemotactic response, but the wild type. In the wt you can observe two halos produced by chemotactic responses to aspartate (bigger halo) and glutamate.</i></p><br />
<br />
<br />
<p>The assay protocol is simple; once the soft agar plates are prepared, a colony is inserted in the agar. Let it grow at 30ºC. The soft agar is a delicate element, so it is important to be careful when moving the plates.</p><br />
<br />
<p>In those plates it could appear different concentric circles which represent chemotaxis to certain attractants. For instance, when two amino acids, which act as chemoattractants, are running out from the medium, two circles will appear. The inner one will show the amino acids limit with low chemotactic response; while the outer one will mean that the amino acid which causes a higher response is running down.</p><br />
<br />
<h2>Optical and Fluorescence Microscopy</h2><br />
<br />
<p>The <strong>microscopy techniques</strong> allow us to see the development of the assay <i>in situ</i> without waiting. In this part we will see how we can carry out an experiment that we could see under the microscope.</p><br />
<br />
<div class="imgLeft"><br />
<img class="ileft" src="https://static.igem.org/mediawiki/2010/4/4e/BacterialCrowdingCapillaryAssay.png" alt="Capillary assay look under a microscope" /><br />
<p class="caption"><i><strong>Fig 2.</strong> Preparation of capillary assay to look under a microscope.</i></p><br />
</div><br />
<br />
<p>Two capillaries are put over a microscope slide which will hold up a cover slip. Then we insert the bacterial dilution between the slide and the cover slip. Two new capillaries are inserted between the slide and the cover slip, inside of the bacterial dilution. One of those capillaries would contain a chemoattractant while the other one would be the control. Under a microscope we could see the difference between both capillaries and we would definitely be able to observe if there is chemotaxis toward this chemoattractant.</p><br />
<br />
<p>Apart from that, we could detect fluorescence emitted by a fluorophore which should be presented in bacteria by using a fluorescent microscope.</p><br />
<br />
<p>This assay can become quantitative too if we spread on agar plates the content of capillaries and count the number of colonies that grew there. Also we could measure the fluorescence inside each capillary, taking to this way other quantitative results.</p><br />
<br />
<div class="clear"></div><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/6/6f/BacterialCrowdingCapillaryAssayMicroscope.png" alt="Capillary assay Pictures"/><br />
<p class="caption"><i><strong>Fig 3.</strong>Results of a capillary assay using microscope techniques. We can see that the chemotatic response toward aspartate is increasing as time passes by. Also there are major differences between the control without aspartate and the control with aspartate.</i></p><br />
<br />
<br />
<h1>Quantitative Assays</h1><br />
<br />
<h2>Capillary assays</h2><br />
<br />
<p>The capillary assays are the most useful to quantify chemotaxis. This team has performed different kinds of capillary assays.</p><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/7/73/BacterialCrowdingCapillaryPictures.png" alt="Capillary assay of E. coli chemotaxis toward aspartate"/><br />
<p class="caption"><i><strong>Fig 4.</strong> On the left, a capillary assay with 96-well PVC microplates and 1&#956;l capillary pipettes; capillaries are inserted through a 2% agarose gel in order to hold it. On the right, a capillary assay using needles and a tip chamber. Both of them were being incubated at 30ºC when pictures was taken.</i></p><br />
<br />
<ul><br />
<li><p><strong>Foundamental Points</strong></p><br />
<p>When a capillary with chemoattractant is put in a bacterial dilution a concentration gradient of chemoatractant is developed according to the <a href="http://en.wikipedia.org/wiki/Fick's_laws_of_diffusion" target="_blank">Fick law</a>. This gradient would be sensed by bacteria that are going from low to high concentration places, thus we should have someThis gradient is sensed by bacteria and they start going from low to high concentration places. This is how some of these bacteria finish into the capillary and so it is possible to demonstrate that capillaries with chemoattractant attract more bacteria than another without any substance (the control), just buffer. The control has to continue the same treatment than the other capillaries. In the same way it could be tested repellent’s efficacy by showing that in its capillary there are less bacteria than in the control.</p><br />
<br />
<div class="table"><br />
<table class="tableBio" cellspacing="0" cellpadding="0"><br />
<thead><br />
<tr><br />
<th>Bacterial dilution recipient or <strong>Chemotaxis Chambers</strong></th><br />
<th>Attractant or Buffer recipient or <strong>Capillaries</strong></th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr><br />
<td>tip chambers</td><br />
<td>syringe’s needles (more or less thin)</td><br />
</tr><br />
<tr><br />
<td>needle’s cups or heated closed 100ml tips</td><br />
<td>Needles</td><br />
</tr><br />
<tr><br />
<td>tip chambers</td><br />
<td>10&#956;l micropipette’s tips</td><br />
</tr><br />
<tr><br />
<td>96-well PVC microplates</td><br />
<td>1&#956;l capillary pipettes</td><br />
</tr><br />
<tr><br />
<td>drop between microscope slide and cover slip</td><br />
<td>1&#956;l capillary pipettes</td><br />
</tr><br />
<tr><br />
<td>Flow-chamber</td><br />
<td>1&#956;l capillary pipettes</td><br />
</tr><br />
</tbody><br />
</table><br />
</div><br />
<br />
<br />
<p class="caption"><i><strong>Table 1</strong>. Several ways to permorm chemotaxis assays we have carried out.</i></p> <br />
<br />
<p>This team has performed those assays on different scales, using differents chemotactic chambers where the bacterial dilution was put inside and with differents capillaries. Some types of these variants are presented in the Table 1. The attractant concentration in the capillary depends on the substance itself, but for aspartate the optimum is reached at 10mM.</p> <br />
<br />
<div class="imgLeft"><br />
<img class="ileft" src="https://static.igem.org/mediawiki/2010/e/ea/BacterialCrowdingFlowChamberAssay.png" alt="Flow-Chamber Capillary Assay" /><br />
<p class="caption"><i><strong>Fig 5. Flow-chamber Capillary Assay</strong>. This picture is shown a three channel flow-chamber, which acts as chemotaxis chamber, with 1&#956;l capillaries fixed in each end of one channel. At that time it was observed with a fluorescence microscope.</i></p><br />
</div><br />
<br />
<br />
<p>One assay that deserves to be explain is the <strong>Multi-capillary assay</strong>. It uses 96-well PVC microplates as chemotaxis chamber and 1μl glass capillaries. You can see a picture of it in figure 4, at the left. We designed this chemotaxis assay when we were looking for a good way to perform numerous and simultaneous capillary assays. The assembly of the assay required to make holes in a microplate lid to allow glass capillaries to go through it. The lid is put upside down over the microplate and it is filled with 2% agarose gel. We also used parafilm to avoid that the gel drip through the lid holes while it was still humid. The function of the gel is to hold capillaries, in its dry way. In orther to fill capillaries we closed one of its ends by heating and heated the glass; then it was introduce in a attractant or buffer suspension and automatically it is filled. It is important to introduce the capillaries in the lid and gel by its closed end to keep the sterility on the other end. Then it is only necessary to put the open ends of capillaries inside the wells of the microplate. Previously these wells should have been filled with a bacterial dilution. At this point the assay continues in the same way than others: 1h incubation, clean outside of capillaries with bidistilled water, dilution of the capillaries content and spread in agar plates.</p><br />
<br />
<p>We got our best results in the last kind of capillary assay showed in the table. It uses <strong>flow-chambers</strong> as chemotaxis chambers and 1&#956;l capillary pipettes as capillaries. This method allows us to quantify bacterial density in two different ways, visualization of flow-chambers by fluorescent microscope techniques and spreading bacteria inside capillaries in LB plates . Actually, we think that this assay leads to less errors than others due to some reasons: when we put a capillary into the flow-chamber the fixing is perfect and the capillary can not move, the exposed part of the capillay inside the chamber is minimal so bacteria can not attach to the outside of the capillary making errors in dilutions, also it is a small device easy to work with.</p><br />
<br />
</li><br />
<br />
<li><p><strong>Protocol</strong></p><br />
<br />
<div class="imgLeft"><br />
<a href="https://static.igem.org/mediawiki/2010/8/80/BacterialCrowdingAllProtocols.pdf"><img class="ileft" src="https://static.igem.org/mediawiki/2010/0/0d/BacterialCrowdingDonwloadProtocols.png" alt="Donwload Bacterial Crowding Protocol" /></a><br />
</div><br />
<br />
<div class="imgRight"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/1/11/BacterialCrowdingCapillaryRepresentation.png" alt="Representation of capillary assay in a tip chambers." /><br />
<p class="caption"><i><strong>Fig 6.</strong>Representation of capillary assay in a tip chambers.</i></p><br />
</div><br />
<br />
<p>The assays can start in two different ways: setting up inocula from the strains we are going to work with into triptone broth or in minimal medium, in a low shaking at 30ºC overnight. High shaking might cause the loss of flagella, and also the production of flagella would not be possible in rich medium since bacteria would not need it. </p><br />
<br />
<p>The following day the inocula should be diluted in the same medium a hundred of times and wait for the growing phase to be the appropriate. For <i>Escherichia Coli</i> it would be necessary to wait for the exponential middle phase, since it is in this phase when flagella develop the flagellar motor. For <i>Pseudomonas</i> instead it would de better to wait for the late exponential phase, as the flagellum is developed later in this organism.</p><br />
<br />
<p>Once the culture is ready, it must be changed to an appropriate medium for mobility and chemotaxis. For that, it is necessary to wash the culture twice with chemotaxis buffer by centrifuging at low speed, since flagella may be lost if culture is treated abruptly.</p><br />
<br />
<p>When the culture is in the right medium, number of bacteria should be adjusted roughly 10<sup>7</sup> fcu/ml. This dilution has to be distributed in chemotaxis chambers where our capillaries will be introduced. The volume of capillaries has not been fully established and usually we used as standard volume 100 &#956;l of diluted chemoattractant in chemotaxis buffer. It is important not to forget controls. Negative controls for chemotaxis assays are filled with chemotaxis buffer. Then chambers and capillaries have to be incubated at 30º during 60 minutes. After that, we have to quantify bacterial popularion contained into capillaries. In order to achieve that we could do it either with dilution and spread in plates or analyzing the fluorescence, supposing that bacteria have any kind of fluorophore. </p><br />
</li><br />
<br />
<li><p><strong>Advices</strong></p><br />
<p>One of the elements we bear in mind is the chemotaxis buffer: chemotaxis medium contains potassium phosphate buffer (pH 7), ethylenediaminetetraacetate (<strong>EDTA</strong>) and <strong>glycerol</strong> (energy source). Glycerol is only necessary in long incubations; meanwhile in short incubations the typical sources of bacteria are enough to maintain the chemotactic machinery. It is all-important to underline that the chemotaxis medium must be free of any other substance which may have chemotactic effects, since this could disturb the results. This is one of the reasons why the carbon source is not glucose, as you may expect. Another important detail to bear in mind is EDTA, this chelation causes the precipitation of magnesium which may dull the movement of bacteria and the flagellar machinery. It would be complicated to be successful in the chemotaxis assays without this chelation. Incubation of bacteria must be carried at 30ºC since it helps motility. Shaking must be low because flagella can be lost in high shaking.</p><br />
<br />
<p>It is crucial to be careful when <strong>choosing the strains</strong> to be used in the chemotaxis assays, since it may not have motility. Strains used in laboratories have normally no motility, as at that point they have usually suffer different screening process in benefit of immobile bacteria. A bacterium which has no motility will not have to invest any source in motility or chemotaxis; this would encourage the creation of a bigger colony and more eye-catching than usual, so Scientifics would be probably leaded to select one of this kind. This problem happened to us and we were trying to attract a non mobile strain toward different attractants until we saw the light.</p><br />
</li><br />
</ul><br />
<br />
<h2>Buridan’s Donkey</h2><br />
<br />
<p>At the beginning of the summer we wanted to test bacterial chemotaxis by using a three-channel device based on <strong>flow-chamber biofilm</strong>. It would be able to produce a linear gradient within narrow tubes that connect the chambers. The linear chemical gradient would be generated by diffusion of the chemoattractant through a dialysis membrane located on the limit of the chamber. This membrane also makes impossible the movement of the chemoattractant-producing bacteria through the tube.</p><br />
<br />
<p>The first assay would involve only chemoattractants, and the second producing bacterias. As result, it would be expected that the movement of the cells in the center chamber was directed to the chamber containing chemoattractant-producing bacterias. It is necessary to clear up that the chemoattractant production would be activated solely by the contact of bacteria with plant cell walls that reside in the same chamber. Bacteria have to “decide” between going toward control empty chamber or going toward chamber with chemoattractant-producing bacteria.</p><br />
<br />
<p>This device could provide some advantages in chemotaxis studies: rapid and easy implementation, parallel and simultaneous test, visual proofs, different assays possibilities. Also some experimental conditions could be changed easily: concentration of bacterial population, chambers distances, bacterial cultures, chemoattractans. Despite its advantages, it maybe requires more than an hour of incubation.</p><br />
<br />
<p>Although we had not enough time to perform this assay, an explaining diagram of this device is provided below.</p><br />
<br />
<h3>Measuring the performance of the chemotaxis circuits (Buridan's donkey assay principle)</h3><br />
<br />
<img class="centerBig" src="https://static.igem.org/mediawiki/2010/a/a8/BacterialCrowdingBuridanDonkey.png" alt="Measuring the performance of the chemotaxis circuits (Buridan's donkey assay principle)"/><br />
<br />
<h3>Buridan's donkey assays with three-channel flow cells</h3><br />
<br />
<img class="centerBig" src="https://static.igem.org/mediawiki/2010/3/38/BacterialCrowdingBuridanDonkeyThree.png" alt="Buridan's donkey assays with three-channel flow cells"/><br />
<br />
<p>Special acknowledgements to Ph.D Parkinson (chemotaxis researcher in University of Utah) who gave us some advices, handed us over some protocols of him, even mobile and mutant <i>E. Coli</i> strains.</p><br />
<br />
<h1>References</h1><br />
<br />
<ul><br />
<li>J. Adler (1972) A Method for Measuring Chemotaxis and Use of the Method to Determine Optimum Conditions for Chemotaxis by Escherichia coli. - Journal of General Microbiology ( I 973), 74, 77-91</li><br />
<br />
<li>Guocheng Han and Joseph J. Cooney (1993) A modified capillary assay for chemotaxis - Journal of Industrial Microbiology, 12 (1993) 396—398</li><br />
<br />
<li>Hanbin Mao, Paul S. Cremer, and Michael D. Manson (2003) A sensitive, versatile microfluidic assay for bacterial chemotaxis - PNAS MICROBIOLOGY vol. 100 no. 9 5449–5454.</li><br />
<br />
<li>Russell Bainer, Heungwon Park, Philippe Cluzel (2003) A high-throughput capillary assay for bacterial chemotaxis - Journal of Microbiological Methods 55 (2003) 315– 319.</li><br />
</ul><br />
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<h1>Chemoattractant Diffusion</h1><br />
<br />
<p><br />
The basic equations for the diffusion of the chemoattractant in the medium are the <a href="http://en.wikipedia.org/wiki/Fick%27s_laws_of_diffusion" target="_blank">Fick laws of diffusion</a>, which govern the variation of the concentration of a substance within a medium.<br />
</p><br />
<br />
<p><br />
The flux <b>J</b> (that is, the amount of substance that flows through a given surface per unit of time mol m<sup>-2</sup>s<sup>-1</sup>) is given by:<br />
</p><br />
<br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/6/6f/UPO-Model-Eq1.png" width="150 "alt="First Fick Law"/><br />
</center><br />
<br />
<br />
<br />
<p><br />
where &phi; is the concentration (mol m<sup>-3</sup>) in a given point. <i>D</i> is a constant called the diffusion coefficient, and that depends on the medium . <br />
</p><br />
<p><br />
Basically, the equation states that the is directed towards places with lower concentration (thus the minus sign). If the concentration is constant in the space (&nabla;&phi;=0) there is no flux.<br />
</p><br />
<br />
<p><br />
If the flux is known, it is possible to determine the amount of substance that goes through a small surface <b>S</b> and a small amount of time <i>dt</i></p><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/f/f8/UPO-Model-Eq2.png" width="170 "alt="First Fick Law"/><br />
</center><br />
<br />
<p><br />
In order to simulate the diffusion, we define the environment and discretize it in very small cells. Each cell determines a given volume <i>V</i>, and has a surface <b>S</b>. At a given time instant, the cell has an amount of substance <i>c</i> (and then a concentration <i>c</i>\<i>V</i>).<br />
</p><br />
<br />
<p><br />
If the cells and time step &Delta;<i>t</i> are small, we can consider that the gradient of concentration can be approximated though the differences in concentration between a cell <i>i</i> and 4 (or 8) neighbors <i>j</i>. Thus:<br />
</p><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/e/ea/UPO-Model-Eq3.png" width="190" alt="First Fick Law"/><br />
</center><br />
<br />
<p><br />
and then, the amount of substance that diffusses from <i>i</i> to <i>j</i>:<br />
</p><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/6/6f/UPO-Model-Eq4.png" width="190" alt="First Fick Law"/><br />
</center><br />
<br />
<p><br />
The following figure illustrates the basic elements of the simulation. The flux <b>J</b> between cells is computed by the difference of concentrations. Then, this flux is used to compute the amount of substance that will flow to the neighbour cell. The amount is proportional to the flux and the common surface between cells.<br />
</p><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/9/97/UPO-Model-diffusion.png" width="300" alt="Basic parameters of the diffusion simulation."/><br />
</center><br />
<br />
<h1>Bacteria motion</h1><br />
<br />
<p><br />
The main actuator of <i>E. coli</i> is a flagellar motor that can rotate clockwise or counterclockwise. Through a set of transmembrane receptors proteins, <i>E. coli</i> is able to detect chemoattractants. Moreover, this detection influences the motion of the flagellar motor <a href=#topp>[Topp and Gallivan, 2007]</a>.<br />
</p><br />
<br />
<p><br />
E. Coli has two main motion modes, which we will name:<br />
<br />
<ol><br />
<li> Random Walk </li><br />
<li> Gradient climbing </li><br />
</ol><br />
</p><br />
<br />
<h2>Random walk mode</h2><br />
<br />
<p><br />
When no gradient of chemoattractant is present, <i>E. coli</i> is in random walk mode. In this case, the bacteria performs smooth runs followed by tumbles. <br />
</p><br />
<br />
<p><br />
Mathematically, we will model this as a Brownian motion:<br />
</p><br />
<br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/b/bc/UPO-Model-Eq5.png" width="300 "alt="First Fick Law"/><br />
</center><br />
<br />
<p><br />
where <b>x</b> is the position of the bacteria and <b>v</b> is the velocity. This (vector) velocity is is defined as:<br />
</p><br />
<br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/7/7d/UPOModel-Eq7.png" width="170 "alt="First Fick Law"/><br />
</center><br />
<br />
<p><br />
where <b>u</b>(t) is an unitary vector and <i>s</i> is the speed. The vector <b>u</b>(t) is randomly sampled from the unit circle. The speed is randomly sampled from a normal distribution:<br />
</p><br />
<br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/b/bd/UPO-Model-Eq6.png" width="170 "alt="First Fick Law"/><br />
</center><br />
<br />
<p><br />
where &mu; is the mean displacement velocity of E.Coli, and &sigma; models the variations from this mean velocity.<br />
</p><br />
<br />
<br />
<br />
<p><br />
The bacteria will move for a time &Delta; <i>t</i><sub>rm</sub> with constant velocity <b>v</b>(<i>t</i>). At the next time instant the bacteria tumbles, selecting a new velocity vector (randomly). <br />
</p><br />
<br />
<br />
<h2>Gradient climbing behaviour</h2><br />
<br />
<p><br />
When a positive difference of concentration (a gradient) on the chemoattractant is detected, the bacteria enters into a new mode that we will call gradient climbing.<br />
</p><br />
<br />
<p><br />
In this mode, the flagellar motor tends to move counterclockwise; as a result, the smooth runs last for more time, and the tumbling frequency decreases.<br />
</p><br />
<br />
In order to model that, we use the same model than above, but a larger tumbling interval (smaller tumbling frequency) &Delta; <i>t</i><sub>gc</sub>;<br />
<br />
<p id="topp"><br />
<small>[Topp and Gallivan, 2007] Topp, S. and Gallivan, J. (2007). Guiding Bacteria with Small Molecules and RNA. J. Am. Chem. Soc., 129:6807–6811.</small><br />
</p><br />
<br />
<br />
<h1>Modeling Tools</h1><br />
<br />
<div class="imgRight"><br />
<a href="https://static.igem.org/mediawiki/2010/3/3f/BacterialCrowdingSimulation.zip"><img src="https://static.igem.org/mediawiki/2010/0/09/BacterialCrowdingDownload.png" alt="Donwload Bacterial Crowding Simulation" /></a><br />
</div><br />
<br />
<div class="clear"><br /></div><br />
<br />
<p>The Dry Lab has created a program using Java to simulate the previous equations and dynamics. The program has a visual interface developed using the graphics library <a href="http://www.interactivepulp.com/pulpcore/" target="_blank">PulpCore</a>.</p><br />
<br />
<p><br />
You can <strong>download the bacterial crowding simulation</strong> <a href="https://static.igem.org/mediawiki/2010/3/3f/BacterialCrowdingSimulation.zip">here</a>. You will find a readme file explaining how to execute it. Moreover, the source code is also included.<br />
</p><br />
<br />
<br />
<p><br />
The simulation allows to control several parameters, like the diffusion coefficient, the velocity of the bacterias, and so on. Moreover, the simulation shows visually the evolution of the concentration of chemoattractant and the position of the bacterias. Below you can see the evolution of a simulation in 5 different time steps. The bacterias are represented in green, while the concrentration is depicted using colors:<br />
</p><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/7/7f/UPOBacterialCrowding-01.png" alt="Bacterial Crowding simulation iteration 1"/><br />
<img class="center" src="https://static.igem.org/mediawiki/2010/3/30/UPOBacterialCrowding-02.png" alt="Bacterial Crowding simulation iteration 2"/><br />
<img class="center" src="https://static.igem.org/mediawiki/2010/c/ce/UPOBacterialCrowding-03.png" alt="Bacterial Crowding simulation iteration 3"/><br />
<img class="center" src="https://static.igem.org/mediawiki/2010/d/d3/UPOBacterialCrowding-04.png" alt="Bacterial Crowding simulation iteration 4"/><br />
<img class="center" src="https://static.igem.org/mediawiki/2010/7/7e/UPOBacterialCrowding-05.png" alt="Bacterial Crowding simulation iteration 5"/><br />
<br />
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<br />
<h1>Chemoattractant Diffusion</h1><br />
<br />
<p><br />
The basic equations for the diffusion of the chemoattractant in the medium are the <a href="http://en.wikipedia.org/wiki/Fick%27s_laws_of_diffusion" target="_blank">Fick laws of diffusion</a>, which govern the variation of the concentration of a substance within a medium.<br />
</p><br />
<br />
<p><br />
The flux <b>J</b> (that is, the amount of substance that flows through a given surface per unit of time mol m<sup>-2</sup>s<sup>-1</sup>) is given by:<br />
</p><br />
<br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/6/6f/UPO-Model-Eq1.png" width="150 "alt="First Fick Law"/><br />
</center><br />
<br />
<br />
<br />
<p><br />
where &phi; is the concentration (mol m<sup>-3</sup>) in a given point. <i>D</i> is a constant called the diffusion coefficient, and that depends on the medium . <br />
</p><br />
<p><br />
Basically, the equation states that the is directed towards places with lower concentration (thus the minus sign). If the concentration is constant in the space (&nabla;&phi;=0) there is no flux.<br />
</p><br />
<br />
<p><br />
If the flux is known, it is possible to determine the amount of substance that goes through a small surface <b>S</b> and a small amount of time <i>dt</i></p><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/f/f8/UPO-Model-Eq2.png" width="170 "alt="First Fick Law"/><br />
</center><br />
<br />
<p><br />
In order to simulate the diffusion, we define the environment and discretize it in very small cells. Each cell determines a given volume <i>V</i>, and has a surface <b>S</b>. At a given time instant, the cell has an amount of substance <i>c</i> (and then a concentration <i>c</i>\<i>V</i>).<br />
</p><br />
<br />
<p><br />
If the cells and time step &Delta;<i>t</i> are small, we can consider that the gradient of concentration can be approximated though the differences in concentration between a cell <i>i</i> and 4 (or 8) neighbors <i>j</i>. Thus:<br />
</p><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/e/ea/UPO-Model-Eq3.png" width="190" alt="First Fick Law"/><br />
</center><br />
<br />
<p><br />
and then, the amount of substance that diffusses from <i>i</i> to <i>j</i>:<br />
</p><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/6/6f/UPO-Model-Eq4.png" width="190" alt="First Fick Law"/><br />
</center><br />
<br />
<p><br />
The following figure illustrates the basic elements of the simulation. The flux <b>J</b> between cells is computed by the difference of concentrations. Then, this flux is used to compute the amount of substance that will flow to the neighbour cell. The amount is proportional to the flux and the common surface between cells.<br />
</p><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/9/97/UPO-Model-diffusion.png" width="300" alt="Basic parameters of the diffusion simulation."/><br />
</center><br />
<br />
<h1>Bacteria motion</h1><br />
<br />
<p><br />
The main actuator of <i>E. coli</i> is a flagellar motor that can rotate clockwise or counterclockwise. Through a set of transmembrane receptors proteins, <i>E. coli</i> is able to detect chemoattractants. Moreover, this detection influences the motion of the flagellar motor <a href=#topp>[Topp and Gallivan, 2007]</a>.<br />
</p><br />
<br />
<p><br />
E. Coli has two main motion modes, which we will name:<br />
<br />
<ol><br />
<li> Random Walk </li><br />
<li> Gradient climbing </li><br />
</ol><br />
</p><br />
<br />
<h2>Random walk mode</h2><br />
<br />
<p><br />
When no gradient of chemoattractant is present, <i>E. coli</i> is in random walk mode. In this case, the bacteria performs smooth runs followed by tumbles. <br />
</p><br />
<br />
<p><br />
Mathematically, we will model this as a Brownian motion:<br />
</p><br />
<br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/b/bc/UPO-Model-Eq5.png" width="300 "alt="First Fick Law"/><br />
</center><br />
<br />
<p><br />
where <b>x</b> is the position of the bacteria and <b>v</b> is the velocity. This (vector) velocity is is defined as:<br />
</p><br />
<br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/7/7d/UPOModel-Eq7.png" width="170 "alt="First Fick Law"/><br />
</center><br />
<br />
<p><br />
where <b>u</b>(t) is an unitary vector and <i>s</i> is the speed. The vector <b>u</b>(t) is randomly sampled from the unit circle. The speed is randomly sampled from a normal distribution:<br />
</p><br />
<br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/b/bd/UPO-Model-Eq6.png" width="170 "alt="First Fick Law"/><br />
</center><br />
<br />
<p><br />
where &mu; is the mean displacement velocity of E.Coli, and &sigma; models the variations from this mean velocity.<br />
</p><br />
<br />
<br />
<br />
<p><br />
The bacteria will move for a time &Delta; <i>t</i><sub>rm</sub> with constant velocity <b>v</b>(<i>t</i>). At the next time instant the bacteria tumbles, selecting a new velocity vector (randomly). <br />
</p><br />
<br />
<br />
<h2>Gradient climbing behaviour</h2><br />
<br />
<p><br />
When a positive difference of concentration (a gradient) on the chemoattractant is detected, the bacteria enters into a new mode that we will call gradient climbing.<br />
</p><br />
<br />
<p><br />
In this mode, the flagellar motor tends to move counterclockwise; as a result, the smooth runs last for more time, and the tumbling frequency decreases.<br />
</p><br />
<br />
In order to model that, we use the same model than above, but a larger tumbling interval (smaller tumbling frequency) &Delta; <i>t</i><sub>gc</sub>;<br />
<br />
<p id="topp"><br />
<small>[Topp and Gallivan, 2007] Topp, S. and Gallivan, J. (2007). Guiding Bacteria with Small Molecules and RNA. J. Am. Chem. Soc., 129:6807–6811.</small><br />
</p><br />
<br />
<br />
<h1>Modeling Tools</h1><br />
<br />
You can <strong>download the bacterial crowding simulation</strong> <a href="https://static.igem.org/mediawiki/2010/3/3f/BacterialCrowdingSimulation.zip">here</a><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/7/7f/UPOBacterialCrowding-01.png" alt="Bacterial Crowding simulation iteration 1"/><br />
<img class="center" src="https://static.igem.org/mediawiki/2010/3/30/UPOBacterialCrowding-02.png" alt="Bacterial Crowding simulation iteration 2"/><br />
<img class="center" src="https://static.igem.org/mediawiki/2010/c/ce/UPOBacterialCrowding-03.png" alt="Bacterial Crowding simulation iteration 3"/><br />
<img class="center" src="https://static.igem.org/mediawiki/2010/d/d3/UPOBacterialCrowding-04.png" alt="Bacterial Crowding simulation iteration 4"/><br />
<img class="center" src="https://static.igem.org/mediawiki/2010/7/7e/UPOBacterialCrowding-05.png" alt="Bacterial Crowding simulation iteration 5"/><br />
<br />
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<br />
<h1>Chemoattractant Diffusion</h1><br />
<br />
<p><br />
The basic equations for the diffusion of the chemoattractant in the medium are the <a href="http://en.wikipedia.org/wiki/Fick%27s_laws_of_diffusion" target="_blank">Fick laws of diffusion</a>, which govern the variation of the concentration of a substance within a medium.<br />
</p><br />
<br />
<p><br />
The flux <b>J</b> (that is, the amount of substance that flows through a given surface per unit of time mol m<sup>-2</sup>s<sup>-1</sup>) is given by:<br />
</p><br />
<br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/6/6f/UPO-Model-Eq1.png" width="150 "alt="First Fick Law"/><br />
</center><br />
<br />
<br />
<br />
<p><br />
where &phi; is the concentration (mol m<sup>-3</sup>) in a given point. <i>D</i> is a constant called the diffusion coefficient, and that depends on the medium . <br />
</p><br />
<p><br />
Basically, the equation states that the is directed towards places with lower concentration (thus the minus sign). If the concentration is constant in the space (&nabla;&phi;=0) there is no flux.<br />
</p><br />
<br />
<p><br />
If the flux is known, it is possible to determine the amount of substance that goes through a small surface <b>S</b> and a small amount of time <i>dt</i></p><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/f/f8/UPO-Model-Eq2.png" width="170 "alt="First Fick Law"/><br />
</center><br />
<br />
<p><br />
In order to simulate the diffusion, we define the environment and discretize it in very small cells. Each cell determines a given volume <i>V</i>, and has a surface <b>S</b>. At a given time instant, the cell has an amount of substance <i>c</i> (and then a concentration <i>c</i>\<i>V</i>).<br />
</p><br />
<br />
<p><br />
If the cells and time step &Delta;<i>t</i> are small, we can consider that the gradient of concentration can be approximated though the differences in concentration between a cell <i>i</i> and 4 (or 8) neighbors <i>j</i>. Thus:<br />
</p><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/e/ea/UPO-Model-Eq3.png" width="190" alt="First Fick Law"/><br />
</center><br />
<br />
<p><br />
and then, the amount of substance that diffusses from <i>i</i> to <i>j</i>:<br />
</p><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/6/6f/UPO-Model-Eq4.png" width="190" alt="First Fick Law"/><br />
</center><br />
<br />
<p><br />
The following figure illustrates the basic elements of the simulation. The flux <b>J</b> between cells is computed by the difference of concentrations. Then, this flux is used to compute the amount of substance that will flow to the neighbour cell. The amount is proportional to the flux and the common surface between cells.<br />
</p><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/9/97/UPO-Model-diffusion.png" width="300" alt="Basic parameters of the diffusion simulation."/><br />
</center><br />
<br />
<h1>Bacteria motion</h1><br />
<br />
<p><br />
The main actuator of <i>E. coli</i> is a flagellar motor that can rotate clockwise or counterclockwise. Through a set of transmembrane receptors proteins, <i>E. coli</i> is able to detect chemoattractants. Moreover, this detection influences the motion of the flagellar motor <a href=#topp>[Topp and Gallivan, 2007]</a>.<br />
</p><br />
<br />
<p><br />
E. Coli has two main motion modes, which we will name:<br />
<br />
<ol><br />
<li> Random Walk </li><br />
<li> Gradient climbing </li><br />
</ol><br />
</p><br />
<br />
<h2>Random walk mode</h2><br />
<br />
<p><br />
When no gradient of chemoattractant is present, <i>E. coli</i> is in random walk mode. In this case, the bacteria performs smooth runs followed by tumbles. <br />
</p><br />
<br />
<p><br />
Mathematically, we will model this as a Brownian motion:<br />
</p><br />
<br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/b/bc/UPO-Model-Eq5.png" width="300 "alt="First Fick Law"/><br />
</center><br />
<br />
<p><br />
where <b>x</b> is the position of the bacteria and <b>v</b> is the velocity. This (vector) velocity is is defined as:<br />
</p><br />
<br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/7/7d/UPOModel-Eq7.png" width="170 "alt="First Fick Law"/><br />
</center><br />
<br />
<p><br />
where <b>u</b>(t) is an unitary vector and <i>s</i> is the speed. The vector <b>u</b>(t) is randomly sampled from the unit circle. The speed is randomly sampled from a normal distribution:<br />
</p><br />
<br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/b/bd/UPO-Model-Eq6.png" width="170 "alt="First Fick Law"/><br />
</center><br />
<br />
<p><br />
where &mu; is the mean displacement velocity of E.Coli, and &sigma; models the variations from this mean velocity.<br />
</p><br />
<br />
<br />
<br />
<p><br />
The bacteria will move for a time &Delta; <i>t</i><sub>rm</sub> with constant velocity <b>v</b>(<i>t</i>). At the next time instant the bacteria tumbles, selecting a new velocity vector (randomly). <br />
</p><br />
<br />
<br />
<h2>Gradient climbing behaviour</h2><br />
<br />
<p><br />
When a positive difference of concentration (a gradient) on the chemoattractant is detected, the bacteria enters into a new mode that we will call gradient climbing.<br />
</p><br />
<br />
<p><br />
In this mode, the flagellar motor tends to move counterclockwise; as a result, the smooth runs last for more time, and the tumbling frequency decreases.<br />
</p><br />
<br />
In order to model that, we use the same model than above, but a larger tumbling interval (smaller tumbling frequency) &Delta; <i>t</i><sub>gc</sub>;<br />
<br />
<p id="topp"><br />
<small>[Topp and Gallivan, 2007] Topp, S. and Gallivan, J. (2007). Guiding Bacteria with Small Molecules and RNA. J. Am. Chem. Soc., 129:6807–6811.</small><br />
</p><br />
<br />
<br />
<h1>Modeling Tools</h1><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/7/7f/UPOBacterialCrowding-01.png" alt="Bacterial Crowding simulation iteration 1"/><br />
<img class="center" src="https://static.igem.org/mediawiki/2010/3/30/UPOBacterialCrowding-02.png" alt="Bacterial Crowding simulation iteration 2"/><br />
<img class="center" src="https://static.igem.org/mediawiki/2010/c/ce/UPOBacterialCrowding-03.png" alt="Bacterial Crowding simulation iteration 3"/><br />
<img class="center" src="https://static.igem.org/mediawiki/2010/d/d3/UPOBacterialCrowding-04.png" alt="Bacterial Crowding simulation iteration 4"/><br />
<img class="center" src="https://static.igem.org/mediawiki/2010/7/7e/UPOBacterialCrowding-05.png" alt="Bacterial Crowding simulation iteration 5"/><br />
<br />
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</div></div>Lepavgomhttp://2010.igem.org/File:BacterialCrowdingSimulation.zipFile:BacterialCrowdingSimulation.zip2010-10-27T14:36:03Z<p>Lepavgom: uploaded a new version of "Image:BacterialCrowdingSimulation.zip"</p>
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<br />
<h1>Models</h1><br />
<br />
The members of the dry lab are simulating the different components of the full system. Three main components can be identified:<br />
<br />
<ol><br />
<li>The difussion of the chemoattractant through the medium. </li><br />
<li>The motion of the bacterias through the medium due to the gradient on the chemoattractant concentration.</li><br />
<li>The circuits and devices for the chemoattractant generation within the bacteria. </li><br />
</ol><br />
<br />
You can download the bacterial crowding simulation <a href="https://static.igem.org/mediawiki/2010/3/3f/BacterialCrowdingSimulation.zip">here</a><br />
<br />
<br />
<div class="center"><br />
<a href="https://2010.igem.org/Team:UPO-Sevilla/Modeling/Chemotaxis"> <img class="subBanner" src="https://static.igem.org/mediawiki/2010/2/24/BacterialCrowdingChemotaxis.png" alt="Chemotaxis" /></a><br />
<a href="https://2010.igem.org/Team:UPO-Sevilla/Modeling/Signaling"><img class="subBanner" src="https://static.igem.org/mediawiki/2010/5/59/BacterialCrowdingSignaling.png" alt="Signaling" /></a><br />
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<br />
<html><br />
<div class=contentBC><br />
<br />
<h1>Models</h1><br />
<br />
The members of the dry lab are simulating the different components of the full system. Three main components can be identified:<br />
<br />
<ol><br />
<li>The difussion of the chemoattractant through the medium. </li><br />
<li>The motion of the bacterias through the medium due to the gradient on the chemoattractant concentration.</li><br />
<li>The circuits and devices for the chemoattractant generation within the bacteria. </li><br />
</ol><br />
<br />
You can download the bacterial crowding simulation <a href="https://static.igem.org/mediawiki/2010/3/3f/BacterialCrowdingSimulation.zip">here</a><br />
<br />
<br />
<div class="center"><br />
<a href="https://2010.igem.org/Team:UPO-Sevilla/Modeling/Chemotaxis"> <img class="subBanner" src="https://static.igem.org/mediawiki/2010/2/24/BacterialCrowdingChemotaxis.png" alt="Chemotaxis" /></a><br />
<a href="https://2010.igem.org/Team:UPO-Sevilla/Modeling/Signaling"><img class="subBanner" src="https://static.igem.org/mediawiki/2010/5/59/BacterialCrowdingSignaling.png" alt="Signaling" /></a><br />
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<br />
<html><br />
<div class=contentBC><br />
<br />
<h1>Models</h1><br />
<br />
The members of the dry lab are simulating the different components of the full system. Three main components can be identified:<br />
<br />
<ol><br />
<li>The difussion of the chemoattractant through the medium. </li><br />
<li>The motion of the bacterias through the medium due to the gradient on the chemoattractant concentration.</li><br />
<li>The circuits and devices for the chemoattractant generation within the bacteria. </li><br />
</ol><br />
<br />
You can download the bacterial crowding simulation <a href="https://static.igem.org/mediawiki/2010/3/3f/BacterialCrowdingSimulation.zip">here</a><br />
<br />
<br />
<div class="center"><br />
<a href="https://2010.igem.org/Team:UPO-Sevilla/Modeling/Chemotaxis"> <img class="subBanner" src="https://static.igem.org/mediawiki/2010/2/24/BacterialCrowdingChemotaxis.png" alt="Chemotaxis" /></a><br />
<a href="https://2010.igem.org/Team:UPO-Sevilla/Modeling/Signaling"><img class="subBanner" src="https://static.igem.org/mediawiki/2010/5/59/BacterialCrowdingSignaling.png" alt="Signaling" /></a><br />
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<div></div>Lepavgomhttp://2010.igem.org/Team:UPO-Sevilla/Modeling/ChemotaxisTeam:UPO-Sevilla/Modeling/Chemotaxis2010-10-26T18:51:45Z<p>Lepavgom: New page: <div class=globalBC> {{:Team:UPO-Sevilla/header}} <!-- --> <html> <script type="text/javascript" language="javascript"> <!-- current("modeling","http://2010.i...</p>
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<h1>Chemoattractant Diffusion</h1><br />
<br />
<p><br />
The basic equations for the diffusion of the chemoattractant in the medium are the <a href="http://en.wikipedia.org/wiki/Fick%27s_laws_of_diffusion" target="_blank">Fick laws of diffusion</a>, which govern the variation of the concentration of a substance within a medium.<br />
</p><br />
<br />
<p><br />
The flux <b>J</b> (that is, the amount of substance that flows through a given surface per unit of time mol m<sup>-2</sup>s<sup>-1</sup>) is given by:<br />
</p><br />
<br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/6/6f/UPO-Model-Eq1.png" width="150 "alt="First Fick Law"/><br />
</center><br />
<br />
<br />
<br />
<p><br />
where &phi; is the concentration (mol m<sup>-3</sup>) in a given point. <i>D</i> is a constant called the diffusion coefficient, and that depends on the medium . <br />
</p><br />
<p><br />
Basically, the equation states that the is directed towards places with lower concentration (thus the minus sign). If the concentration is constant in the space (&nabla;&phi;=0) there is no flux.<br />
</p><br />
<br />
<p><br />
If the flux is known, it is possible to determine the amount of substance that goes through a small surface <b>S</b> and a small amount of time <i>dt</i></p><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/f/f8/UPO-Model-Eq2.png" width="170 "alt="First Fick Law"/><br />
</center><br />
<br />
<p><br />
In order to simulate the diffusion, we define the environment and discretize it in very small cells. Each cell determines a given volume <i>V</i>, and has a surface <b>S</b>. At a given time instant, the cell has an amount of substance <i>c</i> (and then a concentration <i>c</i>\<i>V</i>).<br />
</p><br />
<br />
<p><br />
If the cells and time step &Delta;<i>t</i> are small, we can consider that the gradient of concentration can be approximated though the differences in concentration between a cell <i>i</i> and 4 (or 8) neighbors <i>j</i>. Thus:<br />
</p><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/e/ea/UPO-Model-Eq3.png" width="190" alt="First Fick Law"/><br />
</center><br />
<br />
<p><br />
and then, the amount of substance that diffusses from <i>i</i> to <i>j</i>:<br />
</p><br />
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<center><br />
<img src="https://static.igem.org/mediawiki/2010/6/6f/UPO-Model-Eq4.png" width="190" alt="First Fick Law"/><br />
</center><br />
<br />
<p><br />
The following figure illustrates the basic elements of the simulation. The flux <b>J</b> between cells is computed by the difference of concentrations. Then, this flux is used to compute the amount of substance that will flow to the neighbour cell. The amount is proportional to the flux and the common surface between cells.<br />
</p><br />
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<center><br />
<img src="https://static.igem.org/mediawiki/2010/9/97/UPO-Model-diffusion.png" width="300" alt="Basic parameters of the diffusion simulation."/><br />
</center><br />
<br />
<h1>Bacteria motion</h1><br />
<br />
<p><br />
The main actuator of <i>E. coli</i> is a flagellar motor that can rotate clockwise or counterclockwise. Through a set of transmembrane receptors proteins, <i>E. coli</i> is able to detect chemoattractants. Moreover, this detection influences the motion of the flagellar motor <a href=#topp>[Topp and Gallivan, 2007]</a>.<br />
</p><br />
<br />
<p><br />
E. Coli has two main motion modes, which we will name:<br />
<br />
<ol><br />
<li> Random Walk </li><br />
<li> Gradient climbing </li><br />
</ol><br />
</p><br />
<br />
<h2>Random walk mode</h2><br />
<br />
<p><br />
When no gradient of chemoattractant is present, <i>E. coli</i> is in random walk mode. In this case, the bacteria performs smooth runs followed by tumbles. <br />
</p><br />
<br />
<p><br />
Mathematically, we will model this as a Brownian motion:<br />
</p><br />
<br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/b/bc/UPO-Model-Eq5.png" width="300 "alt="First Fick Law"/><br />
</center><br />
<br />
<p><br />
where <b>x</b> is the position of the bacteria and <b>v</b> is the velocity. This (vector) velocity is randomly sampled from a normal distribution of zero mean and a certain covariance matrix that models the potential . <br />
</p><br />
<br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/b/bd/UPO-Model-Eq6.png" width="150 "alt="First Fick Law"/><br />
</center><br />
<br />
<p><br />
The bacteria will move for a time &Delta; <i>t</i><sub>rm</sub> with constant velocity <b>v</b>(<i>t</i>). At next time instant, a new velocity is (randomly) selected <br />
</p><br />
<br />
<br />
<h2>Gradient climbing behaviour</h2><br />
<br />
<p><br />
When a positive difference of concentration (a gradient) on the chemoattractant is detected, the bacteria enters into a new mode that we will call gradient climbing.<br />
</p><br />
<br />
<p><br />
In this mode, the flagellar motor tends to move counterclockwise; as a result, the smooth runs last for more time, and the tumbling frequency decreases.<br />
</p><br />
<br />
In order to model that, we use the same model than above, but a larger tumbling interval (smaller tumbling frequency) &Delta; <i>t</i><sub>gc</sub>;<br />
<br />
<p id="topp"><br />
<small>[Topp and Gallivan, 2007] Topp, S. and Gallivan, J. (2007). Guiding Bacteria with Small Molecules and RNA. J. Am. Chem. Soc., 129:6807–6811.</small><br />
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<br />
<h1>Models</h1><br />
<br />
The members of the dry lab are simulating the different components of the full system. Three main components can be identified:<br />
<br />
<ol><br />
<li>The difussion of the chemoattractant through the medium. </li><br />
<li>The motion of the bacterias through the medium due to the gradient on the chemoattractant concentration.</li><br />
<li>The circuits and devices for the chemoattractant generation within the bacteria. </li><br />
</ol><br />
<br />
You can download the bacterial crowding simulation <a href="https://static.igem.org/mediawiki/2010/3/3f/BacterialCrowdingSimulation.zip">here</a><br />
<br />
<h2>Chemoattractant Diffusion</h2><br />
<br />
<p><br />
The basic equations for the diffusion of the chemoattractant in the medium are the <a href="http://en.wikipedia.org/wiki/Fick%27s_laws_of_diffusion" target="_blank">Fick laws of diffusion</a>, which govern the variation of the concentration of a substance within a medium.<br />
</p><br />
<br />
<p><br />
The flux <b>J</b> (that is, the amount of substance that flows through a given surface per unit of time mol m<sup>-2</sup>s<sup>-1</sup>) is given by:<br />
</p><br />
<br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/6/6f/UPO-Model-Eq1.png" width="150 "alt="First Fick Law"/><br />
</center><br />
<br />
<br />
<br />
<p><br />
where &phi; is the concentration (mol m<sup>-3</sup>) in a given point. <i>D</i> is a constant called the diffusion coefficient, and that depends on the medium . <br />
</p><br />
<p><br />
Basically, the equation states that the is directed towards places with lower concentration (thus the minus sign). If the concentration is constant in the space (&nabla;&phi;=0) there is no flux.<br />
</p><br />
<br />
<p><br />
If the flux is known, it is possible to determine the amount of substance that goes through a small surface <b>S</b> and a small amount of time <i>dt</i></p><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/f/f8/UPO-Model-Eq2.png" width="170 "alt="First Fick Law"/><br />
</center><br />
<br />
<p><br />
In order to simulate the diffusion, we define the environment and discretize it in very small cells. Each cell determines a given volume <i>V</i>, and has a surface <b>S</b>. At a given time instant, the cell has an amount of substance <i>c</i> (and then a concentration <i>c</i>\<i>V</i>).<br />
</p><br />
<br />
<p><br />
If the cells and time step &Delta;<i>t</i> are small, we can consider that the gradient of concentration can be approximated though the differences in concentration between a cell <i>i</i> and 4 (or 8) neighbors <i>j</i>. Thus:<br />
</p><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/e/ea/UPO-Model-Eq3.png" width="190" alt="First Fick Law"/><br />
</center><br />
<br />
<p><br />
and then, the amount of substance that diffusses from <i>i</i> to <i>j</i>:<br />
</p><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/6/6f/UPO-Model-Eq4.png" width="190" alt="First Fick Law"/><br />
</center><br />
<br />
<p><br />
The following figure illustrates the basic elements of the simulation. The flux <b>J</b> between cells is computed by the difference of concentrations. Then, this flux is used to compute the amount of substance that will flow to the neighbour cell. The amount is proportional to the flux and the common surface between cells.<br />
</p><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/9/97/UPO-Model-diffusion.png" width="300" alt="Basic parameters of the diffusion simulation."/><br />
</center><br />
<br />
<h2> Bacteria motion</h2><br />
<br />
<p><br />
The main actuator of <i>E. coli</i> is a flagellar motor that can rotate clockwise or counterclockwise. Through a set of transmembrane receptors proteins, <i>E. coli</i> is able to detect chemoattractants. Moreover, this detection influences the motion of the flagellar motor <a href=#topp>[Topp and Gallivan, 2007]</a>.<br />
</p><br />
<br />
<p><br />
E. Coli has two main motion modes, which we will name:<br />
<br />
<ol><br />
<li> Random Walk </li><br />
<li> Gradient climbing </li><br />
</ol><br />
</p><br />
<br />
<p><b>Random walk mode</b></p><br />
<br />
<p><br />
When no gradient of chemoattractant is present, <i>E. coli</i> is in random walk mode. In this case, the bacteria performs smooth runs followed by tumbles. <br />
</p><br />
<br />
<p><br />
Mathematically, we will model this as a Brownian motion:<br />
</p><br />
<br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/b/bc/UPO-Model-Eq5.png" width="300 "alt="First Fick Law"/><br />
</center><br />
<br />
<p><br />
where <b>x</b> is the position of the bacteria and <b>v</b> is the velocity. This (vector) velocity is randomly sampled from a normal distribution of zero mean and a certain covariance matrix that models the potential . <br />
</p><br />
<br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/b/bd/UPO-Model-Eq6.png" width="150 "alt="First Fick Law"/><br />
</center><br />
<br />
<p><br />
The bacteria will move for a time &Delta; <i>t</i><sub>rm</sub> with constant velocity <b>v</b>(<i>t</i>). At next time instant, a new velocity is (randomly) selected <br />
</p><br />
<br />
<br />
<b>Gradient climbing behaviour</b><br />
<br />
<p><br />
When a positive difference of concentration (a gradient) on the chemoattractant is detected, the bacteria enters into a new mode that we will call gradient climbing.<br />
</p><br />
<br />
<p><br />
In this mode, the flagellar motor tends to move counterclockwise; as a result, the smooth runs last for more time, and the tumbling frequency decreases.<br />
</p><br />
<br />
In order to model that, we use the same model than above, but a larger tumbling interval (smaller tumbling frequency) &Delta; <i>t</i><sub>gc</sub>;<br />
<br />
<p id="topp"><br />
<small>[Topp and Gallivan, 2007] Topp, S. and Gallivan, J. (2007). Guiding Bacteria with Small Molecules and RNA. J. Am. Chem. Soc., 129:6807–6811.</small><br />
</p><br />
<br />
<h2>Circuits and devices</h2><br />
<br />
<br />
<center><br />
<a href="https://2010.igem.org/Image:IGEMUPO-model-small.png"><br />
<img src="https://static.igem.org/mediawiki/2010/8/8a/IGEMUPO-model-small.png" width="700" alt="Simbiology model"/><br />
</a><br />
</center><br />
<br />
<br />
<h1>Modeling Tools</h1><br />
<br />
</div><br />
</html><br />
<br />
{{:Team:UPO-Sevilla/footer}}<br />
</div></div>Lepavgomhttp://2010.igem.org/File:BacterialCrowdingSimulation.zipFile:BacterialCrowdingSimulation.zip2010-10-26T00:12:52Z<p>Lepavgom: </p>
<hr />
<div></div>Lepavgomhttp://2010.igem.org/Team:UPO-Sevilla/Biobricks/PartsTeam:UPO-Sevilla/Biobricks/Parts2010-10-25T21:04:54Z<p>Lepavgom: </p>
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<h1>Parts</h1><br />
<br />
<p><br />
In this site you can find all the parts iGEM team UPO-Sevilla had been working on.<br />
</p><br />
<p><br />
BioBricks names give you information about the kind of BioBrick and it is a good way to work with a big quantity of parts (<a href="http://partsregistry.org/Help:BioBrick_Part_Names" target="_blank">see more</a>). But it is hard to write these long names in a microcentrifuge tube! That is why we used a second numerical name for each part. Numerical names are easier to work with in the lab and also to organize the work. You will see this codec next to standard names. This will help you to understand other issues of Bacterial Crowding project, as Circuits and Devices.<br />
</p><br />
<div class="table"><br />
<table class="tableBio" cellspacing="0" cellpadding="0"><br />
<caption>New BioBricks</caption><br />
<thead><br />
<tr><br />
<th>&nbsp;</th><br />
<th>Part Number</th><br />
<th>iGEM ID</th><br />
<th>Identity</th><br />
<th>Type</th><br />
<th>Origin</th><br />
<th>Description</th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr><br />
<td class="headRow" rowspan="4">Prh System</td><br />
<td>UPO-04</td><br />
<td class="link"><a href="http://partsregistry.org/Part:BBa_K367000" target="_blank">BBa_K367000</a></td><br />
<td><i>prhA</i></td><br />
<td>Coding sequence</td><br />
<td>Synthesis</td><br />
<td><i>Ralstonia solanacearum</i> gene <i>prhA</i>, encoding an outer membrane protein<br />
that senses a signal on plant cell walls and transduces it through the<br />
bacterial cell envelop to stimulate transcription from several operons.<br />
Optimized sequence to be expressed in <i>Escherichia coli.</i></td><br />
</tr><br />
<tr><br />
<td>UPO-05</td><br />
<td class="link"><a href="http://partsregistry.org/Part:BBa_K367001" target="_blank">BBa_K367001</a></td><br />
<td><i>prhR</i></td><br />
<td>Coding sequence</td><br />
<td>Synthesis</td><br />
<td><i>Ralstonia solanacearum</i> gene <i>prhR</i>, encoding a membrane signal transduction<br />
protein involved in the Prh pathway. The nondiffusible plant cell wall<br />
signal is transduced by the N-terminal extension of PrhA to the C-terminal<br />
part of the transmembrane protein PrhR and then through PrhR across the<br />
cytoplasmic membrane. Optimized sequence to be expressed <i>in Escherichia coli.</i></td><br />
</tr><br />
<tr><br />
<td>UPO-06</td><br />
<td class="link"><a href="http://partsregistry.org/Part:BBa_K367002" target="_blank">BBa_K367002</a></td><br />
<td><i>prhI</i></td><br />
<td>Coding sequence</td><br />
<td>Synthesis</td><br />
<td><i>Ralstonia solanacearum</i> gene <i>prhI</i>, encoding an ECF sigma factor responsible<br />
for transcription dependent on the Prh signal transduction system. PrhI<br />
is activated by PrhR and then the sigma factor induces the expression by<br />
activating P<i>prhJ</i> promoter. Optimized sequence to be expressed in <i>Escherichia coli.</i></td><br />
<br />
</tr><br />
<tr><br />
<td>UPO-11</td><br />
<td class="link"><a href="http://partsregistry.org/Part:BBa_K367008" target="_blank">BBa_K367008</a></td><br />
<td>P<i>prhJ</i></td><br />
<td>Promoter</td><br />
<td>Synthesis</td><br />
<td>The P<i>prhJ</i> promoter region, responsive to plant cell contact via signal transduction<br />
by PrhA and PrhR and activation by ECF sigma factor PrhI. Optimized sequence to be<br />
expressed in <i>Escherichia coli.</i></td><br />
</tr><br />
<tr><br />
<td class="headRow" >Hybrid Protein</td><br />
<td>UPO-07</td><br />
<td class="link"><a href="http://partsregistry.org/Part:BBa_K367006" target="_blank">BBa_K367006</a></td><br />
<td><i>fecA-prhA</i></td><br />
<td>Coding sequence</td><br />
<td>Synthesis</td><br />
<td>Artificial coding sequence spanning the first 92 codons of <i>fecA</i>, encoding the signal<br />
peptide (aa. 1-33), the proposed Ton-box (aa. 54-63), fused to the distal end of<br />
the <i>prhA</i> coding sequence at the conserved GSGL motif (aa. 89-92). This hybrid<br />
protein allows to sense nondifusible signals and to transduce it by the well known<br />
Fec pathway. Optimized sequence to be expressed in <i>Escherichia coli.</i></td><br />
</tr><br />
<tr><br />
<td class="headRow" rowspan="5">Fec System</td><br />
<td>UPO-08</td><br />
<td class="link"><a href="http://partsregistry.org/Part:BBa_K367003" target="_blank">BBa_K367003</a></td><br />
<td><i>fecA</i></td><br />
<td>Coding sequence</td><br />
<td>Finally not made</td><br />
<td>Gene <i>fecA</i> of <i>Escherichia coli</i>, encoding an outer membrane ferric citrate sensor that<br />
initiates signal transduction via FecR and FecI to activate transcription.</td><br />
</tr><br />
<tr><br />
<td>UPO-28</td><br />
<td class="link"><a href="http://partsregistry.org/Part:BBa_K367012" target="_blank">BBa_K367012</a></td><br />
<td><i>fecI</i> & <i>fecR</i></td><br />
<td>Two overlapping coding sequences</td><br />
<td>Finally not made</td><br />
<td><i>fecI</i> and <i>fecR</i> genes of <i>Escherichia coli</i>, encoding an ECF sigma factor and a membrane<br />
signal transduction protein respectively, involved in signal transduction of the ferric<br />
citrate-dependent Fec system. They have been synthesized in the same Biobrick because<br />
of their 4 bp overlap, not to reduce their expression. FecR protein interacts with the<br />
outer membrane sensor FecA and activates the sigma factor FecI, which acts over P<i>fecA</i> promoter region.</td><br />
</tr><br />
<tr><br />
<td>UPO-12</td><br />
<td class="link"><a href="http://partsregistry.org/Part:BBa_K367009" target="_blank">BBa_K367009</a></td><br />
<td>P<i>fecA</i></td><br />
<td>Promoter</td><br />
<td>Made by PCR</td><br />
<td><i>Escherichia coli</i> P<i>fecA</i> promoter region, repressed by Fur under iron excess, and<br />
induced by ferric citrate through the FecA-FecR signal transduction pathway and<br />
the FecI ECF sigma factor.</td><br />
</tr><br />
<tr><br />
<td>UPO-09</td><br />
<td class="link"><a href="http://partsregistry.org/Part:BBa_K367004" target="_blank">BBa_K367004</a></td><br />
<td><i>fecI</i></td><br />
<td>Coding sequence</td><br />
<td>Made by PCR</td><br />
<td>Gene <i>fecI</i> of <i>Escherichia coli</i>, encoding an ECF sigma factor<br />
used to regulate transcription in response to the FecA-FecR<br />
signal transduction pathway in response to ferric citrate</td><br />
</tr><br />
<tr><br />
<td>UPO-10</td><br />
<td class="link"><a href="http://partsregistry.org/Part:BBa_K367005" target="_blank">BBa_K367005</a></td><br />
<td><i>fecR</i></td><br />
<td>Coding sequence</td><br />
<td>Finally not made</td><br />
<td>Gene <i>fecR</i> from <i>Escherichia coli</i>, encoding a membrane protein involved<br />
in signal transduction of the ferric citrate-dependent Fec system to <br />
stimulate transcription via the ECF sigma factor FecI</td><br />
</tr><br />
<tr><br />
<td class="headRow" rowspan="2">Glutamate Synthetase</td><br />
<td>UPO-17</td><br />
<td class="link"><a href="http://partsregistry.org/Part:BBa_K367010" target="_blank">BBa_K367010</a></td><br />
<td><i>gltD</i></td><br />
<td>Coding sequence</td><br />
<td>Finally not made</td><br />
<td><i>Escherichia coli</i> <i>gltD</i> gene, encoding glutamate synthase beta subunit. Glutamate<br />
synthetase converts glutamine + 2-oxoglutarate into glutamate. Glutamate is a<br />
source of amine groups via transamination, and a chemoattractant for <i>E. coli.</i></td><br />
</tr><br />
<tr><br />
<td>UPO-18</td><br />
<td class="link"><a href="http://partsregistry.org/Part:BBa_K367011" target="_blank">BBa_K367011</a></td><br />
<td><i>gltB</i></td><br />
<td>Coding sequence</td><br />
<td>Made by PCR</td><br />
<td><i>Escherichia coli</i> <i>gltB</i> gene, encoding glutamate synthase alpha subunit. Glutamate<br />
synthetase converts glutamine + 2-oxoglutarate into glutamate. Glutamate is a<br />
source of amine groups via transamination, and a chemoattractant for <i>E. coli.</i></td><br />
</tr><br />
</tbody><br />
</table><br />
</div><br />
<br />
<div class="table"><br />
<table class="tableBio" cellspacing="0" cellpadding="0"><br />
<caption>Existing BioBricks</caption><br />
<thead><br />
<tr><br />
<th>&nbsp;</th><br />
<th>Part Number</th><br />
<th>iGEM ID</th><br />
<th>Identity</th><br />
<th>Type</th><br />
<th>Origin</th><br />
<th>Description</th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr><br />
<td class="headRow" rowspan="3">Expression</td><br />
<td>UPO-01</td><br />
<td class="link"><a href="http://partsregistry.org/Part:BBa_J23100" target="_blank">BBa_J23100</a></td><br />
<td>Strong Promoter</td><br />
<td>Promoter</td><br />
<td>Self-annealing primers</td><br />
<td>Constitutive strong promoter. We ordered it like a primer to make our work easier.</tr><br />
<tr><br />
<td>UPO-02</td><br />
<td class="link"><a href="http://partsregistry.org/Part:BBa_B0030" target="_blank">BBa_B0030</a></td><br />
<td>RBS.1 (strong)</td><br />
<td>Shine-Dalgarno</td><br />
<td>Self-annealing primers</td><br />
<td>Strong RBS based on Ron Weiss thesis.</td><br />
</tr><br />
<tr><br />
<td>UPO-03</td><br />
<td class="link"><a href="http://partsregistry.org/Part:BBa_B0015" target="_blank">BBa_B0015</a></td><br />
<td>rrnBT1-T7TE</td><br />
<td>Terminator (double)</td><br />
<td>2010P1; 23L</td><br />
<td>A reliable double transcription terminator.</td><br />
<br />
</tr><br />
<tr><br />
<td class="headRow" rowspan="2">Reporters</td><br />
<td>UPO-13</td><br />
<td class="link"><a href="http://partsregistry.org/Part:BBa_E0040" target="_blank">BBa_E0040</a></td><br />
<td>GFP</td><br />
<td>Coding sequence</td><br />
<td>2010P1; 14K</td><br />
<td>Green fluorescent protein derived from jellyfish <i>Aequeora victoria</i> wild-type.<br />
It is used to report the expression behind some promoters.</td><br />
</tr><br />
<tr><br />
<td>UPO-20</td><br />
<td class="link"><a href="http://partsregistry.org/Part:BBa_I13522" target="_blank">BBa_I13522</a></td><br />
<td>Ptet-SD-GFP-TT-TT</td><br />
<td>Composite (constitutive protein generator)</td><br />
<td>2010P2; 8A</td><br />
<td>Constitutive GFP generator. It allows to see bacteria easier and also quantificating<br />
their amount.</td><br />
</tr><br />
<tr><br />
<td class="headRow" rowspan="2">Chemoattractants Synthesis</td><br />
<td>UPO-16</td><br />
<td class="link"><a href="http://partsregistry.org/Part:BBa_C0083" target="_blank">BBa_C0083</a></td><br />
<td><i>aspA</i></td><br />
<td>Coding sequence</td><br />
<td>2010P2; 17A</td><br />
<td>Coding secuence for Aspartate ammonia-lyase enzyme. AspA aminates fumarate to make<br />
aspartate. Aspartate can be used as a bacterial chemotaxis signal for <i>Escherichia coli.</i></td><br />
</tr><br />
<tr><br />
<td>UPO-19</td><br />
<td class="link"><a href="http://partsregistry.org/Part:BBa_J45319" target="_blank">BBa_J45319</a></td><br />
<td>SD-pchBA-TT-TT</td><br />
<td>Composite (protein generator)</td><br />
<td>2010P2;15I</td><br />
<td>PchA & PchB enzyme generator takes as input a transcriptional signal<br />
(PoPS) and produces as output the PchA and PchB enzymes that catalyze<br />
production of salicylate from the cellular metabolite chorismate. Salicylate<br />
can be used as a bacterial chemotaxis signal for <i>Pseudomonas putidas.</i></td><br />
</tr><br />
</tbody><br />
</table><br />
</div><br />
<div class="table"><br />
<table class="tableBio" cellspacing="0" cellpadding="0"><br />
<caption>Vectors</caption><br />
<thead><br />
<tr><br />
<th>&nbsp;</th><br />
<th>Part Number</th><br />
<th>iGEM ID</th><br />
<th>Type</th><br />
<th>Origin</th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr><br />
<td class="headRow" rowspan="7">High Copy Vectors</td><br />
<td>UPO-24</td><br />
<td class="link"><a href="http://partsregistry.org/Part:pSB1AK3" target="_blank">pSB1AK3</a></td><br />
<td>Plasmid</td><br />
<td>2010P1; 11A</td><br />
<tr><br />
<td>UPO-25</td><br />
<td class="link"><a href="http://partsregistry.org/Part:pSB1AT3" target="_blank">pSB1AT3</a></td><br />
<td>Plasmid</td><br />
<td>2010P1; 13A</td><br />
</tr><br />
<tr><br />
<td>UPO-26</td><br />
<td class="link"><a href="http://partsregistry.org/Part:pSB1AC3" target="_blank">pSB1AC3</a></td><br />
<td>Plasmid</td><br />
<td>2010P1; 9A</td><br />
</tr><br />
<tr><br />
<td>UPO-32</td><br />
<td class="link"><a href="http://partsregistry.org/Part:pSB1K3" target="_blank">pSB1K3</a></td><br />
<td>Plasmid</td><br />
<td>2010P1; 5A</td><br />
</tr><br />
<tr><br />
<td>UPO-33</td><br />
<td class="link"><a href="http://partsregistry.org/Part:pSB1T3" target="_blank">pSB1T3</a></td><br />
<td>Plasmid</td><br />
<td>2010P1; 7A</td><br />
</tr><br />
<tr><br />
<td>UPO-34</td><br />
<td class="link"><a href="http://partsregistry.org/Part:pSB1C3" target="_blank">pSB1C3</a></td><br />
<td>Plasmid</td><br />
<td>2010P1; 3A</td><br />
</tr><br />
<tr><br />
<td>UPO-35</td><br />
<td class="link"><a href="http://partsregistry.org/Part:pSB1A3" target="_blank">pSB1A3</a></td><br />
<td>Plasmid</td><br />
<td>2010P1; 1C</td><br />
</tr><br />
<tr><br />
<td class="headRow" rowspan="5">Low Copy Vectors</td><br />
<td>UPO-36</td><br />
<td class="link"><a href="http://partsregistry.org/Part:pSB3T5" target="_blank">pSB3T5</a></td><br />
<td>Plasmid</td><br />
<td>2010P1; 7C</td><br />
</tr><br />
<tr><br />
<td>UPO-37</td><br />
<td class="link"><a href="http://partsregistry.org/Part:pSB3C5" target="_blank">pSB3C5</a></td><br />
<td>Plasmid</td><br />
<td>2010P1; 3C</td><br />
</tr><br />
<tr><br />
<td>UPO-38</td><br />
<td class="link"><a href="http://partsregistry.org/Part:pSB4K5" target="_blank">pSB4K5</a></td><br />
<td>Plasmid</td><br />
<td>2010P1; 5G</td><br />
</tr><br />
<tr><br />
<td>UPO-39</td><br />
<td class="link"><a href="http://partsregistry.org/Part:pSB4C5" target="_blank">pSB4C5</a></td><br />
<td>Plasmid</td><br />
<td>2010P1; 3E</td><br />
</tr><br />
<tr><br />
<td>UPO-40</td><br />
<td class="link"><a href="http://partsregistry.org/Part:pSB4A5" target="_blank">pSB4A5</a></td><br />
<td>Plasmid</td><br />
<td>2010P1; 1G</td><br />
</tr><br />
</tbody><br />
</table><br />
</div><br />
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</div></div>Lepavgomhttp://2010.igem.org/Team:UPO-Sevilla/Project/SensingTeam:UPO-Sevilla/Project/Sensing2010-10-25T18:27:45Z<p>Lepavgom: </p>
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<h1>Introduction</h1><br />
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<p>Bacteria can sense a lot of different stimuli. They have to detect changes in their environment and to interpret them. Bacteria use different receptors to sense ionic state, chemicals, pH, nutrients, lights… and, when a stimuli is caused, they generate a intracellular signaling pathway and produce a specific response. Usually, signals that bacteria can sense are diffusible-signals. However, we will use the first known system to sense a non-diffusible signal: the Prh system of <i>Ralstonia solanacearum</i>. This system detects an unknown polysaccharide ligand of plant cell walls and activates a signal transduction cascade that, in original system, causes the virulence gene expression. </p><br />
<br />
<p>The aim of the <strong>sensing circuits</strong> is to ensure that our system is able to detect specifically the walls of plant cells and causes the secretion and excretion of a chemoattractant through signal transduction pathways. In order to achieve effective bacterial accumulation around plant cells, we have designed four different circuits that attempt to prevent possible different problems. We have combined different regulated proteins from <i>Escherichia coli</i> and <i>Ralstonia solanacearum</i> with specific biobricks, and we have replaced virulence genes by chemoattranctant genes. So, despite of <i>R. solanacearum</i> toxicity, the fact of working with biobricks avoids us having to work with the security measures required by this strain. </p><br />
<br />
<p>A future improvement for our project would be to use an <strong>adhesin</strong> to keep the bacteria attached to the plant cell wall as has been happening in R.solanacearum. We expect our system works without it.</p><br />
<br />
<br />
<br />
<h3>Brief description of <i>Ralstonia solanacearum</i>:</h3><br />
<br />
<p><i><strong>Ralstonia solanacearum</strong></i> belongs to the family of Gram-negative phyto-pathogens. This bacterium causes great losses in crops worldwide in tropical, subtropical and temperate environments. The genes involved in virulence are known as <i>hrp</i> (hypersensitive response and pathogenicity) and they are induced by contact with various plant species from three different families of dicotyledonous (Solanaceae, Cruciferae and legumes), including both host and non host species. These genes encode a type III secretion system (TTSS), required to develop the disease in the host or the hypersensitive response (HR) in resistant and non-host plants. The HR is a defense mechanism that certain plant species possess, in which cells infected by a pathogen are killed to prevent spread to healthy tissue. </p><br />
<br />
<p>The secretion system is expressed only when there is a <strong>physical interaction</strong> between <i>R. solanacearum</i> and the plant cell. PrhA (plant regulator of hrp genes), the protein that recognizes the plant cell ligand, is an outer membrane receptor that shows homology with some TonB-dependent siderophore receptors. However, PrhA is not involved in the bacteria-plant cell adhesion, but only in sensing. The interaction of bacteria with plant cells occurs in two steps: first, <i>R. solanacearum</i> binds to the cell surface. This union is independent of PrhA or any protein encoded by <i>hrp</i>. Once the union has been established, the PrhA receiver can detect an accessible ligand, which increases the transcription of the <i>hrp</i> regulatory gene. Induction of <i>hrp</i> expression is very fast, around 90 minutes, a period much shorter than generation time of <i>R. solanacearum</i> in optimal conditions. </p><br />
<br />
<p>PrhA fundamental feature is that it is the first known bacterial receptor that can detect a <strong>non-diffusible signal</strong> present in plant cell walls. The possibility of attaching bacteria to a specific tissue was what made us choose the Ralstonia system for our project. The induction of the expression of virulence hrp regulon integrates a complex signaling cascade that begins in the PrhA outer membrane protein. PrhA transduces the contact-dependent signal through a complex regulatory cascade composed of PrhR, PrhI, PrhJ, HrpG and HrpB. Finally, HrpB activates the expression of <i>hrp</i>, comprising the TTSS structural genes and genes that encode effector proteins that travel through the TTSS.</p><br />
<br />
<br />
<br />
<h1>Signal Transduction Circuits</h1><br />
<br />
<h2>Prh system of <i>Ralstonia solanacearum</i></h2><br />
<br />
<h3>Brief description of the original Prh system:</h3><br />
<br />
<p>The <strong>Prh system</strong> integrates the genes involved in the control of expression of <strong><i>hrp</i> virulence genes</strong> from <i>Ralstonia solanacearum</i>. <i>hrp</i> gene encodes a type III secretion system, necessary to develop disease in their hosts. The induction of these genes integrates a complex signaling network that begins when the bacteria and the plant cell contact. This signaling mechanism is composed of PrhA PrhR PrhI proteins and other regulators that, as a last resort, activate the expression of the <i>hrp</i> or <i>hrc</i> (conserved hrp genes) genes.</p><br />
<br />
<p>The induction of virulence genes occurs when PrhA contacts with the plant cell. PrhA is an outer membrane protein that recognizes an unknown non-diffusible signal from the plant cell wall. PrhA-ligand binding causes that the periplasmic exposed N-terminal end of PrhA interacts with the carboxy terminal end of PrhR (an inner membrane protein) in the periplasm, transmitting the signal across the cytoplasmic membrane. In the cytoplasm inactive PrhI is actived by PrhR interaction by a still unkown mechanism.</p><br />
<br />
<p>The <i>prhIR</i> gene expression is induced in coculture with plant cells due to unknown environmental signal PrhA independent. PrhI is an ECF (extracytoplasmic function) sigma factor that, when it is activated, binds to RNA polymerase core enzyme and directs the polimerase to the promoter region of <i>PrhJ</i> gene to initiate transcription. In <i>R. solanacearum</i>, PrhJ protein induces <i>hrpG</i> transcription, which activates expression of <i>hrpB</i> gene and finally expresses <i>hrp</i> and <i>hrc</i> virulence genes.</p><br />
<br />
<p>The <strong>PrhA-PrhR-PrhI</strong> module of <i>Ralstonia</i> works similarly to FecA-FecR-FecI module of <i>E.coli</i>, with both similar sequences. PrhA shows homology with several members of the family of siderophore outer membrane receptors (as is the case of FecA). The three boxes that this family of proteins presents (TonB-box, box II and boxIII) are well conserved and correctly located in PrhA. PrhR has a transmembrane domain (TM) in the same position as FecR and both proteins have a similar orientation. In addition, two of the three tryptophan residues of the N-terminal end of FecR required to activate FecII are present in PrhR. However, unlike most of the siderophores, both <i>prhIR</i> and <i>prhA</i> lack Fur-boxes which are necessary for the regulation in function of the internal iron status.</p><br />
<br />
<p>Another striking difference between PrhAIR and FecAIR is their gene organization: while there is a physical grouping between genes of FecAIR, in PrhAIR system, <i>prhA</i> constitutes a monocistronic operon at the left edge of <i>hrp</i> gene cluster and <i>prhIR</i> is on the right side of cluster, both <i>prhA</i> and <i>prhIR</i> separated by virulence genes. Moreover, in contrast to the Fec system where FecA is activated by FecI and repressed by Fur, PrhA is always expressed a very low level in the presence of the inducing signal and is PrhI independent.</p><br />
<br />
<h3>Circuit 1:</h3><br />
<br />
<div class="imgRight"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/4/4e/BacterialCrowdingCircuit1.png" alt="Bacterial Crowding Circuit 1" /><br />
</div><br />
<br />
<p>In circuit 1 we wanted to use Prh system to transduce plant cell wall signals in chemoattractant synthesis. This first circuit integrates regulatory components PrhA-PrhI-PrhR and PprhJ of <i>R. solanacearum,</i> but they are transfered to <i>E. coli.</i> Genes required for synthesis and excretion of the chemoattractant are under PprhJ promoter control.</p><br />
<br />
<p>Because the Prh system is not fully characterized, unknown elements could be involved in and prevent the signal being correctly transmitted to P<i>prhJ</i>. Also, it is possible that it could not perform its usual answers being express in E.coli, for example it could be problems setting PrhA protein in the outer membrane. For that reasons, we have designed other circuits wich use <i>E. coli</i> proteins in the signal cascade.</p> <br />
<br />
<div class="clear"></div><br />
<br />
<h2>Fec system of <i>Escherichia coli</i></h2><br />
<br />
<h3>Brief description of the original Fec system:</h3><br />
<br />
<p><strong>Fec system</strong> includes genes involved in regulation and expression of <i>E. coli</i> iron transporters. <i>fecABCDE</i> genes express the <strong>ferric citrate transporter</strong> when bacteria iron status is low or deficient. The induction of genes <i>fecABCDE</i> integrates a signaling cascade that begins at the cell surface and is extended to the cytoplasm. To do this, three specific proteins are involved : FecA in outer membrane, FecR in cytoplasmic membrane and FecI in cytoplasm. This module <strong>FecA-FecR-FecI</strong> is known as a signal transduction system between three compartments (outside, periplasm and cytoplasm).</p><br />
<br />
<p>The signaling pathway begins when the outer membrane receptor FecA binds to its ligand, ferric dicitrate. This binding causes structural changes in FecA that allow the interaction of its amino terminal end to the carboxy terminal end of FecR in the periplasm. FecR, a transmembrane protein, transmits the signal to the cytoplasm, where it activates FecI. FecI is an extracitoplasmatic function (ECF) sigma factor that, when activated, binds to core RNA polymerase and directs the complex to the upstream promoter of <i>fecABCDE</i> transport genes to initiate transcription. </p><br />
<br />
<p>In addition, the transcription of regulatory genes <i>fecIR</i> is controlled by the internal iron status through the Fur repressor. When the Fur protein is loaded with Fe 2+, it represses <i>fecIR</i> transcription and prevents the <i>fec</i> gene expression. Therefore, the <i>fec</i> transport gene transcription is subjected to a double control: first, cells detect iron deficiency. Then, regulatory proteins FecI and FecR are synthetised, which, if ferric citrate is available, initiate the transcription of <i>fec</i> transport genes.</p><br />
<br />
<p>Dicitrate ferric transport through the outer membrane requires an energy transduction complex consisting of TonB, ExbB and Exb cytoplasmic membrane proteins.</p><br />
<br />
<h3>Circuit 2:</h3><br />
<br />
<div class="imgRight"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/a/a0/BacterialCrowdingCircuit2.jpg" alt="Bacterial Crowding Circuit 2" /><br />
</div><br />
<br />
<p>In our second circuit, the iron transport genes (<i>fecABCDE</i>) are replaced for genes required for synthesis and excretion of chemoattractant. Those genes remain under control of the promoter PfecABCDE, being regulated by the module FecA-FecI-FecR which is dependent of internal iron status and external ferric citrate concentration.</p><br />
<br />
<p>This circuit has the advantage that, besides being well characterized, is presented in wild type <i>E. coli</i>. However, this system will not be directed specifically to plant tissues and would be regulated by iron status of the bacteria and the environment. Nevertheless the second signal transduction circuit could be use like control of the chemoattractant synthesis. To this way, if the plant cell wall signal is not transduced properly, we could induce the chemoattractant synthesis changing medium conditions.</p><br />
<br />
<div class="clear"></div><br />
<br />
<h2>(FecA/PrhA)-FecI-FecR hybrid protein system</h2><br />
<br />
<h3>Circuit 3:</h3><br />
<br />
<div class="imgRight"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/9/9d/BacterialCrowdingCircuit3.jpg" alt="Bacterial Crowding Circuit 3" /><br />
</div><br />
<br />
<p>Our third circuit uses the FecA and PrhA <strong>sequence homology</strong>. We have designed a <strong>hybrid protein</strong> with the aim that it could detect the plant cell ligand and transmit the signal to FecR. The hybrid protein contains most of PrhA and the N-terminal end of FecA; binding both proteins by a shared sequence near the Ton-box. The signal would be transmited through the interaction between the periplasmic exposed N-terminal extension of FecA and the C-terminal part of FecR.</p><br />
<br />
<p>The third circuit would allow us to sense a non-diffusible signal and to transduce it using an E. coli system without problems with expression and function.</p><br />
<br />
<p>We have focused in this circuit.</p><br />
<br />
<div class="clear"></div><br />
<br />
<h2>PrhA-fecI-FecR hybrid system</h2><br />
<br />
<h3>Circuit 4:</h3><br />
<br />
<div class="imgRight"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/7/73/BacterialCrowdingCircuit4.png" alt="Bacterial Crowding Circuit 4" /><br />
</div><br />
<br />
<p>Due to the proximity in the life tree, the similarities between N-terminal extension of PrhA and FecA is significant, in particular the amino acid sequence Gx10(L,A)L(D,Q,A)G(S,T)L is well conserved. Also PrhR shows sequence similarity with FecR (27% identity, 43% similarity). Cause this information we wanted to test if the interaction between these systems was possible without modification.</p><br />
<br />
<p>However, this construction is largely a test.</p><br />
<br />
<div class="clear"></div><br />
<br />
<h1>Outer Membrane Protein Structures</h1><br />
<br />
<p>Now it is going to be shown the structure of the outer membrane proteins which start the signal transduction in sensing systems described before. It is interesting to study the structure and domains of FecA and PrhA before seeing the hybrid protein, in which we have focused our project mainly.</p><br />
<br />
<h2>Fe(3+) dicitrate transport protein FecA</h2><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/7/78/BacterialCrowndingFecA01.png" alt="Crystal structure of the Outer Membrane Transporter FecA"/><br />
<p class="caption"><b><a src="http://www.pdb.org/pdb/explore/explore.do?pdbId=1KMO" target="_blank">Crystal structure of the Outer Membrane Transporter FecA.</a></b></p><br />
<br />
<p><a href="http://www.uniprot.org/uniprot/P13036" target="_blank">FecA</a> is the outer membrane receptor protein in the Fe(3+) dicitrate transport system of <i>Escherichia coli</i>. It binds and transports ferric citrate, and it is required to initiate transcription of the <i>fecABCDE</i> transport operon but not the regulatory fecIR genes. This is a well-known protein, compound of 773 amino acids, whose main domains are shown below:</p><br />
<br />
<img class="centerBig" src="https://static.igem.org/mediawiki/2010/6/63/BacterialCrowdingFecADomains.png" alt="FecA Domains"/><br />
<br />
<p>The yellow left domain represents a <strong>signal peptide</strong> which takes from 1st to 33rd codon. The cleavage site of the signal peptidase has been found between residues 33 and 34<sup><a href="#Reference_Sensing01">[1]</a></sup>. Its function is to drive FecA protein to the outer membrane of <i>E. coli</i>, where the protein works.</p><br />
<br />
<div class="imgLeft"><br />
<img class="ileft" src="https://static.igem.org/mediawiki/2010/8/8c/BacterialCrowdingFecATonB.png" alt="the structure of the periplasmic signaling domain of FecA by nuclear magnetic resonance" /><br />
</div><br />
<br />
<p>The green illustration represents <strong>Secretin and TonB N-terminus Short Domain</strong> which takes from 57th to 107th codon. This domain is found at the N-terminus of the Secretins of the bacterial type II/III secretory system as well as the TonB-dependent receptor proteins. These proteins are involved in TonB-dependent active uptake of selective substrates. Thus, FecA interacts with TonB, which couples the electrochemical potential of the cytoplasmic membrane to active transport of ferric citrate across the outer membrane. The TonB box undergoes a substrate-induced disorder transition which produces an aqueous exposed, highly disordered protein fragment, which probably regulates transporter–TonB interactions<sup><a href="#Reference_Sensing02">[2]</a></sup>.</p><br />
<br />
<p>It is usual to find the TonB domain nearby signal and Plug domains. It is a common domain organization. At the left it is shown the structure of the periplasmic signaling domain of FecA by nuclear magnetic resonance. </p><br />
<br />
<p>Between both before domains it is a flexible 79-residue domain of FecA termed the <strong>NH2-terminal extension</strong>, which resides entirely within the periplasm. Its function is proposed to be to transmit the liganded status of the receptor to FecR<sup><a href="#Reference_Sensing03">[3]</a></sup>.</p><br />
<br />
<div class="clear"></div><br />
<br />
<p>In red color in the schematic representation it is shown the <strong>TonB-dependent Receptor Plug Domain</strong> which takes from 129th to 244th codon. The Plug domain has been shown to be an independently folding subunit of the TonB-dependent receptors. It acts as the channel gate, blocking the pore until the channel is bound by ligand. At this point it undergoes conformational changes that open the channel. Also ligand induces allosteric transitions which are propagated through the outer membrane by the plug domain, signaling the occupancy of the receptor in the periplasm. The plug domain is located inside a barrel, comprising five helices, two &beta; strands, and a mixed four-stranded &beta; sheet. Also three loops of the Plug domain extend above the plane of the upper leaflet of the outer membrane<sup><a href="#Reference_Sensing03">[3]</a></sup>.</p><br />
<br />
<img class="centerBig" src="https://static.igem.org/mediawiki/2010/0/04/BacterialCrowdingFecAUnligandedandFerric.png" alt="FecA Crystal structure"/><br />
<p class="caption"><b>a.</b>Crystal structure of ferric citrate transporter FecA in the unliganded form <br/><br />
<b>b.</b>Crystal structure of the outer membrane transporter FecA complexed with ferric citrate</p><br />
<br />
<p>Finally in the C-terminus there is a <strong>TonB Dependent Receptor Domain</strong> which takes from 525th to 773rd codon. The TonB dependent receptor domain is included in the 22-stranded &beta; barrel that traverse de outer membrane. The barrel of a TonB dependent receptor is a dynamic entity that actively participates in the energy-dependent siderophore uptake. This barrel has elipsoidal shape as you can see in before representations of FecA. Below it is shown the C-terminal domain of FecA, from 525th codon to the end.</p><br />
<br />
<img class="centerSmall" src="https://static.igem.org/mediawiki/2010/9/9d/BacterialCrowdingFecAC.png" alt="C-terminal domain of FecA"/><br />
<p class="caption">C-Terminal domain of FecA (representation made with RasWin program)</p><br />
<br />
<p>The common domain organization represents TonB dependent receptor domain at the same time as Plug domain because the interaction between the receptor (FecA) and the ligand (dinuclear ferric citrate molecule) is performed by Plug domain and the barrel. Formation of the liganded complex carries out changes on the conformation of the barrel and the Plug domain of FecA.</p><br />
<br />
<h2>Outer membrane receptor protein PrhA</h2><br />
<br />
<p>PrhA is the only known protein able to detect a non-diffusible signal and transduce this information into the cell. It is compound of 770 amino acids and it was found not too long ago. This is why there is not many information about it. Not being well-known is a point to use the hybrid protein FecA/PrhA instead of it. Anyway, their main domains are shown in <a href="http://pfam.sanger.ac.uk/protein?acc=B7ZJG7" target="_blank">Pfam website</a>, but it is not possible to see its structure because it has not been modeled yet.</p><br />
<br />
<img class="centerBig" src="https://static.igem.org/mediawiki/2010/d/dd/BacterialCrowdingPrhADomains.png" alt="PrhA Domains"/><br />
<br />
<p>Like in the case of FecA, PrhA has a putative <strong>signal peptide</strong> which takes from 1st to 35th codon. Its function would be direct PrhA to the outer membrane of <i>Rastonia solanacearum</i>. Despite its existence, you can not see it in the domain summary picture since it has not been well studied.</p><br />
<br />
<p>Next it is a not confirmed domain with unknown function which would take from the beginning of the protein to 130th amino acid. By now, it is called <strong>PfamB PB000342</strong> and its family was generated automatically from an alignment taken from Automatic Domain Decomposition Algorithm (<a href="http://ekhidna.biocenter.helsinki.fi/sqgraph/pairsdb/index_html" target="_blank">ADDA</a>). Since PrhA interacts with PrhR using its periplasmic domain, it is expected that this domain performs that function.</p><br />
<br />
<p>Then, PrhA presents the same domains that FecA: <strong>TonB-dependent Receptor Plug Domain</strong> (154 – 250 aa.) and <strong>TonB-dependent Receptor Domain</strong>(542 – 767 aa.), setting out the high similarities that exist between these two outer membrane proteins. Also their N-terminal extensions are quite similar as it was found by Marenda et al<sup><a href="#Reference_Sensing04">[4]</a></sup>.</p><br />
<br />
<img class="centerBig" src="https://static.igem.org/mediawiki/2010/c/c8/BacterialCrowdingFerrecCitrateTree.png" alt="Ferric Citrate tree"/><br />
<p class="caption"> <strong> Phylogenetic tree of TonB-dependent receptors </strong>. The tree was constructed as described by Rakin et al. (1994). Circled numbers indicate the number of times (from the whole 100) a particular node was supported by bootstrap analysis. The proteins used in this analysis are referenced in Rakin et al. (1994).<sup><a href="#Reference_Sensing04">[4]</a></sup></p><br />
<br />
<p>PrhA has high similarities with TonB-dependent receptors, which need to interact with the TonB protein to perform their functions. It shares two of the three main domains those proteins have. Nevertheless, PrhA is lacking of the periplasmic Secretin and TonB N-terminus Short Domain, the necessary domain to interact with TonB. In its place there is an unknown domain still not well studied. It will be required to continue studying this protein to know if TonB is necessary in its function and understand the evolution that TonB interaction domain suffered.</p><br />
<br />
<h2>Hybrid protein FecA/PrhA</h2><br />
<br />
<p>Taking in advance that Prh system is not naturally expressed in <i>Escherichia coli</i> and that Prh system is not well-known, we decided to create a fusion protein. FecA/PrhA artificial coding sequence spans the first 92 codons of <i>fecA</i>, encoding the signal peptide, NH2 terminal extension and the proposed Ton-box, fused to the distal end of the <i>prhA</i> coding sequence at the conserved GSGL motif (aa. 89-92). We synthesized this biobrick using MrGene services, so we also optimized the sequence to be expressed in <i>Escherichia coli</i>.</p><br />
<br />
<img class="centerBig" src="https://static.igem.org/mediawiki/2010/2/2c/BacterialCrowdingFecAPrhAHybrid.png" alt="PrhA, FecA and Hybrid Domains"/><br />
<br />
<p>Above is shown approximately the point where we fused FecA and PrhA proteins. To this way the hybrid protein include domains from both OM proteins:</p><br />
<br />
<ul><br />
<li><strong>Signal peptide of FecA</strong> which will help to the accurate emplacement of the hybrid protein in the outer membrane of <i>E. coli</i>.</li><br />
<br />
<li><strong>NH2-terminal extension of FecA</strong>. Including this domain of FecA means including the periplasmic signaling domain of this protein. The signal transfer between the OM and the IM proteins is performed between the N-terminus of FecA and the C-terminus of FecR (both are shown in the periplasmic). The hybrid protein includes the N-terminus of FecA so our expectation is that FecA/PrhA protein was able to interact with FecR.</li><br />
<br />
<li>Most of the <strong>Secretin and TonB N-terminus Short Domain of FecA</strong>. This domain helps FecA to interact with TonB. If TonB interaction is required for the OM-IM signal transfer, our hybrid protein includes this domain. Also, doing this fusion the hybrid protein loses the unknown function domain set in the N-terminus of PrhA.<br/><br />
<img class="centerSmall" src="https://static.igem.org/mediawiki/2010/2/21/BacterialCrowdingFecAN.png" alt="N-terminus short domain of FecA"/><br />
<p class="caption"><strong>N-terminus</strong> (aa. 34-92) <strong>of FecA.</strong> Here is shown the FecA contribution to the hybrid protein. Our aim is that this structure was able to interact with FecR without the rest of FecA protein. Representation made with RasWin.</p></li><br />
<br />
<li><strong>TonB-dependent Receptor Plug Domain of PrhA</strong>. In 89<sup>th</sup> codon there is a conserved motif which was used to fuse FecA with PrhA. The function of the Plug domain is to propagate allosteric transitions through the outer membrane signaling the occupancy of the receptor.</li><br />
<br />
<li><strong>TonB Dependent Receptor Domain of PrhA</strong>. From the conserved motif GSGL to the end of the protein amino acids are the same that in PrhA protein. The hybrid protein includes most of the PrhA protein, from 92<sup>sd</sup> codon to the C-terminus. The aim of it is that FecA/PrhA was able to interact with the non-diffusible plant wall signal that PrhA detects. The mechanism of this interaction is unknown.</li><br />
</ul><br />
<br />
<p>As you can see we work with a lot of uncertainty cause of the unknown mechanisms that manage the process we work with. Anyhow, we hope that this hybrid protein allows to sense non-difusible signals (with PrhA domains) and to transduce it by the Fec pathway (using the N-terminus of FecA). If this happened we would not have any problem with other Prh protein because the signal would continue by FecR and FecI in the Fec pathway of <i>E. coli<i>.</p><br />
<br />
<h1>Bibliography</h1><br />
<br />
<ol><br />
<li id="Reference_Sensing01">Uwe Pressler, Horst Staudenmaier, Luitgard Zimmermann, And Volkmar Braun (1988), Genetics of the Iron Dicitrate Transport System of Escherichia coli. JOURNAL OF BACTERIOLOGY, June 1988, p. 2716-2724</li><br />
<br />
<li id="Reference_Sensing02">Miyeon Kim, Gail E. Fanucci, and David S. Cafiso (2007), Substrate-dependent transmembrane signaling in TonB-dependent transporters is not conserved. PNAS, July 17, 2007, vol. 104, no. 29, 11975–11980.</li><br />
<br />
<li id="Reference_Sensing03">Andrew D. Ferguson, et al (2002). Structural Basis of Gating by the Outer Membrane Transporter FecA. Sience 295, 1715.</li> <br />
<br />
<li id="Reference_Sensing04">Marc Marenda, Belen Brito, Didier Callard, Stéphane Genin, Patrick Barberis, Christian Boucher and Matthieu Arlat (1998). PrhA controls a novel regulatory pathway required for the specific induction of Ralstonia solanacearum hrp genes in the presence of plant cells. Molecular Microbiology (1998) 27(2), 437–453.<br/><br />
<br />
</li> <br />
<br />
</ol><br />
<br />
<ul><br />
<li>Brito, B., Marenda, M., Barberis, P., Boucher, C., and Genin, S. 1999. <i>prhJ and hrpG: Two new components of the plant signal-dependent regulatory cascade controlled by PrhA in Ralstonia solanacearum</i>. Mol. Microbiol. 31:237-251.</li><br />
<br />
<li>Marenda, M., Brito, B., Callard, D., Genin, S., Barberis, P., Boucher, C. A., and Arlat, M. 1998. <i>PrhA controls a novel regulatory pathway required for the specific induction of Ralstonia solanacearum hrp genes in the presence of plant cells</i>. Mol. Microbiol. 27:437-453.</li><br />
<br />
<li>Aldon, D., Brito, B., Boucher, C., and Genin, S. 2000. <i>A bacterial sensor of plant cell contact controls the transcriptional induction of Ralstonia solanacearum pathogenicity genes</i>. EMBO (Eur. Mol. Biol. Organ.) J. 19:2304-2314.</li><br />
<br />
<li>Brito, B., Aldon, D., Barberis, P., Boucher, C., and Genin, S. 2002. <i>A Signal Transfer System Through Three Compartments Transduces the Plant Cell Contact-Dependent Signal Controlling Ralstonia solanacearum hrp Genes</i>. Molecular Plant-Microbe Interactions. Vol. 15, No. 2: 109/119</li><br />
<br />
<li>Braun V, Mahren S, Sauter A. <i>Gene regulation by transmembrane signaling</i>. 2006. Biometals. 19(2):103-13</li><br />
<br />
<li>Braun V, Mahren S, Ogierman M. 2003. <i>Regulation of the FecI-type ECF sigma factor by transmembrane signalling</i>. Curr Opin Microbiol. 6(2):173-80.</li><br />
<br />
<li>Enz, S., Brand, H., Orellana, C., Mahren, S., and Braun, V. 2003. <i>Sites of Interaction between the FecA and FecR Signal Transduction Proteins of Ferric Citrate Transport in Escherichia coli</i> K-12. J. Bacteriol. Vol. 185, No: 133745–3752 </li><br />
<br />
<li>Kim, M., Fanucci, G. E., and Cafiso, D. S. 2007. <i>Substrate-dependent transmembrane signaling in TonB-dependent transporters is not conserved</i>. PNAS. Vol. 104 N. 29: 11975/11980</li><br />
<br />
<li>http://www.mikrobio.uni-tuebingen.de/ag_braun/research_areas.html</li><br />
<li>www.uniprot.org </li><br />
<li>http://pfam.sanger.ac.uk/ </li><br />
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</ul><br />
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<h1>Introduction</h1><br />
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<p>Bacteria can sense a lot of different stimuli. They have to detect changes in their environment and to interpret them. Bacteria use different receptors to sense ionic state, chemicals, pH, nutrients, lights… and, when a stimuli is caused, they generate a intracellular signaling pathway and produce a specific response. Usually, signals that bacteria can sense are diffusible-signals. However, we will use the first known system to sense a non-diffusible signal: the Prh system of <i>Ralstonia solanacearum</i>. This system detects an unknown polysaccharide ligand of plant cell walls and activates a signal transduction cascade that, in original system, causes the virulence gene expression. </p><br />
<br />
<p>The aim of the <strong>sensing circuits</strong> is to ensure that our system is able to detect specifically the walls of plant cells and causes the secretion and excretion of a chemoattractant through signal transduction pathways. In order to achieve effective bacterial accumulation around plant cells, we have designed four different circuits that attempt to prevent possible different problems. We have combined different regulated proteins from <i>Escherichia coli</i> and <i>Ralstonia solanacearum</i> with specific biobricks, and we have replaced virulence genes by chemoattranctant genes. So, despite of <i>R. solanacearum</i> toxicity, the fact of working with biobricks avoids us having to work with the security measures required by this strain. </p><br />
<br />
<p>A future improvement for our project would be to use an <strong>adhesin</strong> to keep the bacteria attached to the plant cell wall as has been happening in R.solanacearum. We expect our system works without it.</p><br />
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<br />
<br />
<h3>Brief description of <i>Ralstonia solanacearum</i>:</h3><br />
<br />
<p><i><strong>Ralstonia solanacearum</strong></i> belongs to the family of Gram-negative phyto-pathogens. This bacterium causes great losses in crops worldwide in tropical, subtropical and temperate environments. The genes involved in virulence are known as <i>hrp</i> (hypersensitive response and pathogenicity) and they are induced by contact with various plant species from three different families of dicotyledonous (Solanaceae, Cruciferae and legumes), including both host and non host species. These genes encode a type III secretion system (TTSS), required to develop the disease in the host or the hypersensitive response (HR) in resistant and non-host plants. The HR is a defense mechanism that certain plant species possess, in which cells infected by a pathogen are killed to prevent spread to healthy tissue. </p><br />
<br />
<p>The secretion system is expressed only when there is a <strong>physical interaction</strong> between <i>R. solanacearum</i> and the plant cell. PrhA (plant regulator of hrp genes), the protein that recognizes the plant cell ligand, is an outer membrane receptor that shows homology with some TonB-dependent siderophore receptors. However, PrhA is not involved in the bacteria-plant cell adhesion, but only in sensing. The interaction of bacteria with plant cells occurs in two steps: first, <i>R. solanacearum</i> binds to the cell surface. This union is independent of PrhA or any protein encoded by <i>hrp</i>. Once the union has been established, the PrhA receiver can detect an accessible ligand, which increases the transcription of the <i>hrp</i> regulatory gene. Induction of <i>hrp</i> expression is very fast, around 90 minutes, a period much shorter than generation time of <i>R. solanacearum</i> in optimal conditions. </p><br />
<br />
<p>PrhA fundamental feature is that it is the first known bacterial receptor that can detect a <strong>non-diffusible signal</strong> present in plant cell walls. The possibility of attaching bacteria to a specific tissue was what made us choose the Ralstonia system for our project. The induction of the expression of virulence hrp regulon integrates a complex signaling cascade that begins in the PrhA outer membrane protein. PrhA transduces the contact-dependent signal through a complex regulatory cascade composed of PrhR, PrhI, PrhJ, HrpG and HrpB. Finally, HrpB activates the expression of <i>hrp</i>, comprising the TTSS structural genes and genes that encode effector proteins that travel through the TTSS.</p><br />
<br />
<br />
<br />
<h1>Signal Transduction Circuits</h1><br />
<br />
<h2>Prh system of <i>Ralstonia solanacearum</i></h2><br />
<br />
<h3>Brief description of the original Prh system:</h3><br />
<br />
<p>The <strong>Prh system</strong> integrates the genes involved in the control of expression of <strong><i>hrp</i> virulence genes</strong> from <i>Ralstonia solanacearum</i>. <i>hrp</i> gene encodes a type III secretion system, necessary to develop disease in their hosts. The induction of these genes integrates a complex signaling network that begins when the bacteria and the plant cell contact. This signaling mechanism is composed of PrhA PrhR PrhI proteins and other regulators that, as a last resort, activate the expression of the <i>hrp</i> or <i>hrc</i> (conserved hrp genes) genes.</p><br />
<br />
<p>The induction of virulence genes occurs when PrhA contacts with the plant cell. PrhA is an outer membrane protein that recognizes an unknown non-diffusible signal from the plant cell wall. PrhA-ligand binding causes that the periplasmic exposed N-terminal end of PrhA interacts with the carboxy terminal end of PrhR (an inner membrane protein) in the periplasm, transmitting the signal across the cytoplasmic membrane. In the cytoplasm inactive PrhI is actived by PrhR interaction by a still unkown mechanism.</p><br />
<br />
<p>The <i>prhIR</i> gene expression is induced in coculture with plant cells due to unknown environmental signal PrhA independent. PrhI is an ECF (extracytoplasmic function) sigma factor that, when it is activated, binds to RNA polymerase core enzyme and directs the polimerase to the promoter region of <i>PrhJ</i> gene to initiate transcription. In <i>R. solanacearum</i>, PrhJ protein induces <i>hrpG</i> transcription, which activates expression of <i>hrpB</i> gene and finally expresses <i>hrp</i> and <i>hrc</i> virulence genes.</p><br />
<br />
<p>The <strong>PrhA-PrhR-PrhI</strong> module of <i>Ralstonia</i> works similarly to FecA-FecR-FecI module of <i>E.coli</i>, with both similar sequences. PrhA shows homology with several members of the family of siderophore outer membrane receptors (as is the case of FecA). The three boxes that this family of proteins presents (TonB-box, box II and boxIII) are well conserved and correctly located in PrhA. PrhR has a transmembrane domain (TM) in the same position as FecR and both proteins have a similar orientation. In addition, two of the three tryptophan residues of the N-terminal end of FecR required to activate FecII are present in PrhR. However, unlike most of the siderophores, both <i>prhIR</i> and <i>prhA</i> lack Fur-boxes which are necessary for the regulation in function of the internal iron status.</p><br />
<br />
<p>Another striking difference between PrhAIR and FecAIR is their gene organization: while there is a physical grouping between genes of FecAIR, in PrhAIR system, <i>prhA</i> constitutes a monocistronic operon at the left edge of <i>hrp</i> gene cluster and <i>prhIR</i> is on the right side of cluster, both <i>prhA</i> and <i>prhIR</i> separated by virulence genes. Moreover, in contrast to the Fec system where FecA is activated by FecI and repressed by Fur, PrhA is always expressed a very low level in the presence of the inducing signal and is PrhI independent.</p><br />
<br />
<h3>Circuit 1:</h3><br />
<br />
<div class="imgRight"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/4/4e/BacterialCrowdingCircuit1.png" alt="Bacterial Crowding Circuit 1" /><br />
</div><br />
<br />
<p>In circuit 1 we wanted to use Prh system to transduce plant cell wall signals in chemoattractant synthesis. This first circuit integrates regulatory components PrhA-PrhI-PrhR and PprhJ of <i>R. solanacearum,</i> but they are transfered to <i>E. coli.</i> Genes required for synthesis and excretion of the chemoattractant are under PprhJ promoter control.</p><br />
<br />
<p>Because the Prh system is not fully characterized, unknown elements could be involved in and prevent the signal being correctly transmitted to P<i>prhJ</i>. Also, it is possible that it could not perform its usual answers being express in E.coli, for example it could be problems setting PrhA protein in the outer membrane. For that reasons, we have designed other circuits wich use <i>E. coli</i> proteins in the signal cascade.</p> <br />
<br />
<div class="clear"></div><br />
<br />
<h2>Fec system of <i>Escherichia coli</i></h2><br />
<br />
<h3>Brief description of the original Fec system:</h3><br />
<br />
<p><strong>Fec system</strong> includes genes involved in regulation and expression of <i>E. coli</i> iron transporters. <i>fecABCDE</i> genes express the <strong>ferric citrate transporter</strong> when bacteria iron status is low or deficient. The induction of genes <i>fecABCDE</i> integrates a signaling cascade that begins at the cell surface and is extended to the cytoplasm. To do this, three specific proteins are involved : FecA in outer membrane, FecR in cytoplasmic membrane and FecI in cytoplasm. This module <strong>FecA-FecR-FecI</strong> is known as a signal transduction system between three compartments (outside, periplasm and cytoplasm).</p><br />
<br />
<p>The signaling pathway begins when the outer membrane receptor FecA binds to its ligand, ferric dicitrate. This binding causes structural changes in FecA that allow the interaction of its amino terminal end to the carboxy terminal end of FecR in the periplasm. FecR, a transmembrane protein, transmits the signal to the cytoplasm, where it activates FecI. FecI is an extracitoplasmatic function (ECF) sigma factor that, when activated, binds to core RNA polymerase and directs the complex to the upstream promoter of <i>fecABCDE</i> transport genes to initiate transcription. </p><br />
<br />
<p>In addition, the transcription of regulatory genes <i>fecIR</i> is controlled by the internal iron status through the Fur repressor. When the Fur protein is loaded with Fe 2+, it represses <i>fecIR</i> transcription and prevents the <i>fec</i> gene expression. Therefore, the <i>fec</i> transport gene transcription is subjected to a double control: first, cells detect iron deficiency. Then, regulatory proteins FecI and FecR are synthetised, which, if ferric citrate is available, initiate the transcription of <i>fec</i> transport genes.</p><br />
<br />
<p>Dicitrate ferric transport through the outer membrane requires an energy transduction complex consisting of TonB, ExbB and Exb cytoplasmic membrane proteins.</p><br />
<br />
<h3>Circuit 2:</h3><br />
<br />
<div class="imgRight"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/a/a0/BacterialCrowdingCircuit2.jpg" alt="Bacterial Crowding Circuit 2" /><br />
</div><br />
<br />
<p>In our second circuit, the iron transport genes (<i>fecABCDE</i>) are replaced for genes required for synthesis and excretion of chemoattractant. Those genes remain under control of the promoter PfecABCDE, being regulated by the module FecA-FecI-FecR which is dependent of internal iron status and external ferric citrate concentration.</p><br />
<br />
<p>This circuit has the advantage that, besides being well characterized, is presented in wild type <i>E. coli</i>. However, this system will not be directed specifically to plant tissues and would be regulated by iron status of the bacteria and the environment. Nevertheless the second signal transduction circuit could be use like control of the chemoattractant synthesis. To this way, if the plant cell wall signal is not transduced properly, we could induce the chemoattractant synthesis changing medium conditions.</p><br />
<br />
<div class="clear"></div><br />
<br />
<h2>(FecA/PrhA)-FecI-FecR hybrid protein system</h2><br />
<br />
<h3>Circuit 3:</h3><br />
<br />
<div class="imgRight"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/9/9d/BacterialCrowdingCircuit3.jpg" alt="Bacterial Crowding Circuit 3" /><br />
</div><br />
<br />
<p>Our third circuit uses the FecA and PrhA <strong>sequence homology</strong>. We have designed a <strong>hybrid protein</strong> with the aim that it could detect the plant cell ligand and transmit the signal to FecR. The hybrid protein contains most of PrhA and the N-terminal end of FecA; binding both proteins by a shared sequence near the Ton-box. The signal would be transmited through the interaction between the periplasmic exposed N-terminal extension of FecA and the C-terminal part of FecR.</p><br />
<br />
<p>The third circuit would allow us to sense a non-diffusible signal and to transduce it using an E. coli system without problems with expression and function.</p><br />
<br />
<p>We have focused in this circuit.</p><br />
<br />
<h2>PrhA-fecI-FecR hybrid system</h2><br />
<br />
<h3>Circuit 4:</h3><br />
<br />
<div class="imgRight"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/7/73/BacterialCrowdingCircuit4.png" alt="Bacterial Crowding Circuit 4" /><br />
</div><br />
<br />
<p>Due to the proximity in the life tree, the similarities between N-terminal extension of PrhA and FecA is significant, in particular the amino acid sequence Gx10(L,A)L(D,Q,A)G(S,T)L is well conserved. Also PrhR shows sequence similarity with FecR (27% identity, 43% similarity). Cause this information we wanted to test if the interaction between these systems was possible without modification.</p><br />
<br />
<p>However, this construction is largely a test.</p><br />
<br />
<div class="clear"></div><br />
<br />
<h1>Outer Membrane Protein Structures</h1><br />
<br />
<p>Now it is going to be shown the structure of the outer membrane proteins which start the signal transduction in sensing systems described before. It is interesting to study the structure and domains of FecA and PrhA before seeing the hybrid protein, in which we have focused our project mainly.</p><br />
<br />
<h2>Fe(3+) dicitrate transport protein FecA</h2><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/7/78/BacterialCrowndingFecA01.png" alt="Crystal structure of the Outer Membrane Transporter FecA"/><br />
<p class="caption"><b><a src="http://www.pdb.org/pdb/explore/explore.do?pdbId=1KMO" target="_blank">Crystal structure of the Outer Membrane Transporter FecA.</a></b></p><br />
<br />
<p><a href="http://www.uniprot.org/uniprot/P13036" target="_blank">FecA</a> is the outer membrane receptor protein in the Fe(3+) dicitrate transport system of <i>Escherichia coli</i>. It binds and transports ferric citrate, and it is required to initiate transcription of the <i>fecABCDE</i> transport operon but not the regulatory fecIR genes. This is a well-known protein, compound of 773 amino acids, whose main domains are shown below:</p><br />
<br />
<img class="centerBig" src="https://static.igem.org/mediawiki/2010/6/63/BacterialCrowdingFecADomains.png" alt="FecA Domains"/><br />
<br />
<p>The yellow left domain represents a <strong>signal peptide</strong> which takes from 1st to 33rd codon. The cleavage site of the signal peptidase has been found between residues 33 and 34<sup><a href="#Reference_Sensing01">[1]</a></sup>. Its function is to drive FecA protein to the outer membrane of <i>E. coli</i>, where the protein works.</p><br />
<br />
<div class="imgLeft"><br />
<img class="ileft" src="https://static.igem.org/mediawiki/2010/8/8c/BacterialCrowdingFecATonB.png" alt="the structure of the periplasmic signaling domain of FecA by nuclear magnetic resonance" /><br />
</div><br />
<br />
<p>The green illustration represents <strong>Secretin and TonB N-terminus Short Domain</strong> which takes from 57th to 107th codon. This domain is found at the N-terminus of the Secretins of the bacterial type II/III secretory system as well as the TonB-dependent receptor proteins. These proteins are involved in TonB-dependent active uptake of selective substrates. Thus, FecA interacts with TonB, which couples the electrochemical potential of the cytoplasmic membrane to active transport of ferric citrate across the outer membrane. The TonB box undergoes a substrate-induced disorder transition which produces an aqueous exposed, highly disordered protein fragment, which probably regulates transporter–TonB interactions<sup><a href="#Reference_Sensing02">[2]</a></sup>.</p><br />
<br />
<p>It is usual to find the TonB domain nearby signal and Plug domains. It is a common domain organization. At the left it is shown the structure of the periplasmic signaling domain of FecA by nuclear magnetic resonance. </p><br />
<br />
<p>Between both before domains it is a flexible 79-residue domain of FecA termed the <strong>NH2-terminal extension</strong>, which resides entirely within the periplasm. Its function is proposed to be to transmit the liganded status of the receptor to FecR<sup><a href="#Reference_Sensing03">[3]</a></sup>.</p><br />
<br />
<div class="clear"></div><br />
<br />
<p>In red color in the schematic representation it is shown the <strong>TonB-dependent Receptor Plug Domain</strong> which takes from 129th to 244th codon. The Plug domain has been shown to be an independently folding subunit of the TonB-dependent receptors. It acts as the channel gate, blocking the pore until the channel is bound by ligand. At this point it undergoes conformational changes that open the channel. Also ligand induces allosteric transitions which are propagated through the outer membrane by the plug domain, signaling the occupancy of the receptor in the periplasm. The plug domain is located inside a barrel, comprising five helices, two &beta; strands, and a mixed four-stranded &beta; sheet. Also three loops of the Plug domain extend above the plane of the upper leaflet of the outer membrane<sup><a href="#Reference_Sensing03">[3]</a></sup>.</p><br />
<br />
<img class="centerBig" src="https://static.igem.org/mediawiki/2010/0/04/BacterialCrowdingFecAUnligandedandFerric.png" alt="FecA Crystal structure"/><br />
<p class="caption"><b>a.</b>Crystal structure of ferric citrate transporter FecA in the unliganded form <br/><br />
<b>b.</b>Crystal structure of the outer membrane transporter FecA complexed with ferric citrate</p><br />
<br />
<p>Finally in the C-terminus there is a <strong>TonB Dependent Receptor Domain</strong> which takes from 525th to 773rd codon. The TonB dependent receptor domain is included in the 22-stranded &beta; barrel that traverse de outer membrane. The barrel of a TonB dependent receptor is a dynamic entity that actively participates in the energy-dependent siderophore uptake. This barrel has elipsoidal shape as you can see in before representations of FecA. Below it is shown the C-terminal domain of FecA, from 525th codon to the end.</p><br />
<br />
<img class="centerSmall" src="https://static.igem.org/mediawiki/2010/9/9d/BacterialCrowdingFecAC.png" alt="C-terminal domain of FecA"/><br />
<p class="caption">C-Terminal domain of FecA (representation made with RasWin program)</p><br />
<br />
<p>The common domain organization represents TonB dependent receptor domain at the same time as Plug domain because the interaction between the receptor (FecA) and the ligand (dinuclear ferric citrate molecule) is performed by Plug domain and the barrel. Formation of the liganded complex carries out changes on the conformation of the barrel and the Plug domain of FecA.</p><br />
<br />
<h2>Outer membrane receptor protein PrhA</h2><br />
<br />
<p>PrhA is the only known protein able to detect a non-diffusible signal and transduce this information into the cell. It is compound of 770 amino acids and it was found not too long ago. This is why there is not many information about it. Not being well-known is a point to use the hybrid protein FecA/PrhA instead of it. Anyway, their main domains are shown in <a href="http://pfam.sanger.ac.uk/protein?acc=B7ZJG7" target="_blank">Pfam website</a>, but it is not possible to see its structure because it has not been modeled yet.</p><br />
<br />
<img class="centerBig" src="https://static.igem.org/mediawiki/2010/d/dd/BacterialCrowdingPrhADomains.png" alt="PrhA Domains"/><br />
<br />
<p>Like in the case of FecA, PrhA has a putative <strong>signal peptide</strong> which takes from 1st to 35th codon. Its function would be direct PrhA to the outer membrane of <i>Rastonia solanacearum</i>. Despite its existence, you can not see it in the domain summary picture since it has not been well studied.</p><br />
<br />
<p>Next it is a not confirmed domain with unknown function which would take from the beginning of the protein to 130th amino acid. By now, it is called <strong>PfamB PB000342</strong> and its family was generated automatically from an alignment taken from Automatic Domain Decomposition Algorithm (<a href="http://ekhidna.biocenter.helsinki.fi/sqgraph/pairsdb/index_html" target="_blank">ADDA</a>). Since PrhA interacts with PrhR using its periplasmic domain, it is expected that this domain performs that function.</p><br />
<br />
<p>Then, PrhA presents the same domains that FecA: <strong>TonB-dependent Receptor Plug Domain</strong> (154 – 250 aa.) and <strong>TonB-dependent Receptor Domain</strong>(542 – 767 aa.), setting out the high similarities that exist between these two outer membrane proteins. Also their N-terminal extensions are quite similar as it was found by Marenda et al<sup><a href="#Reference_Sensing04">[4]</a></sup>.</p><br />
<br />
<img class="centerBig" src="https://static.igem.org/mediawiki/2010/c/c8/BacterialCrowdingFerrecCitrateTree.png" alt="Ferric Citrate tree"/><br />
<p class="caption"> <strong> Phylogenetic tree of TonB-dependent receptors </strong>. The tree was constructed as described by Rakin et al. (1994). Circled numbers indicate the number of times (from the whole 100) a particular node was supported by bootstrap analysis. The proteins used in this analysis are referenced in Rakin et al. (1994).<sup><a href="#Reference_Sensing04">[4]</a></sup></p><br />
<br />
<p>PrhA has high similarities with TonB-dependent receptors, which need to interact with the TonB protein to perform their functions. It shares two of the three main domains those proteins have. Nevertheless, PrhA is lacking of the periplasmic Secretin and TonB N-terminus Short Domain, the necessary domain to interact with TonB. In its place there is an unknown domain still not well studied. It will be required to continue studying this protein to know if TonB is necessary in its function and understand the evolution that TonB interaction domain suffered.</p><br />
<br />
<h2>Hybrid protein FecA/PrhA</h2><br />
<br />
<p>Taking in advance that Prh system is not naturally expressed in <i>Escherichia coli</i> and that Prh system is not well-known, we decided to create a fusion protein. FecA/PrhA artificial coding sequence spans the first 92 codons of <i>fecA</i>, encoding the signal peptide, NH2 terminal extension and the proposed Ton-box, fused to the distal end of the <i>prhA</i> coding sequence at the conserved GSGL motif (aa. 89-92). We synthesized this biobrick using MrGene services, so we also optimized the sequence to be expressed in <i>Escherichia coli</i>.</p><br />
<br />
<img class="centerBig" src="https://static.igem.org/mediawiki/2010/2/2c/BacterialCrowdingFecAPrhAHybrid.png" alt="PrhA, FecA and Hybrid Domains"/><br />
<br />
<p>Above is shown approximately the point where we fused FecA and PrhA proteins. To this way the hybrid protein include domains from both OM proteins:</p><br />
<br />
<ul><br />
<li><strong>Signal peptide of FecA</strong> which will help to the accurate emplacement of the hybrid protein in the outer membrane of <i>E. coli</i>.</li><br />
<br />
<li><strong>NH2-terminal extension of FecA</strong>. Including this domain of FecA means including the periplasmic signaling domain of this protein. The signal transfer between the OM and the IM proteins is performed between the N-terminus of FecA and the C-terminus of FecR (both are shown in the periplasmic). The hybrid protein includes the N-terminus of FecA so our expectation is that FecA/PrhA protein was able to interact with FecR.</li><br />
<br />
<li>Most of the <strong>Secretin and TonB N-terminus Short Domain of FecA</strong>. This domain helps FecA to interact with TonB. If TonB interaction is required for the OM-IM signal transfer, our hybrid protein includes this domain. Also, doing this fusion the hybrid protein loses the unknown function domain set in the N-terminus of PrhA.<br/><br />
<img class="centerSmall" src="https://static.igem.org/mediawiki/2010/2/21/BacterialCrowdingFecAN.png" alt="N-terminus short domain of FecA"/><br />
<p class="caption"><strong>N-terminus</strong> (aa. 34-92) <strong>of FecA.</strong> Here is shown the FecA contribution to the hybrid protein. Our aim is that this structure was able to interact with FecR without the rest of FecA protein. Representation made with RasWin.</p></li><br />
<br />
<li><strong>TonB-dependent Receptor Plug Domain of PrhA</strong>. In 89<sup>th</sup> codon there is a conserved motif which was used to fuse FecA with PrhA. The function of the Plug domain is to propagate allosteric transitions through the outer membrane signaling the occupancy of the receptor.</li><br />
<br />
<li><strong>TonB Dependent Receptor Domain of PrhA</strong>. From the conserved motif GSGL to the end of the protein amino acids are the same that in PrhA protein. The hybrid protein includes most of the PrhA protein, from 92<sup>sd</sup> codon to the C-terminus. The aim of it is that FecA/PrhA was able to interact with the non-diffusible plant wall signal that PrhA detects. The mechanism of this interaction is unknown.</li><br />
</ul><br />
<br />
<p>As you can see we work with a lot of uncertainty cause of the unknown mechanisms that manage the process we work with. Anyhow, we hope that this hybrid protein allows to sense non-difusible signals (with PrhA domains) and to transduce it by the Fec pathway (using the N-terminus of FecA). If this happened we would not have any problem with other Prh protein because the signal would continue by FecR and FecI in the Fec pathway of <i>E. coli<i>.</p><br />
<br />
<h1>Bibliography</h1><br />
<br />
<ol><br />
<li id="Reference_Sensing01">Uwe Pressler, Horst Staudenmaier, Luitgard Zimmermann, And Volkmar Braun (1988), Genetics of the Iron Dicitrate Transport System of Escherichia coli. JOURNAL OF BACTERIOLOGY, June 1988, p. 2716-2724</li><br />
<br />
<li id="Reference_Sensing02">Miyeon Kim, Gail E. Fanucci, and David S. Cafiso (2007), Substrate-dependent transmembrane signaling in TonB-dependent transporters is not conserved. PNAS, July 17, 2007, vol. 104, no. 29, 11975–11980.</li><br />
<br />
<li id="Reference_Sensing03">Andrew D. Ferguson, et al (2002). Structural Basis of Gating by the Outer Membrane Transporter FecA. Sience 295, 1715.</li> <br />
<br />
<li id="Reference_Sensing04">Marc Marenda, Belen Brito, Didier Callard, Stéphane Genin, Patrick Barberis, Christian Boucher and Matthieu Arlat (1998). PrhA controls a novel regulatory pathway required for the specific induction of Ralstonia solanacearum hrp genes in the presence of plant cells. Molecular Microbiology (1998) 27(2), 437–453.<br/><br />
<br />
</li> <br />
<br />
</ol><br />
<br />
<ul><br />
<li>Brito, B., Marenda, M., Barberis, P., Boucher, C., and Genin, S. 1999. <i>prhJ and hrpG: Two new components of the plant signal-dependent regulatory cascade controlled by PrhA in Ralstonia solanacearum</i>. Mol. Microbiol. 31:237-251.</li><br />
<br />
<li>Marenda, M., Brito, B., Callard, D., Genin, S., Barberis, P., Boucher, C. A., and Arlat, M. 1998. <i>PrhA controls a novel regulatory pathway required for the specific induction of Ralstonia solanacearum hrp genes in the presence of plant cells</i>. Mol. Microbiol. 27:437-453.</li><br />
<br />
<li>Aldon, D., Brito, B., Boucher, C., and Genin, S. 2000. <i>A bacterial sensor of plant cell contact controls the transcriptional induction of Ralstonia solanacearum pathogenicity genes</i>. EMBO (Eur. Mol. Biol. Organ.) J. 19:2304-2314.</li><br />
<br />
<li>Brito, B., Aldon, D., Barberis, P., Boucher, C., and Genin, S. 2002. <i>A Signal Transfer System Through Three Compartments Transduces the Plant Cell Contact-Dependent Signal Controlling Ralstonia solanacearum hrp Genes</i>. Molecular Plant-Microbe Interactions. Vol. 15, No. 2: 109/119</li><br />
<br />
<li>Braun V, Mahren S, Sauter A. <i>Gene regulation by transmembrane signaling</i>. 2006. Biometals. 19(2):103-13</li><br />
<br />
<li>Braun V, Mahren S, Ogierman M. 2003. <i>Regulation of the FecI-type ECF sigma factor by transmembrane signalling</i>. Curr Opin Microbiol. 6(2):173-80.</li><br />
<br />
<li>Enz, S., Brand, H., Orellana, C., Mahren, S., and Braun, V. 2003. <i>Sites of Interaction between the FecA and FecR Signal Transduction Proteins of Ferric Citrate Transport in Escherichia coli</i> K-12. J. Bacteriol. Vol. 185, No: 133745–3752 </li><br />
<br />
<li>Kim, M., Fanucci, G. E., and Cafiso, D. S. 2007. <i>Substrate-dependent transmembrane signaling in TonB-dependent transporters is not conserved</i>. PNAS. Vol. 104 N. 29: 11975/11980</li><br />
<br />
<li>http://www.mikrobio.uni-tuebingen.de/ag_braun/research_areas.html</li><br />
<li>www.uniprot.org </li><br />
<li>http://pfam.sanger.ac.uk/ </li><br />
<br />
</ul><br />
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<h1>Introduction</h1><br />
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<p>Bacteria can sense a lot of different stimuli. They have to detect changes in their environment and to interpret them. Bacteria use different receptors to sense ionic state, chemicals, pH, nutrients, lights… and, when a stimuli is caused, they generate a intracellular signaling pathway and produce a specific response. Usually, signals that bacteria can sense are diffusible-signals. However, we will use the first known system to sense a non-diffusible signal: the Prh system of <i>Ralstonia solanacearum</i>. This system detects an unknown polysaccharide ligand of plant cell walls and activates a signal transduction cascade that, in original system, causes the virulence gene expression. </p><br />
<br />
<p>The aim of the <strong>sensing circuits</strong> is to ensure that our system is able to detect specifically the walls of plant cells and causes the secretion and excretion of a chemoattractant through signal transduction pathways. In order to achieve effective bacterial accumulation around plant cells, we have designed four different circuits that attempt to prevent possible different problems. We have combined different regulated proteins from <i>Escherichia coli</i> and <i>Ralstonia solanacearum</i> with specific biobricks, and we have replaced virulence genes by chemoattranctant genes. So, despite of <i>R. solanacearum</i> toxicity, the fact of working with biobricks avoids us having to work with the security measures required by this strain. </p><br />
<br />
<p>A future improvement for our project would be to use an <strong>adhesin</strong> to keep the bacteria attached to the plant cell wall as has been happening in R.solanacearum. We expect our system works without it.</p><br />
<br />
<br />
<br />
<h3>Brief description of <i>Ralstonia solanacearum</i>:</h3><br />
<br />
<p><i><strong>Ralstonia solanacearum</strong></i> belongs to the family of Gram-negative phyto-pathogens. This bacterium causes great losses in crops worldwide in tropical, subtropical and temperate environments. The genes involved in virulence are known as <i>hrp</i> (hypersensitive response and pathogenicity) and they are induced by contact with various plant species from three different families of dicotyledonous (Solanaceae, Cruciferae and legumes), including both host and non host species. These genes encode a type III secretion system (TTSS), required to develop the disease in the host or the hypersensitive response (HR) in resistant and non-host plants. The HR is a defense mechanism that certain plant species possess, in which cells infected by a pathogen are killed to prevent spread to healthy tissue. </p><br />
<br />
<p>The secretion system is expressed only when there is a <strong>physical interaction</strong> between <i>R. solanacearum</i> and the plant cell. PrhA (plant regulator of hrp genes), the protein that recognizes the plant cell ligand, is an outer membrane receptor that shows homology with some TonB-dependent siderophore receptors. However, PrhA is not involved in the bacteria-plant cell adhesion, but only in sensing. The interaction of bacteria with plant cells occurs in two steps: first, <i>R. solanacearum</i> binds to the cell surface. This union is independent of PrhA or any protein encoded by <i>hrp</i>. Once the union has been established, the PrhA receiver can detect an accessible ligand, which increases the transcription of the <i>hrp</i> regulatory gene. Induction of <i>hrp</i> expression is very fast, around 90 minutes, a period much shorter than generation time of <i>R. solanacearum</i> in optimal conditions. </p><br />
<br />
<p>PrhA fundamental feature is that it is the first known bacterial receptor that can detect a <strong>non-diffusible signal</strong> present in plant cell walls. The possibility of attaching bacteria to a specific tissue was what made us choose the Ralstonia system for our project. The induction of the expression of virulence hrp regulon integrates a complex signaling cascade that begins in the PrhA outer membrane protein. PrhA transduces the contact-dependent signal through a complex regulatory cascade composed of PrhR, PrhI, PrhJ, HrpG and HrpB. Finally, HrpB activates the expression of <i>hrp</i>, comprising the TTSS structural genes and genes that encode effector proteins that travel through the TTSS.</p><br />
<br />
<br />
<br />
<h1>Signal Transduction Circuits</h1><br />
<br />
<h2>Prh system of <i>Ralstonia solanacearum</i></h2><br />
<br />
<h3>Brief description of the original Prh system:</h3><br />
<br />
<p>The <strong>Prh system</strong> integrates the genes involved in the control of expression of <strong><i>hrp</i> virulence genes</strong> from <i>Ralstonia solanacearum</i>. <i>hrp</i> gene encodes a type III secretion system, necessary to develop disease in their hosts. The induction of these genes integrates a complex signaling network that begins when the bacteria and the plant cell contact. This signaling mechanism is composed of PrhA PrhR PrhI proteins and other regulators that, as a last resort, activate the expression of the <i>hrp</i> or <i>hrc</i> (conserved hrp genes) genes.</p><br />
<br />
<p>The induction of virulence genes occurs when PrhA contacts with the plant cell. PrhA is an outer membrane protein that recognizes an unknown non-diffusible signal from the plant cell wall. PrhA-ligand binding causes that the periplasmic exposed N-terminal end of PrhA interacts with the carboxy terminal end of PrhR (an inner membrane protein) in the periplasm, transmitting the signal across the cytoplasmic membrane. In the cytoplasm inactive PrhI is actived by PrhR interaction by a still unkown mechanism.</p><br />
<br />
<p>The <i>prhIR</i> gene expression is induced in coculture with plant cells due to unknown environmental signal PrhA independent. PrhI is an ECF (extracytoplasmic function) sigma factor that, when it is activated, binds to RNA polymerase core enzyme and directs the polimerase to the promoter region of <i>PrhJ</i> gene to initiate transcription. In <i>R. solanacearum</i>, PrhJ protein induces <i>hrpG</i> transcription, which activates expression of <i>hrpB</i> gene and finally expresses <i>hrp</i> and <i>hrc</i> virulence genes.</p><br />
<br />
<p>The <strong>PrhA-PrhR-PrhI</strong> module of <i>Ralstonia</i> works similarly to FecA-FecR-FecI module of <i>E.coli</i>, with both similar sequences. PrhA shows homology with several members of the family of siderophore outer membrane receptors (as is the case of FecA). The three boxes that this family of proteins presents (TonB-box, box II and boxIII) are well conserved and correctly located in PrhA. PrhR has a transmembrane domain (TM) in the same position as FecR and both proteins have a similar orientation. In addition, two of the three tryptophan residues of the N-terminal end of FecR required to activate FecII are present in PrhR. However, unlike most of the siderophores, both <i>prhIR</i> and <i>prhA</i> lack Fur-boxes which are necessary for the regulation in function of the internal iron status.</p><br />
<br />
<p>Another striking difference between PrhAIR and FecAIR is their gene organization: while there is a physical grouping between genes of FecAIR, in PrhAIR system, <i>prhA</i> constitutes a monocistronic operon at the left edge of <i>hrp</i> gene cluster and <i>prhIR</i> is on the right side of cluster, both <i>prhA</i> and <i>prhIR</i> separated by virulence genes. Moreover, in contrast to the Fec system where FecA is activated by FecI and repressed by Fur, PrhA is always expressed a very low level in the presence of the inducing signal and is PrhI independent.</p><br />
<br />
<h3>Circuit 1:</h3><br />
<br />
<div class="imgRight"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/4/4e/BacterialCrowdingCircuit1.png" alt="Bacterial Crowding Circuit 1" /><br />
</div><br />
<br />
<p>In circuit 1 we wanted to use Prh system to transduce plant cell wall signals in chemoattractant synthesis. This first circuit integrates regulatory components PrhA-PrhI-PrhR and PprhJ of <i>R. solanacearum,</i> but they are transfered to <i>E. coli.</i> Genes required for synthesis and excretion of the chemoattractant are under PprhJ promoter control.</p><br />
<br />
<p>Because the Prh system is not fully characterized, unknown elements could be involved in and prevent the signal being correctly transmitted to P<i>prhJ</i>. Also, it is possible that it could not perform its usual answers being express in E.coli, for example it could be problems setting PrhA protein in the outer membrane. For that reasons, we have designed other circuits wich use <i>E. coli</i> proteins in the signal cascade.</p> <br />
<br />
<div class="clear"></div><br />
<br />
<h2>Fec system of <i>Escherichia coli</i></h2><br />
<br />
<h3>Brief description of the original Fec system:</h3><br />
<br />
<p><strong>Fec system</strong> includes genes involved in regulation and expression of <i>E. coli</i> iron transporters. <i>fecABCDE</i> genes express the <strong>ferric citrate transporter</strong> when bacteria iron status is low or deficient. The induction of genes <i>fecABCDE</i> integrates a signaling cascade that begins at the cell surface and is extended to the cytoplasm. To do this, three specific proteins are involved : FecA in outer membrane, FecR in cytoplasmic membrane and FecI in cytoplasm. This module <strong>FecA-FecR-FecI</strong> is known as a signal transduction system between three compartments (outside, periplasm and cytoplasm).</p><br />
<br />
<p>The signaling pathway begins when the outer membrane receptor FecA binds to its ligand, ferric dicitrate. This binding causes structural changes in FecA that allow the interaction of its amino terminal end to the carboxy terminal end of FecR in the periplasm. FecR, a transmembrane protein, transmits the signal to the cytoplasm, where it activates FecI. FecI is an extracitoplasmatic function (ECF) sigma factor that, when activated, binds to core RNA polymerase and directs the complex to the upstream promoter of <i>fecABCDE</i> transport genes to initiate transcription. </p><br />
<br />
<p>In addition, the transcription of regulatory genes <i>fecIR</i> is controlled by the internal iron status through the Fur repressor. When the Fur protein is loaded with Fe 2+, it represses <i>fecIR</i> transcription and prevents the <i>fec</i> gene expression. Therefore, the <i>fec</i> transport gene transcription is subjected to a double control: first, cells detect iron deficiency. Then, regulatory proteins FecI and FecR are synthetised, which, if ferric citrate is available, initiate the transcription of <i>fec</i> transport genes.</p><br />
<br />
<p>Dicitrate ferric transport through the outer membrane requires an energy transduction complex consisting of TonB, ExbB and Exb cytoplasmic membrane proteins.</p><br />
<br />
<h3>Circuit 2:</h3><br />
<br />
<div class="imgRight"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/a/a0/BacterialCrowdingCircuit2.jpg" alt="Bacterial Crowding Circuit 2" /><br />
</div><br />
<br />
<p>In our second circuit, the iron transport genes (<i>fecABCDE</i>) are replaced for genes required for synthesis and excretion of chemoattractant. Those genes remain under control of the promoter PfecABCDE, being regulated by the module FecA-FecI-FecR which is dependent of internal iron status and external ferric citrate concentration.</p><br />
<br />
<p>This circuit has the advantage that, besides being well characterized, is presented in wild type <i>E. coli</i>. However, this system will not be directed specifically to plant tissues and would be regulated by iron status of the bacteria and the environment. Nevertheless the second signal transduction circuit could be use like control of the chemoattractant synthesis. To this way, if the plant cell wall signal is not transduced properly, we could induce the chemoattractant synthesis changing medium conditions.</p><br />
<br />
<div class="clear"></div><br />
<br />
<h2>(FecA/PrhA)-FecI-FecR hybrid protein system</h2><br />
<br />
<h3>Circuit 3:</h3><br />
<br />
<p>Our third circuit uses the FecA and PrhA <strong>sequence homology</strong>. We have designed a <strong>hybrid protein</strong> with the aim that it could detect the plant cell ligand and transmit the signal to FecR. The hybrid protein contains most of PrhA and the N-terminal end of FecA; binding both proteins by a shared sequence near the Ton-box. The signal would be transmited through the interaction between the periplasmic exposed N-terminal extension of FecA and the C-terminal part of FecR.</p><br />
<br />
<p>The third circuit would allow us to sense a non-diffusible signal and to transduce it using an E. coli system without problems with expression and function.</p><br />
<br />
<p>We have focused in this circuit.</p><br />
<br />
<h2>PrhA-fecI-FecR hybrid system</h2><br />
<br />
<h3>Circuit 4:</h3><br />
<br />
<div class="imgRight"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/7/73/BacterialCrowdingCircuit4.png" alt="Bacterial Crowding Circuit 4" /><br />
</div><br />
<br />
<p>Due to the proximity in the life tree, the similarities between N-terminal extension of PrhA and FecA is significant, in particular the amino acid sequence Gx10(L,A)L(D,Q,A)G(S,T)L is well conserved. Also PrhR shows sequence similarity with FecR (27% identity, 43% similarity). Cause this information we wanted to test if the interaction between these systems was possible without modification.</p><br />
<br />
<p>However, this construction is largely a test.</p><br />
<br />
<div class="clear"></div><br />
<br />
<h1>Outer Membrane Protein Structures</h1><br />
<br />
<p>Now it is going to be shown the structure of the outer membrane proteins which start the signal transduction in sensing systems described before. It is interesting to study the structure and domains of FecA and PrhA before seeing the hybrid protein, in which we have focused our project mainly.</p><br />
<br />
<h2>Fe(3+) dicitrate transport protein FecA</h2><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/7/78/BacterialCrowndingFecA01.png" alt="Crystal structure of the Outer Membrane Transporter FecA"/><br />
<p class="caption"><b><a src="http://www.pdb.org/pdb/explore/explore.do?pdbId=1KMO" target="_blank">Crystal structure of the Outer Membrane Transporter FecA.</a></b></p><br />
<br />
<p><a href="http://www.uniprot.org/uniprot/P13036" target="_blank">FecA</a> is the outer membrane receptor protein in the Fe(3+) dicitrate transport system of <i>Escherichia coli</i>. It binds and transports ferric citrate, and it is required to initiate transcription of the <i>fecABCDE</i> transport operon but not the regulatory fecIR genes. This is a well-known protein, compound of 773 amino acids, whose main domains are shown below:</p><br />
<br />
<img class="centerBig" src="https://static.igem.org/mediawiki/2010/6/63/BacterialCrowdingFecADomains.png" alt="FecA Domains"/><br />
<br />
<p>The yellow left domain represents a <strong>signal peptide</strong> which takes from 1st to 33rd codon. The cleavage site of the signal peptidase has been found between residues 33 and 34<sup><a href="#Reference_Sensing01">[1]</a></sup>. Its function is to drive FecA protein to the outer membrane of <i>E. coli</i>, where the protein works.</p><br />
<br />
<div class="imgLeft"><br />
<img class="ileft" src="https://static.igem.org/mediawiki/2010/8/8c/BacterialCrowdingFecATonB.png" alt="the structure of the periplasmic signaling domain of FecA by nuclear magnetic resonance" /><br />
</div><br />
<br />
<p>The green illustration represents <strong>Secretin and TonB N-terminus Short Domain</strong> which takes from 57th to 107th codon. This domain is found at the N-terminus of the Secretins of the bacterial type II/III secretory system as well as the TonB-dependent receptor proteins. These proteins are involved in TonB-dependent active uptake of selective substrates. Thus, FecA interacts with TonB, which couples the electrochemical potential of the cytoplasmic membrane to active transport of ferric citrate across the outer membrane. The TonB box undergoes a substrate-induced disorder transition which produces an aqueous exposed, highly disordered protein fragment, which probably regulates transporter–TonB interactions<sup><a href="#Reference_Sensing02">[2]</a></sup>.</p><br />
<br />
<p>It is usual to find the TonB domain nearby signal and Plug domains. It is a common domain organization. At the left it is shown the structure of the periplasmic signaling domain of FecA by nuclear magnetic resonance. </p><br />
<br />
<p>Between both before domains it is a flexible 79-residue domain of FecA termed the <strong>NH2-terminal extension</strong>, which resides entirely within the periplasm. Its function is proposed to be to transmit the liganded status of the receptor to FecR<sup><a href="#Reference_Sensing03">[3]</a></sup>.</p><br />
<br />
<div class="clear"></div><br />
<br />
<p>In red color in the schematic representation it is shown the <strong>TonB-dependent Receptor Plug Domain</strong> which takes from 129th to 244th codon. The Plug domain has been shown to be an independently folding subunit of the TonB-dependent receptors. It acts as the channel gate, blocking the pore until the channel is bound by ligand. At this point it undergoes conformational changes that open the channel. Also ligand induces allosteric transitions which are propagated through the outer membrane by the plug domain, signaling the occupancy of the receptor in the periplasm. The plug domain is located inside a barrel, comprising five helices, two &beta; strands, and a mixed four-stranded &beta; sheet. Also three loops of the Plug domain extend above the plane of the upper leaflet of the outer membrane<sup><a href="#Reference_Sensing03">[3]</a></sup>.</p><br />
<br />
<img class="centerBig" src="https://static.igem.org/mediawiki/2010/0/04/BacterialCrowdingFecAUnligandedandFerric.png" alt="FecA Crystal structure"/><br />
<p class="caption"><b>a.</b>Crystal structure of ferric citrate transporter FecA in the unliganded form <br/><br />
<b>b.</b>Crystal structure of the outer membrane transporter FecA complexed with ferric citrate</p><br />
<br />
<p>Finally in the C-terminus there is a <strong>TonB Dependent Receptor Domain</strong> which takes from 525th to 773rd codon. The TonB dependent receptor domain is included in the 22-stranded &beta; barrel that traverse de outer membrane. The barrel of a TonB dependent receptor is a dynamic entity that actively participates in the energy-dependent siderophore uptake. This barrel has elipsoidal shape as you can see in before representations of FecA. Below it is shown the C-terminal domain of FecA, from 525th codon to the end.</p><br />
<br />
<img class="centerSmall" src="https://static.igem.org/mediawiki/2010/9/9d/BacterialCrowdingFecAC.png" alt="C-terminal domain of FecA"/><br />
<p class="caption">C-Terminal domain of FecA (representation made with RasWin program)</p><br />
<br />
<p>The common domain organization represents TonB dependent receptor domain at the same time as Plug domain because the interaction between the receptor (FecA) and the ligand (dinuclear ferric citrate molecule) is performed by Plug domain and the barrel. Formation of the liganded complex carries out changes on the conformation of the barrel and the Plug domain of FecA.</p><br />
<br />
<h2>Outer membrane receptor protein PrhA</h2><br />
<br />
<p>PrhA is the only known protein able to detect a non-diffusible signal and transduce this information into the cell. It is compound of 770 amino acids and it was found not too long ago. This is why there is not many information about it. Not being well-known is a point to use the hybrid protein FecA/PrhA instead of it. Anyway, their main domains are shown in <a href="http://pfam.sanger.ac.uk/protein?acc=B7ZJG7" target="_blank">Pfam website</a>, but it is not possible to see its structure because it has not been modeled yet.</p><br />
<br />
<img class="centerBig" src="https://static.igem.org/mediawiki/2010/d/dd/BacterialCrowdingPrhADomains.png" alt="PrhA Domains"/><br />
<br />
<p>Like in the case of FecA, PrhA has a putative <strong>signal peptide</strong> which takes from 1st to 35th codon. Its function would be direct PrhA to the outer membrane of <i>Rastonia solanacearum</i>. Despite its existence, you can not see it in the domain summary picture since it has not been well studied.</p><br />
<br />
<p>Next it is a not confirmed domain with unknown function which would take from the beginning of the protein to 130th amino acid. By now, it is called <strong>PfamB PB000342</strong> and its family was generated automatically from an alignment taken from Automatic Domain Decomposition Algorithm (<a href="http://ekhidna.biocenter.helsinki.fi/sqgraph/pairsdb/index_html" target="_blank">ADDA</a>). Since PrhA interacts with PrhR using its periplasmic domain, it is expected that this domain performs that function.</p><br />
<br />
<p>Then, PrhA presents the same domains that FecA: <strong>TonB-dependent Receptor Plug Domain</strong> (154 – 250 aa.) and <strong>TonB-dependent Receptor Domain</strong>(542 – 767 aa.), setting out the high similarities that exist between these two outer membrane proteins. Also their N-terminal extensions are quite similar as it was found by Marenda et al<sup><a href="#Reference_Sensing04">[4]</a></sup>.</p><br />
<br />
<img class="centerBig" src="https://static.igem.org/mediawiki/2010/c/c8/BacterialCrowdingFerrecCitrateTree.png" alt="Ferric Citrate tree"/><br />
<p class="caption"> <strong> Phylogenetic tree of TonB-dependent receptors </strong>. The tree was constructed as described by Rakin et al. (1994). Circled numbers indicate the number of times (from the whole 100) a particular node was supported by bootstrap analysis. The proteins used in this analysis are referenced in Rakin et al. (1994).<sup><a href="#Reference_Sensing04">[4]</a></sup></p><br />
<br />
<p>PrhA has high similarities with TonB-dependent receptors, which need to interact with the TonB protein to perform their functions. It shares two of the three main domains those proteins have. Nevertheless, PrhA is lacking of the periplasmic Secretin and TonB N-terminus Short Domain, the necessary domain to interact with TonB. In its place there is an unknown domain still not well studied. It will be required to continue studying this protein to know if TonB is necessary in its function and understand the evolution that TonB interaction domain suffered.</p><br />
<br />
<h2>Hybrid protein FecA/PrhA</h2><br />
<br />
<p>Taking in advance that Prh system is not naturally expressed in <i>Escherichia coli</i> and that Prh system is not well-known, we decided to create a fusion protein. FecA/PrhA artificial coding sequence spans the first 92 codons of <i>fecA</i>, encoding the signal peptide, NH2 terminal extension and the proposed Ton-box, fused to the distal end of the <i>prhA</i> coding sequence at the conserved GSGL motif (aa. 89-92). We synthesized this biobrick using MrGene services, so we also optimized the sequence to be expressed in <i>Escherichia coli</i>.</p><br />
<br />
<img class="centerBig" src="https://static.igem.org/mediawiki/2010/2/2c/BacterialCrowdingFecAPrhAHybrid.png" alt="PrhA, FecA and Hybrid Domains"/><br />
<br />
<p>Above is shown approximately the point where we fused FecA and PrhA proteins. To this way the hybrid protein include domains from both OM proteins:</p><br />
<br />
<ul><br />
<li><strong>Signal peptide of FecA</strong> which will help to the accurate emplacement of the hybrid protein in the outer membrane of <i>E. coli</i>.</li><br />
<br />
<li><strong>NH2-terminal extension of FecA</strong>. Including this domain of FecA means including the periplasmic signaling domain of this protein. The signal transfer between the OM and the IM proteins is performed between the N-terminus of FecA and the C-terminus of FecR (both are shown in the periplasmic). The hybrid protein includes the N-terminus of FecA so our expectation is that FecA/PrhA protein was able to interact with FecR.</li><br />
<br />
<li>Most of the <strong>Secretin and TonB N-terminus Short Domain of FecA</strong>. This domain helps FecA to interact with TonB. If TonB interaction is required for the OM-IM signal transfer, our hybrid protein includes this domain. Also, doing this fusion the hybrid protein loses the unknown function domain set in the N-terminus of PrhA.<br/><br />
<img class="centerSmall" src="https://static.igem.org/mediawiki/2010/2/21/BacterialCrowdingFecAN.png" alt="N-terminus short domain of FecA"/><br />
<p class="caption"><strong>N-terminus</strong> (aa. 34-92) <strong>of FecA.</strong> Here is shown the FecA contribution to the hybrid protein. Our aim is that this structure was able to interact with FecR without the rest of FecA protein. Representation made with RasWin.</p></li><br />
<br />
<li><strong>TonB-dependent Receptor Plug Domain of PrhA</strong>. In 89<sup>th</sup> codon there is a conserved motif which was used to fuse FecA with PrhA. The function of the Plug domain is to propagate allosteric transitions through the outer membrane signaling the occupancy of the receptor.</li><br />
<br />
<li><strong>TonB Dependent Receptor Domain of PrhA</strong>. From the conserved motif GSGL to the end of the protein amino acids are the same that in PrhA protein. The hybrid protein includes most of the PrhA protein, from 92<sup>sd</sup> codon to the C-terminus. The aim of it is that FecA/PrhA was able to interact with the non-diffusible plant wall signal that PrhA detects. The mechanism of this interaction is unknown.</li><br />
</ul><br />
<br />
<p>As you can see we work with a lot of uncertainty cause of the unknown mechanisms that manage the process we work with. Anyhow, we hope that this hybrid protein allows to sense non-difusible signals (with PrhA domains) and to transduce it by the Fec pathway (using the N-terminus of FecA). If this happened we would not have any problem with other Prh protein because the signal would continue by FecR and FecI in the Fec pathway of <i>E. coli<i>.</p><br />
<br />
<h1>Bibliography</h1><br />
<br />
<ol><br />
<li id="Reference_Sensing01">Uwe Pressler, Horst Staudenmaier, Luitgard Zimmermann, And Volkmar Braun (1988), Genetics of the Iron Dicitrate Transport System of Escherichia coli. JOURNAL OF BACTERIOLOGY, June 1988, p. 2716-2724</li><br />
<br />
<li id="Reference_Sensing02">Miyeon Kim, Gail E. Fanucci, and David S. Cafiso (2007), Substrate-dependent transmembrane signaling in TonB-dependent transporters is not conserved. PNAS, July 17, 2007, vol. 104, no. 29, 11975–11980.</li><br />
<br />
<li id="Reference_Sensing03">Andrew D. Ferguson, et al (2002). Structural Basis of Gating by the Outer Membrane Transporter FecA. Sience 295, 1715.</li> <br />
<br />
<li id="Reference_Sensing04">Marc Marenda, Belen Brito, Didier Callard, Stéphane Genin, Patrick Barberis, Christian Boucher and Matthieu Arlat (1998). PrhA controls a novel regulatory pathway required for the specific induction of Ralstonia solanacearum hrp genes in the presence of plant cells. Molecular Microbiology (1998) 27(2), 437–453.<br/><br />
<br />
</li> <br />
<br />
</ol><br />
<br />
<ul><br />
<li>Brito, B., Marenda, M., Barberis, P., Boucher, C., and Genin, S. 1999. <i>prhJ and hrpG: Two new components of the plant signal-dependent regulatory cascade controlled by PrhA in Ralstonia solanacearum</i>. Mol. Microbiol. 31:237-251.</li><br />
<br />
<li>Marenda, M., Brito, B., Callard, D., Genin, S., Barberis, P., Boucher, C. A., and Arlat, M. 1998. <i>PrhA controls a novel regulatory pathway required for the specific induction of Ralstonia solanacearum hrp genes in the presence of plant cells</i>. Mol. Microbiol. 27:437-453.</li><br />
<br />
<li>Aldon, D., Brito, B., Boucher, C., and Genin, S. 2000. <i>A bacterial sensor of plant cell contact controls the transcriptional induction of Ralstonia solanacearum pathogenicity genes</i>. EMBO (Eur. Mol. Biol. Organ.) J. 19:2304-2314.</li><br />
<br />
<li>Brito, B., Aldon, D., Barberis, P., Boucher, C., and Genin, S. 2002. <i>A Signal Transfer System Through Three Compartments Transduces the Plant Cell Contact-Dependent Signal Controlling Ralstonia solanacearum hrp Genes</i>. Molecular Plant-Microbe Interactions. Vol. 15, No. 2: 109/119</li><br />
<br />
<li>Braun V, Mahren S, Sauter A. <i>Gene regulation by transmembrane signaling</i>. 2006. Biometals. 19(2):103-13</li><br />
<br />
<li>Braun V, Mahren S, Ogierman M. 2003. <i>Regulation of the FecI-type ECF sigma factor by transmembrane signalling</i>. Curr Opin Microbiol. 6(2):173-80.</li><br />
<br />
<li>Enz, S., Brand, H., Orellana, C., Mahren, S., and Braun, V. 2003. <i>Sites of Interaction between the FecA and FecR Signal Transduction Proteins of Ferric Citrate Transport in Escherichia coli</i> K-12. J. Bacteriol. Vol. 185, No: 133745–3752 </li><br />
<br />
<li>Kim, M., Fanucci, G. E., and Cafiso, D. S. 2007. <i>Substrate-dependent transmembrane signaling in TonB-dependent transporters is not conserved</i>. PNAS. Vol. 104 N. 29: 11975/11980</li><br />
<br />
<li>http://www.mikrobio.uni-tuebingen.de/ag_braun/research_areas.html</li><br />
<li>www.uniprot.org </li><br />
<li>http://pfam.sanger.ac.uk/ </li><br />
<br />
</ul><br />
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<h1>Introduction</h1><br />
<br />
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<p>Bacteria can sense a lot of different stimuli. They have to detect changes in their environment and to interpret them. Bacteria use different receptors to sense ionic state, chemicals, pH, nutrients, lights… and, when a stimuli is caused, they generate a intracellular signaling pathway and produce a specific response. Usually, signals that bacteria can sense are diffusible-signals. However, we will use the first known system to sense a non-diffusible signal: the Prh system of <i>Ralstonia solanacearum</i>. This system detects an unknown polysaccharide ligand of plant cell walls and activates a signal transduction cascade that, in original system, causes the virulence gene expression. </p><br />
<br />
<p>The aim of the <strong>sensing circuits</strong> is to ensure that our system is able to detect specifically the walls of plant cells and causes the secretion and excretion of a chemoattractant through signal transduction pathways. In order to achieve effective bacterial accumulation around plant cells, we have designed four different circuits that attempt to prevent possible different problems. We have combined different regulated proteins from <i>Escherichia coli</i> and <i>Ralstonia solanacearum</i> with specific biobricks, and we have replaced virulence genes by chemoattranctant genes. So, despite of <i>R. solanacearum</i> toxicity, the fact of working with biobricks avoids us having to work with the security measures required by this strain. </p><br />
<br />
<p>A future improvement for our project would be to use an <strong>adhesin</strong> to keep the bacteria attached to the plant cell wall as has been happening in R.solanacearum. We expect our system works without it.</p><br />
<br />
<br />
<br />
<h3>Brief description of <i>Ralstonia solanacearum</i>:</h3><br />
<br />
<p><i><strong>Ralstonia solanacearum</strong></i> belongs to the family of Gram-negative phyto-pathogens. This bacterium causes great losses in crops worldwide in tropical, subtropical and temperate environments. The genes involved in virulence are known as <i>hrp</i> (hypersensitive response and pathogenicity) and they are induced by contact with various plant species from three different families of dicotyledonous (Solanaceae, Cruciferae and legumes), including both host and non host species. These genes encode a type III secretion system (TTSS), required to develop the disease in the host or the hypersensitive response (HR) in resistant and non-host plants. The HR is a defense mechanism that certain plant species possess, in which cells infected by a pathogen are killed to prevent spread to healthy tissue. </p><br />
<br />
<p>The secretion system is expressed only when there is a <strong>physical interaction</strong> between <i>R. solanacearum</i> and the plant cell. PrhA (plant regulator of hrp genes), the protein that recognizes the plant cell ligand, is an outer membrane receptor that shows homology with some TonB-dependent siderophore receptors. However, PrhA is not involved in the bacteria-plant cell adhesion, but only in sensing. The interaction of bacteria with plant cells occurs in two steps: first, <i>R. solanacearum</i> binds to the cell surface. This union is independent of PrhA or any protein encoded by <i>hrp</i>. Once the union has been established, the PrhA receiver can detect an accessible ligand, which increases the transcription of the <i>hrp</i> regulatory gene. Induction of <i>hrp</i> expression is very fast, around 90 minutes, a period much shorter than generation time of <i>R. solanacearum</i> in optimal conditions. </p><br />
<br />
<p>PrhA fundamental feature is that it is the first known bacterial receptor that can detect a <strong>non-diffusible signal</strong> present in plant cell walls. The possibility of attaching bacteria to a specific tissue was what made us choose the Ralstonia system for our project. The induction of the expression of virulence hrp regulon integrates a complex signaling cascade that begins in the PrhA outer membrane protein. PrhA transduces the contact-dependent signal through a complex regulatory cascade composed of PrhR, PrhI, PrhJ, HrpG and HrpB. Finally, HrpB activates the expression of <i>hrp</i>, comprising the TTSS structural genes and genes that encode effector proteins that travel through the TTSS.</p><br />
<br />
<br />
<br />
<h1>Signal Transduction Circuits</h1><br />
<br />
<h2>Prh system of <i>Ralstonia solanacearum</i></h2><br />
<br />
<h3>Brief description of the original Prh system:</h3><br />
<br />
<p>The <strong>Prh system</strong> integrates the genes involved in the control of expression of <strong><i>hrp</i> virulence genes</strong> from <i>Ralstonia solanacearum</i>. <i>hrp</i> gene encodes a type III secretion system, necessary to develop disease in their hosts. The induction of these genes integrates a complex signaling network that begins when the bacteria and the plant cell contact. This signaling mechanism is composed of PrhA PrhR PrhI proteins and other regulators that, as a last resort, activate the expression of the <i>hrp</i> or <i>hrc</i> (conserved hrp genes) genes.</p><br />
<br />
<p>The induction of virulence genes occurs when PrhA contacts with the plant cell. PrhA is an outer membrane protein that recognizes an unknown non-diffusible signal from the plant cell wall. PrhA-ligand binding causes that the periplasmic exposed N-terminal end of PrhA interacts with the carboxy terminal end of PrhR (an inner membrane protein) in the periplasm, transmitting the signal across the cytoplasmic membrane. In the cytoplasm inactive PrhI is actived by PrhR interaction by a still unkown mechanism.</p><br />
<br />
<p>The <i>prhIR</i> gene expression is induced in coculture with plant cells due to unknown environmental signal PrhA independent. PrhI is an ECF (extracytoplasmic function) sigma factor that, when it is activated, binds to RNA polymerase core enzyme and directs the polimerase to the promoter region of <i>PrhJ</i> gene to initiate transcription. In <i>R. solanacearum</i>, PrhJ protein induces <i>hrpG</i> transcription, which activates expression of <i>hrpB</i> gene and finally expresses <i>hrp</i> and <i>hrc</i> virulence genes.</p><br />
<br />
<p>The <strong>PrhA-PrhR-PrhI</strong> module of <i>Ralstonia</i> works similarly to FecA-FecR-FecI module of <i>E.coli</i>, with both similar sequences. PrhA shows homology with several members of the family of siderophore outer membrane receptors (as is the case of FecA). The three boxes that this family of proteins presents (TonB-box, box II and boxIII) are well conserved and correctly located in PrhA. PrhR has a transmembrane domain (TM) in the same position as FecR and both proteins have a similar orientation. In addition, two of the three tryptophan residues of the N-terminal end of FecR required to activate FecII are present in PrhR. However, unlike most of the siderophores, both <i>prhIR</i> and <i>prhA</i> lack Fur-boxes which are necessary for the regulation in function of the internal iron status.</p><br />
<br />
<p>Another striking difference between PrhAIR and FecAIR is their gene organization: while there is a physical grouping between genes of FecAIR, in PrhAIR system, <i>prhA</i> constitutes a monocistronic operon at the left edge of <i>hrp</i> gene cluster and <i>prhIR</i> is on the right side of cluster, both <i>prhA</i> and <i>prhIR</i> separated by virulence genes. Moreover, in contrast to the Fec system where FecA is activated by FecI and repressed by Fur, PrhA is always expressed a very low level in the presence of the inducing signal and is PrhI independent.</p><br />
<br />
<h3>Circuit 1:</h3><br />
<br />
<div class="imgRight"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/4/4e/BacterialCrowdingCircuit1.png" alt="Bacterial Crowding Circuit 1" /><br />
</div><br />
<br />
<p>In circuit 1 we wanted to use Prh system to transduce plant cell wall signals in chemoattractant synthesis. This first circuit integrates regulatory components PrhA-PrhI-PrhR and PprhJ of <i>R. solanacearum,</i> but they are transfered to <i>E. coli.</i> Genes required for synthesis and excretion of the chemoattractant are under PprhJ promoter control.</p><br />
<br />
<p>Because the Prh system is not fully characterized, unknown elements could be involved in and prevent the signal being correctly transmitted to P<i>prhJ</i>. Also, it is possible that it could not perform its usual answers being express in E.coli, for example it could be problems setting PrhA protein in the outer membrane. For that reasons, we have designed other circuits wich use <i>E. coli</i> proteins in the signal cascade.</p> <br />
<br />
<div class="clear"></div><br />
<br />
<h2>Fec system of <i>Escherichia coli</i></h2><br />
<br />
<h3>Brief description of the original Fec system:</h3><br />
<br />
<p><strong>Fec system</strong> includes genes involved in regulation and expression of <i>E. coli</i> iron transporters. <i>fecABCDE</i> genes express the <strong>ferric citrate transporter</strong> when bacteria iron status is low or deficient. The induction of genes <i>fecABCDE</i> integrates a signaling cascade that begins at the cell surface and is extended to the cytoplasm. To do this, three specific proteins are involved : FecA in outer membrane, FecR in cytoplasmic membrane and FecI in cytoplasm. This module <strong>FecA-FecR-FecI</strong> is known as a signal transduction system between three compartments (outside, periplasm and cytoplasm).</p><br />
<br />
<p>The signaling pathway begins when the outer membrane receptor FecA binds to its ligand, ferric dicitrate. This binding causes structural changes in FecA that allow the interaction of its amino terminal end to the carboxy terminal end of FecR in the periplasm. FecR, a transmembrane protein, transmits the signal to the cytoplasm, where it activates FecI. FecI is an extracitoplasmatic function (ECF) sigma factor that, when activated, binds to core RNA polymerase and directs the complex to the upstream promoter of <i>fecABCDE</i> transport genes to initiate transcription. </p><br />
<br />
<p>In addition, the transcription of regulatory genes <i>fecIR</i> is controlled by the internal iron status through the Fur repressor. When the Fur protein is loaded with Fe 2+, it represses <i>fecIR</i> transcription and prevents the <i>fec</i> gene expression. Therefore, the <i>fec</i> transport gene transcription is subjected to a double control: first, cells detect iron deficiency. Then, regulatory proteins FecI and FecR are synthetised, which, if ferric citrate is available, initiate the transcription of <i>fec</i> transport genes.</p><br />
<br />
<p>Dicitrate ferric transport through the outer membrane requires an energy transduction complex consisting of TonB, ExbB and Exb cytoplasmic membrane proteins.</p><br />
<br />
<h3>Circuit 2:</h3><br />
<br />
<p>In our second circuit, the iron transport genes (<i>fecABCDE</i>) are replaced for genes required for synthesis and excretion of chemoattractant. Those genes remain under control of the promoter PfecABCDE, being regulated by the module FecA-FecI-FecR which is dependent of internal iron status and external ferric citrate concentration.</p><br />
<br />
<p>This circuit has the advantage that, besides being well characterized, is presented in wild type <i>E. coli</i>. However, this system will not be directed specifically to plant tissues and would be regulated by iron status of the bacteria and the environment. Nevertheless the second signal transduction circuit could be use like control of the chemoattractant synthesis. To this way, if the plant cell wall signal is not transduced properly, we could induce the chemoattractant synthesis changing medium conditions.</p><br />
<br />
<h2>(FecA/PrhA)-FecI-FecR hybrid protein system</h2><br />
<br />
<h3>Circuit 3:</h3><br />
<br />
<p>Our third circuit uses the FecA and PrhA <strong>sequence homology</strong>. We have designed a <strong>hybrid protein</strong> with the aim that it could detect the plant cell ligand and transmit the signal to FecR. The hybrid protein contains most of PrhA and the N-terminal end of FecA; binding both proteins by a shared sequence near the Ton-box. The signal would be transmited through the interaction between the periplasmic exposed N-terminal extension of FecA and the C-terminal part of FecR.</p><br />
<br />
<p>The third circuit would allow us to sense a non-diffusible signal and to transduce it using an E. coli system without problems with expression and function.</p><br />
<br />
<p>We have focused in this circuit.</p><br />
<br />
<h2>PrhA-fecI-FecR hybrid system</h2><br />
<br />
<h3>Circuit 4:</h3><br />
<br />
<div class="imgRight"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/7/73/BacterialCrowdingCircuit4.png" alt="Bacterial Crowding Circuit 4" /><br />
</div><br />
<br />
<p>Due to the proximity in the life tree, the similarities between N-terminal extension of PrhA and FecA is significant, in particular the amino acid sequence Gx10(L,A)L(D,Q,A)G(S,T)L is well conserved. Also PrhR shows sequence similarity with FecR (27% identity, 43% similarity). Cause this information we wanted to test if the interaction between these systems was possible without modification.</p><br />
<br />
<p>However, this construction is largely a test.</p><br />
<br />
<div class="clear"></div><br />
<br />
<h1>Outer Membrane Protein Structures</h1><br />
<br />
<p>Now it is going to be shown the structure of the outer membrane proteins which start the signal transduction in sensing systems described before. It is interesting to study the structure and domains of FecA and PrhA before seeing the hybrid protein, in which we have focused our project mainly.</p><br />
<br />
<h2>Fe(3+) dicitrate transport protein FecA</h2><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/7/78/BacterialCrowndingFecA01.png" alt="Crystal structure of the Outer Membrane Transporter FecA"/><br />
<p class="caption"><b><a src="http://www.pdb.org/pdb/explore/explore.do?pdbId=1KMO" target="_blank">Crystal structure of the Outer Membrane Transporter FecA.</a></b></p><br />
<br />
<p><a href="http://www.uniprot.org/uniprot/P13036" target="_blank">FecA</a> is the outer membrane receptor protein in the Fe(3+) dicitrate transport system of <i>Escherichia coli</i>. It binds and transports ferric citrate, and it is required to initiate transcription of the <i>fecABCDE</i> transport operon but not the regulatory fecIR genes. This is a well-known protein, compound of 773 amino acids, whose main domains are shown below:</p><br />
<br />
<img class="centerBig" src="https://static.igem.org/mediawiki/2010/6/63/BacterialCrowdingFecADomains.png" alt="FecA Domains"/><br />
<br />
<p>The yellow left domain represents a <strong>signal peptide</strong> which takes from 1st to 33rd codon. The cleavage site of the signal peptidase has been found between residues 33 and 34<sup><a href="#Reference_Sensing01">[1]</a></sup>. Its function is to drive FecA protein to the outer membrane of <i>E. coli</i>, where the protein works.</p><br />
<br />
<div class="imgLeft"><br />
<img class="ileft" src="https://static.igem.org/mediawiki/2010/8/8c/BacterialCrowdingFecATonB.png" alt="the structure of the periplasmic signaling domain of FecA by nuclear magnetic resonance" /><br />
</div><br />
<br />
<p>The green illustration represents <strong>Secretin and TonB N-terminus Short Domain</strong> which takes from 57th to 107th codon. This domain is found at the N-terminus of the Secretins of the bacterial type II/III secretory system as well as the TonB-dependent receptor proteins. These proteins are involved in TonB-dependent active uptake of selective substrates. Thus, FecA interacts with TonB, which couples the electrochemical potential of the cytoplasmic membrane to active transport of ferric citrate across the outer membrane. The TonB box undergoes a substrate-induced disorder transition which produces an aqueous exposed, highly disordered protein fragment, which probably regulates transporter–TonB interactions<sup><a href="#Reference_Sensing02">[2]</a></sup>.</p><br />
<br />
<p>It is usual to find the TonB domain nearby signal and Plug domains. It is a common domain organization. At the left it is shown the structure of the periplasmic signaling domain of FecA by nuclear magnetic resonance. </p><br />
<br />
<p>Between both before domains it is a flexible 79-residue domain of FecA termed the <strong>NH2-terminal extension</strong>, which resides entirely within the periplasm. Its function is proposed to be to transmit the liganded status of the receptor to FecR<sup><a href="#Reference_Sensing03">[3]</a></sup>.</p><br />
<br />
<div class="clear"></div><br />
<br />
<p>In red color in the schematic representation it is shown the <strong>TonB-dependent Receptor Plug Domain</strong> which takes from 129th to 244th codon. The Plug domain has been shown to be an independently folding subunit of the TonB-dependent receptors. It acts as the channel gate, blocking the pore until the channel is bound by ligand. At this point it undergoes conformational changes that open the channel. Also ligand induces allosteric transitions which are propagated through the outer membrane by the plug domain, signaling the occupancy of the receptor in the periplasm. The plug domain is located inside a barrel, comprising five helices, two &beta; strands, and a mixed four-stranded &beta; sheet. Also three loops of the Plug domain extend above the plane of the upper leaflet of the outer membrane<sup><a href="#Reference_Sensing03">[3]</a></sup>.</p><br />
<br />
<img class="centerBig" src="https://static.igem.org/mediawiki/2010/0/04/BacterialCrowdingFecAUnligandedandFerric.png" alt="FecA Crystal structure"/><br />
<p class="caption"><b>a.</b>Crystal structure of ferric citrate transporter FecA in the unliganded form <br/><br />
<b>b.</b>Crystal structure of the outer membrane transporter FecA complexed with ferric citrate</p><br />
<br />
<p>Finally in the C-terminus there is a <strong>TonB Dependent Receptor Domain</strong> which takes from 525th to 773rd codon. The TonB dependent receptor domain is included in the 22-stranded &beta; barrel that traverse de outer membrane. The barrel of a TonB dependent receptor is a dynamic entity that actively participates in the energy-dependent siderophore uptake. This barrel has elipsoidal shape as you can see in before representations of FecA. Below it is shown the C-terminal domain of FecA, from 525th codon to the end.</p><br />
<br />
<img class="centerSmall" src="https://static.igem.org/mediawiki/2010/9/9d/BacterialCrowdingFecAC.png" alt="C-terminal domain of FecA"/><br />
<p class="caption">C-Terminal domain of FecA (representation made with RasWin program)</p><br />
<br />
<p>The common domain organization represents TonB dependent receptor domain at the same time as Plug domain because the interaction between the receptor (FecA) and the ligand (dinuclear ferric citrate molecule) is performed by Plug domain and the barrel. Formation of the liganded complex carries out changes on the conformation of the barrel and the Plug domain of FecA.</p><br />
<br />
<h2>Outer membrane receptor protein PrhA</h2><br />
<br />
<p>PrhA is the only known protein able to detect a non-diffusible signal and transduce this information into the cell. It is compound of 770 amino acids and it was found not too long ago. This is why there is not many information about it. Not being well-known is a point to use the hybrid protein FecA/PrhA instead of it. Anyway, their main domains are shown in <a href="http://pfam.sanger.ac.uk/protein?acc=B7ZJG7" target="_blank">Pfam website</a>, but it is not possible to see its structure because it has not been modeled yet.</p><br />
<br />
<img class="centerBig" src="https://static.igem.org/mediawiki/2010/d/dd/BacterialCrowdingPrhADomains.png" alt="PrhA Domains"/><br />
<br />
<p>Like in the case of FecA, PrhA has a putative <strong>signal peptide</strong> which takes from 1st to 35th codon. Its function would be direct PrhA to the outer membrane of <i>Rastonia solanacearum</i>. Despite its existence, you can not see it in the domain summary picture since it has not been well studied.</p><br />
<br />
<p>Next it is a not confirmed domain with unknown function which would take from the beginning of the protein to 130th amino acid. By now, it is called <strong>PfamB PB000342</strong> and its family was generated automatically from an alignment taken from Automatic Domain Decomposition Algorithm (<a href="http://ekhidna.biocenter.helsinki.fi/sqgraph/pairsdb/index_html" target="_blank">ADDA</a>). Since PrhA interacts with PrhR using its periplasmic domain, it is expected that this domain performs that function.</p><br />
<br />
<p>Then, PrhA presents the same domains that FecA: <strong>TonB-dependent Receptor Plug Domain</strong> (154 – 250 aa.) and <strong>TonB-dependent Receptor Domain</strong>(542 – 767 aa.), setting out the high similarities that exist between these two outer membrane proteins. Also their N-terminal extensions are quite similar as it was found by Marenda et al<sup><a href="#Reference_Sensing04">[4]</a></sup>.</p><br />
<br />
<img class="centerBig" src="https://static.igem.org/mediawiki/2010/c/c8/BacterialCrowdingFerrecCitrateTree.png" alt="Ferric Citrate tree"/><br />
<p class="caption"> <strong> Phylogenetic tree of TonB-dependent receptors </strong>. The tree was constructed as described by Rakin et al. (1994). Circled numbers indicate the number of times (from the whole 100) a particular node was supported by bootstrap analysis. The proteins used in this analysis are referenced in Rakin et al. (1994).<sup><a href="#Reference_Sensing04">[4]</a></sup></p><br />
<br />
<p>PrhA has high similarities with TonB-dependent receptors, which need to interact with the TonB protein to perform their functions. It shares two of the three main domains those proteins have. Nevertheless, PrhA is lacking of the periplasmic Secretin and TonB N-terminus Short Domain, the necessary domain to interact with TonB. In its place there is an unknown domain still not well studied. It will be required to continue studying this protein to know if TonB is necessary in its function and understand the evolution that TonB interaction domain suffered.</p><br />
<br />
<h2>Hybrid protein FecA/PrhA</h2><br />
<br />
<p>Taking in advance that Prh system is not naturally expressed in <i>Escherichia coli</i> and that Prh system is not well-known, we decided to create a fusion protein. FecA/PrhA artificial coding sequence spans the first 92 codons of <i>fecA</i>, encoding the signal peptide, NH2 terminal extension and the proposed Ton-box, fused to the distal end of the <i>prhA</i> coding sequence at the conserved GSGL motif (aa. 89-92). We synthesized this biobrick using MrGene services, so we also optimized the sequence to be expressed in <i>Escherichia coli</i>.</p><br />
<br />
<img class="centerBig" src="https://static.igem.org/mediawiki/2010/2/2c/BacterialCrowdingFecAPrhAHybrid.png" alt="PrhA, FecA and Hybrid Domains"/><br />
<br />
<p>Above is shown approximately the point where we fused FecA and PrhA proteins. To this way the hybrid protein include domains from both OM proteins:</p><br />
<br />
<ul><br />
<li><strong>Signal peptide of FecA</strong> which will help to the accurate emplacement of the hybrid protein in the outer membrane of <i>E. coli</i>.</li><br />
<br />
<li><strong>NH2-terminal extension of FecA</strong>. Including this domain of FecA means including the periplasmic signaling domain of this protein. The signal transfer between the OM and the IM proteins is performed between the N-terminus of FecA and the C-terminus of FecR (both are shown in the periplasmic). The hybrid protein includes the N-terminus of FecA so our expectation is that FecA/PrhA protein was able to interact with FecR.</li><br />
<br />
<li>Most of the <strong>Secretin and TonB N-terminus Short Domain of FecA</strong>. This domain helps FecA to interact with TonB. If TonB interaction is required for the OM-IM signal transfer, our hybrid protein includes this domain. Also, doing this fusion the hybrid protein loses the unknown function domain set in the N-terminus of PrhA.<br/><br />
<img class="centerSmall" src="https://static.igem.org/mediawiki/2010/2/21/BacterialCrowdingFecAN.png" alt="N-terminus short domain of FecA"/><br />
<p class="caption"><strong>N-terminus</strong> (aa. 34-92) <strong>of FecA.</strong> Here is shown the FecA contribution to the hybrid protein. Our aim is that this structure was able to interact with FecR without the rest of FecA protein. Representation made with RasWin.</p></li><br />
<br />
<li><strong>TonB-dependent Receptor Plug Domain of PrhA</strong>. In 89<sup>th</sup> codon there is a conserved motif which was used to fuse FecA with PrhA. The function of the Plug domain is to propagate allosteric transitions through the outer membrane signaling the occupancy of the receptor.</li><br />
<br />
<li><strong>TonB Dependent Receptor Domain of PrhA</strong>. From the conserved motif GSGL to the end of the protein amino acids are the same that in PrhA protein. The hybrid protein includes most of the PrhA protein, from 92<sup>sd</sup> codon to the C-terminus. The aim of it is that FecA/PrhA was able to interact with the non-diffusible plant wall signal that PrhA detects. The mechanism of this interaction is unknown.</li><br />
</ul><br />
<br />
<p>As you can see we work with a lot of uncertainty cause of the unknown mechanisms that manage the process we work with. Anyhow, we hope that this hybrid protein allows to sense non-difusible signals (with PrhA domains) and to transduce it by the Fec pathway (using the N-terminus of FecA). If this happened we would not have any problem with other Prh protein because the signal would continue by FecR and FecI in the Fec pathway of <i>E. coli<i>.</p><br />
<br />
<h1>Bibliography</h1><br />
<br />
<ol><br />
<li id="Reference_Sensing01">Uwe Pressler, Horst Staudenmaier, Luitgard Zimmermann, And Volkmar Braun (1988), Genetics of the Iron Dicitrate Transport System of Escherichia coli. JOURNAL OF BACTERIOLOGY, June 1988, p. 2716-2724</li><br />
<br />
<li id="Reference_Sensing02">Miyeon Kim, Gail E. Fanucci, and David S. Cafiso (2007), Substrate-dependent transmembrane signaling in TonB-dependent transporters is not conserved. PNAS, July 17, 2007, vol. 104, no. 29, 11975–11980.</li><br />
<br />
<li id="Reference_Sensing03">Andrew D. Ferguson, et al (2002). Structural Basis of Gating by the Outer Membrane Transporter FecA. Sience 295, 1715.</li> <br />
<br />
<li id="Reference_Sensing04">Marc Marenda, Belen Brito, Didier Callard, Stéphane Genin, Patrick Barberis, Christian Boucher and Matthieu Arlat (1998). PrhA controls a novel regulatory pathway required for the specific induction of Ralstonia solanacearum hrp genes in the presence of plant cells. Molecular Microbiology (1998) 27(2), 437–453.<br/><br />
<br />
</li> <br />
<br />
</ol><br />
<br />
<ul><br />
<li>Brito, B., Marenda, M., Barberis, P., Boucher, C., and Genin, S. 1999. <i>prhJ and hrpG: Two new components of the plant signal-dependent regulatory cascade controlled by PrhA in Ralstonia solanacearum</i>. Mol. Microbiol. 31:237-251.</li><br />
<br />
<li>Marenda, M., Brito, B., Callard, D., Genin, S., Barberis, P., Boucher, C. A., and Arlat, M. 1998. <i>PrhA controls a novel regulatory pathway required for the specific induction of Ralstonia solanacearum hrp genes in the presence of plant cells</i>. Mol. Microbiol. 27:437-453.</li><br />
<br />
<li>Aldon, D., Brito, B., Boucher, C., and Genin, S. 2000. <i>A bacterial sensor of plant cell contact controls the transcriptional induction of Ralstonia solanacearum pathogenicity genes</i>. EMBO (Eur. Mol. Biol. Organ.) J. 19:2304-2314.</li><br />
<br />
<li>Brito, B., Aldon, D., Barberis, P., Boucher, C., and Genin, S. 2002. <i>A Signal Transfer System Through Three Compartments Transduces the Plant Cell Contact-Dependent Signal Controlling Ralstonia solanacearum hrp Genes</i>. Molecular Plant-Microbe Interactions. Vol. 15, No. 2: 109/119</li><br />
<br />
<li>Braun V, Mahren S, Sauter A. <i>Gene regulation by transmembrane signaling</i>. 2006. Biometals. 19(2):103-13</li><br />
<br />
<li>Braun V, Mahren S, Ogierman M. 2003. <i>Regulation of the FecI-type ECF sigma factor by transmembrane signalling</i>. Curr Opin Microbiol. 6(2):173-80.</li><br />
<br />
<li>Enz, S., Brand, H., Orellana, C., Mahren, S., and Braun, V. 2003. <i>Sites of Interaction between the FecA and FecR Signal Transduction Proteins of Ferric Citrate Transport in Escherichia coli</i> K-12. J. Bacteriol. Vol. 185, No: 133745–3752 </li><br />
<br />
<li>Kim, M., Fanucci, G. E., and Cafiso, D. S. 2007. <i>Substrate-dependent transmembrane signaling in TonB-dependent transporters is not conserved</i>. PNAS. Vol. 104 N. 29: 11975/11980</li><br />
<br />
<li>http://www.mikrobio.uni-tuebingen.de/ag_braun/research_areas.html</li><br />
<li>www.uniprot.org </li><br />
<li>http://pfam.sanger.ac.uk/ </li><br />
<br />
</ul><br />
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<h1>Introduction</h1><br />
<br />
<p><strong>Motility</strong> is one of the most readily demonstrated bacterial characters, and <strong>chemotaxis</strong> is one of the most studied bacterial behaviors. Motile organisms are attracted by certain chemicals and repelled by others (positive and negative chemotaxis). Quantification of chemotactic motion is necessary to identify chemoeffectors and to determine the structure of bacterial communities.</p><br />
<br />
<p>Current methods of quantifying chemotaxis use chemotactic bacteria such as <i>Escherichia Coli</i>, which is assayed by measuring the number of organisms attracted into a capillary tube containing an attractant.</p><br />
<br />
<p>UPO-Sevilla team has carried out different experimental prototypes that are better to achieve. The goal of the group was designing different assays that allow us to study this effect in both point of views, <strong>qualitative</strong> and <strong>quantitative</strong>. Also we have to test induction of sensing systems and chemoattractant production. Anyway, these processes are highly related with the chemotaxis process in Bacterial Crowding project. This is why we measured chemoattractant production counting the range of attracted bacteria using chemotaxis assays. The induction of sensing systems could be tested by using GFP measures when its promoter is <i>PfecA</i> or <i>PprhJ</i>; or also due to the levels of chemoattractant production. </p><br />
<br />
<h1>Qualitative Assays</h1><br />
<br />
<h2>Agar Soft Plates</h2><br />
<br />
<p>Our qualitative assays were made in <strong>soft agar</strong> thanks to the protocols that we had received from Mr. Parkinson (University of Utah).</p><br />
<br />
<p>This kind of plates allows bacteria to swim trough the agar freely and showing their chemotactic capacities. A colony, inserted in soft agar plate, starts to grow while running out the environmental sources. For this reason bacteria would move to places where the sources are not limited. That phenomenon produces a number of <strong>halos</strong> which are spread within the plate and increase in volume as the sources are lowered.</p><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/3/36/BacterialCrowdingSoftAgarPlate.png" alt="Soft Agar Plate Assay"/><br />
<p class="caption"><strong>Fig 1. Soft agar plate assay.</strong> Different strains are shown. Every strain carries out a mutation that affects its chemotactic response, every strain but the wild type. In the wt you can observe two halos produced by chemotactic responses to aspartate and glutamate.</p><br />
<br />
<br />
<p>The assay protocol is simple; once the soft agar plates are prepared, a colony is inserted in the previous plate. Let it grow in 30ºC. The soft agar is a delicate element, so it is important to be careful when moving the plates.</p><br />
<br />
<p>In those plates it might appear different concentric circles which represent chemotaxis to a certain attractant. For instance, when two amino acids are run out from the medium, two circles will appear. The inner one will show the amino acids limit with low chemotactic response; while the outer one will mean that the amino acid which causes a higher response is running down.</p><br />
<br />
<h2>Optical and Fluorescence Microscopy</h2><br />
<br />
<p>The <strong>microscopy techniques</strong> allow us to see the development of the assay <i>in situ</i> without any wait. In this part we will see how we can carry out an experiment that we could see under the microscope.</p><br />
<br />
<div class="imgLeft"><br />
<img class="ileft" src="https://static.igem.org/mediawiki/2010/4/4e/BacterialCrowdingCapillaryAssay.png" alt="Capillary assay yo look under a microscope" /><br />
<p class="caption"><strong>Fig 2.</strong> Preparation of capillary assay to look under a microscope.</p><br />
</div><br />
<br />
<p>Over a microscope slide two capillaries are put which will hold up a cover slip. Then we insert the bacterial dilution between the slide and the cover slip. Two new capillaries are inserted between the slide and the cover slip inside of the bacterial dilution. One of those capillaries will contain a chemoattractant while the other one will be the control. Under the microscope we can see the different between both capillaries and we will definitely be able to observe if there is chemotaxis toward this chemoattractant.</p><br />
<br />
<p>Apart from that, we can detect the fluorescence emitted by the fluorophore which is present in bacteria using a fluorescent microscope.</p><br />
<br />
<p>This assay can be quantitative too if we spread on agar plates the content of capillaries and count the number of colonies that grew there.</p><br />
<br />
<div class="clear"></div><br />
<br />
<img class="centerBig" src="https://static.igem.org/mediawiki/2010/6/6f/BacterialCrowdingCapillaryAssayMicroscope.png" alt="Capillary assay Pictures"/><br />
<p class="caption"><strong>Fig 5.</strong>On the left, a capillary assay with 96-well PVC microplates and 1&#956;l capillary pipettes;<br />
capillaries are inserted through a 2% agarose gel. On the right, a capillary assay using needles and a tip chamber. Both of them were being incubated at 30oC when pictures was taken.</p><br />
<br />
<br />
<h1>Quantitative Assays</h1><br />
<br />
<h2>Capillary assays</h2><br />
<br />
<p>The capillary assays are the most useful to quantify chemotaxis. Although with some problems, this team has performed a capillary assay.</p><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/7/73/BacterialCrowdingCapillaryPictures.png" alt="Capillary assay of E. coli chemotaxis toward aspartate"/><br />
<p class="caption"><strong>Fig 3.</strong> Results of a capillary assay using microscope techniques. We can see that the chemotatic response toward aspartate is increasing as time passes by. Also there are major differences between the control without aspartate and the control with aspartate.</p><br />
<br />
<ul><br />
<li><p><strong>Foundations</strong></p><br />
<p>A capillary, which is put in a bacterial dilution, makes a concentration gradient of chemoatractant, produced by thr flow that goes from capillary to the medium according to the <strong>Fick law</strong> (see http://en.wikipedia.org/wiki/Fick's_laws_of_diffusion). This gradient would be sensed by bacteria that are going from low to high concentration places thus we should have some bacteria into the capillary. We can demonstrate that when we compare a capillary chemoattractant with another without any substance (the control), just the buffer. The control has to continue the same protocol than the others. In the same way the efficacy of a repellent could be tested since the capillary with the repellent will have less bacteria than the control.</p><br />
<br />
<div class="table"><br />
<table class="tableBio" cellspacing="0" cellpadding="0"><br />
<thead><br />
<tr><br />
<th>Bacterial dilution recipient or <strong>Chemotaxis Chambers</strong></th><br />
<th>Attractant or Buffer recipient or <strong>Capillaries</strong></th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr><br />
<td>tip chambers</td><br />
<td>syringe’s needles (more or less thin)</td><br />
</tr><br />
<tr><br />
<td>needle’s cups or heated closed 100ml tips</td><br />
<td>Needles</td><br />
</tr><br />
<tr><br />
<td>tip chambers</td><br />
<td>10&#956;l micropipette’s tips</td><br />
</tr><br />
<tr><br />
<td>96-well PVC microplates</td><br />
<td>1&#956;l capillary pipettes</td><br />
</tr><br />
<tr><br />
<td>drop between microscope slide and cover slip</td><br />
<td>1&#956;l capillary pipettes</td><br />
</tr><br />
<tr><br />
<td>Flow-chamber</td><br />
<td>1&#956;l capillary pipettes</td><br />
</tr><br />
</tbody><br />
</table><br />
</div><br />
<br />
<br />
<p class="caption"><strong>Table 1</strong>. Several ways we have carried out using capillary methods in chemotaxis assays.</p> <br />
<br />
<p>This team has performed this assay in different scales using differents chemotactic chambers where the bacterial dilution was put inside and differents capillaries. Some types of that are reflected in the Table 1. The attractant concentration in the capillary depends on the substance itself.</p> <br />
</li><br />
<br />
<li><p><strong>Protocol</strong></p><br />
<br />
<div class="imgRight"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/1/11/BacterialCrowdingCapillaryRepresentation.png" alt="Representation of capillary assay in a tip chambers." /><br />
<p class="caption"><strong>Fig 4.</strong>Representation of capillary assay in a tip chambers.</p><br />
</div><br />
<br />
<p>The experiment may start in two different ways; putting inocula from the strains we are going to work with into triptone broth or in minimal medium in a low shaking at 30ºC overnight. A high shaking might provoke the loss of flagella. The production of flagella wouldn’t be possible in a rich medium since bacteria wouldn’t need it.</p><br />
<br />
<p>The following day the inocula should be diluted in the same medium a hundred of times and wait for the growing phase to be the appropriate. For <i>Escherichia Coli</i> it would be necessary to wait for the exponential middle phase since it is this phase when flagella develop the flagellar motor. For <i>Pseudomonas</i> instead it would better wait for the late exponential phase, as the flagellum is developed later in this organism.</p><br />
<br />
<p>Once the culture is ready, it must be changed in an appropriate medium for mobility and chemotaxis. For that, it is necessary to wash the culture twice with chemotaxis buffer centrifuging in a low speed since flagella may be lost if it is treated abruptly.</p><br />
<br />
<p>When the culture is in the right medium the number of bacteria is adjusted roughly to 10<sup>7</sup> fcu/ml. This dilution has to be distributed in chemotaxis chambers where our capillaries will be introduced in it. The volume of capillaries can be unsettled; we have used as a standard volume 100 &#956;l of diluted chemoattractant in chemotaxis buffer. Mind controls, they will be capillaries thanks to the chemotaxis buffer.</p><br />
<br />
<p>Incubate the experiment at 30º during 60 minutes, after that we have to quantify bacteria that are contained into the capillaries. In order to achieve that we could do it either with dilution and spread in plates or analyzing the fluorescence, supposing that bacteria have any kind of fluorophore.</p><br />
</li><br />
<br />
<li><p><strong>Advice</strong></p><br />
<p>One of the elements we bear in mind is the chemotaxis buffer: chemotaxis medium contain potassium potassium phospgate buffer (pH 7), ethylenediaminetetraacetate (<strong>EDTA</strong>) and <strong>glycerol</strong> (energy source). The glycerol is only necessary in long incubations; meanwhile in short incubations the typical sources of bacteria are enough to maintain the chemotactic machinery. It is all-important to underline that the chemotaxis medium must be free of any other substance which may have chemotactic effects, since this could disturb the results. This is one of the reasons why the carbon source is not glucose, how you may expect. Other important detail to bear in mind is EDTA, this chelation provoke the precipitation of magnesium which may dull the movement of bacteria and the flagellar machinery. It would be complicated to success in the chemotaxis assays without this chelation. Incubation of bacteria must be carried at 30ºC since it helps motility. Shaking must be low as flagella can be lost in high shaking.</p><br />
<br />
<p>It is crucial to be careful when <strong>choosing the strains</strong> to be used in the chemotaxis assays, since it may not have motility. Strains used in laboratories have normally no motility, as at that point they have usually suffer different screening process in benefit of immobile bacteria. A bacterium which has no motility won’t have to invest in any source in motility or chemotaxis; this would encourage the creation of a colony bigger and more eye-catching than usual, so scientifics would be probably leaded to select one of this kind. . This issue happened to us and we were trying to attract a non mobile strain toward different attractants until we realised.</p><br />
</li><br />
</ul><br />
<br />
<h2>Buridan’s Donkey</h2><br />
<br />
<p>To test bacterial chemotaxis we have used a three-channel device based on <strong>flow-chamber biofilm</strong>. It would be able to produce a linear gradient within narrow tubes that connect the chambers. The linear chemical gradient would be generated by diffusion of the chemoattractant through a dialysis membrane located in the limit of the chamber. This membrane also makes impossible the movement of the chemoattractant-producing bacteria through the tube.</p><br />
<br />
<p>The first assay involves only chemoattractants, and the second one includes producing bacterias. As result, it is expected that the movement of the cells in the center chamber was directed to the chamber containing chemoattractant-producing bacterias, for the cells chemotactic response, but not in the control chamber, in the opposite side. It is necessary to clear up that the chemoattractant production would be activated solely by the contact of bacteria with plant cell walls that reside in the same chamber. Bacteria have to “decide” between going toward control empty chamber or going toward chamber with chemoattractant-producing bacteria.</p><br />
<br />
<p>This device could provide a lot of advantages in the study of chemotaxis: rapid and easy implementation, parallel and simultaneous test, visual proofs, different assays possibilities. Also some experimental conditions could be changed easily, for instance: concentration of bacterial population, chambers distances, bacterial cultures, chemoattractans.</p><br />
<br />
<p>An explaining diagram of this device is provided below.</p><br />
<br />
<h3>Measuring the performance of the chemotaxis circuits (Buridan's donkey assay principle)</h3><br />
<br />
<img class="centerBig" src="https://static.igem.org/mediawiki/2010/a/a8/BacterialCrowdingBuridanDonkey.png" alt="Measuring the performance of the chemotaxis circuits (Buridan's donkey assay principle)"/><br />
<br />
<h3>Buridan's donkey assays with three-channel flow cells</h3><br />
<br />
<img class="centerBig" src="https://static.igem.org/mediawiki/2010/3/38/BacterialCrowdingBuridanDonkeyThree.png" alt="Buridan's donkey assays with three-channel flow cells"/><br />
<br />
<p><strong>Special acknowledgements to Ph.D Parkinson (University of Utah) who gave us some advices, handed us over some protocols of him, even mobile and mutant <i>E. Coli</i> strains. </strong></p><br />
<br />
<h1>References</h1><br />
<br />
<ul><br />
<li>J. Adler (1972) A Method for Measuring Chemotaxis and Use of the Method to Determine Optimum Conditions for Chemotaxis by Escherichia coli. - Journal of General Microbiology ( I 973), 74, 77-91</li><br />
<br />
<li>Guocheng Han and Joseph J. Cooney (1993) A modified capillary assay for chemotaxis - Journal of Industrial Microbiology, 12 (1993) 396—398</li><br />
<br />
<li>Hanbin Mao, Paul S. Cremer, and Michael D. Manson (2003) A sensitive, versatile microfluidic assay for bacterial chemotaxis - PNAS MICROBIOLOGY vol. 100 no. 9 5449–5454.</li><br />
<br />
<li>Russell Bainer, Heungwon Park, Philippe Cluzel (2003) A high-throughput capillary assay for bacterial chemotaxis - Journal of Microbiological Methods 55 (2003) 315– 319.</li><br />
</ul><br />
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<h1>Introduction</h1><br />
<br />
<p><strong>Motility</strong> is one of the most readily demonstrated bacterial characters, and <strong>chemotaxis</strong> is one of the most studied bacterial behaviors. Motile organisms are attracted by certain chemicals and repelled by others (positive and negative chemotaxis). Quantification of chemotactic motion is necessary to identify chemoeffectors and to determine the structure of bacterial communities.</p><br />
<br />
<p>Current methods of quantifying chemotaxis use chemotactic bacteria such as <i>Escherichia Coli</i>, which is assayed by measuring the number of organisms attracted into a capillary tube containing an attractant.</p><br />
<br />
<p>UPO-Sevilla team has carried out different experimental prototypes that are better to achieve. The goal of the group was designing different assays that allow us to study this effect in both point of views, <strong>qualitative</strong> and <strong>quantitative</strong>. Also we have to test induction of sensing systems and chemoattractant production. Anyway, these processes are highly related with the chemotaxis process in Bacterial Crowding project. This is why we measured chemoattractant production counting the range of attracted bacteria using chemotaxis assays. The induction of sensing systems could be tested by using GFP measures when its promoter is <i>PfecA</i> or <i>PprhJ</i>; or also due to the levels of chemoattractant production. </p><br />
<br />
<h1>Qualitative Assays</h1><br />
<br />
<h2>Agar Soft Plates</h2><br />
<br />
<p>Our qualitative assays were made in <strong>soft agar</strong> thanks to the protocols that we had received from Mr. Parkinson (University of Utah).</p><br />
<br />
<p>This kind of plates allows bacteria to swim trough the agar freely and showing their chemotactic capacities. A colony, inserted in soft agar plate, starts to grow while running out the environmental sources. For this reason bacteria would move to places where the sources are not limited. That phenomenon produces a number of <strong>halos</strong> which are spread within the plate and increase in volume as the sources are lowered.</p><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/3/36/BacterialCrowdingSoftAgarPlate.png" alt="Soft Agar Plate Assay"/><br />
<p class="caption"><strong>Fig 1. Soft agar plate assay.</strong> Different strains are shown. Every strain carries out a mutation that affects its chemotactic response, every strain but the wild type. In the wt you can observe two halos produced by chemotactic responses to aspartate and glutamate.</p><br />
<br />
<br />
<p>The assay protocol is simple; once the soft agar plates are prepared, a colony is inserted in the previous plate. Let it grow in 30ºC. The soft agar is a delicate element, so it is important to be careful when moving the plates.</p><br />
<br />
<p>In those plates it might appear different concentric circles which represent chemotaxis to a certain attractant. For instance, when two amino acids are run out from the medium, two circles will appear. The inner one will show the amino acids limit with low chemotactic response; while the outer one will mean that the amino acid which causes a higher response is running down.</p><br />
<br />
<h2>Optical and Fluorescence Microscopy</h2><br />
<br />
<p>The <strong>microscopy techniques</strong> allow us to see the development of the assay <i>in situ</i> without any wait. In this part we will see how we can carry out an experiment that we could see under the microscope.</p><br />
<br />
<div class="imgLeft"><br />
<img class="ileft" src="https://static.igem.org/mediawiki/2010/4/4e/BacterialCrowdingCapillaryAssay.png" alt="Capillary assay yo look under a microscope" /><br />
<p class="caption"><strong>Fig 2.</strong> Preparation of capillary assay to look under a microscope.</p><br />
</div><br />
<br />
<p>Over a microscope slide two capillaries are put which will hold up a cover slip. Then we insert the bacterial dilution between the slide and the cover slip. Two new capillaries are inserted between the slide and the cover slip inside of the bacterial dilution. One of those capillaries will contain a chemoattractant while the other one will be the control. Under the microscope we can see the different between both capillaries and we will definitely be able to observe if there is chemotaxis toward this chemoattractant.</p><br />
<br />
<p>Apart from that, we can detect the fluorescence emitted by the fluorophore which is present in bacteria using a fluorescent microscope.</p><br />
<br />
<p>This assay can be quantitative too if we spread on agar plates the content of capillaries and count the number of colonies that grew there.</p><br />
<br />
<div class="clear"></div><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/6/6f/BacterialCrowdingCapillaryAssayMicroscope.png" alt="Capillary assay Pictures"/><br />
<p class="caption"><strong>Fig 5.</strong>On the left, a capillary assay with 96-well PVC microplates and 1&#956;l capillary pipettes;<br />
capillaries are inserted through a 2% agarose gel. On the right, a capillary assay using needles and a tip chamber. Both of them were being incubated at 30oC when pictures was taken.</p><br />
<br />
<br />
<h1>Quantitative Assays</h1><br />
<br />
<h2>Capillary assays</h2><br />
<br />
<p>The capillary assays are the most useful to quantify chemotaxis. Although with some problems, this team has performed a capillary assay.</p><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/7/73/BacterialCrowdingCapillaryPictures.png" alt="Capillary assay of E. coli chemotaxis toward aspartate"/><br />
<p class="caption"><strong>Fig 3.</strong> Results of a capillary assay using microscope techniques. We can see that the chemotatic response toward aspartate is increasing as time passes by. Also there are major differences between the control without aspartate and the control with aspartate.</p><br />
<br />
<ul><br />
<li><p><strong>Foundations</strong></p><br />
<p>A capillary, which is put in a bacterial dilution, makes a concentration gradient of chemoatractant, produced by thr flow that goes from capillary to the medium according to the <strong>Fick law</strong> (see http://en.wikipedia.org/wiki/Fick's_laws_of_diffusion). This gradient would be sensed by bacteria that are going from low to high concentration places thus we should have some bacteria into the capillary. We can demonstrate that when we compare a capillary chemoattractant with another without any substance (the control), just the buffer. The control has to continue the same protocol than the others. In the same way the efficacy of a repellent could be tested since the capillary with the repellent will have less bacteria than the control.</p><br />
<br />
<div class="table"><br />
<table class="tableBio" cellspacing="0" cellpadding="0"><br />
<thead><br />
<tr><br />
<th>Bacterial dilution recipient or <strong>Chemotaxis Chambers</strong></th><br />
<th>Attractant or Buffer recipient or <strong>Capillaries</strong></th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr><br />
<td>tip chambers</td><br />
<td>syringe’s needles (more or less thin)</td><br />
</tr><br />
<tr><br />
<td>needle’s cups or heated closed 100ml tips</td><br />
<td>Needles</td><br />
</tr><br />
<tr><br />
<td>tip chambers</td><br />
<td>10&#956;l micropipette’s tips</td><br />
</tr><br />
<tr><br />
<td>96-well PVC microplates</td><br />
<td>1&#956;l capillary pipettes</td><br />
</tr><br />
<tr><br />
<td>drop between microscope slide and cover slip</td><br />
<td>1&#956;l capillary pipettes</td><br />
</tr><br />
<tr><br />
<td>Flow-chamber</td><br />
<td>1&#956;l capillary pipettes</td><br />
</tr><br />
</tbody><br />
</table><br />
</div><br />
<br />
<br />
<p class="caption"><strong>Table 1</strong>. Several ways we have carried out using capillary methods in chemotaxis assays.</p> <br />
<br />
<p>This team has performed this assay in different scales using differents chemotactic chambers where the bacterial dilution was put inside and differents capillaries. Some types of that are reflected in the Table 1. The attractant concentration in the capillary depends on the substance itself.</p> <br />
</li><br />
<br />
<li><p><strong>Protocol</strong></p><br />
<br />
<div class="imgRight"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/1/11/BacterialCrowdingCapillaryRepresentation.png" alt="Representation of capillary assay in a tip chambers." /><br />
<p class="caption"><strong>Fig 4.</strong>Representation of capillary assay in a tip chambers.</p><br />
</div><br />
<br />
<p>The experiment may start in two different ways; putting inocula from the strains we are going to work with into triptone broth or in minimal medium in a low shaking at 30ºC overnight. A high shaking might provoke the loss of flagella. The production of flagella wouldn’t be possible in a rich medium since bacteria wouldn’t need it.</p><br />
<br />
<p>The following day the inocula should be diluted in the same medium a hundred of times and wait for the growing phase to be the appropriate. For <i>Escherichia Coli</i> it would be necessary to wait for the exponential middle phase since it is this phase when flagella develop the flagellar motor. For <i>Pseudomonas</i> instead it would better wait for the late exponential phase, as the flagellum is developed later in this organism.</p><br />
<br />
<p>Once the culture is ready, it must be changed in an appropriate medium for mobility and chemotaxis. For that, it is necessary to wash the culture twice with chemotaxis buffer centrifuging in a low speed since flagella may be lost if it is treated abruptly.</p><br />
<br />
<p>When the culture is in the right medium the number of bacteria is adjusted roughly to 10<sup>7</sup> fcu/ml. This dilution has to be distributed in chemotaxis chambers where our capillaries will be introduced in it. The volume of capillaries can be unsettled; we have used as a standard volume 100 &#956;l of diluted chemoattractant in chemotaxis buffer. Mind controls, they will be capillaries thanks to the chemotaxis buffer.</p><br />
<br />
<p>Incubate the experiment at 30º during 60 minutes, after that we have to quantify bacteria that are contained into the capillaries. In order to achieve that we could do it either with dilution and spread in plates or analyzing the fluorescence, supposing that bacteria have any kind of fluorophore.</p><br />
</li><br />
<br />
<li><p><strong>Advice</strong></p><br />
<p>One of the elements we bear in mind is the chemotaxis buffer: chemotaxis medium contain potassium potassium phospgate buffer (pH 7), ethylenediaminetetraacetate (<strong>EDTA</strong>) and <strong>glycerol</strong> (energy source). The glycerol is only necessary in long incubations; meanwhile in short incubations the typical sources of bacteria are enough to maintain the chemotactic machinery. It is all-important to underline that the chemotaxis medium must be free of any other substance which may have chemotactic effects, since this could disturb the results. This is one of the reasons why the carbon source is not glucose, how you may expect. Other important detail to bear in mind is EDTA, this chelation provoke the precipitation of magnesium which may dull the movement of bacteria and the flagellar machinery. It would be complicated to success in the chemotaxis assays without this chelation. Incubation of bacteria must be carried at 30ºC since it helps motility. Shaking must be low as flagella can be lost in high shaking.</p><br />
<br />
<p>It is crucial to be careful when <strong>choosing the strains</strong> to be used in the chemotaxis assays, since it may not have motility. Strains used in laboratories have normally no motility, as at that point they have usually suffer different screening process in benefit of immobile bacteria. A bacterium which has no motility won’t have to invest in any source in motility or chemotaxis; this would encourage the creation of a colony bigger and more eye-catching than usual, so scientifics would be probably leaded to select one of this kind. . This issue happened to us and we were trying to attract a non mobile strain toward different attractants until we realised.</p><br />
</li><br />
</ul><br />
<br />
<h2>Buridan’s Donkey</h2><br />
<br />
<p>To test bacterial chemotaxis we have used a three-channel device based on <strong>flow-chamber biofilm</strong>. It would be able to produce a linear gradient within narrow tubes that connect the chambers. The linear chemical gradient would be generated by diffusion of the chemoattractant through a dialysis membrane located in the limit of the chamber. This membrane also makes impossible the movement of the chemoattractant-producing bacteria through the tube.</p><br />
<br />
<p>The first assay involves only chemoattractants, and the second one includes producing bacterias. As result, it is expected that the movement of the cells in the center chamber was directed to the chamber containing chemoattractant-producing bacterias, for the cells chemotactic response, but not in the control chamber, in the opposite side. It is necessary to clear up that the chemoattractant production would be activated solely by the contact of bacteria with plant cell walls that reside in the same chamber. Bacteria have to “decide” between going toward control empty chamber or going toward chamber with chemoattractant-producing bacteria.</p><br />
<br />
<p>This device could provide a lot of advantages in the study of chemotaxis: rapid and easy implementation, parallel and simultaneous test, visual proofs, different assays possibilities. Also some experimental conditions could be changed easily, for instance: concentration of bacterial population, chambers distances, bacterial cultures, chemoattractans.</p><br />
<br />
<p>An explaining diagram of this device is provided below.</p><br />
<br />
<h3>Measuring the performance of the chemotaxis circuits (Buridan's donkey assay principle)</h3><br />
<br />
<img class="centerBig" src="https://static.igem.org/mediawiki/2010/a/a8/BacterialCrowdingBuridanDonkey.png" alt="Measuring the performance of the chemotaxis circuits (Buridan's donkey assay principle)"/><br />
<br />
<h3>Buridan's donkey assays with three-channel flow cells</h3><br />
<br />
<img class="centerBig" src="https://static.igem.org/mediawiki/2010/3/38/BacterialCrowdingBuridanDonkeyThree.png" alt="Buridan's donkey assays with three-channel flow cells"/><br />
<br />
<p><strong>Special acknowledgements to Ph.D Parkinson (University of Utah) who gave us some advices, handed us over some protocols of him, even mobile and mutant <i>E. Coli</i> strains. </strong></p><br />
<br />
<h1>References</h1><br />
<br />
<ul><br />
<li>J. Adler (1972) A Method for Measuring Chemotaxis and Use of the Method to Determine Optimum Conditions for Chemotaxis by Escherichia coli. - Journal of General Microbiology ( I 973), 74, 77-91</li><br />
<br />
<li>Guocheng Han and Joseph J. Cooney (1993) A modified capillary assay for chemotaxis - Journal of Industrial Microbiology, 12 (1993) 396—398</li><br />
<br />
<li>Hanbin Mao, Paul S. Cremer, and Michael D. Manson (2003) A sensitive, versatile microfluidic assay for bacterial chemotaxis - PNAS MICROBIOLOGY vol. 100 no. 9 5449–5454.</li><br />
<br />
<li>Russell Bainer, Heungwon Park, Philippe Cluzel (2003) A high-throughput capillary assay for bacterial chemotaxis - Journal of Microbiological Methods 55 (2003) 315– 319.</li><br />
</ul><br />
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<h1>Introduction</h1><br />
<br />
<p><strong>Motility</strong> is one of the most readily demonstrated bacterial characters, and <strong>chemotaxis</strong> is one of the most studied bacterial behaviors. Motile organisms are attracted by certain chemicals and repelled by others (positive and negative chemotaxis). Quantification of chemotactic motion is necessary to identify chemoeffectors and to determine the structure of bacterial communities.</p><br />
<br />
<p>Current methods of quantifying chemotaxis use chemotactic bacteria such as <i>Escherichia Coli</i>, which is assayed by measuring the number of organisms attracted into a capillary tube containing an attractant.</p><br />
<br />
<p>UPO-Sevilla team has carried out different experimental prototypes that are better to achieve. The goal of the group was designing different assays that allow us to study this effect in both point of views, <strong>qualitative</strong> and <strong>quantitative</strong>. Also we have to test induction of sensing systems and chemoattractant production. Anyway, these processes are highly related with the chemotaxis process in Bacterial Crowding project. This is why we measured chemoattractant production counting the range of attracted bacteria using chemotaxis assays. The induction of sensing systems could be tested by using GFP measures when its promoter is <i>PfecA</i> or <i>PprhJ</i>; or also due to the levels of chemoattractant production. </p><br />
<br />
<h1>Qualitative Assays</h1><br />
<br />
<h2>Agar Soft Plates</h2><br />
<br />
<p>Our qualitative assays were made in <strong>soft agar</strong> thanks to the protocols that we had received from Mr. Parkinson (University of Utah).</p><br />
<br />
<p>This kind of plates allows bacteria to swim trough the agar freely and showing their chemotactic capacities. A colony, inserted in soft agar plate, starts to grow while running out the environmental sources. For this reason bacteria would move to places where the sources are not limited. That phenomenon produces a number of <strong>halos</strong> which are spread within the plate and increase in volume as the sources are lowered.</p><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/3/36/BacterialCrowdingSoftAgarPlate.png" alt="Soft Agar Plate Assay"/><br />
<p class="caption"><strong>Fig 1. Soft agar plate assay.</strong> Different strains are shown. Every strain carries out a mutation that affects its chemotactic response, every strain but the wild type. In the wt you can observe two halos produced by chemotactic responses to aspartate and glutamate.</p><br />
<br />
<br />
<p>The assay protocol is simple; once the soft agar plates are prepared, a colony is inserted in the previous plate. Let it grow in 30ºC. The soft agar is a delicate element, so it is important to be careful when moving the plates.</p><br />
<br />
<p>In those plates it might appear different concentric circles which represent chemotaxis to a certain attractant. For instance, when two amino acids are run out from the medium, two circles will appear. The inner one will show the amino acids limit with low chemotactic response; while the outer one will mean that the amino acid which causes a higher response is running down.</p><br />
<br />
<h2>Optical and Fluorescence Microscopy</h2><br />
<br />
<p>The <strong>microscopy techniques</strong> allow us to see the development of the assay <i>in situ</i> without any wait. In this part we will see how we can carry out an experiment that we could see under the microscope.</p><br />
<br />
<div class="imgLeft"><br />
<img class="ileft" src="https://static.igem.org/mediawiki/2010/4/4e/BacterialCrowdingCapillaryAssay.png" alt="Capillary assay yo look under a microscope" /><br />
<p class="caption"><strong>Fig 2.</strong> Preparation of capillary assay to look under a microscope.</p><br />
</div><br />
<br />
<p>Over a microscope slide two capillaries are put which will hold up a cover slip. Then we insert the bacterial dilution between the slide and the cover slip. Two new capillaries are inserted between the slide and the cover slip inside of the bacterial dilution. One of those capillaries will contain a chemoattractant while the other one will be the control. Under the microscope we can see the different between both capillaries and we will definitely be able to observe if there is chemotaxis toward this chemoattractant.</p><br />
<br />
<p>Apart from that, we can detect the fluorescence emitted by the fluorophore which is present in bacteria using a fluorescent microscope.</p><br />
<br />
<p>This assay can be quantitative too if we spread on agar plates the content of capillaries and count the number of colonies that grew there.</p><br />
<br />
<div class="clear"></div><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/6/6f/BacterialCrowdingCapillaryAssayMicroscope.png" alt="Capillary assay Pictures"/><br />
<p class="caption"><strong>Fig 5.</strong>On the left, a capillary assay with 96-well PVC microplates and 1&#956;l capillary pipettes;<br />
capillaries are inserted through a 2% agarose gel. On the right, a capillary assay using needles and a tip chamber. Both of them were being incubated at 30oC when pictures was taken.</p><br />
<br />
<br />
<h1>Quantitative Assays</h1><br />
<br />
<h2>Capillary assays</h2><br />
<br />
<p>The capillary assays are the most useful to quantify chemotaxis. Although with some problems, this team has performed a capillary assay.</p><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/7/73/BacterialCrowdingCapillaryPictures.png" alt="Capillary assay of E. coli chemotaxis toward aspartate"/><br />
<p class="caption"><strong>Fig 3.</strong> Results of a capillary assay using microscope techniques. We can see that the chemotatic response toward aspartate is increasing as time passes by. Also there are major differences between the control without aspartate and the control with aspartate.</p><br />
<br />
<ul><br />
<li><p><strong>Foundations</strong></p><br />
<p>A capillary, which is put in a bacterial dilution, makes a concentration gradient of chemoatractant, produced by thr flow that goes from capillary to the medium according to the <strong>Fick law</strong> (see http://en.wikipedia.org/wiki/Fick's_laws_of_diffusion). This gradient would be sensed by bacteria that are going from low to high concentration places thus we should have some bacteria into the capillary. We can demonstrate that when we compare a capillary chemoattractant with another without any substance (the control), just the buffer. The control has to continue the same protocol than the others. In the same way the efficacy of a repellent could be tested since the capillary with the repellent will have less bacteria than the control.</p><br />
<br />
<div class="table"><br />
<table class="tableBio" cellspacing="0" cellpadding="0"><br />
<thead><br />
<tr><br />
<th>Bacterial dilution recipient or <strong>Chemotaxis Chambers</strong></th><br />
<th>Attractant or Buffer recipient or <strong>Capillaries</strong></th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr><br />
<td>tip chambers</td><br />
<td>syringe’s needles (more or less thin)</td><br />
</tr><br />
<tr><br />
<td>needle’s cups or heated closed 100ml tips</td><br />
<td>Needles</td><br />
</tr><br />
<tr><br />
<td>tip chambers</td><br />
<td>10&#956;l micropipette’s tips</td><br />
</tr><br />
<tr><br />
<td>96-well PVC microplates</td><br />
<td>1&#956;l capillary pipettes</td><br />
</tr><br />
<tr><br />
<td>drop between microscope slide and cover slip</td><br />
<td>1&#956;l capillary pipettes</td><br />
</tr><br />
<tr><br />
<td>Flow-chamber</td><br />
<td>1&#956;l capillary pipettes</td><br />
</tr><br />
</tbody><br />
</table><br />
</div><br />
<br />
<br />
<p><strong>Table 1</strong>. Several ways we have carried out using capillary methods in chemotaxis assays.</p> <br />
<br />
<p>This team has performed this assay in different scales using differents chemotactic chambers where the bacterial dilution was put inside and differents capillaries. Some types of that are reflected in the Table 1. The attractant concentration in the capillary depends on the substance itself.</p> <br />
</li><br />
<br />
<li><p><strong>Protocol</strong></p><br />
<br />
<div class="imgRight"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/1/11/BacterialCrowdingCapillaryRepresentation.png" alt="Representation of capillary assay in a tip chambers." /><br />
<p class="caption"><strong>Fig 4.</strong>Representation of capillary assay in a tip chambers.</p><br />
</div><br />
<br />
<p>The experiment may start in two different ways; putting inocula from the strains we are going to work with into triptone broth or in minimal medium in a low shaking at 30ºC overnight. A high shaking might provoke the loss of flagella. The production of flagella wouldn’t be possible in a rich medium since bacteria wouldn’t need it.</p><br />
<br />
<p>The following day the inocula should be diluted in the same medium a hundred of times and wait for the growing phase to be the appropriate. For <i>Escherichia Coli</i> it would be necessary to wait for the exponential middle phase since it is this phase when flagella develop the flagellar motor. For <i>Pseudomonas</i> instead it would better wait for the late exponential phase, as the flagellum is developed later in this organism.</p><br />
<br />
<p>Once the culture is ready, it must be changed in an appropriate medium for mobility and chemotaxis. For that, it is necessary to wash the culture twice with chemotaxis buffer centrifuging in a low speed since flagella may be lost if it is treated abruptly.</p><br />
<br />
<p>When the culture is in the right medium the number of bacteria is adjusted roughly to 10<sup>7</sup> fcu/ml. This dilution has to be distributed in chemotaxis chambers where our capillaries will be introduced in it. The volume of capillaries can be unsettled; we have used as a standard volume 100 &#956;l of diluted chemoattractant in chemotaxis buffer. Mind controls, they will be capillaries thanks to the chemotaxis buffer.</p><br />
<br />
<p>Incubate the experiment at 30º during 60 minutes, after that we have to quantify bacteria that are contained into the capillaries. In order to achieve that we could do it either with dilution and spread in plates or analyzing the fluorescence, supposing that bacteria have any kind of fluorophore.</p><br />
</li><br />
<br />
<li><p><strong>Advice</strong></p><br />
<p>One of the elements we bear in mind is the chemotaxis buffer: chemotaxis medium contain potassium potassium phospgate buffer (pH 7), ethylenediaminetetraacetate (<strong>EDTA</strong>) and <strong>glycerol</strong> (energy source). The glycerol is only necessary in long incubations; meanwhile in short incubations the typical sources of bacteria are enough to maintain the chemotactic machinery. It is all-important to underline that the chemotaxis medium must be free of any other substance which may have chemotactic effects, since this could disturb the results. This is one of the reasons why the carbon source is not glucose, how you may expect. Other important detail to bear in mind is EDTA, this chelation provoke the precipitation of magnesium which may dull the movement of bacteria and the flagellar machinery. It would be complicated to success in the chemotaxis assays without this chelation. Incubation of bacteria must be carried at 30ºC since it helps motility. Shaking must be low as flagella can be lost in high shaking.</p><br />
<br />
<p>It is crucial to be careful when <strong>choosing the strains</strong> to be used in the chemotaxis assays, since it may not have motility. Strains used in laboratories have normally no motility, as at that point they have usually suffer different screening process in benefit of immobile bacteria. A bacterium which has no motility won’t have to invest in any source in motility or chemotaxis; this would encourage the creation of a colony bigger and more eye-catching than usual, so scientifics would be probably leaded to select one of this kind. . This issue happened to us and we were trying to attract a non mobile strain toward different attractants until we realised.</p><br />
</li><br />
</ul><br />
<br />
<h2>Buridan’s Donkey</h2><br />
<br />
<p>To test bacterial chemotaxis we have used a three-channel device based on <strong>flow-chamber biofilm</strong>. It would be able to produce a linear gradient within narrow tubes that connect the chambers. The linear chemical gradient would be generated by diffusion of the chemoattractant through a dialysis membrane located in the limit of the chamber. This membrane also makes impossible the movement of the chemoattractant-producing bacteria through the tube.</p><br />
<br />
<p>The first assay involves only chemoattractants, and the second one includes producing bacterias. As result, it is expected that the movement of the cells in the center chamber was directed to the chamber containing chemoattractant-producing bacterias, for the cells chemotactic response, but not in the control chamber, in the opposite side. It is necessary to clear up that the chemoattractant production would be activated solely by the contact of bacteria with plant cell walls that reside in the same chamber. Bacteria have to “decide” between going toward control empty chamber or going toward chamber with chemoattractant-producing bacteria.</p><br />
<br />
<p>This device could provide a lot of advantages in the study of chemotaxis: rapid and easy implementation, parallel and simultaneous test, visual proofs, different assays possibilities. Also some experimental conditions could be changed easily, for instance: concentration of bacterial population, chambers distances, bacterial cultures, chemoattractans.</p><br />
<br />
<p>An explaining diagram of this device is provided below.</p><br />
<br />
<h3>Measuring the performance of the chemotaxis circuits (Buridan's donkey assay principle)</h3><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/a/a8/BacterialCrowdingBuridanDonkey.png" alt="Measuring the performance of the chemotaxis circuits (Buridan's donkey assay principle)"/><br />
<br />
<h3>Buridan's donkey assays with three-channel flow cells</h3><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/3/38/BacterialCrowdingBuridanDonkeyThree.png" alt="Buridan's donkey assays with three-channel flow cells"/><br />
<br />
<p><strong>Special acknowledgements to Ph.D Parkinson (University of Utah) who gave us some advices, handed us over some protocols of him, even mobile and mutant <i>E. Coli</i> strains. </strong></p><br />
<br />
<h1>References</h1><br />
<br />
<ul><br />
<li>J. Adler (1972) A Method for Measuring Chemotaxis and Use of the Method to Determine Optimum Conditions for Chemotaxis by Escherichia coli. - Journal of General Microbiology ( I 973), 74, 77-91</li><br />
<br />
<li>Guocheng Han and Joseph J. Cooney (1993) A modified capillary assay for chemotaxis - Journal of Industrial Microbiology, 12 (1993) 396—398</li><br />
<br />
<li>Hanbin Mao, Paul S. Cremer, and Michael D. Manson (2003) A sensitive, versatile microfluidic assay for bacterial chemotaxis - PNAS MICROBIOLOGY vol. 100 no. 9 5449–5454.</li><br />
<br />
<li>Russell Bainer, Heungwon Park, Philippe Cluzel (2003) A high-throughput capillary assay for bacterial chemotaxis - Journal of Microbiological Methods 55 (2003) 315– 319.</li><br />
</ul><br />
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<h1>Introduction</h1><br />
<br />
<p><strong>Motility</strong> is one of the most readily demonstrated bacterial characters, and <strong>chemotaxis</strong> is one of the most studied bacterial behaviors. Motile organisms are attracted by certain chemicals and repelled by others (positive and negative chemotaxis). Quantification of chemotactic motion is necessary to identify chemoeffectors and to determine the structure of bacterial communities.</p><br />
<br />
<p>Current methods of quantifying chemotaxis use chemotactic bacteria such as <i>Escherichia Coli</i>, which is assayed by measuring the number of organisms attracted into a capillary tube containing an attractant.</p><br />
<br />
<p>UPO-Sevilla team has carried out different experimental prototypes that are better to achieve. The goal of the group was designing different assays that allow us to study this effect in both point of views, <strong>qualitative</strong> and <strong>quantitative</strong>. Also we have to test induction of sensing systems and chemoattractant production. Anyway, these processes are highly related with the chemotaxis process in Bacterial Crowding project. This is why we measured chemoattractant production counting the range of attracted bacteria using chemotaxis assays. The induction of sensing systems could be tested by using GFP measures when its promoter is <i>PfecA</i> or <i>PprhJ</i>; or also due to the levels of chemoattractant production. </p><br />
<br />
<h1>Qualitative Assays</h1><br />
<br />
<h2>Agar Soft Plates</h2><br />
<br />
<p>Our qualitative assays were made in <strong>soft agar</strong> thanks to the protocols that we had received from Mr. Parkinson (University of Utah).</p><br />
<br />
<p>This kind of plates allows bacteria to swim trough the agar freely and showing their chemotactic capacities. A colony, inserted in soft agar plate, starts to grow while running out the environmental sources. For this reason bacteria would move to places where the sources are not limited. That phenomenon produces a number of <strong>halos</strong> which are spread within the plate and increase in volume as the sources are lowered.</p><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/3/36/BacterialCrowdingSoftAgarPlate.png" alt="Soft Agar Plate Assay"/><br />
<p class="caption"><strong>Fig 1. Soft agar plate assay.</strong> Different strains are shown. Every strain carries out a mutation that affects its chemotactic response, every strain but the wild type. In the wt you can observe two halos produced by chemotactic responses to aspartate and glutamate.</p><br />
<br />
<br />
<p>The assay protocol is simple; once the soft agar plates are prepared, a colony is inserted in the previous plate. Let it grow in 30ºC. The soft agar is a delicate element, so it is important to be careful when moving the plates.</p><br />
<br />
<p>In those plates it might appear different concentric circles which represent chemotaxis to a certain attractant. For instance, when two amino acids are run out from the medium, two circles will appear. The inner one will show the amino acids limit with low chemotactic response; while the outer one will mean that the amino acid which causes a higher response is running down.</p><br />
<br />
<h2>Optical and Fluorescence Microscopy</h2><br />
<br />
<p>The <strong>microscopy techniques</strong> allow us to see the development of the assay <i>in situ</i> without any wait. In this part we will see how we can carry out an experiment that we could see under the microscope.</p><br />
<br />
<div class="imgLeft"><br />
<img class="ileft" src="https://static.igem.org/mediawiki/2010/4/4e/BacterialCrowdingCapillaryAssay.png" alt="Capillary assay yo look under a microscope" /><br />
<p class="caption"><strong>Fig 2.</strong> Preparation of capillary assay to look under a microscope.</p><br />
</div><br />
<br />
<p>Over a microscope slide two capillaries are put which will hold up a cover slip. Then we insert the bacterial dilution between the slide and the cover slip. Two new capillaries are inserted between the slide and the cover slip inside of the bacterial dilution. One of those capillaries will contain a chemoattractant while the other one will be the control. Under the microscope we can see the different between both capillaries and we will definitely be able to observe if there is chemotaxis toward this chemoattractant.</p><br />
<br />
<p>Apart from that, we can detect the fluorescence emitted by the fluorophore which is present in bacteria using a fluorescent microscope.</p><br />
<br />
<p>This assay can be quantitative too if we spread on agar plates the content of capillaries and count the number of colonies that grew there.</p><br />
<br />
<div class="clear"></div><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/6/6f/BacterialCrowdingCapillaryAssayMicroscope.png" alt="Capillary assay Pictures"/><br />
<p class="caption"><strong>Fig 5.</strong>On the left, a capillary assay with 96-well PVC microplates and 1&#956;l capillary pipettes;<br />
capillaries are inserted through a 2% agarose gel. On the right, a capillary assay using needles and a tip chamber. Both of them were being incubated at 30oC when pictures was taken.</p><br />
<br />
<br />
<h1>Quantitative Assays</h1><br />
<br />
<h2>Capillary assays</h2><br />
<br />
<p>The capillary assays are the most useful to quantify chemotaxis. Although with some problems, this team has performed a capillary assay.</p><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/7/73/BacterialCrowdingCapillaryPictures.png" alt="Capillary assay of E. coli chemotaxis toward aspartate"/><br />
<p class="caption"><strong>Fig 3.</strong> Results of a capillary assay using microscope techniques. We can see that the chemotatic response toward aspartate is increasing as time passes by. Also there are major differences between the control without aspartate and the control with aspartate.</p><br />
<br />
<ul><br />
<li><p><strong>Foundations</strong></p><br />
<p>A capillary, which is put in a bacterial dilution, makes a concentration gradient of chemoatractant, produced by thr flow that goes from capillary to the medium according to the <strong>Fick law</strong> (see http://en.wikipedia.org/wiki/Fick's_laws_of_diffusion). This gradient would be sensed by bacteria that are going from low to high concentration places thus we should have some bacteria into the capillary. We can demonstrate that when we compare a capillary chemoattractant with another without any substance (the control), just the buffer. The control has to continue the same protocol than the others. In the same way the efficacy of a repellent could be tested since the capillary with the repellent will have less bacteria than the control.</p><br />
<br />
<div class="table"><br />
<table class="tableBio" cellspacing="0" cellpadding="0"><br />
<thead><br />
<tr><br />
<th>Bacterial dilution recipient or <strong>Chemotaxis Chambers</strong></th><br />
<th>Attractant or Buffer recipient or <strong>Capillaries</strong></th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr><br />
<td>tip chambers</td><br />
<td>syringe’s needles (more or less thin)</td><br />
</tr><br />
<tr><br />
<td>needle’s cups or heated closed 100ml tips</td><br />
<td>Needles</td><br />
</tr><br />
<tr><br />
<td>tip chambers</td><br />
<td>10&#956;l micropipette’s tips</td><br />
</tr><br />
<tr><br />
<td>96-well PVC microplates</td><br />
<td>1&#956;l capillary pipettes</td><br />
</tr><br />
<tr><br />
<td>drop between microscope slide and cover slip</td><br />
<td>1&#956;l capillary pipettes</td><br />
</tr><br />
<tr><br />
<td>Flow-chamber</td><br />
<td>1&#956;l capillary pipettes</td><br />
</tr><br />
</tbody><br />
</table><br />
</div><br />
<br />
<br />
<p><strong>Table 1</strong>. Several ways we have carried out using capillary methods in chemotaxis assays.</p> <br />
<br />
<p>This team has performed this assay in different scales using differents chemotactic chambers where the bacterial dilution was put inside and differents capillaries. Some types of that are reflected in the Table 1. The attractant concentration in the capillary depends on the substance itself.</p> <br />
</li><br />
<br />
<li><p><strong>Protocol</strong></p><br />
<br />
<div class="imgRight"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/1/11/BacterialCrowdingCapillaryRepresentation.png" alt="Representation of capillary assay in a tip chambers." /><br />
<p class="caption"><strong>Fig 4.</strong>Representation of capillary assay in a tip chambers.</p><br />
</div><br />
<br />
<p>The experiment may start in two different ways; putting inocula from the strains we are going to work with into triptone broth or in minimal medium in a low shaking at 30ºC overnight. A high shaking might provoke the loss of flagella. The production of flagella wouldn’t be possible in a rich medium since bacteria wouldn’t need it.</p><br />
<br />
<p>The following day the inocula should be diluted in the same medium a hundred of times and wait for the growing phase to be the appropriate. For <i>Escherichia Coli</i> it would be necessary to wait for the exponential middle phase since it is this phase when flagella develop the flagellar motor. For <i>Pseudomonas</i> instead it would better wait for the late exponential phase, as the flagellum is developed later in this organism.</p><br />
<br />
<p>Once the culture is ready, it must be changed in an appropriate medium for mobility and chemotaxis. For that, it is necessary to wash the culture twice with chemotaxis buffer centrifuging in a low speed since flagella may be lost if it is treated abruptly.</p><br />
<br />
<p>When the culture is in the right medium the number of bacteria is adjusted roughly to 10<sup>7</sup> fcu/ml. This dilution has to be distributed in chemotaxis chambers where our capillaries will be introduced in it. The volume of capillaries can be unsettled; we have used as a standard volume 100 &#956;l of diluted chemoattractant in chemotaxis buffer. Mind controls, they will be capillaries thanks to the chemotaxis buffer.</p><br />
<br />
<p>Incubate the experiment at 30º during 60 minutes, after that we have to quantify bacteria that are contained into the capillaries. In order to achieve that we could do it either with dilution and spread in plates or analyzing the fluorescence, supposing that bacteria have any kind of fluorophore.</p><br />
</li><br />
<br />
<li><p><strong>Advice</strong></p><br />
<p>One of the elements we bear in mind is the chemotaxis buffer: chemotaxis medium contain potassium potassium phospgate buffer (pH 7), ethylenediaminetetraacetate (<strong>EDTA</strong>) and <strong>glycerol</strong> (energy source). The glycerol is only necessary in long incubations; meanwhile in short incubations the typical sources of bacteria are enough to maintain the chemotactic machinery. It is all-important to underline that the chemotaxis medium must be free of any other substance which may have chemotactic effects, since this could disturb the results. This is one of the reasons why the carbon source is not glucose, how you may expect. Other important detail to bear in mind is EDTA, this chelation provoke the precipitation of magnesium which may dull the movement of bacteria and the flagellar machinery. It would be complicated to success in the chemotaxis assays without this chelation. Incubation of bacteria must be carried at 30ºC since it helps motility. Shaking must be low as flagella can be lost in high shaking.</p><br />
<br />
<p>It is crucial to be careful when <strong>choosing the strains</strong> to be used in the chemotaxis assays, since it may not have motility. Strains used in laboratories have normally no motility, as at that point they have usually suffer different screening process in benefit of immobile bacteria. A bacterium which has no motility won’t have to invest in any source in motility or chemotaxis; this would encourage the creation of a colony bigger and more eye-catching than usual, so scientifics would be probably leaded to select one of this kind. . This issue happened to us and we were trying to attract a non mobile strain toward different attractants until we realised.</p><br />
</li><br />
</ul><br />
<br />
<h2>Buridan’s Donkey</h2><br />
<br />
<p>To test bacterial chemotaxis we have used a three-channel device based on <strong>flow-chamber biofilm</strong>. It would be able to produce a linear gradient within narrow tubes that connect the chambers. The linear chemical gradient would be generated by diffusion of the chemoattractant through a dialysis membrane located in the limit of the chamber. This membrane also makes impossible the movement of the chemoattractant-producing bacteria through the tube.</p><br />
<br />
<p>The first assay involves only chemoattractants, and the second one includes producing bacterias. As result, it is expected that the movement of the cells in the center chamber was directed to the chamber containing chemoattractant-producing bacterias, for the cells chemotactic response, but not in the control chamber, in the opposite side. It is necessary to clear up that the chemoattractant production would be activated solely by the contact of bacteria with plant cell walls that reside in the same chamber. Bacteria have to “decide” between going toward control empty chamber or going toward chamber with chemoattractant-producing bacteria.</p><br />
<br />
<p>This device could provide a lot of advantages in the study of chemotaxis: rapid and easy implementation, parallel and simultaneous test, visual proofs, different assays possibilities. Also some experimental conditions could be changed easily, for instance: concentration of bacterial population, chambers distances, bacterial cultures, chemoattractans.</p><br />
<br />
<p>An explaining diagram of this device is provided below.</p><br />
<br />
<p><strong>Special acknowledgements to Ph.D Parkinson (University of Utah) who gave us some advices, handed us over some protocols of him, even mobile and mutant <i>E. Coli</i> strains. </strong></p><br />
<br />
<h1>References</h1><br />
<br />
<ul><br />
<li>J. Adler (1972) A Method for Measuring Chemotaxis and Use of the Method to Determine Optimum Conditions for Chemotaxis by Escherichia coli. - Journal of General Microbiology ( I 973), 74, 77-91</li><br />
<br />
<li>Guocheng Han and Joseph J. Cooney (1993) A modified capillary assay for chemotaxis - Journal of Industrial Microbiology, 12 (1993) 396—398</li><br />
<br />
<li>Hanbin Mao, Paul S. Cremer, and Michael D. Manson (2003) A sensitive, versatile microfluidic assay for bacterial chemotaxis - PNAS MICROBIOLOGY vol. 100 no. 9 5449–5454.</li><br />
<br />
<li>Russell Bainer, Heungwon Park, Philippe Cluzel (2003) A high-throughput capillary assay for bacterial chemotaxis - Journal of Microbiological Methods 55 (2003) 315– 319.</li><br />
</ul><br />
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<h1>Introduction</h1><br />
<br />
<p><strong>Motility</strong> is one of the most readily demonstrated bacterial characters, and <strong>chemotaxis</strong> is one of the most studied bacterial behaviors. Motile organisms are attracted by certain chemicals and repelled by others (positive and negative chemotaxis). Quantification of chemotactic motion is necessary to identify chemoeffectors and to determine the structure of bacterial communities.</p><br />
<br />
<p>Current methods of quantifying chemotaxis use chemotactic bacteria such as <i>Escherichia Coli</i>, which is assayed by measuring the number of organisms attracted into a capillary tube containing an attractant.</p><br />
<br />
<p>UPO-Sevilla team has carried out different experimental prototypes that are better to achieve. The goal of the group was designing different assays that allow us to study this effect in both point of views, <strong>qualitative</strong> and <strong>quantitative</strong>. Also we have to test induction of sensing systems and chemoattractant production. Anyway, these processes are highly related with the chemotaxis process in Bacterial Crowding project. This is why we measured chemoattractant production counting the range of attracted bacteria using chemotaxis assays. The induction of sensing systems could be tested by using GFP measures when its promoter is <i>PfecA</i> or <i>PprhJ</i>; or also due to the levels of chemoattractant production. </p><br />
<br />
<h1>Qualitative Assays</h1><br />
<br />
<h2>Agar Soft Plates</h2><br />
<br />
<p>Our qualitative assays were made in <strong>soft agar</strong> thanks to the protocols that we had received from Mr. Parkinson (University of Utah).</p><br />
<br />
<p>This kind of plates allows bacteria to swim trough the agar freely and showing their chemotactic capacities. A colony, inserted in soft agar plate, starts to grow while running out the environmental sources. For this reason bacteria would move to places where the sources are not limited. That phenomenon produces a number of <strong>halos</strong> which are spread within the plate and increase in volume as the sources are lowered.</p><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/3/36/BacterialCrowdingSoftAgarPlate.png" alt="Soft Agar Plate Assay"/><br />
<p class="caption"><strong>Fig 1. Soft agar plate assay.</strong> Different strains are shown. Every strain carries out a mutation that affects its chemotactic response, every strain but the wild type. In the wt you can observe two halos produced by chemotactic responses to aspartate and glutamate.</p><br />
<br />
<br />
<p>The assay protocol is simple; once the soft agar plates are prepared, a colony is inserted in the previous plate. Let it grow in 30ºC. The soft agar is a delicate element, so it is important to be careful when moving the plates.</p><br />
<br />
<p>In those plates it might appear different concentric circles which represent chemotaxis to a certain attractant. For instance, when two amino acids are run out from the medium, two circles will appear. The inner one will show the amino acids limit with low chemotactic response; while the outer one will mean that the amino acid which causes a higher response is running down.</p><br />
<br />
<h2>Optical and Fluorescence Microscopy</h2><br />
<br />
<p>The <strong>microscopy techniques</strong> allow us to see the development of the assay <i>in situ</i> without any wait. In this part we will see how we can carry out an experiment that we could see under the microscope.</p><br />
<br />
<div class="imgLeft"><br />
<img class="ileft" src="https://static.igem.org/mediawiki/2010/4/4e/BacterialCrowdingCapillaryAssay.png" alt="Capillary assay yo look under a microscope" /><br />
<p class="caption"><strong>Fig 2.</strong> Preparation of capillary assay to look under a microscope.</p><br />
</div><br />
<br />
<p>Over a microscope slide two capillaries are put which will hold up a cover slip. Then we insert the bacterial dilution between the slide and the cover slip. Two new capillaries are inserted between the slide and the cover slip inside of the bacterial dilution. One of those capillaries will contain a chemoattractant while the other one will be the control. Under the microscope we can see the different between both capillaries and we will definitely be able to observe if there is chemotaxis toward this chemoattractant.</p><br />
<br />
<p>Apart from that, we can detect the fluorescence emitted by the fluorophore which is present in bacteria using a fluorescent microscope.</p><br />
<br />
<p>This assay can be quantitative too if we spread on agar plates the content of capillaries and count the number of colonies that grew there.</p><br />
<br />
<div class="clear"></div><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/6/6f/BacterialCrowdingCapillaryAssayMicroscope.png" alt="Capillary assay of E. coli chemotaxis toward aspartate"/><br />
<p class="caption"><strong>Fig 3.</strong> Results of a capillary assay using microscope techniques. We can see that the chemotatic response toward aspartate is increasing as time passes by. Also there are major differences between the control without aspartate and the control with aspartate.</p><br />
<br />
<br />
<h1>Quantitative Assays</h1><br />
<br />
<h2>Capillary assays</h2><br />
<br />
<p>The capillary assays are the most useful to quantify chemotaxis. Although with some problems, this team has performed a capillary assay.</p><br />
<br />
<ul><br />
<li><p><strong>Foundations</strong></p><br />
<p>A capillary, which is put in a bacterial dilution, makes a concentration gradient of chemoatractant, produced by thr flow that goes from capillary to the medium according to the <strong>Fick law</strong> (see http://en.wikipedia.org/wiki/Fick's_laws_of_diffusion). This gradient would be sensed by bacteria that are going from low to high concentration places thus we should have some bacteria into the capillary. We can demonstrate that when we compare a capillary chemoattractant with another without any substance (the control), just the buffer. The control has to continue the same protocol than the others. In the same way the efficacy of a repellent could be tested since the capillary with the repellent will have less bacteria than the control.</p><br />
<br />
<div class="table"><br />
<table class="tableBio" cellspacing="0" cellpadding="0"><br />
<thead><br />
<tr><br />
<th>Bacterial dilution recipient or <strong>Chemotaxis Chambers</strong></th><br />
<th>Attractant or Buffer recipient or <strong>Capillaries</strong></th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr><br />
<td>tip chambers</td><br />
<td>syringe’s needles (more or less thin)</td><br />
</tr><br />
<tr><br />
<td>needle’s cups or heated closed 100ml tips</td><br />
<td>Needles</td><br />
</tr><br />
<tr><br />
<td>tip chambers</td><br />
<td>10&#956;l micropipette’s tips</td><br />
</tr><br />
<tr><br />
<td>96-well PVC microplates</td><br />
<td>1&#956;l capillary pipettes</td><br />
</tr><br />
<tr><br />
<td>drop between microscope slide and cover slip</td><br />
<td>1&#956;l capillary pipettes</td><br />
</tr><br />
<tr><br />
<td>Flow-chamber</td><br />
<td>1&#956;l capillary pipettes</td><br />
</tr><br />
</tbody><br />
</table><br />
</div><br />
<br />
<br />
<p><strong>Table 1</strong>. Several ways we have carried out using capillary methods in chemotaxis assays.</p> <br />
<br />
<p>This team has performed this assay in different scales using differents chemotactic chambers where the bacterial dilution was put inside and differents capillaries. Some types of that are reflected in the Table 1. The attractant concentration in the capillary depends on the substance itself.</p> <br />
</li><br />
<br />
<li><p><strong>Protocol</strong></p><br />
<br />
<div class="imgRight"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/1/11/BacterialCrowdingCapillaryRepresentation.png" alt="Representation of capillary assay in a tip chambers." /><br />
<p class="caption"><strong>Fig 4.</strong>Representation of capillary assay in a tip chambers.</p><br />
</div><br />
<br />
<p>The experiment may start in two different ways; putting inocula from the strains we are going to work with into triptone broth or in minimal medium in a low shaking at 30ºC overnight. A high shaking might provoke the loss of flagella. The production of flagella wouldn’t be possible in a rich medium since bacteria wouldn’t need it.</p><br />
<br />
<p>The following day the inocula should be diluted in the same medium a hundred of times and wait for the growing phase to be the appropriate. For <i>Escherichia Coli</i> it would be necessary to wait for the exponential middle phase since it is this phase when flagella develop the flagellar motor. For <i>Pseudomonas</i> instead it would better wait for the late exponential phase, as the flagellum is developed later in this organism.</p><br />
<br />
<p>Once the culture is ready, it must be changed in an appropriate medium for mobility and chemotaxis. For that, it is necessary to wash the culture twice with chemotaxis buffer centrifuging in a low speed since flagella may be lost if it is treated abruptly.</p><br />
<br />
<p>When the culture is in the right medium the number of bacteria is adjusted roughly to 10<sup>7</sup> fcu/ml. This dilution has to be distributed in chemotaxis chambers where our capillaries will be introduced in it. The volume of capillaries can be unsettled; we have used as a standard volume 100 &#956;l of diluted chemoattractant in chemotaxis buffer. Mind controls, they will be capillaries thanks to the chemotaxis buffer.</p><br />
<br />
<p>Incubate the experiment at 30º during 60 minutes, after that we have to quantify bacteria that are contained into the capillaries. In order to achieve that we could do it either with dilution and spread in plates or analyzing the fluorescence, supposing that bacteria have any kind of fluorophore.</p><br />
</li><br />
<br />
<li><p><strong>Advice</strong></p><br />
<p>One of the elements we bear in mind is the chemotaxis buffer: chemotaxis medium contain potassium potassium phospgate buffer (pH 7), ethylenediaminetetraacetate (<strong>EDTA</strong>) and <strong>glycerol</strong> (energy source). The glycerol is only necessary in long incubations; meanwhile in short incubations the typical sources of bacteria are enough to maintain the chemotactic machinery. It is all-important to underline that the chemotaxis medium must be free of any other substance which may have chemotactic effects, since this could disturb the results. This is one of the reasons why the carbon source is not glucose, how you may expect. Other important detail to bear in mind is EDTA, this chelation provoke the precipitation of magnesium which may dull the movement of bacteria and the flagellar machinery. It would be complicated to success in the chemotaxis assays without this chelation. Incubation of bacteria must be carried at 30ºC since it helps motility. Shaking must be low as flagella can be lost in high shaking.</p><br />
<br />
<p>It is crucial to be careful when <strong>choosing the strains</strong> to be used in the chemotaxis assays, since it may not have motility. Strains used in laboratories have normally no motility, as at that point they have usually suffer different screening process in benefit of immobile bacteria. A bacterium which has no motility won’t have to invest in any source in motility or chemotaxis; this would encourage the creation of a colony bigger and more eye-catching than usual, so scientifics would be probably leaded to select one of this kind. . This issue happened to us and we were trying to attract a non mobile strain toward different attractants until we realised.</p><br />
</li><br />
</ul><br />
<br />
<h2>Buridan’s Donkey</h2><br />
<br />
<p>To test bacterial chemotaxis we have used a three-channel device based on <strong>flow-chamber biofilm</strong>. It would be able to produce a linear gradient within narrow tubes that connect the chambers. The linear chemical gradient would be generated by diffusion of the chemoattractant through a dialysis membrane located in the limit of the chamber. This membrane also makes impossible the movement of the chemoattractant-producing bacteria through the tube.</p><br />
<br />
<p>The first assay involves only chemoattractants, and the second one includes producing bacterias. As result, it is expected that the movement of the cells in the center chamber was directed to the chamber containing chemoattractant-producing bacterias, for the cells chemotactic response, but not in the control chamber, in the opposite side. It is necessary to clear up that the chemoattractant production would be activated solely by the contact of bacteria with plant cell walls that reside in the same chamber. Bacteria have to “decide” between going toward control empty chamber or going toward chamber with chemoattractant-producing bacteria.</p><br />
<br />
<p>This device could provide a lot of advantages in the study of chemotaxis: rapid and easy implementation, parallel and simultaneous test, visual proofs, different assays possibilities. Also some experimental conditions could be changed easily, for instance: concentration of bacterial population, chambers distances, bacterial cultures, chemoattractans.</p><br />
<br />
<p>An explaining diagram of this device is provided below.</p><br />
<br />
<p><strong>Special acknowledgements to Ph.D Parkinson (University of Utah) who gave us some advices, handed us over some protocols of him, even mobile and mutant <i>E. Coli</i> strains. </strong></p><br />
<br />
<h1>References</h1><br />
<br />
<ul><br />
<li>J. Adler (1972) A Method for Measuring Chemotaxis and Use of the Method to Determine Optimum Conditions for Chemotaxis by Escherichia coli. - Journal of General Microbiology ( I 973), 74, 77-91</li><br />
<br />
<li>Guocheng Han and Joseph J. Cooney (1993) A modified capillary assay for chemotaxis - Journal of Industrial Microbiology, 12 (1993) 396—398</li><br />
<br />
<li>Hanbin Mao, Paul S. Cremer, and Michael D. Manson (2003) A sensitive, versatile microfluidic assay for bacterial chemotaxis - PNAS MICROBIOLOGY vol. 100 no. 9 5449–5454.</li><br />
<br />
<li>Russell Bainer, Heungwon Park, Philippe Cluzel (2003) A high-throughput capillary assay for bacterial chemotaxis - Journal of Microbiological Methods 55 (2003) 315– 319.</li><br />
</ul><br />
<br />
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<h1>Introduction</h1><br />
<br />
<p><strong>Motility</strong> is one of the most readily demonstrated bacterial characters, and <strong>chemotaxis</strong> is one of the most studied bacterial behaviors. Motile organisms are attracted by certain chemicals and repelled by others (positive and negative chemotaxis). Quantification of chemotactic motion is necessary to identify chemoeffectors and to determine the structure of bacterial communities.</p><br />
<br />
<p>Current methods of quantifying chemotaxis use chemotactic bacteria such as <i>Escherichia Coli</i>, which is assayed by measuring the number of organisms attracted into a capillary tube containing an attractant.</p><br />
<br />
<p>UPO-Sevilla team has carried out different experimental prototypes that are better to achieve. The goal of the group was designing different assays that allow us to study this effect in both point of views, <strong>qualitative</strong> and <strong>quantitative</strong>. Also we have to test induction of sensing systems and chemoattractant production. Anyway, these processes are highly related with the chemotaxis process in Bacterial Crowding project. This is why we measured chemoattractant production counting the range of attracted bacteria using chemotaxis assays. The induction of sensing systems could be tested by using GFP measures when its promoter is <i>PfecA</i> or <i>PprhJ</i>; or also due to the levels of chemoattractant production. </p><br />
<br />
<h1>Qualitative Assays</h1><br />
<br />
<h2>Agar Soft Plates</h2><br />
<br />
<p>Our qualitative assays were made in <strong>soft agar</strong> thanks to the protocols that we had received from Mr. Parkinson (University of Utah).</p><br />
<br />
<p>This kind of plates allows bacteria to swim trough the agar freely and showing their chemotactic capacities. A colony, inserted in soft agar plate, starts to grow while running out the environmental sources. For this reason bacteria would move to places where the sources are not limited. That phenomenon produces a number of <strong>halos</strong> which are spread within the plate and increase in volume as the sources are lowered.</p><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/3/36/BacterialCrowdingSoftAgarPlate.png" alt="Soft Agar Plate Assay"/><br />
<p class="caption"><strong>Fig 1. Soft agar plate assay.</strong> Different strains are shown. Every strain carries out a mutation that affects its chemotactic response, every strain but the wild type. In the wt you can observe two halos produced by chemotactic responses to aspartate and glutamate.</p><br />
<br />
<br />
<p>The assay protocol is simple; once the soft agar plates are prepared, a colony is inserted in the previous plate. Let it grow in 30ºC. The soft agar is a delicate element, so it is important to be careful when moving the plates.</p><br />
<br />
<p>In those plates it might appear different concentric circles which represent chemotaxis to a certain attractant. For instance, when two amino acids are run out from the medium, two circles will appear. The inner one will show the amino acids limit with low chemotactic response; while the outer one will mean that the amino acid which causes a higher response is running down.</p><br />
<br />
<h2>Optical and Fluorescence Microscopy</h2><br />
<br />
<p>The <strong>microscopy techniques</strong> allow us to see the development of the assay <i>in situ</i> without any wait. In this part we will see how we can carry out an experiment that we could see under the microscope.</p><br />
<br />
<div class="imgLeft"><br />
<img class="ileft" src="https://static.igem.org/mediawiki/2010/4/4e/BacterialCrowdingCapillaryAssay.png" alt="Capillary assay yo look under a microscope" /><br />
<p class="caption"><strong>Fig 2.</strong> Preparation of capillary assay to look under a microscope.</p><br />
</div><br />
<br />
<p>Over a microscope slide two capillaries are put which will hold up a cover slip. Then we insert the bacterial dilution between the slide and the cover slip. Two new capillaries are inserted between the slide and the cover slip inside of the bacterial dilution. One of those capillaries will contain a chemoattractant while the other one will be the control. Under the microscope we can see the different between both capillaries and we will definitely be able to observe if there is chemotaxis toward this chemoattractant.</p><br />
<br />
<p>Apart from that, we can detect the fluorescence emitted by the fluorophore which is present in bacteria using a fluorescent microscope.</p><br />
<br />
<p>This assay can be quantitative too if we spread on agar plates the content of capillaries and count the number of colonies that grew there.</p><br />
<br />
<div class="clear"></div><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/6/6f/BacterialCrowdingCapillaryAssayMicroscope.png" alt="Capillary assay of E. coli chemotaxis toward aspartate"/><br />
<p class="caption"><strong>Fig 3.</strong> Results of a capillary assay using microscope techniques. We can see that the chemotatic response toward aspartate is increasing as time passes by. Also there are major differences between the control without aspartate and the control with aspartate.</p><br />
<br />
<br />
<h1>Quantitative Assays</h1><br />
<br />
<h2>Capillary assays</h2><br />
<br />
<p>The capillary assays are the most useful to quantify chemotaxis. Although with some problems, this team has performed a capillary assay.</p><br />
<br />
<ul><br />
<li><p><strong>Foundations</strong></p><br />
<p>A capillary, which is put in a bacterial dilution, makes a concentration gradient of chemoatractant, produced by thr flow that goes from capillary to the medium according to the <strong>Fick law</strong> (see http://en.wikipedia.org/wiki/Fick's_laws_of_diffusion). This gradient would be sensed by bacteria that are going from low to high concentration places thus we should have some bacteria into the capillary. We can demonstrate that when we compare a capillary chemoattractant with another without any substance (the control), just the buffer. The control has to continue the same protocol than the others. In the same way the efficacy of a repellent could be tested since the capillary with the repellent will have less bacteria than the control.</p><br />
<br />
<div class="table"><br />
<table class="tableBio" cellspacing="0" cellpadding="0"><br />
<thead><br />
<tr><br />
<th>Bacterial dilution recipient or <strong>Chemotaxis Chambers</strong></th><br />
<th>Attractant or Buffer recipient or <strong>Capillaries</strong></th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr><br />
<td>tip chambers</td><br />
<td>syringe’s needles (more or less thin)</td><br />
</tr><br />
<tr><br />
<td>needle’s cups or heated closed 100ml tips</td><br />
<td>Needles</td><br />
</tr><br />
<tr><br />
<td>tip chambers</td><br />
<td>10&#956;l micropipette’s tips</td><br />
</tr><br />
<tr><br />
<td>96-well PVC microplates</td><br />
<td>1&#956;l capillary pipettes</td><br />
</tr><br />
<tr><br />
<td>drop between microscope slide and cover slip</td><br />
<td>1&#956;l capillary pipettes</td><br />
</tr><br />
<tr><br />
<td>Flow-chamber</td><br />
<td>1&#956;l capillary pipettes</td><br />
</tr><br />
</tbody><br />
</table><br />
</div><br />
<br />
<br />
<p><strong>Table 1</strong>. Several ways we have carried out using capillary methods in chemotaxis assays.</p> <br />
<br />
<p>This team has performed this assay in different scales using differents chemotactic chambers where the bacterial dilution was put inside and differents capillaries. Some types of that are reflected in the Table 1. The attractant concentration in the capillary depends on the substance itself.</p> <br />
</li><br />
<br />
<li><p><strong>Protocol</strong></p><br />
<p>The experiment may start in two different ways; putting inocula from the strains we are going to work with into triptone broth or in minimal medium in a low shaking at 30ºC overnight. A high shaking might provoke the loss of flagella. The production of flagella wouldn’t be possible in a rich medium since bacteria wouldn’t need it.</p><br />
<br />
<p>The following day the inocula should be diluted in the same medium a hundred of times and wait for the growing phase to be the appropriate. For <i>Escherichia Coli</i> it would be necessary to wait for the exponential middle phase since it is this phase when flagella develop the flagellar motor. For <i>Pseudomonas</i> instead it would better wait for the late exponential phase, as the flagellum is developed later in this organism.</p><br />
<br />
<p>Once the culture is ready, it must be changed in an appropriate medium for mobility and chemotaxis. For that, it is necessary to wash the culture twice with chemotaxis buffer centrifuging in a low speed since flagella may be lost if it is treated abruptly.</p><br />
<br />
<div class="imgRight"><br />
<img class="right" src="https://static.igem.org/mediawiki/2010/1/11/BacterialCrowdingCapillaryRepresentation.png" alt="Representation of capillary assay in a tip chambers." /><br />
<p class="caption"><strong>Fig 4.</strong>Representation of capillary assay in a tip chambers.</p><br />
</div><br />
<br />
<p>When the culture is in the right medium the number of bacteria is adjusted roughly to 10<sup>7</sup> fcu/ml. This dilution has to be distributed in chemotaxis chambers where our capillaries will be introduced in it. The volume of capillaries can be unsettled; we have used as a standard volume 100 &#956;l of diluted chemoattractant in chemotaxis buffer. Mind controls, they will be capillaries thanks to the chemotaxis buffer.</p><br />
<br />
<p>Incubate the experiment at 30º during 60 minutes, after that we have to quantify bacteria that are contained into the capillaries. In order to achieve that we could do it either with dilution and spread in plates or analyzing the fluorescence, supposing that bacteria have any kind of fluorophore.</p><br />
</li><br />
<br />
<li><p><strong>Advice</strong></p><br />
<p>One of the elements we bear in mind is the chemotaxis buffer: chemotaxis medium contain potassium potassium phospgate buffer (pH 7), ethylenediaminetetraacetate (<strong>EDTA</strong>) and <strong>glycerol</strong> (energy source). The glycerol is only necessary in long incubations; meanwhile in short incubations the typical sources of bacteria are enough to maintain the chemotactic machinery. It is all-important to underline that the chemotaxis medium must be free of any other substance which may have chemotactic effects, since this could disturb the results. This is one of the reasons why the carbon source is not glucose, how you may expect. Other important detail to bear in mind is EDTA, this chelation provoke the precipitation of magnesium which may dull the movement of bacteria and the flagellar machinery. It would be complicated to success in the chemotaxis assays without this chelation. Incubation of bacteria must be carried at 30ºC since it helps motility. Shaking must be low as flagella can be lost in high shaking.</p><br />
<br />
<p>It is crucial to be careful when <strong>choosing the strains</strong> to be used in the chemotaxis assays, since it may not have motility. Strains used in laboratories have normally no motility, as at that point they have usually suffer different screening process in benefit of immobile bacteria. A bacterium which has no motility won’t have to invest in any source in motility or chemotaxis; this would encourage the creation of a colony bigger and more eye-catching than usual, so scientifics would be probably leaded to select one of this kind. . This issue happened to us and we were trying to attract a non mobile strain toward different attractants until we realised.</p><br />
</li><br />
</ul><br />
<br />
<h2>Buridan’s Donkey</h2><br />
<br />
<p>To test bacterial chemotaxis we have used a three-channel device based on <strong>flow-chamber biofilm</strong>. It would be able to produce a linear gradient within narrow tubes that connect the chambers. The linear chemical gradient would be generated by diffusion of the chemoattractant through a dialysis membrane located in the limit of the chamber. This membrane also makes impossible the movement of the chemoattractant-producing bacteria through the tube.</p><br />
<br />
<p>The first assay involves only chemoattractants, and the second one includes producing bacterias. As result, it is expected that the movement of the cells in the center chamber was directed to the chamber containing chemoattractant-producing bacterias, for the cells chemotactic response, but not in the control chamber, in the opposite side. It is necessary to clear up that the chemoattractant production would be activated solely by the contact of bacteria with plant cell walls that reside in the same chamber. Bacteria have to “decide” between going toward control empty chamber or going toward chamber with chemoattractant-producing bacteria.</p><br />
<br />
<p>This device could provide a lot of advantages in the study of chemotaxis: rapid and easy implementation, parallel and simultaneous test, visual proofs, different assays possibilities. Also some experimental conditions could be changed easily, for instance: concentration of bacterial population, chambers distances, bacterial cultures, chemoattractans.</p><br />
<br />
<p>An explaining diagram of this device is provided below.</p><br />
<br />
<p><strong>Special acknowledgements to Ph.D Parkinson (University of Utah) who gave us some advices, handed us over some protocols of him, even mobile and mutant <i>E. Coli</i> strains. </strong></p><br />
<br />
<h1>References</h1><br />
<br />
<ul><br />
<li>J. Adler (1972) A Method for Measuring Chemotaxis and Use of the Method to Determine Optimum Conditions for Chemotaxis by Escherichia coli. - Journal of General Microbiology ( I 973), 74, 77-91</li><br />
<br />
<li>Guocheng Han and Joseph J. Cooney (1993) A modified capillary assay for chemotaxis - Journal of Industrial Microbiology, 12 (1993) 396—398</li><br />
<br />
<li>Hanbin Mao, Paul S. Cremer, and Michael D. Manson (2003) A sensitive, versatile microfluidic assay for bacterial chemotaxis - PNAS MICROBIOLOGY vol. 100 no. 9 5449–5454.</li><br />
<br />
<li>Russell Bainer, Heungwon Park, Philippe Cluzel (2003) A high-throughput capillary assay for bacterial chemotaxis - Journal of Microbiological Methods 55 (2003) 315– 319.</li><br />
</ul><br />
<br />
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<h1>Introduction</h1><br />
<br />
<p><strong>Motility</strong> is one of the most readily demonstrated bacterial characters, and <strong>chemotaxis</strong> is one of the most studied bacterial behaviors. Motile organisms are attracted by certain chemicals and repelled by others (positive and negative chemotaxis). Quantification of chemotactic motion is necessary to identify chemoeffectors and to determine the structure of bacterial communities.</p><br />
<br />
<p>Current methods of quantifying chemotaxis use chemotactic bacteria such as <i>Escherichia Coli</i>, which is assayed by measuring the number of organisms attracted into a capillary tube containing an attractant.</p><br />
<br />
<p>UPO-Sevilla team has carried out different experimental prototypes that are better to achieve. The goal of the group was designing different assays that allow us to study this effect in both point of views, <strong>qualitative</strong> and <strong>quantitative</strong>. Also we have to test induction of sensing systems and chemoattractant production. Anyway, these processes are highly related with the chemotaxis process in Bacterial Crowding project. This is why we measured chemoattractant production counting the range of attracted bacteria using chemotaxis assays. The induction of sensing systems could be tested by using GFP measures when its promoter is <i>PfecA</i> or <i>PprhJ</i>; or also due to the levels of chemoattractant production. </p><br />
<br />
<h1>Qualitative Assays</h1><br />
<br />
<h2>Agar Soft Plates</h2><br />
<br />
<p>Our qualitative assays were made in <strong>soft agar</strong> thanks to the protocols that we had received from Mr. Parkinson (University of Utah).</p><br />
<br />
<p>This kind of plates allows bacteria to swim trough the agar freely and showing their chemotactic capacities. A colony, inserted in soft agar plate, starts to grow while running out the environmental sources. For this reason bacteria would move to places where the sources are not limited. That phenomenon produces a number of <strong>halos</strong> which are spread within the plate and increase in volume as the sources are lowered.</p><br />
<br />
<img class="center" src="https://static.igem.org/mediawiki/2010/3/36/BacterialCrowdingSoftAgarPlate.png" alt="Soft Agar Plate Assay"/><br />
<p class="caption"><strong>Fig 1. Soft agar plate assay.</strong> Different strains are shown. Every strain carries out a mutation that affects its chemotactic response, every strain but the wild type. In the wt you can observe two halos produced by chemotactic responses to aspartate and glutamate.</p><br />
<br />
<br />
<p>The assay protocol is simple; once the soft agar plates are prepared, a colony is inserted in the previous plate. Let it grow in 30ºC. The soft agar is a delicate element, so it is important to be careful when moving the plates.</p><br />
<br />
<p>In those plates it might appear different concentric circles which represent chemotaxis to a certain attractant. For instance, when two amino acids are run out from the medium, two circles will appear. The inner one will show the amino acids limit with low chemotactic response; while the outer one will mean that the amino acid which causes a higher response is running down.</p><br />
<br />
<h2>Optical and Fluorescence Microscopy</h2><br />
<br />
<p>The <strong>microscopy techniques</strong> allow us to see the development of the assay <i>in situ</i> without any wait. In this part we will see how we can carry out an experiment that we could see under the microscope.</p><br />
<br />
<div class="imgLeft"><br />
<img class="ileft" src="https://static.igem.org/mediawiki/2010/4/4e/BacterialCrowdingCapillaryAssay.png" alt="Capillary assay yo look under a microscope" /><br />
<p class="caption"><strong>Fig 2.</strong> Preparation of capillary assay to look under a microscope.</p><br />
</div><br />
<br />
<p>Over a microscope slide two capillaries are put which will hold up a cover slip. Then we insert the bacterial dilution between the slide and the cover slip. Two new capillaries are inserted between the slide and the cover slip inside of the bacterial dilution. One of those capillaries will contain a chemoattractant while the other one will be the control. Under the microscope we can see the different between both capillaries and we will definitely be able to observe if there is chemotaxis toward this chemoattractant.</p><br />
<br />
<p>Apart from that, we can detect the fluorescence emitted by the fluorophore which is present in bacteria using a fluorescent microscope.</p><br />
<br />
<p>This assay can be quantitative too if we spread on agar plates the content of capillaries and count the number of colonies that grew there.</p><br />
<br />
<div class="clear"></div><br />
<br />
<h1>Quantitative Assays</h1><br />
<br />
<h2>Capillary assays</h2><br />
<br />
<p>The capillary assays are the most useful to quantify chemotaxis. Although with some problems, this team has performed a capillary assay.</p><br />
<br />
<ul><br />
<li><p><strong>Foundations</strong></p><br />
<p>A capillary, which is put in a bacterial dilution, makes a concentration gradient of chemoatractant, produced by thr flow that goes from capillary to the medium according to the <strong>Fick law</strong> (see http://en.wikipedia.org/wiki/Fick's_laws_of_diffusion). This gradient would be sensed by bacteria that are going from low to high concentration places thus we should have some bacteria into the capillary. We can demonstrate that when we compare a capillary chemoattractant with another without any substance (the control), just the buffer. The control has to continue the same protocol than the others. In the same way the efficacy of a repellent could be tested since the capillary with the repellent will have less bacteria than the control.</p><br />
<br />
<div class="table"><br />
<table class="tableBio" cellspacing="0" cellpadding="0"><br />
<thead><br />
<tr><br />
<th>Bacterial dilution recipient or <strong>Chemotaxis Chambers</strong></th><br />
<th>Attractant or Buffer recipient or <strong>Capillaries</strong></th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr><br />
<td>tip chambers</td><br />
<td>syringe’s needles (more or less thin)</td><br />
</tr><br />
<tr><br />
<td>needle’s cups or heated closed 100ml tips</td><br />
<td>Needles</td><br />
</tr><br />
<tr><br />
<td>tip chambers</td><br />
<td>10&#956;l micropipette’s tips</td><br />
</tr><br />
<tr><br />
<td>96-well PVC microplates</td><br />
<td>1&#956;l capillary pipettes</td><br />
</tr><br />
<tr><br />
<td>drop between microscope slide and cover slip</td><br />
<td>1&#956;l capillary pipettes</td><br />
</tr><br />
<tr><br />
<td>Flow-chamber</td><br />
<td>1&#956;l capillary pipettes</td><br />
</tr><br />
</tbody><br />
</table><br />
</div><br />
<br />
<br />
<p><strong>Table 1</strong>. Several ways we have carried out using capillary methods in chemotaxis assays.</p> <br />
<br />
<p>This team has performed this assay in different scales using differents chemotactic chambers where the bacterial dilution was put inside and differents capillaries. Some types of that are reflected in the Table 1. The attractant concentration in the capillary depends on the substance itself.</p> <br />
</li><br />
<br />
<li><p><strong>Protocol</strong></p><br />
<p>The experiment may start in two different ways; putting inocula from the strains we are going to work with into triptone broth or in minimal medium in a low shaking at 30ºC overnight. A high shaking might provoke the loss of flagella. The production of flagella wouldn’t be possible in a rich medium since bacteria wouldn’t need it.</p><br />
<br />
<p>The following day the inocula should be diluted in the same medium a hundred of times and wait for the growing phase to be the appropriate. For <i>Escherichia Coli</i> it would be necessary to wait for the exponential middle phase since it is this phase when flagella develop the flagellar motor. For <i>Pseudomonas</i> instead it would better wait for the late exponential phase, as the flagellum is developed later in this organism.</p><br />
<br />
<p>Once the culture is ready, it must be changed in an appropriate medium for mobility and chemotaxis. For that, it is necessary to wash the culture twice with chemotaxis buffer centrifuging in a low speed since flagella may be lost if it is treated abruptly.</p><br />
<br />
<p>When the culture is in the right medium the number of bacteria is adjusted roughly to 10<sup>7</sup> fcu/ml. This dilution has to be distributed in chemotaxis chambers where our capillaries will be introduced in it. The volume of capillaries can be unsettled; we have used as a standard volume 100 &#956;l of diluted chemoattractant in chemotaxis buffer. Mind controls, they will be capillaries thanks to the chemotaxis buffer.</p><br />
<br />
<p>Incubate the experiment at 30º during 60 minutes, after that we have to quantify bacteria that are contained into the capillaries. In order to achieve that we could do it either with dilution and spread in plates or analyzing the fluorescence, supposing that bacteria have any kind of fluorophore.</p><br />
</li><br />
<br />
<li><p><strong>Advice</strong></p><br />
<p>One of the elements we bear in mind is the chemotaxis buffer: chemotaxis medium contain potassium potassium phospgate buffer (pH 7), ethylenediaminetetraacetate (<strong>EDTA</strong>) and <strong>glycerol</strong> (energy source). The glycerol is only necessary in long incubations; meanwhile in short incubations the typical sources of bacteria are enough to maintain the chemotactic machinery. It is all-important to underline that the chemotaxis medium must be free of any other substance which may have chemotactic effects, since this could disturb the results. This is one of the reasons why the carbon source is not glucose, how you may expect. Other important detail to bear in mind is EDTA, this chelation provoke the precipitation of magnesium which may dull the movement of bacteria and the flagellar machinery. It would be complicated to success in the chemotaxis assays without this chelation. Incubation of bacteria must be carried at 30ºC since it helps motility. Shaking must be low as flagella can be lost in high shaking.</p><br />
<br />
<p>It is crucial to be careful when <strong>choosing the strains</strong> to be used in the chemotaxis assays, since it may not have motility. Strains used in laboratories have normally no motility, as at that point they have usually suffer different screening process in benefit of immobile bacteria. A bacterium which has no motility won’t have to invest in any source in motility or chemotaxis; this would encourage the creation of a colony bigger and more eye-catching than usual, so scientifics would be probably leaded to select one of this kind. . This issue happened to us and we were trying to attract a non mobile strain toward different attractants until we realised.</p><br />
</li><br />
</ul><br />
<br />
<h2>Buridan’s Donkey</h2><br />
<br />
<p>To test bacterial chemotaxis we have used a three-channel device based on <strong>flow-chamber biofilm</strong>. It would be able to produce a linear gradient within narrow tubes that connect the chambers. The linear chemical gradient would be generated by diffusion of the chemoattractant through a dialysis membrane located in the limit of the chamber. This membrane also makes impossible the movement of the chemoattractant-producing bacteria through the tube.</p><br />
<br />
<p>The first assay involves only chemoattractants, and the second one includes producing bacterias. As result, it is expected that the movement of the cells in the center chamber was directed to the chamber containing chemoattractant-producing bacterias, for the cells chemotactic response, but not in the control chamber, in the opposite side. It is necessary to clear up that the chemoattractant production would be activated solely by the contact of bacteria with plant cell walls that reside in the same chamber. Bacteria have to “decide” between going toward control empty chamber or going toward chamber with chemoattractant-producing bacteria.</p><br />
<br />
<p>This device could provide a lot of advantages in the study of chemotaxis: rapid and easy implementation, parallel and simultaneous test, visual proofs, different assays possibilities. Also some experimental conditions could be changed easily, for instance: concentration of bacterial population, chambers distances, bacterial cultures, chemoattractans.</p><br />
<br />
<p>An explaining diagram of this device is provided below.</p><br />
<br />
<p><strong>Special acknowledgements to Ph.D Parkinson (University of Utah) who gave us some advices, handed us over some protocols of him, even mobile and mutant <i>E. Coli</i> strains. </strong></p><br />
<br />
<h1>References</h1><br />
<br />
<ul><br />
<li>J. Adler (1972) A Method for Measuring Chemotaxis and Use of the Method to Determine Optimum Conditions for Chemotaxis by Escherichia coli. - Journal of General Microbiology ( I 973), 74, 77-91</li><br />
<br />
<li>Guocheng Han and Joseph J. Cooney (1993) A modified capillary assay for chemotaxis - Journal of Industrial Microbiology, 12 (1993) 396—398</li><br />
<br />
<li>Hanbin Mao, Paul S. Cremer, and Michael D. Manson (2003) A sensitive, versatile microfluidic assay for bacterial chemotaxis - PNAS MICROBIOLOGY vol. 100 no. 9 5449–5454.</li><br />
<br />
<li>Russell Bainer, Heungwon Park, Philippe Cluzel (2003) A high-throughput capillary assay for bacterial chemotaxis - Journal of Microbiological Methods 55 (2003) 315– 319.</li><br />
</ul><br />
<br />
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