Team:Groningen/Project/Motility

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<h1>Heat Motility</h1>
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<h1>Stay Warm, Stay Close</h1>
<p>
<p>
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In case of a low yield we want a targeted secretion only near our (that we want to coat with silk).
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Our initial idea was to let the bacteria produce the silk in a bath and when the implant is put into the bath, the implant will be coated with silk. This is a wasteful and inelegant method. A low production yield can be expected and a solution is needed to overcome this problem. Therefore the heat motility was developed. With the help of heat motility the silk will be produced on site, this will also save energy in the form of nutrition and energy of heating the bath. An overview of our 'Coating GEM' is shown in figure 1.
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In order to achieve this we want to have a bacillus that will move towards heat. If the implant is heated it will attract our silk secreting bacillus.
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</p>
</p>
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<br>
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<h2>The cold sensor DesK</h2>
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<h2>Native Motility</h2>
<p>
<p>
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Use of DesK as an sensor for temperature. DesK is a membrane protein (a kinase), it is part of a
+
The Motility in most bacteria is governed by the Che proteins [1], they control if the bacteria is swimming straight or tumbling (changing directions). They do this by controlling the flagella. If the flagella spin counter-clockwise (CCW) they will group together in one pole of the bacterium, causing straight swimming.  On the other hand if the flagella are spinning clockwise (CW) then the flagella will disperse over the membrane and cause tumbling.
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cascade that has to maintain the membrane liquidity. It does this by phosphorylating DesR, this
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<br>
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phosphorylated DesR activates a promotor (pdes) that expresses the des gene, changing the
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<h3>CheY</h3>
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membrane in the process.
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The Che proteins are present in many motile bacteria, however they can have different effects depending on the species [1]. The CheY protein in <i>Bacillus subtilis</i>, for example, has the complete opposite effect as in <i>Escherichia coli</i>. CheY is an important protein for controlling the spinning of the flagella. When the concentration of phosphorylated CheY (CheY-p) is sufficiently high the flagella turn CCW (straight swimming), but when the concentration of CheY decreases the chance of tumbling also increases, and the bacterium will reorient themselves more often.
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The article mentions that DesK is most active onces the temperature drops below 30 C,
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however the autokinase activity is still present at 42 C though drastically less. DesK is a real cold
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sensor, which is natively present in bacillus.
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We want to use the DesK in our heat sensing construct (how we do this is under motility), by
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PCR'ing the pdes promotor out of B. subtilis and Hereby activating the motility gene that we fuse
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after it.
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</p>
</p>
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<h3>References</h3>
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<h3>Attractant Receptor</h3>
<p>
<p>
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Mariana Martin and Diego de Mendoza , of Bacillus subtilis DesK thermosensor by lipids , Biochemical Journal (2013), Vol 451 No 2, 269–275
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The chemotaxis process is initiated at the receptor, which can sense the concentration of an attractant (or repellent) [2]. Increasing concentrations of attractant correspond to an increased chance of swimming. If the concentration decreases, the bacteria will start to tumble more frequently, and will reorient its swimming direction, hopefully to more desirable regions.
</p>
</p>
 +
<h3>cheA & CheC-CheD</h3>
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Binding of the attractant receptor causes straight swimming via a small cascade [2]. When the receptor is bound, the CheA protein (which is attached to it) gets phosphorylated. The CheA-p phophorylates cheY, which then causes straight swimming. The protein complex CheC-CheD causes dephophorylation of cheY-p (when receptor is bound) resulting in a negative feedback. Two more negative feedback systems are also activated following the binding of attractant, which also result in decreased values of CheY-p. In such a way, CheY-p adaption occurs, and <i>B. subtilis</i> is ready to sense new changes in its environment. For more information about how this pathway works please visit the <a href="https://2013.igem.org/Team:Groningen/Navigation/Heatmotility#six">heat motility section</a>.
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</p>
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<br>
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<h2>Motility</h2>
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<div align="left">
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<table id="layout" width="90%">
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<tr>
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<td>
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<img src="http://img844.imageshack.us/img844/289/kwv.png" width="100%" >
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</td>
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</tr>
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<tr>
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<td>
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<font size="1">Figure 1: The scheme shows <i>B. subtilis</i> containing knockouts of <i>cheY</i>, <i>cheC</i> and <i>des</i>. It also shows the motility gene and the silk gene positively controlled by cold and heat respectively. </font>
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</td>
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</tr>
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</table>
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</div>
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<br>
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<br>
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<h2>Controllable motility</h2>
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<h3 id = "special"><i>cheY</i> Knockout</h3>
<p>
<p>
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The motility part of our construct is based on two articles, a very old (1995) article with general
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To make our coating mechanism a success we need to have control over the motility. This is achieved by knocking out <i>cheY</i>. This might sound strange since CheY-p is controlling straight swimming. However since we know that a CheY null mutant is immobile due to excessive tumbling. This makes it possible to insert <i>cheY</i> with a promoter of our choosing, and make it the sole producer of CheY. Also a <i>cheC</i> knockout is made in order to prevent negative feedback. How this is effecting the cell is explained in more detail with our <a href="https://2013.igem.org/Team:Groningen/Navigation/Heatmotility#six">model</a>.
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information about motility and a newer one focusing on the attractant/repellent sensor cascade.
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<h3>General:</h3>
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Bacterial movement based on flagella (tail like structures) and utilizes CCW(counter-clockwise)
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and CW (clockwise) movement. When the flagella spin CCW they gather in one area resulting in
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bacteria that move strait. When the flagella move CW they disperse all over the cell membrane,
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resulting in the bacteria spinning in random directions (tumbling). When the bacteria senses an
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attractant it will go CCW, till the concentration gets lower after which it will go CW resulting in a
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change in direction. Bacteria will move towards attractants and away from repellents.
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</p>
</p>
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<h3>The receptor and our Idea:</h3>
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<h3>DesK pathway</h3>
<p>
<p>
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The receptor is displayed in the figure on the page below. The letters are all Che proteins, with this
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We envision a bacterium that moves towards a heat source, in order to do this it needs a temperature sensor. <i>B. subtilis</i> natively has a protein that fits this requirement nicely. It is called DesK, it is a membrane protein that senses cold [3][4] (25&deg;C). When the environment is cold, DesK autophophorylates, after which it phosphorylates DesR. DesR in turn activates the promoter of the des gene (P<sub><i>des</i></sub>), which would be the promoter we are looking for. The <i>des</i> gene expresses a protein that provides negative feedback to the system, so we need to knockout this des gene to keep our promoter active.
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cascade of proteins motility is coordinated. When a attractant is bound to the receptor, CheA
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phosphorylizes CheY into CheY-P. CheY-P causes the CCW motility in the flagella, it also causes
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(by forming a CheY-p/CheC complex) that CheD is pulled off the receptor. CheD and CheC forms
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also a complex which dephosphorylizes CheY-p into CheY thus resetting the receptor.
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What we want is to knock out the natively CheY gene, which the article from 1995 mentions so
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it is possible. And then fuse a CheY gene to the pdes promoter. The result is that CheY is only
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present when the temperature is low (below 30 C). Causing a very particular movement, two ways
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of movements are theorized.
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</p>
</p>
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<h3>Claudio's theory:</h3>
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<br>
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<h3>Thermal control of fatty acid synthesis.</h3>
<p>
<p>
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The DesK activates when cold, so the bacteria could swim forward when it is still cold. This is
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In order to maintain the fluidity of the cell membrane when the environmental temperature is decreasing, <i>B. subtilis</i> (among other bacteria) adapts the membrane by increasing the fraction of unsaturated phospholipids acyl chains.
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dependent on the fact if the attractant receptor is bound at the moment of expression of CheY.
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</p>
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Causing a pseudo random movement.  As soon as it gets warmer the bacteria will stop being able to
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move cause no CheY will be present, therefore staying in place. The result will be a bacteria that
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will stay in a warmer area’s, cause as soon it gets cold again it will be able to move again.
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</p>
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<h3>Inne's theory:</h3>
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<p>
<p>
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The pseudo random movement claudio proposes is correct, however could be improved by
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The desaturation of the membrane starts with the membrane protein, DesK. DesK senses temperature of its environment and when the temperature is 30&deg;C, DesK autophosphorylates its conserved histidine. Sequentially the phosphoryl group is transferred to the aspartate residue in desR that activates the promoter of <i>des</i>. The gene <i>des</i> is translated into a fatty acid desaturase (&Delta;5-Des), that changes the fluidity of the membrane by introducing
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continually stimulating a repellent receptor. This will cause the Bacillus to move away as soon it
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double bonds into pre-existing saturated fatty acyl chains.
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gets into a cold area. It guarantees that CheY will be phosphorylated as soon as it is transcribed/
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translated, and therefore the bacteria can move. Both systems are not directly influencing
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movement, but cause the organism to have a bias towards warmer area's.
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Note: chemotaxis is kind of confusing when applied to different organisms, e.coli for example uses
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the same proteins but they have the effect of these proteins is the total opposite. Namely CheY
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causing tumbling.
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</p>
</p>
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<h3>References</h3>
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<br>
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<h4>The promoter activity of <i>des</i></h4>
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<br>
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<div align="left">
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<table id="layout" width="50%">
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<tr>
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<td>
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<img src="https://static.igem.org/mediawiki/2013/d/d7/Promoter-des-activity.jpg" width="100%">
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</td>
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</tr>
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<tr>
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<td>
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<font size="1">Figure 2: Pattern of P<i>des</i>-<i>lacZ</i> expression on a temperature downshift.</b>(a) <i>B. subtilis</i> AKP3 cells were grown at 37&deg;C to an optical density of 0.4 at 525 nm and then divided into two fractions. The first was transferred to 25&deg;C (&#9679;) and the second was kept at 37&deg;C (&#9675;). (b) Pattern of P<i>des</i>-<i>lacZ</i> expression in a <i>des</i>&#8254; background. <i>B. subtilis</i> AKP4 cells were grown at 37&deg;C to an optical density of 0.4 at 525 nm and then divided into two fractions. One fraction was transferred to 25&deg;C (&#9679;) while the other was kept at 37&deg;C (&#9675;). (c). Effect of exogenous fatty acids on P<i>des</i>-<i>lacZ</i> expression pattern. <i>B. subtilis</i> AKP4 cells were grown at 37&deg;C to an optical density of 0.4 at 525 nm and then divided into two fractions. Each fraction was supplemented with palmitic (&#9679;) or oleic acid (&#9632;) and growth was continued at 25&deg;C. (d) Effect of <i>desKR</i> disruption on P<i>des</i>-<i>lacZ</i> expression. <i>B. subtilis</i> AKP21 cells were grown at 37&deg;C to an optical density of 0.4 at 525 nm and then divided into two fractions. One of the fractions was transferred to 25&deg;C (&#9679;) and the other one was kept at 37&deg;C (&#9675;). Optical density at 525 nm (inserts) and &beta;-galactosidase specific activity were determined at the indicated times (a, b, c, or d). </font>
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</td>
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</table>
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</div>
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<br>
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<br>
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<table id="normal">
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<tr>
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<th>Strain</th>
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<th>Description</th>
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</tr>
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<tr>
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<td>JH642</td>
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<td><i>trpC2 pheA1</i></td>
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</tr>
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<tr>
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<td>AKP3</td>
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<td>JH642 <i>amyE</i>::[P<i>des</i>(-2269 to +31)<i>lacZ</i>]</td>
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</tr>
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<tr>
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<td>AKP4</td>
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<td>AKP3 <i>des</i>::<i>kan</i></td>
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</tr>
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<tr>
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<td>AKP21</td>
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<td>AKP3 <i>desKR</i>::<i>kan</i></td>
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</tr>
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</table>
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<b>Bredeston <i>et al.</i> 2011</b>
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</p>
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<Br><br>
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<!--
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<h2>Motility</h2>
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<h3>The principle</h3>
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CheY is a mayor factor in spinning the flagella CCW. When <i>cheY</i> is absent, cells are significant less motile[ref]. Because the promoter of <i>des</i> is active at low temperatures (25&deg;C) we placed <i>cheY</i> under control of the promoter of <i>des</i> (figure 2).   
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<br>
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-->
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<br>
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<h2>The coating</h2>
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The silk proteins are secreted with a Strep tag. The implant will be covered with biotin (vitamine B8). The Strep tag will automatically form a Van der Waals binding with the biotin and thus, if the silk is secreted closely the implant will be coated automatically. <i>B.subtilis</i> has a tendency to form a biofilm on a structure, or in our case an implant (insert proof that biofilm can grow in plastic/titanium) . To coat the implant we have thought of to option 1 via secretion and 2 via a biofilm formation. The secretion process is the most elegant and best option, but secretion of large proteins is proven to be difficult and has never been done before with silk. So a ‘backup’ plan was needed.
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<br>
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<br>
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<h2>Animation</h2>
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<video  controls width="40%">
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<source src="https://static.igem.org/mediawiki/igem.org/d/d0/Animation-secretion_VP8.webm" type="video/webm">
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</video>
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<br>
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<h3>Motility of the knockout strains</h3>
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To observe whether or not the mutant strains are less motile than the wild type strain different tests are done. One of the tests is performed using microscopy. 
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<h4>Microscope movies</h4>
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Microscope movies (4x real time) are made for the wildtype, the &Delta;cheY and the &Delta;cheY&Delta;des strain. These movies show that the wildtype strain (Movie 1) is more motile than both mutant strains. The comparison between the movie of the &Delta;cheY (Movie 2) and &Delta;cheY&Delta;des (Movie 3) strain shows just as seen in the motility assay, that the &Delta;cheY&Delta;des strain is less motile than the &Delta;cheY strain. 
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<br>
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<br><iframe width="420" height="315" src="//www.youtube.com/embed/vRjSmewTEDc" frameborder="0" allowfullscreen></iframe>
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<br><font size="1">Movie 1: Motility of the wild type strain</font>
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<br>
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<br><iframe width="420" height="315" src="//www.youtube.com/embed/Seilf16g3OI" frameborder="0" allowfullscreen></iframe>
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<br><font size="1">Movie 2: Motility of &Delta;cheY</font>
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<br>
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<br><iframe width="420" height="315" src="//www.youtube.com/embed/Y_qiqkzbAj8" frameborder="0" allowfullscreen></iframe>
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<br><font size="1">Movie 3: Motility of &Delta;cheY&Delta;des</font>
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<br>
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<br>
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<h2>References</h2>
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<font size="1,5">
<p>
<p>
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Christopher V. Rao, George D. Glekas and George W. Ordal, three adaptation systems of Bacillus subtilis chemotaxis , Trends in Biology (2008), Vol. 16 No 10, pp. 480-487.
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[1] Liam F. Garrity and George W. Ordal, Chemotaxis in <i>Bacillus subtilis</i>: How bacteria monitor environmental signals, <i>Pharmacology and Therepeutics</i> (1995), Vol. 68 No.1, pp. 87-104.
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<br>Liam F. Garrity and George W. Ordal, Pharmacology and Therepeutics. (1995), Chemotaxis in Bacillus Subtilis: How bacteria monitor environmental signals, Vol. 68 No.1, pp. 87-104.
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<br>[2] Christopher V. Rao, George D. Glekas and George W. Ordal, The three adaptation systems of <i>Bacillus subtilis</i> chemotaxis, <i>Trends in Biology</i> (2008), Vol. 16 No 10, pp. 480-487.
 +
<br>[3] Mariana Martin and Diego de Mendoza, Regulation of <i>Bacillus subtilis</i> DesK thermosensor by lipids, <i>Biochemical Journal</i> (2013), Vol 451 No 2, pp. 269–275.
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<br>[4] Thermal Regulation of Membrane Lipid Fluidity by a Two-Component System in <i>Bacillus subtilis</i>, January 31 2011, BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Vol. 39, No. 5, pp. 362–366, 2011.
</p>
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</html>
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Latest revision as of 03:53, 5 October 2013

Stay Warm, Stay Close

Our initial idea was to let the bacteria produce the silk in a bath and when the implant is put into the bath, the implant will be coated with silk. This is a wasteful and inelegant method. A low production yield can be expected and a solution is needed to overcome this problem. Therefore the heat motility was developed. With the help of heat motility the silk will be produced on site, this will also save energy in the form of nutrition and energy of heating the bath. An overview of our 'Coating GEM' is shown in figure 1.


Native Motility

The Motility in most bacteria is governed by the Che proteins [1], they control if the bacteria is swimming straight or tumbling (changing directions). They do this by controlling the flagella. If the flagella spin counter-clockwise (CCW) they will group together in one pole of the bacterium, causing straight swimming. On the other hand if the flagella are spinning clockwise (CW) then the flagella will disperse over the membrane and cause tumbling.

CheY

The Che proteins are present in many motile bacteria, however they can have different effects depending on the species [1]. The CheY protein in Bacillus subtilis, for example, has the complete opposite effect as in Escherichia coli. CheY is an important protein for controlling the spinning of the flagella. When the concentration of phosphorylated CheY (CheY-p) is sufficiently high the flagella turn CCW (straight swimming), but when the concentration of CheY decreases the chance of tumbling also increases, and the bacterium will reorient themselves more often.

Attractant Receptor

The chemotaxis process is initiated at the receptor, which can sense the concentration of an attractant (or repellent) [2]. Increasing concentrations of attractant correspond to an increased chance of swimming. If the concentration decreases, the bacteria will start to tumble more frequently, and will reorient its swimming direction, hopefully to more desirable regions.

cheA & CheC-CheD

Binding of the attractant receptor causes straight swimming via a small cascade [2]. When the receptor is bound, the CheA protein (which is attached to it) gets phosphorylated. The CheA-p phophorylates cheY, which then causes straight swimming. The protein complex CheC-CheD causes dephophorylation of cheY-p (when receptor is bound) resulting in a negative feedback. Two more negative feedback systems are also activated following the binding of attractant, which also result in decreased values of CheY-p. In such a way, CheY-p adaption occurs, and B. subtilis is ready to sense new changes in its environment. For more information about how this pathway works please visit the heat motility section.


Figure 1: The scheme shows B. subtilis containing knockouts of cheY, cheC and des. It also shows the motility gene and the silk gene positively controlled by cold and heat respectively.


Controllable motility

cheY Knockout

To make our coating mechanism a success we need to have control over the motility. This is achieved by knocking out cheY. This might sound strange since CheY-p is controlling straight swimming. However since we know that a CheY null mutant is immobile due to excessive tumbling. This makes it possible to insert cheY with a promoter of our choosing, and make it the sole producer of CheY. Also a cheC knockout is made in order to prevent negative feedback. How this is effecting the cell is explained in more detail with our model.

DesK pathway

We envision a bacterium that moves towards a heat source, in order to do this it needs a temperature sensor. B. subtilis natively has a protein that fits this requirement nicely. It is called DesK, it is a membrane protein that senses cold [3][4] (25°C). When the environment is cold, DesK autophophorylates, after which it phosphorylates DesR. DesR in turn activates the promoter of the des gene (Pdes), which would be the promoter we are looking for. The des gene expresses a protein that provides negative feedback to the system, so we need to knockout this des gene to keep our promoter active.


Thermal control of fatty acid synthesis.

In order to maintain the fluidity of the cell membrane when the environmental temperature is decreasing, B. subtilis (among other bacteria) adapts the membrane by increasing the fraction of unsaturated phospholipids acyl chains.

The desaturation of the membrane starts with the membrane protein, DesK. DesK senses temperature of its environment and when the temperature is 30°C, DesK autophosphorylates its conserved histidine. Sequentially the phosphoryl group is transferred to the aspartate residue in desR that activates the promoter of des. The gene des is translated into a fatty acid desaturase (Δ5-Des), that changes the fluidity of the membrane by introducing double bonds into pre-existing saturated fatty acyl chains.


The promoter activity of des


Figure 2: Pattern of Pdes-lacZ expression on a temperature downshift.(a) B. subtilis AKP3 cells were grown at 37°C to an optical density of 0.4 at 525 nm and then divided into two fractions. The first was transferred to 25°C (●) and the second was kept at 37°C (○). (b) Pattern of Pdes-lacZ expression in a des‾ background. B. subtilis AKP4 cells were grown at 37°C to an optical density of 0.4 at 525 nm and then divided into two fractions. One fraction was transferred to 25°C (●) while the other was kept at 37°C (○). (c). Effect of exogenous fatty acids on Pdes-lacZ expression pattern. B. subtilis AKP4 cells were grown at 37°C to an optical density of 0.4 at 525 nm and then divided into two fractions. Each fraction was supplemented with palmitic (●) or oleic acid (■) and growth was continued at 25°C. (d) Effect of desKR disruption on Pdes-lacZ expression. B. subtilis AKP21 cells were grown at 37°C to an optical density of 0.4 at 525 nm and then divided into two fractions. One of the fractions was transferred to 25°C (●) and the other one was kept at 37°C (○). Optical density at 525 nm (inserts) and β-galactosidase specific activity were determined at the indicated times (a, b, c, or d).


Strain Description
JH642 trpC2 pheA1
AKP3 JH642 amyE::[Pdes(-2269 to +31)lacZ]
AKP4 AKP3 des::kan
AKP21 AKP3 desKR::kan
Bredeston et al. 2011




The coating

The silk proteins are secreted with a Strep tag. The implant will be covered with biotin (vitamine B8). The Strep tag will automatically form a Van der Waals binding with the biotin and thus, if the silk is secreted closely the implant will be coated automatically. B.subtilis has a tendency to form a biofilm on a structure, or in our case an implant (insert proof that biofilm can grow in plastic/titanium) . To coat the implant we have thought of to option 1 via secretion and 2 via a biofilm formation. The secretion process is the most elegant and best option, but secretion of large proteins is proven to be difficult and has never been done before with silk. So a ‘backup’ plan was needed.

Animation


Motility of the knockout strains

To observe whether or not the mutant strains are less motile than the wild type strain different tests are done. One of the tests is performed using microscopy.

Microscope movies

Microscope movies (4x real time) are made for the wildtype, the ΔcheY and the ΔcheYΔdes strain. These movies show that the wildtype strain (Movie 1) is more motile than both mutant strains. The comparison between the movie of the ΔcheY (Movie 2) and ΔcheYΔdes (Movie 3) strain shows just as seen in the motility assay, that the ΔcheYΔdes strain is less motile than the ΔcheY strain.


Movie 1: Motility of the wild type strain


Movie 2: Motility of ΔcheY


Movie 3: Motility of ΔcheYΔdes

References

[1] Liam F. Garrity and George W. Ordal, Chemotaxis in Bacillus subtilis: How bacteria monitor environmental signals, Pharmacology and Therepeutics (1995), Vol. 68 No.1, pp. 87-104.
[2] Christopher V. Rao, George D. Glekas and George W. Ordal, The three adaptation systems of Bacillus subtilis chemotaxis, Trends in Biology (2008), Vol. 16 No 10, pp. 480-487.
[3] Mariana Martin and Diego de Mendoza, Regulation of Bacillus subtilis DesK thermosensor by lipids, Biochemical Journal (2013), Vol 451 No 2, pp. 269–275.
[4] Thermal Regulation of Membrane Lipid Fluidity by a Two-Component System in Bacillus subtilis, January 31 2011, BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Vol. 39, No. 5, pp. 362–366, 2011.

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