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Team:SYSU-China/project bacterial migration
From 2011.igem.org
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<p>Ionizing radiation activates the SOS repair system of bacteria through DNA damage. The single-strand DNA breaks leads to the activation of protein RecA, which leads to proteolysis of the repressor protein LexA, resulting in the increased transcription of about 20 genes, including <i>recA</i> and <i>recN</i> (Fig.1). Therefore, <i>recAp</i> and <i>recNp</i> are radiation-inducible and can be utilized to control gene expression induced by ionizing radiation. </p> | <p>Ionizing radiation activates the SOS repair system of bacteria through DNA damage. The single-strand DNA breaks leads to the activation of protein RecA, which leads to proteolysis of the repressor protein LexA, resulting in the increased transcription of about 20 genes, including <i>recA</i> and <i>recN</i> (Fig.1). Therefore, <i>recAp</i> and <i>recNp</i> are radiation-inducible and can be utilized to control gene expression induced by ionizing radiation. </p> | ||
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| - | <p>The rotational direction of bacteria is controlled by the flagellar motor system, of which CheY plays a pivotal role. When CheY is phosphorylated (CheY-P), it binds to the flagellar switch protein FliM and induces the flagellum to rotate clockwise, which causes the cell to tumble. Smooth swimming is restored by the phosphatase CheZ, which dephosphorylates CheY-P and causes the flagellum to rotate counterclockwise (Fig.2). E.coli lacking the <i>cheZ</i> gene (<i>∆cheZ</i>, strain RP1616, non-motile) cannot dephosphorylate CheY-P, tumble incessantly, and are thus nonmotile. So, if we controls the expression of <i>cheZ< | + | <p>The rotational direction of bacteria is controlled by the flagellar motor system, of which CheY plays a pivotal role. When CheY is phosphorylated (CheY-P), it binds to the flagellar switch protein FliM and induces the flagellum to rotate clockwise, which causes the cell to tumble. Smooth swimming is restored by the phosphatase CheZ, which dephosphorylates CheY-P and causes the flagellum to rotate counterclockwise (Fig.2). E.coli lacking the <i>cheZ</i> gene (<i>∆cheZ</i>, strain RP1616, non-motile) cannot dephosphorylate CheY-P, tumble incessantly, and are thus nonmotile. So, if we controls the expression of <i>cheZ</i> in strain RP1616, it's possible to control the migration of bacteria.</p> |
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<p>Accordingly, in order to construct bacteria that move directionally towards ionizing radiation, we can place gene <i>cheZ</i> in the downstream of <i>recAp</i>. </p> | <p>Accordingly, in order to construct bacteria that move directionally towards ionizing radiation, we can place gene <i>cheZ</i> in the downstream of <i>recAp</i>. </p> | ||
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<li> <a href="https://static.igem.org/mediawiki/2011/d/d7/BM_03_Fig_1.jpg" title="Fig.7 Plasmid RecA-CheZ-pET28a(without T7p)."><img src="https://static.igem.org/mediawiki/2011/e/ef/BM_03_Fig_1_small.jpg" width="150" height="94" alt="Flower" /></a> | <li> <a href="https://static.igem.org/mediawiki/2011/d/d7/BM_03_Fig_1.jpg" title="Fig.7 Plasmid RecA-CheZ-pET28a(without T7p)."><img src="https://static.igem.org/mediawiki/2011/e/ef/BM_03_Fig_1_small.jpg" width="150" height="94" alt="Flower" /></a> | ||
Fig.1 | Fig.1 | ||
| - | <p> Plasmid RecA-CheZ-pET28a(without T7p).</p> | + | <p> Plasmid RecA-CheZ-pET28a(without <i>T7p</i>).</p> |
</li> | </li> | ||
<li> <a href="https://static.igem.org/mediawiki/igem.org/thumb/4/40/Function.jpg/600px-Function.jpg" title="Fig.2. Migration of cheZ knockout E.coli on semi-solid media. Ionizing radiation was replced by UV radiation because of limited conditions. After exposing to a certain intensity of UV(Power: 19W UV lamp, 43.5cm between the lamp and the culture dish; Time: 2h), the experiment group showed an obviously faster migration of bacteria than the control."><img src="https://static.igem.org/mediawiki/igem.org/5/5b/Function_small.jpg" width="150" height="150" alt="Flower" /></a> | <li> <a href="https://static.igem.org/mediawiki/igem.org/thumb/4/40/Function.jpg/600px-Function.jpg" title="Fig.2. Migration of cheZ knockout E.coli on semi-solid media. Ionizing radiation was replced by UV radiation because of limited conditions. After exposing to a certain intensity of UV(Power: 19W UV lamp, 43.5cm between the lamp and the culture dish; Time: 2h), the experiment group showed an obviously faster migration of bacteria than the control."><img src="https://static.igem.org/mediawiki/igem.org/5/5b/Function_small.jpg" width="150" height="150" alt="Flower" /></a> | ||
Fig.2 | Fig.2 | ||
| - | <p> Migration of cheZ knockout E.coli on semi-solid media.</p></li> | + | <p> Migration of <i>cheZ</i> knockout <i>E.coli</i> on semi-solid media.</p></li> |
<li> <a href="https://static.igem.org/mediawiki/igem.org/e/ed/Function_b.jpg" title="Fig.3. Migration of E.coli with the gradient of UV intensity. A gradient of UV intensity was created from the left to the right. The colonies expanded significantly with the increase of UV intensity, which means E.coli exposed to a higher UV intensity obtained a fater migration."><img src="https://static.igem.org/mediawiki/igem.org/5/5d/Function_b_small.jpg" width="150" height="172" alt="Flower" /></a> | <li> <a href="https://static.igem.org/mediawiki/igem.org/e/ed/Function_b.jpg" title="Fig.3. Migration of E.coli with the gradient of UV intensity. A gradient of UV intensity was created from the left to the right. The colonies expanded significantly with the increase of UV intensity, which means E.coli exposed to a higher UV intensity obtained a fater migration."><img src="https://static.igem.org/mediawiki/igem.org/5/5d/Function_b_small.jpg" width="150" height="172" alt="Flower" /></a> | ||
Fig.3 | Fig.3 | ||
| - | <p> Migration of E.coli with the gradient of UV intensity.</p> | + | <p> Migration of <i>E.coli</i> with the gradient of UV intensity.</p> |
</li> | </li> | ||
<li> <a href="https://static.igem.org/mediawiki/2011/d/d7/BM_03_Fig_1.jpg" title="Fig.4. Migration rate of E.coli with an increasing or decreasing UV intensity. When the intensity of UV increased, E.coli moved faster; when the intensity decreased, it moved much slower. These results indicate that the constructed E.coli migrates towards a higher intensity of UV light."><img src="https://static.igem.org/mediawiki/igem.org/thumb/c/c7/Zhexiantu.png/697px-Zhexiantu.png" width="150" height="129" alt="Flower" /></a> | <li> <a href="https://static.igem.org/mediawiki/2011/d/d7/BM_03_Fig_1.jpg" title="Fig.4. Migration rate of E.coli with an increasing or decreasing UV intensity. When the intensity of UV increased, E.coli moved faster; when the intensity decreased, it moved much slower. These results indicate that the constructed E.coli migrates towards a higher intensity of UV light."><img src="https://static.igem.org/mediawiki/igem.org/thumb/c/c7/Zhexiantu.png/697px-Zhexiantu.png" width="150" height="129" alt="Flower" /></a> | ||
Fig.4 | Fig.4 | ||
| - | <p> Migration rate of E.coli with an increasing or decreasing UV intensity.</p> | + | <p> Migration rate of <i>E.coli</i> with an increasing or decreasing UV intensity.</p> |
</li> | </li> | ||
</ul> | </ul> | ||
Latest revision as of 19:45, 28 October 2011
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Background
Ionizing radiation activates the SOS repair system of bacteria through DNA damage. The single-strand DNA breaks leads to the activation of protein RecA, which leads to proteolysis of the repressor protein LexA, resulting in the increased transcription of about 20 genes, including recA and recN (Fig.1). Therefore, recAp and recNp are radiation-inducible and can be utilized to control gene expression induced by ionizing radiation.
The rotational direction of bacteria is controlled by the flagellar motor system, of which CheY plays a pivotal role. When CheY is phosphorylated (CheY-P), it binds to the flagellar switch protein FliM and induces the flagellum to rotate clockwise, which causes the cell to tumble. Smooth swimming is restored by the phosphatase CheZ, which dephosphorylates CheY-P and causes the flagellum to rotate counterclockwise (Fig.2). E.coli lacking the cheZ gene (∆cheZ, strain RP1616, non-motile) cannot dephosphorylate CheY-P, tumble incessantly, and are thus nonmotile. So, if we controls the expression of cheZ in strain RP1616, it's possible to control the migration of bacteria.
Accordingly, in order to construct bacteria that move directionally towards ionizing radiation, we can place gene cheZ in the downstream of recAp.
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Fig.1The SOS repair system.
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Fig.2 The flagellar motor system.
Modules
We tested the expression of cheZ on protein level. Within the inducement of 0.1 mM IPTG at 18℃ for 18h, we extracted the total proteins of the E.coli transformed with plasmid CheZ-pET28a (Fig.1). Western Blot results showed that the expression of protein CheZ significantly increased after the inducement, indicating that cheZ expressed well (Fig.2). Additionally, sicne a basal expression caused by lacp in pET28a, the control group also showed a low expression of CheZ.
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Fig.1
Plasmid CheZ-pET28a.
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Fig.2
Western Blot results of CheZ .
Function
We planned to test the function of gene cheZ in bacterial migration towards ionizing radiation. Because of limited conditions, we replaced ionizing radiation with UV radiation, which has the same effect of DNA damage to activate the SOS repair system. The ∆cheZ E.coli strain RP1616 was transformed with plasmid RecA-CheZ-pET28a (Fig.1) (RP1616(RecA-CheZ-pET28a)) whose original T7p was digested by restriction endonuclease.
After exposing to certain intensities (which couldn't be measured for a lack of apposite apparatus but could merely be described roughly) of UV radiation, we observed the behavior of both wild-type (RP437, motile) and ∆cheZ (RP1616) with RecA-CheZ-pET28a. The results indicate that RP1616(RecA-CheZ-pET28a) moves directionally towards higher UV intensity (Fig.2, 3& 4).
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Fig.1
Plasmid RecA-CheZ-pET28a(without T7p).
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Fig.2
Migration of cheZ knockout E.coli on semi-solid media.
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Fig.3
Migration of E.coli with the gradient of UV intensity.
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Fig.4
Migration rate of E.coli with an increasing or decreasing UV intensity.
