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Team:SYSU-China/project bacterial migration
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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 recA and recN (Fig.1). Therefore, <i | + | <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 recA and recN (Fig.1). Therefore, <i>recAp<i> and recNp 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 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 movement of bacteria.</p> | <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 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 movement of bacteria.</p> | ||
Revision as of 01:29, 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 movement 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 The racAp testing plasmid
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Fig.3
Plasmid CheZ-pET28a.
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Fig.4
The expression of cheZ.
Function
Finally, 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, 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 a certain intensity (which couldn't be measured for a lack of apposite apparatus) of UV, 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).
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Fig.7 Plasmid RecA-CheZ-pET28a(without T7p).
