Team:XMU-China/Project Application OscillationTimer

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     <span style="font-size:27px;font-family:Arial">Oscillation Timer</span>
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     <span style="font-size:27px;font-family:Arial">OSCILLATION TIMER</span>
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     <span style="font-family:Arial; font-size: 20px;">--Ameliorate last project by chemotaxis</span>
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     <span style="font-family:Arial; font-size: 21px;">--Ameliorate last project by chemotaxis</span>
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     iGEM13_XMU-China has tried to construct oscillation system by standard biobricks. The synchronized oscillation system used in that study(</span><span style="font-family: Times New Roman; font-weight: 700;">Figure 1</span><span style="font-family: Times New Roman; font-weight: 700;">A</span><span style="font-family:Arial">) is based on the quorum sensing machineries in </span><span style="font-family: Times New Roman; font-style: italic;">Vibrio fischeri</span> and <span style="font-family: Times New Roman; font-style: italic;">Bacillus thurigensis</span>. Three identical <span style="font-family: Times New Roman; font-style: italic;">luxI</span><span style="font-family:Arial"> promoters are in charge of </span><span style="font-family: Times New Roman; font-style: italic;">luxI</span><span style="font-family:Arial"> (from </span><span style="font-family: Times New Roman; font-style: italic;">V. fischeri</span><span style="font-family:Arial">), </span><span style="font-family: Times New Roman; font-style: italic;">aiiA</span><span style="font-family:Arial"> (from </span><span style="font-family: Times New Roman; font-style: italic;">B.thurigensis</span><span style="font-family:Arial">) and </span><span style="font-family: Times New Roman; font-style: italic;">gfp</span><span style="font-family:Arial"> genes separately. The LuxI synthase generates an acyl-homoserine-lactone (AHL), which can spread across the cell membrane and mediate intercellular coupling. AHL then binds </span><span style="font-family:Arial">to LuxR produced intracellularly</span><span style="font-family:Arial">, and the LuxR-AHL complex would activate the <i>luxI</i> promoter. AiiA catalyzes the degradation of AHL as the negative feedback in the circuit. Therefore, both the activator AHL and the repressor AiiA of the network are activated by the <i>luxI</i> promoter simultaneously.</span>
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     <a href="https://2013.igem.org/Team:XMU-China/Background" target="_blank">iGEM13_XMU-China</a> has tried to construct oscillation system by standard biobricks. The synchronized oscillation system used in that study(</span><span style="font-family: Times New Roman; font-weight: 700;">Figure 1</span><span style="font-family: Times New Roman; font-weight: 700;">A</span><span style="font-family:Arial">) is based on the quorum sensing machineries in </span><span style="font-family: Times New Roman; font-style: italic;">Vibrio fischeri</span> and <span style="font-family: Times New Roman; font-style: italic;">Bacillus thurigensis</span>. Three identical <span style="font-family: Times New Roman; font-style: italic;">luxI</span><span style="font-family:Arial"> promoters are in charge of </span><span style="font-family: Times New Roman; font-style: italic;">luxI</span><span style="font-family:Arial"> (from </span><span style="font-family: Times New Roman; font-style: italic;">V. fischeri</span><span style="font-family:Arial">), </span><span style="font-family: Times New Roman; font-style: italic;">aiiA</span><span style="font-family:Arial"> (from </span><span style="font-family: Times New Roman; font-style: italic;">B.thurigensis</span><span style="font-family:Arial">) and </span><span style="font-family: Times New Roman; font-style: italic;">gfp</span><span style="font-family:Arial"> genes separately. The LuxI synthase generates an acyl-homoserine-lactone (AHL), which can spread across the cell membrane and mediate intercellular coupling. AHL then binds </span><span style="font-family:Arial">to LuxR produced intracellularly</span><span style="font-family:Arial">, and the LuxR-AHL complex would activate the <i>luxI</i> promoter. AiiA catalyzes the degradation of AHL as the negative feedback in the circuit. Therefore, both the activator AHL and the repressor AiiA of the network are activated by the <i>luxI</i> promoter simultaneously.</span>
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                     <span style="font-family: Times New Roman; font-weight: 700;">A</span>
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                     <span style="font-family: Times New Roman; font-weight: 700;"><b>A</b></span>
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                     <span style="font-family: Times New Roman; font-weight: 700;">B</span><img width="337" height="248" style="font-family:Arial" src="https://static.igem.org/mediawiki/parts/b/b6/Xmu_project_application_OscillationTimer02.png"/>
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                     <span style="font-family: Times New Roman; font-weight: 700;"><b>B</b></span><br><img width="337" height="248" style="font-family:Arial" src="https://static.igem.org/mediawiki/parts/b/b6/Xmu_project_application_OscillationTimer02.png"/>
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                     <span style="font-family: Times New Roman; font-weight: 700;">Figure 1A</span>Schematic of the oscillation based on quorum sensing system.</span><span style="font-family: Times New Roman; font-weight: 700;">1B.</span>Two oscillation cycles were observed within 500 minutes by microplate reader.</span>
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                     <span style="font-family: Times New Roman; font-weight: 700;">Figure 1A. </span>Schematic of the oscillation based on quorum sensing system.</span><span style="font-family: Times New Roman; font-weight: 700;">1B.</span>Two oscillation cycles were observed within 500 minutes by microplate reader.</span>
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     <span style="font-family:Arial">Based on above principle, one published paper has already realized synchronized oscillations under microfluidic</span><span style="font-family:Arial"> </span><span style="font-family:Arial">device.</span><span style="font-family: Times New Roman; valign: sup;">[1]</span><span style="font-family:Arial"> However</span><span style="font-family:Arial">,</span><span style="font-family:Arial"> </span><span style="font-family:Arial">iGEM13_XMU-China</span><span style="font-family:Arial"> can’</span><span style="font-family:Arial">t get </span><span style="font-family:Arial">synchronized </span><span style="font-family:Arial">oscillation</span><span style="font-family:Arial"> on microfluidics</span><span style="font-family:Arial">, and that will be discussed later. </span><span style="font-family:Arial">Through calculating fluorescence </span><span style="font-family:Arial">on 96-microwell plate e</span><span style="font-family:Arial">very 15 minutes, they got two oscillation cycles within 500 minutes (</span><span style="font-family: Times New Roman; font-weight: 700;">Figure 1B</span><span style="font-family:Arial">).</span>
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     <span style="font-size: 16px;font-family:arial, helvetica, sans-serif">Based on above principles, one published paper has already realized synchronized oscillations under microfluidic device <sup>[1]</sup>. However, <a href="https://2013.igem.org/Team:XMU-China/Background" target="_blank">iGEM13_XMU-China</a> can’t get synchronized oscillation on microfluidics, which is discussed in <a href="https://2014.igem.org/Team:XMU-China/Project_PSystem" target="_blank">P
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SYSTEM</a href="https://2014.igem.org/Team:XMU-China/Project_PSystem" target="_blank">. Through calculated fluorescence on 96-microwell plate every 15 minutes, it got two oscillation cycles within 500 minutes (<strong>Figure 1B</strong>).</span>
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     <span style="font-weight: 700;">Circuit design</span>
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     <span style="font-size:21px;font-weight: 700;">Circuit design</span>
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     <span style="font-family:Arial">Based on that, we construct our circuit by replacing </span><span style="font-family: Times New Roman; font-style: italic;">GFP</span><span style="font-family:Arial"> </span><span style="font-family:Arial">with</span><span style="font-family:Arial"> </span><span style="font-family: Times New Roman; font-style: italic;">CheZ</span><span style="font-family: Times New Roman; font-style: italic;"> </span><span style="font-family:Arial">(</span><span style="font-family: Times New Roman; font-weight: 700;">Figure 2</span><span style="font-family:Arial">)</span><span style="font-family:Arial">.</span><span style="font-family:Arial"> As the expression strength of </span><span style="font-family: Times New Roman; font-style: italic;">CheZ</span><span style="font-family:Arial"> </span><span style="font-family:Arial">is </span><span style="font-family:Arial">oscillatory</span><span style="font-family:Arial"> fluctuating</span><span style="font-family:Arial">, </span><span style="font-family:Arial">the motile ability will change periodically.</span><span style="font-family:Arial"> </span><span style="font-family:Arial">Cells</span><span style="font-family:Arial"> will have the strongest motile ability at wave crest while even be </span><span style="font-family:Arial">non-motile</span><span style="font-family:Arial"> at wave tr</span><span style="font-family:Arial">ough. Thus, periodical </span><span style="font-family:Arial">change of motile ability leads</span><span style="font-family:Arial"> to periodical change in swimming velocity</span><span style="font-family:Arial">. </span><span style="font-family:Arial">A</span><span style="font-family:Arial">t non-motile period, </span><span style="font-family:Arial">cells</span><span style="font-family:Arial"> will aggregate together leading to the formation of </span><span style="font-family:Arial">growth</span><span style="font-family:Arial">-ring</span><span style="font-family:Arial">-like</span><span style="font-family:Arial"> </span><span style="font-family:Arial">patterns which could be distinguished by naked eyes.</span>
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     <span style="font-size: 16px;font-family:arial, helvetica, sans-serif">Based on that, we constructed our circuit by replacing <em>gfp</em> with <em>cheZ </em>(<strong>Figure 2</strong>). As the expression strength of <em>cheZ</em> is oscillatory fluctuating, the motile ability will change periodically. Cells will have the strongest motile ability at wave crest while even be <span style="font-family:arial, helvetica, sans-serif"></span><span style="font-family:arial, helvetica, sans-serif">non-motile</span> at wave trough. Thus, periodical change of motile ability leads to periodical change in swimming velocity. At non-motile period, cells will aggregate together leading to the formation of growth-ring-like patterns which could be distinguished by naked eyes.</span>
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                     <span style="font-family: Times New Roman; font-weight: 700;">Figure 2.</span><span style="font-family: Times New Roman;">Schematic of the growth-ring formation circuit. Derived from quorum sensing oscillator by replacing</span><span style="font-family: Times New Roman; font-style: italic;"> GFP</span> with <span style="font-family: Times New Roman; font-style: italic;">CheZ</span><span style="font-family:Arial">.</span>
                     <span style="font-family: Times New Roman; font-weight: 700;">Figure 2.</span><span style="font-family: Times New Roman;">Schematic of the growth-ring formation circuit. Derived from quorum sensing oscillator by replacing</span><span style="font-family: Times New Roman; font-style: italic;"> GFP</span> with <span style="font-family: Times New Roman; font-style: italic;">CheZ</span><span style="font-family:Arial">.</span>
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     <span style="font-family:Arial">Many trees in temperate zones make one growth ring each year, with the newest adjacent to the bark. We can tell </span><span style="font-family:Arial">a tree’s </span><span style="font-family:Arial">age b</span><span style="font-family:Arial">y counting the number of growth </span><span style="font-family:Arial">ring</span><span style="font-family:Arial">s</span><span style="font-family:Arial">. Analogously, bacteria rings could also be formed by gene oscillator. </span><span style="font-family:Arial">Multiply</span><span style="font-family:Arial"> the period </span><span style="font-family:Arial">by</span><span style="font-family:Arial"> the quantity of bacteria rings, we c</span><span style="font-family:Arial">an tell how much time has passed.</span>
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     <span style="font-family:Arial">Many trees in temperate zones make one growth ring each year, with the newest adjacent to the bark. We can tell a tree’s age by counting the number of growth rings. Analogously, bacteria rings formed by gene oscillator can tell us how much time has passed through the quantity of bacteria rings.</span>
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     <span style="font-family: Arial; font-weight: 700;">Characterization of circuit</span>
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     <span style="font-family: Arial;font-size:21px; font-weight: 700;">Characterization of circuit</span>
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     <span style="font-family:Arial">Experiments show that bacteria could just form several rings in 48 hours. Then, no bacteria ring formed while bacteria kept spreading evenly from the inside out. As bacteria formed the rings (<span style="font-family: Times New Roman; font-weight: 700;">Figure 3A</span>)which are quiet different from wild-type (<span style="font-family: Times New Roman; font-weight: 700;">Figure 3B</span><span style="font-family:Arial">).</span>
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     <span style="font-size: 16px;font-family:arial, helvetica, sans-serif">Experiments showed that bacteria could just form several rings in 48 hours. Then, no bacteria rings formed while bacteria kept spreading evenly from the inside out. As bacteria formed the rings (<strong>Figure 3A</strong>) which are quite different from wild-type (<strong>Figure 3B</strong>), we can conclude that chemotaxis is reprogrammed successfully, however, not as expected.</span>
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                     <img width="186" height="176" style="font-family:Arial" src="https://static.igem.org/mediawiki/parts/7/7d/Xmu_project_application_OscillationTimer04.png"/>
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                     <img width=300 style="font-family:Arial" src="https://static.igem.org/mediawiki/parts/7/7d/Xmu_project_application_OscillationTimer04.png"/>
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                     <img width="185" height="179" style="font-family:Arial" src="https://static.igem.org/mediawiki/parts/e/ef/Wild-type_E.coli%28CL-M%29.png"/>
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                     <img width=300 style="font-family:Arial" src="https://static.igem.org/mediawiki/parts/e/ef/Wild-type_E.coli%28CL-M%29.png"/>
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                     <span style="font-family: Times New Roman; font-weight: 700;">Figure 3A. </span><span style="font-family: Times New Roman">Bacteria rings formed by CL-1 with above oscillation circuit, it has several bacteria rings in the beginning.</span><span style="font-family: Times New Roman; font-weight: 700;">3B. </span><span style="font-family: Times New Roman">Bacteria rings formed by wild-type E.coli (</span><span style="font-family: Times New Roman; font-style: italic;">CL-M</span><span style="font-family: Times New Roman">). It is different from the right picture. </span>
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                     <span style="font-family: Times New Roman; font-weight: 700;line-height:1.2;">Figure 3A. </span><span style="font-family: Times New Roman">Bacteria rings formed by CL-1 with above oscillation circuit, it has several bacteria rings in the beginning.</span><span style="font-family: Times New Roman; font-weight: 700;">3B. </span><span style="font-family: Times New Roman">Bacteria rings formed by wild-type <i>E. coli</i> (</span><span style="font-family: Times New Roman;">CL-M</span><span style="font-family: Times New Roman">). It is different from the right picture. </span>
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     <span style="font-family:arial, helvetica, sans-serif">We make sure that chemotaxis is reprogrammed successfully but not as expected. We try to make use of the grown time (as X axis) and radius of grown (as Y axis) to draw a curve to see whether the amplification rate of chemotaxis radius are equal over a period of time (</span><strong><span style="font-family:times new roman">Figure 4A, 4B</span></strong><span style="font-family:arial, helvetica, sans-serif">). We notice that the rate of two curves kept stable expect the beginning 40 hours. So why the bacteria cannot form rings afterwards and the grown rate becomes stable? We thought that there must be something wrong with the negative feedback in the circuit. Circuit can’t generate enough feedback to repress the chemotactic ability so that swimming speed keeps constant which is actually maximum speed. Hence, combining the experimental results of iGEM13_XMU-China with ours, we are still trying to find out why the oscillation could just keep several periods
+
     <span style="font-size: 16px;font-family:arial, helvetica, sans-serif">Therefore, we tried to make use of the grown time (as X axis) and radius of grown (as Y axis) to draw a curve to see whether the amplification rate of chemotaxis radius are equal over a period of time (<strong>Figure 4A, 4B</strong>). We noticed that the rate of two curves keep stable expect the beginning 40 hours. So why the bacteria cannot form rings afterwards but the grown rate becomes stable? We thought that there must be something wrong with the negative feedback in the circuit. Circuit can’t generate enough feedback to repress the chemotactic ability so that swimming speed keeps constant which is actually maximum speed. Hence, combining the experimental results of <a href="https://2013.igem.org/Team:XMU-China/Background" target="_blank">iGEM13_XMU-China</a> with ours, we are still trying to find out why the oscillation could just keep several periods, and we got a reasonable conclusion that may help us make it clear.&nbsp;</span>
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, and we got a reasonable conclusion that may help us make it clear.</span>
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     <span style="font-family:times new roman"><strong>Figure 4A</strong> Cultivating <em>CL-1</em> in on semisolid culture medium with chloramphenicol and tetracycline (halve the concentration of LB) and make use of the grown time (as X axis) and radius of grown (as Y axis) to draw a curve, the curve show that the rate is stable. <strong>4B</strong> Cultivating <em>CL-1</em> on semisolid culture medium with chloramphenicol and tetracycline and draw a curve with the grown time (as X axis) and radius of grown (as Y axis), the curve show that the rate is stable.</span>
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     <span style="font-family:times new roman"><strong><span style="font-size: 16px;"><b>Figure 4A.</b></span></strong><span style="font-size: 16px;"> The plot of chemotactic diameter versus time. Cultivated CL-1 on semisolid culture medium with chloramphenicol and tetracycline (halve concentration) and made use of the grown time (as X axis) and chemotaxis diameter (as Y axis) to draw a curve, the curve show that the diffusion rate is stable. <strong> </strong></span><strong><span style="font-size: 16px;">4B. </span></strong><span style="font-size: 16px;"> A parallel experiment to Figure 4A</span></span>
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<p>When we were improving the project of iGEM13-XMU-China, we creatively put forward an assumption to a problem mentioned in <a href="https://2014.igem.org/Team:XMU-China/Project_PSystem" target="_blank">P SYSTEM</a href="https://2014.igem.org/Team:XMU-China/Project_PSystem" target="_blank"> which has been ignored for a long time.</p>
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<p>Want to see more applications: <a href="https://2014.igem.org/Team:XMU-China/Project_Application_RBSpromoter" target="_blank">Promoter Yardstick</a href="https://2014.igem.org/Team:XMU-China/Project_Application_RBSpromoter" target="_blank">,<a href="https://2014.igem.org/Team:XMU-China/Project_Application_BlackHole" target="_blank">Black Hole</a href="https://2014.igem.org/Team:XMU-China/Project_Application_BlackHole" target="_blank">, <a href="https://2014.igem.org/Team:XMU-China/Project_Application_Aptamer" target="_blank">Aptamer Key-Lock</a href="https://2014.igem.org/Team:XMU-China/Project_Application_Aptamer" target="_blank">.</p>
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     <span style="font-family: Times New Roman; font-size: 21px; font-weight: 700;">Reference</span><span style="font-family: Times New Roman; font-size: 21px; font-weight: 700;">s</span>
     <span style="font-family: Times New Roman; font-size: 21px; font-weight: 700;">Reference</span><span style="font-family: Times New Roman; font-size: 21px; font-weight: 700;">s</span>
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    <span style="font-family:Arial">1. </span><span style=" color:#8ccfff; font-family: Times New Roman; font-size: 13px;">Danino T, Mondragón-Palomino O, Tsimring L, et al. </span><span style=" color:#8ccfff; font-family: Times New Roman; font-size: 13px;">A synchronized quorum of genetic clocks[J]. </span><span style=" color:#8ccfff; font-family: Times New Roman; font-size: 13px;">Nature, 2010, 463(7279): 326-330.</span>
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     <span style="color: rgb(0, 0, 255); font-family: Times New Roman; text-decoration: underline; styleName: Default Paragraph Font;"><a href="http://www.nature.com/nature/journal/v463/n7279/full/nature08753.html" target="_self">http://www.nature.com/nature/journal/v463/n7279/full/nature08753.html</a></span>
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     <span style="color: rgb(0, 0, 255); font-family: arial; text-decoration: underline; styleName: Default Paragraph Font;"><a href="http://www.nature.com/nature/journal/v463/n7279/full/nature08753.html" target="_blank">1. Danino T, Mondragón-Palomino O, Tsimring L, et al. A synchronized quorum of genetic clocks[J]. Nature, 2010, 463(7279): 326-330.</a></span>
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Latest revision as of 03:25, 18 October 2014

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OSCILLATION TIMER

--Ameliorate last project by chemotaxis


iGEM13_XMU-China has tried to construct oscillation system by standard biobricks. The synchronized oscillation system used in that study(Figure 1A) is based on the quorum sensing machineries in Vibrio fischeri and Bacillus thurigensis. Three identical luxI promoters are in charge of luxI (from V. fischeri), aiiA (from B.thurigensis) and gfp genes separately. The LuxI synthase generates an acyl-homoserine-lactone (AHL), which can spread across the cell membrane and mediate intercellular coupling. AHL then binds to LuxR produced intracellularly, and the LuxR-AHL complex would activate the luxI promoter. AiiA catalyzes the degradation of AHL as the negative feedback in the circuit. Therefore, both the activator AHL and the repressor AiiA of the network are activated by the luxI promoter simultaneously.

 

A

B

Figure 1A. Schematic of the oscillation based on quorum sensing system.1B.Two oscillation cycles were observed within 500 minutes by microplate reader.

 

Based on above principles, one published paper has already realized synchronized oscillations under microfluidic device [1]. However, iGEM13_XMU-China can’t get synchronized oscillation on microfluidics, which is discussed in P SYSTEM. Through calculated fluorescence on 96-microwell plate every 15 minutes, it got two oscillation cycles within 500 minutes (Figure 1B).

 

Circuit design

Based on that, we constructed our circuit by replacing gfp with cheZ (Figure 2). As the expression strength of cheZ is oscillatory fluctuating, the motile ability will change periodically. Cells will have the strongest motile ability at wave crest while even be non-motile at wave trough. Thus, periodical change of motile ability leads to periodical change in swimming velocity. At non-motile period, cells will aggregate together leading to the formation of growth-ring-like patterns which could be distinguished by naked eyes.

Figure 2.Schematic of the growth-ring formation circuit. Derived from quorum sensing oscillator by replacing GFP with CheZ.

 

Many trees in temperate zones make one growth ring each year, with the newest adjacent to the bark. We can tell a tree’s age by counting the number of growth rings. Analogously, bacteria rings formed by gene oscillator can tell us how much time has passed through the quantity of bacteria rings.

 

Characterization of circuit

Experiments showed that bacteria could just form several rings in 48 hours. Then, no bacteria rings formed while bacteria kept spreading evenly from the inside out. As bacteria formed the rings (Figure 3A) which are quite different from wild-type (Figure 3B), we can conclude that chemotaxis is reprogrammed successfully, however, not as expected.

 

A

B

Figure 3A. Bacteria rings formed by CL-1 with above oscillation circuit, it has several bacteria rings in the beginning.3B. Bacteria rings formed by wild-type E. coli (CL-M). It is different from the right picture.

 

Therefore, we tried to make use of the grown time (as X axis) and radius of grown (as Y axis) to draw a curve to see whether the amplification rate of chemotaxis radius are equal over a period of time (Figure 4A, 4B). We noticed that the rate of two curves keep stable expect the beginning 40 hours. So why the bacteria cannot form rings afterwards but the grown rate becomes stable? We thought that there must be something wrong with the negative feedback in the circuit. Circuit can’t generate enough feedback to repress the chemotactic ability so that swimming speed keeps constant which is actually maximum speed. Hence, combining the experimental results of iGEM13_XMU-China with ours, we are still trying to find out why the oscillation could just keep several periods, and we got a reasonable conclusion that may help us make it clear. 


 

A

B

Figure 4A. The plot of chemotactic diameter versus time. Cultivated CL-1 on semisolid culture medium with chloramphenicol and tetracycline (halve concentration) and made use of the grown time (as X axis) and chemotaxis diameter (as Y axis) to draw a curve, the curve show that the diffusion rate is stable. 4B. A parallel experiment to Figure 4A

When we were improving the project of iGEM13-XMU-China, we creatively put forward an assumption to a problem mentioned in P SYSTEM which has been ignored for a long time.


Want to see more applications: Promoter Yardstick,Black Hole, Aptamer Key-Lock.


 

References

1. Danino T, Mondragón-Palomino O, Tsimring L, et al. A synchronized quorum of genetic clocks[J]. Nature, 2010, 463(7279): 326-330.