Team:XMU-China/Project Application

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<a href="https://2014.igem.org/Team:XMU-China/Project_Application_RBSpromoter"><div class="Project_Application_sub" id="Project_Application_sub01"><p style="font-size:40px;position:absolute;top:25px;left:50px;">RBS promoter</p></div></a>
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<a href="https://2014.igem.org/Team:XMU-China/Project_Application_BlackHole"><div class="Project_Application_sub" id="Project_Application_sub02"><p style="font-size:40px;position:absolute;top:25px;left:50px;">Black Hole</p></div></a>
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    Bacterial chemotaxis, which is universal in </span><span style="font-style: italic;">E.coli</span>, is defined as bacteria cells migration in response to a chemical stimulus. The natural </span><span style="font-style: italic;">E.coli</span> chemotaxis has limited receptor proteins which can respond to only six kinds of amino acid. Nevertheless, the reprogrammed chemotaxis named pseudotaxis makes </span><span style="font-style: italic;">E.coli</span> able to respond to molecules, whose receptor proteins do not exist in classical </span><span style="font-style: italic;">E.coli</span>, such as IPTG and L-arabinose, etc.
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    <span style="font-style: italic;">E.coli</span> have several flagella per cell (4–10 typically), which can rotate in two ways </span><span style="valign: sup;">[1]: counterclockwise (CCW) and clockwise (CW)</span><span style="valign: sup;"> [</span><span style="valign: sup;">2</span><span style="valign: sup;">]. The former aligns the flagella into a single rotating bundle, causing the bacterium to swim in line, while the later breaks the flagella bundle apart such that each flagellum points in a different direction, causing the bacterium to tumble. The motility is determined by the phosphorylation state of CheY protein governed by CheZ</span> protein. In the presence of CheZ</span> protein, CheY-P is dephosphorylated and produce CheY, thus CheY lead the flagellar motor to rotate CCW resulting in swimming. In the absence of CheZ</span>, CheY is phosphorylated into CheY-P which can bind to the flagellar switch protein FliM resulting in tumbling (</span><span style="font-weight: 700;">Figure 1). </span><span style="valign: sup;">[</span><span style="valign: sup;">2</span><span style="valign: sup;">] </span>
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                    <span ><b>Figure 1</b>. </span><span>Chemotaxis mechanism of</span><span style="font-style: italic;"> E.coli</span>. </span><span>The direction of rotation of the flagellar motor is controlled by the protein CheY. If the CheY is phosphorylated (CheY-P), it can bind to the flagellar motor protein FliM, c</span><span>ausing the cell to tumble. When</span><span> CheY is not phosphorylated, the flagellar motor</span><span> rotates counterclockwise (CCW). </span><span style="valign: sup;">[1]</span>
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    <span >Therefore, if no CheZ</span> is expressed (such as </span><span style="font-style: italic;">E.coli</span> CL-1 with </span><span style="font-style: italic;">CheZ</span> gene knocked out of genome), CheY-P couldn’t be dephosphorylated so that flagella keep CW, thus </span><span style="font-style: italic;">E.coli</span> keep tumbling and perform non-motile ability on semi-solid culture medium (</span><span style="font-weight: 700;">Figure 2</span> left). With enough <span style="font-style: italic;">CheZ</span> expressed, <span style="font-style: italic;">E.coli</span> regain chemotaxis ability on semi-solid culture medium (<span style="font-weight: 700;">Figure 2</span> right). If one kind of molecule (such as IPTG) could stimulate circuit to express<span style="font-style: italic;"> CheZ</span>, reprogrammed </span><span style="font-style: italic;">E.coli</span> will have the tendency to migrate to it. We named the reprogrammed chemotaxis pseudotaxis. Therefore, we are able to reprogram bacterial chemotaxis by knocking </span><span style="font-style: italic;">CheZ</span> gene out of the wild-type genome to control the expression of </span><span style="font-style: italic;">CheZ</span> by logic gene circuit.</span>
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                    <span style="color: #fff; font-size: 16px;">BBa_K1412000</span>
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                    <span style="color:#fff; font-size: 16px; font-weight: 400;">BBa_J04450</span>
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                    <span style="font-weight: 700;">Figure 2</span><span style="font-weight: 700;">.</span><span style="font-weight: 700;"> </span><span style="font-style: italic;">CL-1</span>&nbsp;could express&nbsp;<span style="font-style: italic;">CheZ</span>&nbsp;with&nbsp;BBa_K1412000&nbsp;to regain chemotaxis ability (the right colony). While with&nbsp;BBa_J04450 (the left colony)&nbsp;for comparison, no chemotaxis ability could be observed.&nbsp;</span>
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    <span >Besides, as aptamers have the potential to respond to almost all kinds of molecules and have already been used to regulate gene expression such as </span><span style="font-style: italic;">CheZ</span> to reprogram chemotaxis (<span style="font-weight: 700;">Figure 3</span>). We are also developing a new mechanism which combines aptamers with RNA-lock system to regulate target gene.
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                    <span><b>Figure 3</b>. </span><span>Me</span><span>chanism of how aptamers control</span><span> the translation of CheZ</span> protein. In the absence of target molecules (theophylline as an example). The mRNA’s ribosome binding site is paired, which inhibits the translation of CheZ</span> protein. In the absence of CheZ</span>, CheY-P will remain phosphorylated and the cells tumble in place. While in the presence of theophylline, the mRNA’s ribosome binding site will expose and the CheZ</span> can be expressed, allowing the cells to run and tumble. </span><span style="valign: sup;">[1]</span>
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    <span >Characterizing the circuit we constructed, we combine mathematical modeling with experiments, using modeling to guide experiments and to explain experimental phenomena. We have got reasonable results for a broader range of applications. As we have proved that the chemotaxis of </span><span style="font-style: italic;">E.coli</span> could be well reprogrammed, we try to apply reprogrammed chemotaxis into practice. For example, we have already demonstrated that motile ability is positively associate with the expression strength of </span><span style="font-style: italic;">CheZ</span>, thus we can characterize the efficiency of RBS or promoter by migration distance. At the same time, we also develop a biosafety system which relies on reprogrammed chemotaxis.</span>
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    <span >Last but not the least, we apply mathematical principles in our project. Mathematics is the simplest and clearest language, whose value to the development of human civilization is now widely recognized because of its extensive application of science, society and daily life. However, the mathematical laws in life sciences is still unclear and even in chaos. Luckily, synthetic biology can overcome these shortcomings on some level. Based on this, we design a gene circuit and expect mathematical regularities to realize the regulation and control of life activities. We hope our work can inspire people&#39;s interests to combine mathematics with synthetic biology.</span>
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    <span style="font-size: 18px; font-weight: 700;">R</span><span style="font-size: 18px; font-weight: 700;">eference</span><span style="font-size: 18px; font-weight: 700;">s</span>
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    <span >1. </span><span style="color: rgb(0, 0, 255); text-decoration: underline; styleName: Default Paragraph Font;">http://en.wikipedia.org/wiki/Chemotaxis</span>
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    <span >2. Topp S, Gallivan J P. Guiding bacteria with small molecules and RNA[J]. Journal of the American Chemical Society, 2007, 129(21): 6807-6811.</span>
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    <span style="color: rgb(0, 0, 255); text-decoration: underline; styleName: Default Paragraph Font;">http://pubs.acs.org/doi/abs/10.1021/ja0692480</span>
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Revision as of 18:27, 15 October 2014

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