Team:HIT-Harbin/Design

From 2014.igem.org

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                     <p>   AhR（arylhydrocarbon receptor）是在生命体内的二恶英及其类似物的结合受体，它能在二恶英的诱导下，通过膜转运与生物体内DNA相关序列结合，促使下游xenobiotic metabolizing enzymes(XMEs) 家族中的 CYP1A1 gene 表达，从而对生物体代谢进行不同程度的调控。 其具体的调控方式如图~所示，In the absence of ligand, AhR is present in the cytosol in a complex with Hsp90, XAP2 and p23 proteins. Upon binding to a ligand, the AhR complex translocates into the nucleus and the AhR dissociates from Hsp90 complex to form a heterodimer with its partner molecule, Arnt. Thus, the formed AhR/Arnt heterodimer recognizes an enhancer DNA element designated xenobiotic responsive element (XRE) sequence located in the promoter region of CYP1A1gene, resulting in the enhanced expression of the gene[1].</p>
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                     <p>   We combined yellow fluorescent protein induced by dioxins mentioned above together with DNA binding auxiliary sequence of lexAop and mdr521-805. Through the transformation of this genetic sequence, our device can express the yellow fluorescent protein massively and rapidly if the result of dioxin moleculesd detection is positive. In addition, as a result of the positive feedback effect, after the dioxin is removed, the device can still express the yellow fluorescent protein steadily, achieving the function of signal enhancement and memorization. In the absence of ligand, AhR is present in the cytosol in a complex with Hsp90, XAP2 and p23 proteins. Upon binding to a ligand, the AhR complex translocates into the nucleus and the AhR dissociates from Hsp90 complex to form a heterodimer with its partner molecule, Arnt. Thus, the formed AhR/Arnt heterodimer recognizes an enhancer DNA element designated xenobiotic responsive element (XRE) sequence located in the promoter region of CYP1A1gene, resulting in the enhanced expression of the gene[1].</p>
<img id="Family" width="911px" height="682px" src="https://static.igem.org/mediawiki/2014/7/75/Design1.png">
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                     <p>小鼠的AHR蛋白是由805个氨基酸序列组成，其中如图所示，包含bHLH (basic helix – loop – helix)、 PAS (Per – Arnt– Sim) domain、(A and B) PAS A and B repeats Q-rich (glutamine rich) region,其中PAS domain能够在hsp90存在的情况下与目标物质dioxin及其类似物结合并在ANRT的帮助下通过bHLH序列与DNA结合，诱导下游基因的表达。我们用lexA DBD蛋白将AHR的第1-82位的氨基酸换掉，使得融合的蛋白质能够与下游基因的增强子lexAoperator相结合，出发cyc1 promoter对下游黄色荧光蛋白的表达。从而使融合蛋白能够在酵母中起到对二噁英的检测功能。
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                     <p>We combined yellow fluorescent protein induced by dioxins mentioned above together with DNA binding auxiliary sequence of lexAop and mdr521-805. Through the transformation of this genetic sequence, our device can express the yellow fluorescent protein massively and rapidly if the result of dioxin moleculesd detection is positive. In addition, as a result of the positive feedback effect, after the dioxin is removed, the device can still express the yellow fluorescent protein steadily, achieving the function of signal enhancement and memorization.
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                     <p>Here we add a rational design of cellular memory in yeast that employs autoregulatory transcriptional positive feedback .我们在表达的上述由二噁英诱导的黄色荧光蛋白后融合了lexAop及mdr521-805这段DNA绑定的辅助序列。通过这段基因序列的改造，装置在探测到二噁英分子后，就能快速大量的表达黄色荧光蛋白，并且由于正反馈的作用，当二噁英不存在后，装置仍能稳定的表达黄色荧光蛋白，实现了信号增强及记忆的功能。</p>
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                     <p>Here we add a rational design of cellular memory in yeast that employs autoregulatory transcriptional positive feedback .We combined yellow fluorescent protein induced by dioxins mentioned above together with DNA binding auxiliary sequence of lexAop and mdr521-805. Through the transformation of this genetic sequence, our device can express the yellow fluorescent protein massively and rapidly if the result of dioxin moleculesd detection is positive. In addition, as a result of the positive feedback effect, after the dioxin is removed, the device can still express the yellow fluorescent protein steadily, achieving the function of signal enhancement and memorization.</p>
<img id="Family" width="911px" height="682px" src="https://static.igem.org/mediawiki/2014/3/34/Design4.png">
<img id="Family" width="911px" height="682px" src="https://static.igem.org/mediawiki/2014/3/34/Design4.png">

Revision as of 14:23, 15 October 2014

Design

DIOXIN DETECTIVE

DIOXIN SENSOR

AhR RECEPTOR

We combined yellow fluorescent protein induced by dioxins mentioned above together with DNA binding auxiliary sequence of lexAop and mdr521-805. Through the transformation of this genetic sequence, our device can express the yellow fluorescent protein massively and rapidly if the result of dioxin moleculesd detection is positive. In addition, as a result of the positive feedback effect, after the dioxin is removed, the device can still express the yellow fluorescent protein steadily, achieving the function of signal enhancement and memorization. In the absence of ligand, AhR is present in the cytosol in a complex with Hsp90, XAP2 and p23 proteins. Upon binding to a ligand, the AhR complex translocates into the nucleus and the AhR dissociates from Hsp90 complex to form a heterodimer with its partner molecule, Arnt. Thus, the formed AhR/Arnt heterodimer recognizes an enhancer DNA element designated xenobiotic responsive element (XRE) sequence located in the promoter region of CYP1A1gene, resulting in the enhanced expression of the gene[1].

Reference：[1] Functional role of AhR in the expression of toxic effects by TCDD

lexA DBD/Mdr

We combined yellow fluorescent protein induced by dioxins mentioned above together with DNA binding auxiliary sequence of lexAop and mdr521-805. Through the transformation of this genetic sequence, our device can express the yellow fluorescent protein massively and rapidly if the result of dioxin moleculesd detection is positive. In addition, as a result of the positive feedback effect, after the dioxin is removed, the device can still express the yellow fluorescent protein steadily, achieving the function of signal enhancement and memorization.

MEMORY SYSTEM

Here we add a rational design of cellular memory in yeast that employs autoregulatory transcriptional positive feedback .We combined yellow fluorescent protein induced by dioxins mentioned above together with DNA binding auxiliary sequence of lexAop and mdr521-805. Through the transformation of this genetic sequence, our device can express the yellow fluorescent protein massively and rapidly if the result of dioxin moleculesd detection is positive. In addition, as a result of the positive feedback effect, after the dioxin is removed, the device can still express the yellow fluorescent protein steadily, achieving the function of signal enhancement and memorization.

DIOXIN DEGRADEE
DIOXIN CONCENTRATION
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Click here and get more details about our project.

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