Difference between revisions of "Team:HUST-China/Proof"

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                        <li><a  href="https://2016.igem.org/Team:HUST-China">HOME</a></li>
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<p>
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                        <li class="dropdown1"><a class="down-scroll active" href="#">PROJECT</a>
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iGEM teams are great at making things work! We value teams not only doing an incredible job with theoretical models and experiments, but also in taking the first steps to make their project real.  
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                            <ul class="dropdown2">
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                                <li><a href="https://2016.igem.org/Team:HUST-China/project/background">Background</a></li>
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                                <li><a href="https://2016.igem.org/Team:HUST-China/Description">Description</a></li>
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                                <li><a href="https://2016.igem.org/Team:HUST-China/Proof">Proof of concept</a></li>
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<h4> What should we do for our proof of concept? </h4>
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                                <li><a href="https://2016.igem.org/Team:HUST-China/Demonstrate">Application</a></li>
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<p>  
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                                <li><a href="https://2016.igem.org/Team:HUST-China/project/inprovement">Previous improvement</a></li>
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You can assemble a device from BioBricks and show it works. You could build some equipment if you're competing for the hardware award. You can create a working model of your software for the software award. Please note that this not an exhaustive list of activities you can do to fulfill the gold medal criterion. As always, your aim is to impress the judges!
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                                <li><a href="https://2016.igem.org/Team:HUST-China/Safety">Safety</a></li>
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                                <li><a href="https://2016.igem.org/Team:HUST-China/Experiments">Experiments</a></li>
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                                <li><a href="https://2016.igem.org/Team:HUST-China/Results">Results</a></li>
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                                <li><a href="https://2016.igem.org/Team:HUST-China/InterLab">Interlab</a></li>
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                                <li><a href="https://2016.igem.org/Team:HUST-China/wetlab/protocols">Protocols</a></li>
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                        <li class="dropdown1"><a class="down-scroll" href="https://2016.igem.org/Team:HUST-China/Model">MODELING</a>
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                    <li><a href="https://2016.igem.org/Team:HUST-China/Model">Overview</a></li>
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                    <li><a href="https://2016.igem.org/Team:HUST-China/Model/model-pro">Prokaryotic circuit</a></li>
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                    <li><a href="https://2016.igem.org/Team:HUST-China/Model/model-euk">Eukaryotic circuit</a></li>
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                    <li><a href="https://2016.igem.org/Team:HUST-China/Model/model-app">Application circuit</a></li>
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                </ul>
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                        <li class="dropdown1"><a class="down-scroll" href="">PARTS</a>
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                                <li><a href="https://2016.igem.org/Team:HUST-China/Parts">Summary</a></li>
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                                <li><a href="https://2016.igem.org/Team:HUST-China/Basic_Part">Basic part</a></li>
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                                <li><a href="https://2016.igem.org/Team:HUST-China/Composite_Part">Composite part</a></li>
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                                <li><a href="https://2016.igem.org/Team:HUST-China/Part_Collection">Part Collection</a></li>
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                                <li><a href="https://2016.igem.org/Team:HUST-China/Integrated_Practices">Integrated HP</a></li>
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                                <li><a href="https://2016.igem.org/Team:HUST-China/Collaborations">Collaborations</a></li>
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                                <li><a href="https://2016.igem.org/Team:HUST-China/Team">Team Roster</a></li>
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                                <li><a href="https://2016.igem.org/Team:HUST-China/Attributions">Attributions</a></li>
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                    This year, we want to offer a handy, adjustable and useful tool kit to everyone working on Synthetic Biology--Signal Filter. The core circuit is based on a positive feedback bi-stable or tri-stable system with the capacity of reducing noise and converting pulse signal into robust and persistent signal. To adapt to different experimental requirements,we also developed two versions of our Signal Filter--prokaryotic one and eukaryotic one.
 +
                </p>
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                <!-- h3二级标题 -->
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                <h2>Prokaryotic version:</h2>
 +
                <p>
 +
                    It is a tri-stable system adapted from bacteriophage λ operon. In the operon, promoter RE is activated by CII trascriptional activator. Ftsh is an ATP-dependent host metalloprotease which will normally degrade CII, while CIII serves as an inhibitor of Ftsh to free CII. And CI can function as a inhibitor to block pR, while Cro can bind pRE to stop the downstream gene’s expression. The positive feedback control under pRE is used to enhance pulse signal 1 and convert it into robust stable signal.
 +
                </p>
 +
                <!-- 图片 -->
 +
                <img src="https://static.igem.org/mediawiki/2016/8/88/T--HUST-China--Description-Fig-Eukaryote.png" alt="" class="img-responsive">
 +
                <p class="text-center">Fig*: circuit of prokaryote version serving as tri-stable signal filter</p>
 +
                <!-- <h2>Creation based on Signal Filter</h2> -->
 +
                <!-- 文字 -->
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                <p>
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                    When signal 1 comes,a double promoter structure will help induce Cro and CII downstream pRE. At the same time constitutively produced CIII will guarantee enough CII to enhance the transcription downstream the pRE. Thus,there forms a positive feedback loop to direct a fast and strong expression of cro. A quantity of Cro represses transcription under pRM by binding to Cro binding site and blocks gene of interest 2’s expression, so it turns to a stable state of expressing gene of interest 1.
 +
                </p>
 +
                <!-- <img src="" alt=""> -->
 +
                <p>When signal 2 comes, under a certain inducible promoter,CI will be expressed and then binds to cI binding site within pR promoter,blocking gene of interest 1 and cIII’s expressions. With CIII’s reduction, FtsH gradually degrades CII to interrupt the stable state. Therefore, Cro’s expression immediately drop down , allowing transcription from pRM. The system turn to another stable state of gene of interest 2‘s expression.
 +
                </p>
 +
                <p>
 +
                    If there is no signal.Both genes of interest will be expressed. And if both of the two signal exist,the expression state will depend on the intensity of the initial signal input.
 +
                </p>
 +
                <h2>Eukaryotic version:</h2>
 +
                <p>It is a bi-stable system derived from Arabidopsis thaliana stress response system. Enzyme catalysis are core of this design to increase the efficiency of state transition.
 +
                </p>
 +
                <img src="https://static.igem.org/mediawiki/2016/1/1e/T--HUST-China--Description-Fig-prokaryote.png" alt="" class="img-responsive">
 +
                <p class="text-center">Fig*: circuit of Eukaryote version serving as bii-stable signal filter</p>
 +
                <p>Clade A protein phosphatases type 2C(PP2CA) and SUCROSE NONFERMENTING1-RELATED SUBFAMILY2(SnRK2s) protein kinase are both components of Abscisic acid signaling network in Arabidopsis thaliana. ABF2 is a leucine zipper transcription factor which basically binds ABA-response element (ABRE). ABF2 can be phosphorylated by SnRK2s and be efficiently dephosphorylated by PP2CA. Also, in the absence of Abscisic acid,SnRK2 kinases can be inactivated by PP2Cs thus shut down the gene expression efficiently.
 +
                </p>
 +
                <p>When signal 1(ON) comes, promoter RD29A drives expression of SnRK2.2, which can phosphorylate ABF2 (constitutively expressed). Comparing to protein co-facter association ,enzyme catalysis can produce a large quantity of phosphorylated ABF2 in a short time, then enhances pRD29A to turn on the gene of interest’s expression. When signal 2(OFF) comes,gene PP2CA expresses, dephosphorylating ABF2 and inactivating SnRK2.2, thus turning the system into OFF state.
 +
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Revision as of 11:43, 10 October 2016

Application

Description

OVERVIEW

This year, we want to offer a handy, adjustable and useful tool kit to everyone working on Synthetic Biology--Signal Filter. The core circuit is based on a positive feedback bi-stable or tri-stable system with the capacity of reducing noise and converting pulse signal into robust and persistent signal. To adapt to different experimental requirements,we also developed two versions of our Signal Filter--prokaryotic one and eukaryotic one.

Prokaryotic version:

It is a tri-stable system adapted from bacteriophage λ operon. In the operon, promoter RE is activated by CII trascriptional activator. Ftsh is an ATP-dependent host metalloprotease which will normally degrade CII, while CIII serves as an inhibitor of Ftsh to free CII. And CI can function as a inhibitor to block pR, while Cro can bind pRE to stop the downstream gene’s expression. The positive feedback control under pRE is used to enhance pulse signal 1 and convert it into robust stable signal.

Fig*: circuit of prokaryote version serving as tri-stable signal filter

When signal 1 comes,a double promoter structure will help induce Cro and CII downstream pRE. At the same time constitutively produced CIII will guarantee enough CII to enhance the transcription downstream the pRE. Thus,there forms a positive feedback loop to direct a fast and strong expression of cro. A quantity of Cro represses transcription under pRM by binding to Cro binding site and blocks gene of interest 2’s expression, so it turns to a stable state of expressing gene of interest 1.

When signal 2 comes, under a certain inducible promoter,CI will be expressed and then binds to cI binding site within pR promoter,blocking gene of interest 1 and cIII’s expressions. With CIII’s reduction, FtsH gradually degrades CII to interrupt the stable state. Therefore, Cro’s expression immediately drop down , allowing transcription from pRM. The system turn to another stable state of gene of interest 2‘s expression.

If there is no signal.Both genes of interest will be expressed. And if both of the two signal exist,the expression state will depend on the intensity of the initial signal input.

Eukaryotic version:

It is a bi-stable system derived from Arabidopsis thaliana stress response system. Enzyme catalysis are core of this design to increase the efficiency of state transition.

Fig*: circuit of Eukaryote version serving as bii-stable signal filter

Clade A protein phosphatases type 2C(PP2CA) and SUCROSE NONFERMENTING1-RELATED SUBFAMILY2(SnRK2s) protein kinase are both components of Abscisic acid signaling network in Arabidopsis thaliana. ABF2 is a leucine zipper transcription factor which basically binds ABA-response element (ABRE). ABF2 can be phosphorylated by SnRK2s and be efficiently dephosphorylated by PP2CA. Also, in the absence of Abscisic acid,SnRK2 kinases can be inactivated by PP2Cs thus shut down the gene expression efficiently.

When signal 1(ON) comes, promoter RD29A drives expression of SnRK2.2, which can phosphorylate ABF2 (constitutively expressed). Comparing to protein co-facter association ,enzyme catalysis can produce a large quantity of phosphorylated ABF2 in a short time, then enhances pRD29A to turn on the gene of interest’s expression. When signal 2(OFF) comes,gene PP2CA expresses, dephosphorylating ABF2 and inactivating SnRK2.2, thus turning the system into OFF state.