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

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{{HUST-China}}
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<p>This page is used by the judges to evaluate your team for the <a href="https://2016.igem.org/Judging/Medals">gold medal criterion for proof of concept</a>. </p>
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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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                                <li><a href="https://2016.igem.org/Team:HUST-China/Basic_Part">Basic part</a></li>
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    <div class="container-fluid member-head">
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            <h2 class="text-center">Proof of Concept</h2>
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        </div>
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    </div>
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    <div class="container-fluid">
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        <div class="text-content container" id="text-content">
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            <div class="ref"></div>
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            <article>
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                <!-- h2一级标题 -->
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                <h2>OVERVIEW</h2>
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                <!-- 文字 -->
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                <p>
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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 developed two versions of our Signal Filter: prokaryotic one and eukaryotic one.
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                </p>
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                <p>dfwef</p>
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                <h3>Hello world</h3>
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                <p>fdwfewf</p> -->
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                <h2 id="location_Pro_ver">Prokaryotic version</h2>
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                <p>
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                    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. CI can function as an inhibitor to block pR, while Cro can bind pRM to stop the expression of downstream gene. The positive feedback control under pRE is used to enhance pulse signal 1 and convert it into robust stable signal.
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                </p>
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                <!-- 图片 -->
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                <img src="https://static.igem.org/mediawiki/2016/8/88/T--HUST-China--Description-Fig-Eukaryote.png" alt="" class="img-responsive">
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                <p  style="text-align:center">Fig 1: circuit of prokaryote version serving as tri-stable signal filter</p>
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                <!-- <h2>Creation based on Signal Filter</h2> -->
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                <!-- 文字 -->
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                <p>
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                    When pulse 1 is sensed, 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 downstream transcription of 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.
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                </p>
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                <!-- <img src="" alt=""> -->
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                <p>When pulse 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, expression of cro immediately drops down, allowing transcription from pRM. The system turns to another stable state of gene of interest 2’s expression.
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                </p>
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                <p>
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                    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.
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                </p>
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                <div class="container-fluid index-details1 index-details">
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                                                <p style="margin-left:100px">
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                                                    According to the Flourescence measurement curve, we can see clearly that GFP level increased over time and it showed significant difference from CK. See more details in our Results page.
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                                                </p>
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                                                <p><a href="https://2016.igem.org/Team:HUST-China/Results#location_pro&pro" class="btn btn-primary" role="button">More details</a></p>
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                                            </div>
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                                            <img src="https://static.igem.org/mediawiki/2016/e/ef/T--HUST-China--CII-pRE_plate.png" alt="" class="img-responsive">
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                                        </div>
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                                    </div>
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                                </div>
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                            </div>
  
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<p> Delete this box in order to be evaluated for this medal. See more information at <a href="https://2016.igem.org/Judging/Pages_for_Awards/Instructions"> Instructions for Pages for awards</a>.</p>
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                                                    We did Fluorescence microscope detection after 30, 120 and 240 minutes induction. According to the figture below, we can tell qualitively that pRE leakage are at relative low level and CII can efficiently activate the promoter. See more details in our Results page.
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                                                </p>
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                                                <p><a href="https://2016.igem.org/Team:HUST-China/Results#location_pro&pro" class="btn btn-primary" role="button">More details</a></p>
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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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                                            <img src="https://static.igem.org/mediawiki/2016/4/4b/T--HUST-China--Experiments-CII-pRE_Flou-detec.png" alt="" class="img-responsive">
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<h4> What should we do for our proof of concept? </h4>
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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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As the Relative Fluorescent intensity measurement data shows, CI can inhibit pR in minor degree but the leakage expression under pR can’t be ignored, so we should consider to increase the binding sites within pR or the amount of CI coding sequence in the circuit. See more details in our Results page.
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                                                <p><a href="https://2016.igem.org/Team:HUST-China/Results#location_pro&pro" class="btn btn-primary" role="button">More details</a></p>
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                                            <img src="https://static.igem.org/mediawiki/2016/d/d1/T--HUST-China--Experiments-CI-pR_plate.png" alt="" class="img-responsive">
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We detected GFP reporter in E.coli after induction of 20minute, 120minutes and 240minutes through 20 times of amplification. See more details in our Results page.
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                                                </p>
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                                                <p><a href="https://2016.igem.org/Team:HUST-China/Results#location_pro&pro" class="btn btn-primary" role="button">More details</a></p>
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                                            <img src="https://static.igem.org/mediawiki/2016/6/63/T--HUST-China--Experiments-CI-pR_Flou-detec.png" alt="" class="img-responsive">
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We characterized cro and pRM inhibition by the same method as CI and pR’s. See more details in our Results page.
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                                                </p>
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                                                <p><a href="https://2016.igem.org/Team:HUST-China/Results#location_pro&pro" class="btn btn-primary" role="button">More details</a></p>
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                                            <img src="https://static.igem.org/mediawiki/2016/1/15/T--HUST-China--CI-pR_inhibition.png" alt="" class="img-responsive">
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We use IPTG with final concentration of 1mM to induce the GFP-LVAssrAtag and measure the relative fluorescence through plate reader with Excitation light 495nm. See more details in our Results page.
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                                                </p>
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                                                <p><a href="https://2016.igem.org/Team:HUST-China/Results#location_tri_fun" class="btn btn-primary" role="button">More details</a></p>
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                                            <img src="https://static.igem.org/mediawiki/2016/f/fa/T--HUST-China--Experiments-LVAssrA.png" alt="" class="img-responsive">
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                <h2 id="location_Euk_ver">Eukaryotic version</h2>
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                <p>It is a bi-stable system derived from Arabidopsis thaliana stress response system. Enzyme catalysis is the core of this design to increase the efficiency of state transition.
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                </p>
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                <img src="https://static.igem.org/mediawiki/2016/1/1e/T--HUST-China--Description-Fig-prokaryote.png" alt="" class="img-responsive">
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                <p  style="text-align:center">Fig 2: circuit of Eukaryote version serving as bii-stable signal filter</p>
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                <p>Clade A protein phosphatases type 2C (PP2CA) and Sucrose Nonfermenting1 Related Subfamily 2 (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.
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                </p>
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                <p>When signal 1 (ON) comes, promoter RD29A drives expression of SnRK2.2, which can phosphorylate ABF2 (constitutively expressed). Compared with protein co-facter association, enzyme catalysis can produce a large quantity of phosphorylated ABF2 in a short time, then enhance pRD29A to turn on the gene of interest’s expression. When signal 2 (OFF) comes, gene PP2CA expresses, dephosphorylates ABF2 and inactivates SnRK2.2, thus turning the system into OFF state.
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                                                    We ran a SDS-PAGE of the culture supernatants of induced cells to identify the three recombinant proteins. See more details in our Results page.
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                                                </p>
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                                                <p><a href="https://2016.igem.org/Team:HUST-China/Results#location_pro_exp" class="btn btn-primary" role="button">More details</a></p>
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                                        We simulated our project in cellular level and Intestinal level to help us figure out the feasibility of it and guide us the direction of further improvement. See more results in our Model page.
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                                    <p><a href="https://2016.igem.org/Team:HUST-China/Model" class="btn btn-primary" role="button">More details</a></p>
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Latest revision as of 02:45, 20 October 2016

Proof

Proof of Concept

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 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. CI can function as an inhibitor to block pR, while Cro can bind pRM to stop the expression of downstream gene. The positive feedback control under pRE is used to enhance pulse signal 1 and convert it into robust stable signal.

Fig 1: circuit of prokaryote version serving as tri-stable signal filter

When pulse 1 is sensed, 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 downstream transcription of 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 pulse 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, expression of cro immediately drops down, allowing transcription from pRM. The system turns 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 is the core of this design to increase the efficiency of state transition.

Fig 2: circuit of Eukaryote version serving as bii-stable signal filter

Clade A protein phosphatases type 2C (PP2CA) and Sucrose Nonfermenting1 Related Subfamily 2 (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). Compared with protein co-facter association, enzyme catalysis can produce a large quantity of phosphorylated ABF2 in a short time, then enhance pRD29A to turn on the gene of interest’s expression. When signal 2 (OFF) comes, gene PP2CA expresses, dephosphorylates ABF2 and inactivates SnRK2.2, thus turning the system into OFF state.