Team:BIT/project.html

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<div style=" float:right;width:180px; height:100px; background:none;" ><a href="/Main_Page" title="Main Page"><img style="width: 150px; height: 60px;margin-top: 30px;margin-right: 25px;" src="https://static.igem.org/mediawiki/2014/e/e4/BIT_igem_logo.png"></a></div>
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<div id="project_content" style="margin:15px 0 0 35px;" >
<div id="project_content" style="margin:15px 0 0 35px;" >
     <img id="BIT_project_logo" src="https://static.igem.org/mediawiki/2014/f/f4/BIT_project_logo.png" style="display:block; margin-left:25px;"/>
     <img id="BIT_project_logo" src="https://static.igem.org/mediawiki/2014/f/f4/BIT_project_logo.png" style="display:block; margin-left:25px;"/>
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<div id="Abstract" style=" background:url(../img/BIT_project_overview.png);margin:10px 0 30px 10px;">
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<h1>Abstract</h1>
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        <p>There are many types of radiation around people. In recent years,more and more organisms are suffering from the radiation damage, such as ionizing radiation, Ultraviolet radiation, electromagnetic radiation, etc . Although we can detect the different types of radiation by some instruments, It is still hard for human being to understand how serious the endangerment through the number on the screen is. So here we are. We create a biological system to detect the dosage of  radiation, so that we can easily know how much damage there is.
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The mechanism of our project by detecting the damage caused by radiation thus to measure the dosage of radiation. We construct 2 sensor systems separately. Both of them aim at the low dosage radiation damage, besides some other specific main targets.
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Sensor A is based on a toggle switch system, involving two types of promoters: recA and lac, which dividedly regulate the expression of the gene sequence of repressor lacl and lexA, whose product suppress promoter lac and recA. As upstream and downstream genes promote and mutually repress, the whole system has two stable output state considering the robust of the toggle switch system itself.
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Sensor B is based on the positive feedback existed with pSox, an oxidative stimulus inducible promoter.
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The amplifier system we assembly origins from the quorum sensing system existed in P.Aeruginosa.
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We combine the amplifier system and sensor system with microfluidics and device platform, which can precisely detect the extent of damage. </p>
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</div>
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<div id="sensorA" style=" background:url(../img/BIT_project_overview.png);margin:10px 0 30px 10px;">
<div id="sensorA" style=" background:url(../img/BIT_project_overview.png);margin:10px 0 30px 10px;">
<h1>SensorA</h1>
<h1>SensorA</h1>
         <p>The mechanism of Sensor A engineering bacteria</p>
         <p>The mechanism of Sensor A engineering bacteria</p>
<img src="https://static.igem.org/mediawiki/2014/2/29/BIT_sensorA.gif">
<img src="https://static.igem.org/mediawiki/2014/2/29/BIT_sensorA.gif">
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<p>This gene circuit applies bistable mechanism, involving two types of promoters: recA and lac, which dividedly regulate the expression of the gene sequence of repressor lacl and lexA, whose product suppress promoter lac and recA. The reporter gene of lacl and lexA thereafter bring out RFP<b>(red fluorescent protein)</b> and GFP<b>(green fluorescent protein)</b>, both of which could ~~~~. As upstream and downstream gene promote and mutually repress, the whole system has two stable output state:</p>
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<p>This gene circuit applies bistable mechanism, involving two types of promoters: recA and lac, which dividedly regulate the expression of the gene sequence of repressor lacl and lexA, whose product suppress promoter lac and recA. The reporter gene of lacl and lexA thereafter bring out RFP<b>(red fluorescent protein)</b> and GFP<b>(green fluorescent protein)</b>, both of which could be output signals that represent the current state of the system. As upstream and downstream gene promote and mutually repress, the whole system has two stable output state:</p>
<p>State A: As promoter precA works, repressor lacl is expressed, then the product lexArepresses promoter lac, and repressor lexA is not expressed, thus cannot exert depressant action to promoter precA. When promoter recA works while promoter plac is shut down, the whole system remains perfectly stable. Afterwards, the downstream reporter gene of promoter precA, GFP, is turned on, then product can be detected.</p>
<p>State A: As promoter precA works, repressor lacl is expressed, then the product lexArepresses promoter lac, and repressor lexA is not expressed, thus cannot exert depressant action to promoter precA. When promoter recA works while promoter plac is shut down, the whole system remains perfectly stable. Afterwards, the downstream reporter gene of promoter precA, GFP, is turned on, then product can be detected.</p>
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<div id="sensorB" style=" background:url(../img/BIT_background.png);margin:10px 0 30px 10px;">
<div id="sensorB" style=" background:url(../img/BIT_background.png);margin:10px 0 30px 10px;">
<h1>SensorB</h1>
<h1>SensorB</h1>
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         <p>Sensor B is a low-dosage-radiation-sensitive biological sensor. As back plan, its mechanism and effect is relatively uncomplicated and direct, which involves radiation response and grouping amplification. </p>
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         <p>Sensor B is a low-dosage-radiation-sensitive biological part, which involves both radiation response and quorum sensing. Sensor B applies a mechanism, completely from that of Sensor A. </p>
          
          
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         <p>Sensor B applies a completely different responsive mechanism, in order to supplement the one of cell radiation.
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         <p>The whole circuit is made up with SoxS P-LuxI-LuxPL-LuxR-LuxPR-LuxI-LuxPR-LasI, and LasI works as the final report gene produce protein PAI1 which is the signal of amplifier part. Sensor B can be divided into three functional units: Sensing, Amplifying, Reporting,4321bp long in all.</p>
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Sensor B composes of three parts, altogether 4321bp long. In sequence, the whole circuit is made up with SoxS P-LuxI-LuxPL-LuxR-LuxPR-LuxI-LuxPR-GFP, and GFP works as the final reporter gene.</p>
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<p>The first part is assembled by SoxS P-LuxI,while SoxS is a oxidation-mark-sensitive promoter. When cell gets radiated, it will produce a oxidation mark, which induces SoxS P to promote, thus making luxl produce AHL synthetase, catalyzing and synthesizing autoinducer information molecule AHL, and interacting with next part.</p>
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<p>The first unit consist of SoxS P-LuxI, while SoxS P is a oxidation stress-sensitive promoter. When cell gets radiated, it will produce an oxidation stress, which induces SoxS P and further promote luxl expression. As a result, AHL synthetase is produced, it will then catalyze and synthesize autoinducer information molecule AHL.</p>
      
      
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<p>The second part composes of LuxPL-LuxR-LuxPR-LuxI. LuxPL makes LuxR express. When cell got radiated, protein LuxR integrates with autoinduer AHL and form complex, which induces LuxPR to start expression of Luxl, and then comes to produce more autoinducer AHL and complex, therefore amplifying the effect of gene expreesion.</p>
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<p>The second unit composes of LuxPL-LuxR-LuxPR-LuxI. Generally, LuxPL gives a weak constitutive expression of luxR. When cell got radiated, protein LuxR integrates with autoinduer AHL and forms a complex, which subsequently increases the transcriptional rate of the lux pR promoter while also decreasing the transcriptional rate of the lux pL promoter. While the repression is much slighter generally. LuxPR starts the expression of Luxl, and then comes to produce more autoinducer AHL. In combination with LuxR, AHL thus increases the production of AHL. The whole unit is a positive feedback loop.</p>
      
      
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<p>The third part applies LuxPR-GFP structure, working as reporter gene in this sensor. Through the interaction and amplification in the first two parts, the complex will exert effect on promoter LuxPR and induce the expression of GFP. According to the fluorescence intensity, the radiation intensity could be detected directly and accurately after collecting and analyzing data.</p>
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<p>The third unit contains LuxPR-LasI, working as a reporter unit in this sensor. Through the interaction and amplification in the first two parts, the complex will exert effect on promoter LuxPR and induce the expression of LasI.</p>
<img src="https://static.igem.org/mediawiki/2014/9/91/BIT_sensorB.gif">
<img src="https://static.igem.org/mediawiki/2014/9/91/BIT_sensorB.gif">
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<img src="https://static.igem.org/mediawiki/2014/2/2d/BIT_amplifier.gif">
<img src="https://static.igem.org/mediawiki/2014/2/2d/BIT_amplifier.gif">
<p>In sensor, the amount of promoter expression stimulated by external condition can be detected by the expression amount of fluorescent protein, but most of all invisible. So, in order to increase it several times of growth, we need amplification effect. In common practice, quorum sensing, which is a grouping bacteria behavior regulation mechanism, is always applicable. It shows that bacteria respond to colony density and environmental change by sensing autoinducer. When the number of bacterial colonies reaches a certain quorum, it starts a series of gene expression to regulate group reaction. At the very beginning, we originally applied lux system in Vibrio Fischeri, for its mechanism is relatively simpler but innovatively harder. Then, we switched to las system and rhl system in pseudomonas aeruginosa.</p>
<p>In sensor, the amount of promoter expression stimulated by external condition can be detected by the expression amount of fluorescent protein, but most of all invisible. So, in order to increase it several times of growth, we need amplification effect. In common practice, quorum sensing, which is a grouping bacteria behavior regulation mechanism, is always applicable. It shows that bacteria respond to colony density and environmental change by sensing autoinducer. When the number of bacterial colonies reaches a certain quorum, it starts a series of gene expression to regulate group reaction. At the very beginning, we originally applied lux system in Vibrio Fischeri, for its mechanism is relatively simpler but innovatively harder. Then, we switched to las system and rhl system in pseudomonas aeruginosa.</p>
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<img src="https://static.igem.org/mediawiki/2014/a/ad/BIT_AM1.png" style="width: 300px;">
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<p>In the plasmid of amplification system, two activating transcription factors are in constant expression and remain in a certain concentration. With a certain level of autoinducer PAO1, pao1 integrates with lasR, the complex of which are bond to the target site of las promoter, then activating it and control the expression of the downstream reporter gene GFP in accord with the concentration of the amount of combination. In addition, this complex can also work on the promoter rhl as transcriptional activator of rhl system, and afterwards activating it.<br/>
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<p>In las system, inducer PAT1 enters cells and then integrates with LasR protein, and the conjugate combines the promoter of las1, then setting up the downstream expression.  
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The efficiency of complexes activating two promoters are relatively different. Theoretically, it is more efficient for the complex to take care of its own promoter than to work on the thl promoter as a transcription activator.<br/>
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On one hand, las1 gene expression brings out las1 protein, which could produce autoinducer PAT1, and then making fluorescent protein constantly expressed by cascade amplification effect and visually visible.</p>
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<p>On the other hand, it exerts amplification effect on prhl1 in rhl system. In rhl system, when autoinducer PAT2 enters cell and integrates with rhlR protein, and the conjugate combines the promoter of las1, then setting up the downstream gene expression, producing Rh1l protein, which gives out autoinducer PA12. Afterwards it makes fluorescent protein constantly expressed by cascade amplification effect.</p>
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<p>Concerning the large number of gene in these two systems, we construct two sections instead of one on two plasmids, for these two cascade amplification systems are interactive. The topological structure are as follows:</p>
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Actually, las and rhl system are not orthogonal, while las system disturbs the rhl system. PAO1 can not only bind to the lasR protein, also thlR protein, even in a very low level. Furthermore, the complex PAO1-rhlR activates promoter to express RFP.  
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<p>The efficiency of complexes activating two promoters are relatively different. Theoretically, it is more efficient for the complex to take care of its own promoter than to work on the thl promoter as a transcription activator.</p>
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<div id="Micro-fluidic chip" style=" background:url(../img/BIT_project_overview.png);margin:10px 0 30px 10px;">
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<p>Actually, las and rhl system are not orthogonal, while las system disturbs the rhl system. PAO1 can not only bind to the lasR protein, also thlR protein, even in a very low level. Furthermore, the complex PAO1-rhlR activates promoter to express RFP. </p>
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<h1>Micro-fluidic chip</h1>
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<h2>Material</h2>
 
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<p> PDMS glue; glass; semipermeable membrane (specifics are as follows)</p>
 
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<p>Sensor bacteria produces AHL molecules when receiving radiation, and transferred AHL to amplifier bacteria, which gives out fluorescent light, whose intensity can be detected by measuring the light intensity.</p>
 
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<p>Based on the simplified mechanism, the basic function of micro-fluidic chip contains:</p>
 
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<p>1/separate culture of sensor and amplified bacteria</p>
 
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<p>2/deliver the metabolite from sensor to amplifier, and mix it with amplifier bacteria as much as possible</p>
 
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<p>3/fluorescent light given out by bacteria B can be detected.</p>
 
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<p>4/detect multiple samples at a time</p>
 
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<br>
 
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<h2>Design of chips</h2>
 
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<p>Single channel pattern</p>
 
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<p>And the chip model to meet the needs of multiple data at one time is as follows.</p>
 
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<p>Through making model, pouring, punching and bonding, a perfect chip is made up.</p>
 
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<h2>Function</h2>
 
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<p>Sensor bacteria above receives radiation, and metabolizes AHL molecules, which pass the semipermeable membrane and transmit the amplifier to the detection hole.</p>
 
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<p>The transmission method remains unknown for we cannot make sure of the transmembrane way of AHL.</p>
 
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<div id="device" style=" background:url(../img/BIT_project_overview.png);margin:10px 0 30px 10px;">
 
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<h1>Device</h1>
 
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<h2>This device composes of three parts</h2>
 
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<p>1、 Internal circuit: “brain” and “heart” of the whole device, single chip microcomputer(SCM) as processing core. Control and sampled signals are received and processed in this unit, then making it possible to control device, switch signals, process algorithm and display on the screen.
 
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<p>2、 Optical structure: this is designed to detect these samples which are excited by laser and make out fluorescent light, then detector will receive it and make high-precision measurement.
 
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<p>3、 Appearance design: out product is not only beautifully designed, and also functionally sufficient.
 
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<h2>Characteristics</h2>
 
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<p>Biological sample detection is a long tough process, which costs a great amount of money and labor. However, our device possesses great character of high-throughput and high-precision, also simplifies conductive process, visualizes detection result, and even substantially lowers the cost. Even though this device serves for project engineering, which can be applied only in our program, its electric cores and optical structure could be easily transferred to other relevant detection. This is greatly significant in further device refinement.</p>
 
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       <label for="toggle1" class="animate">MENU<i class="fa fa-bars float-right"></i></label>
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        <li class="animate" id="project_logo" onclick="javascript:goto('BIT_project_logo');return false;"><a href="#BIT_project_overview"><p>SensorA
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      <li class="animate" id="Abstract"> <a href="#Abstract"><p>Abstract
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         <li class="animate" id="project_gene_circuit" onclick="javascript:goto('BIT_gene_circuit');return false;"><a href="#BIT_gene_circuit"><p> Amplifier</p></a></li>
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         <li class="animate" id="Amplifier"><a href="#amplifier"><p> Amplifier</p></a></li>
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        <li class="animate" id="project_gene_circuit" onclick="javascript:goto('BIT_gene_circuit');return false;"><a href="#BIT_gene_circuit"><p> Micro-fluidic chip</p></a></li>
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        <li class="animate" id="project_gene_circuit" onclick="javascript:goto('BIT_gene_circuit');return false;"><a href="#BIT_gene_circuit"><p> Device</p></a></li>
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        <li class="animate" id="achievement" ><a href="https://2014.igem.org/Team:BIT/achievement.html"><p>Achievement</p></a></li>
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           <p>Radiation as a natural part of our environment. Annually, worldwide, more than 3,600 million X-ray examinations are performed, 37 million nuclear medicine procedures are carried </p>
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           <p>[1]Pseudomonas aeruginosa Autoinducer Enters and Functions in Mammalian Cells. Simon C. Williams,Erin K. Patterson,Nancy L. Carty,3John A. Griswold,Abdul N. Hamood,and Kendra P. Rumbaugh.JOURNAL OF BACTERIOLOGY, Apr. 2004, p. 2281-2287</br>
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[2]Regulation of las and rhl Quorum Sensing in Pseudomonas aeruginosa. EVERETT C. PESCI, JAMES P. PEARSON, PATRICK C. SEED, BARBARA H. IGLEWSKI.JOURNAL OF BACTERIOLOGY, May 1997, p. 3127–3132 </br>
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[3]Functional Analysis of the Pseudomonas aeruginosa Autoinducer PAI. LUCIANO PASSADOR,KENNETH D. TUCKER,KEVIN R. GUERTIN,MICHEL P. JOURNET,ANDREW S. KENDE,BARBARA H. IGLEWSKI.JOURNAL OF BACTERIOLOGY, Oct. 1996, p. 5995–6000 </br>
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[4]Dimerization of the quorum sensing regulator RhlR: development of a method using EGFP fluorescence anisotropy.Isabelle Ventre,Fouzia Ledgham,Valérie Prima, Andrée Lazdunski, Maryline Foglino and James N. Sturgis.Molecular Microbiology (2003) 48(1), 187–198</br>
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[5] Quorum sensing: cell-to-cell communication in bacteria.Waters CM1, Bassler BL. Annu Rev Cell Dev Biol. 2005;21:319-46.</br>
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[6]Signal-amplifying genetic circuit enables in vivo observation of weak promoter activation in the Rhl quorum sensing system. Karig D1, Weiss R. Biotechnol Bioeng. 2005 Mar 20;89(6):709-18.</br>
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[7] A New Transcriptional Repressor of the Pseudomonas aeruginosa Quorum Sensing Receptor Gene lasR.Longo, F; Rampioni, G; Bondi, R; Imperi, F; Fimia, GM; Visca, P; Zennaro, E; Leoni, L; Leoni, L (reprint author), Univ Roma Tre, Dept Sci, Rome, Italy. PLOS ONE, 2013; 8 (7)</br>
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          <p>out, and 7.5 million radiotherapy treatments are given.”-from World Health Organization Website.
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Radiation correlates with us tightly. With technique development, the use of radiation in our life ascends. However inappropriate dosage may jeopardize h</p>
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          <p>ealth condition potentially. Most of the public are nevertheless not conscious of the latent risks.
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Therefore an engineered bio-system was constructed based on synthetic biology.  
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The system is composed of amplifier and sensor. Once the sensor wa</p>
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           <p>s exposed to low dose radiation, it would produce signal molecules continuously. The signals would be conveyed to amplifier then and brought the amplifier into operation. Thus the amplifier would produce high massive signals for precise detection. And aiming at precise measurements, we equip the sensor with a switch to pause, hence we can detect the stable signal of the system.s exposed to low dose radiation, it would produce signal molecules continuously. The signals would be conveyed to amplifier then and brought the amplifier into operation. Thus the amplifier would produce high massive signals for precise detection. And aiming at precise measurements, we equip the sensor with a switch to pause, hence we can detect the stable signal of the system.s exposed to low dose radiation, it would produce signal molecules continuously. The signals would be conveyed to amplifier then and brought the amplifier into operation. Thus the amplifier would produce high massive signals for precise detection. And aiming at precise measurements, we equip the sensor with a switch to pause, hence we can detect the stable signal of the system.</p>
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           <p>Experiment</br>
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2014.5~6 </br>
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Look up for references/Brainstrom</br>
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2014.6.15-2014.7.15</br>
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SensorA  Plasmid  Construction</br>
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2014.7.16-2014.10.10</br>
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SensorB & Amplifier  Plasmids Construction</br>
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Pre-experiment</br>
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1.sensorA :IPTG-induce experiment for 8 times</br>
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2.sensorA :UV radiation experiment for 8 times</br>
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3.sensorA :Ionizing radiation/γ-ray radiation experiment once</br>
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4.amplifier :Autoinducer PAO1 (time and concentration gradient) induced twice</br>
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</br>
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Data</br>
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Fluorescence  Detection  of  SensorA & Amplifier’s  pre-experiment</br>
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2014.9.15-2014.9.25</br>
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Mathematical  Model  Construction</br>
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Humanpractice:</br>
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8.22-8.23  CCIC</br>
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8.28  Academic Exchange with BUCT</br>
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9.29  LZU</br>
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8.11 am: Environmental Protection Bureau(EPB) Interview</br>
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pm: Radiology Department Interview</br>
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10.11 </br>
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The freshmen mobilization</br>
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png"><span>blala</span></td>
 
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        <td>abubuvljvlugliuhjhlohhoinonion.hoinabubuvljvlugliuhjhlohhoinonion.hoinabubuvljvlugliuhjhl</td>
 
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            <td>abubuvljvlugliuhjhlohhoinonion.hoinabubuvljvlugliuhjhlohhoinonion.hoinabubuvljvlugliuhjhl</td>
 
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Latest revision as of 03:39, 18 October 2014

radiation measurement

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