Tianjin/DIODE

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==Design==
==Design==
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Bio-diode: The diode, one of the most significant parts in the electric integrate circuit and electronic equipment, functions as allowing the current get through when the diode is positive-biased, and cut off while it is negative-biased. When the negative voltage increases to a threshold, the diode tends to be negatively broken down. In this project, the molecular signal AHL is considered as the current of electric circuit, the generator, amplifiers, blocks and detector cells are constructed with the parts provided by MIT and then are equipped in series in order to achieve the cellular and molecular biological diode.
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<font size="5" color="#60ff00">Brief Introduction of Bio-diode</font>
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<br><br>
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[[Image:TJU1DD001.gif|500px]]<br>
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Generator: Generator cells could produce AHL signal constantly which could be sensed by special gene sequence to activate particular function. In our experiment, the production of AHL is controlled by LuxI promoter and could be promoted by the addition of IPTG and the function of Generator could be verified by the detector cells.
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Reporter cell: this cell is designed to test the concentration of AHL signal. In this cell, LuxR gene under the control of tet promoter could produce LuxR protein which could activite the following Lux pR promoter when binding to certain amount of AHL signal, thus GFP expressed to make this cell green within the view of a fluoresence microscope. The intensity of fluoresence could be measured by a fluoresence spectrophometer to decide the concentration of AHL signal roughly.
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<font size="3" color="#ffff00" rowspan=2 > The diode, one of the most significant parts in the electric integrate circuit and electronic equipment, functions as allowing the current get through when the diode is positive-biased, and cut off while it is negative-biased. When the negative voltage increases to a threshold, the diode tends to be negatively broken down. In this project, the molecular signal AHL is considered as the current of electric circuit.Three types of cells: [[Tianjin/DIODE/Generator cells|Generator cells]]、[[Tianjin/DIODE/Amplifier cells|Amplifier cells]] and [[Tianjin/DIODE/Block cells|Block cells]] are immobilized into small beads with a diameter of 0.5 millimeter. Then different beads are settled into particular parts of our specially designed equipment sequentially and respectively.The diode is built up with the amplifier cells and the block cells.The generator cells are thought as the electrical source and there are detector cells,which are considered as the lamp ,to detect the AHL.
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Amplifier cells: Also containing a LuxR protein producer together with an AHL producer controlled by a Lux pR promoter, the amplifier cells could generate more AHL signal when it combines with the environmental AHL signal.  
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Block cells: Block cells contain a gene coding for a special enzyme digesting the AHL signal. The concentration of AHL signals in liquid culture could dramatically decrease after passing through the block cell congeries.
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<br>The liquid culture flows through the generator cells and carries the AHL signal into the amplifier cells which are activated to make more AHL signals. <br>Then the concentration of AHL accumulates to such a level that block cells could not eliminate them completely, and the rest could be detected by [[Tianjin/DIODE/Reporter cells|Detector cells]], a condition called ‘positive-biased pass’.</font><br>
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Mechanism: The three types of cells, generator cell、amplifier cells and block cells are immobilized into small beads with a diameter of 0.5 millimeter, then different beads are settled into particular parts of our specially designed equipment sequentially and respectively. Firstly, the liquid culture flow through the generator cells and carry the AHL signal into the amplifier cells which are activated to make more AHL signals. Finally, the concentration of AHL accumulates to such a level that block cells could not eliminate them completely, and the rest could be detected by reporter cells, a condition called ‘pass’ On the contrary, if the positions of amplifier cells and block cells in the equipment are reversed, the AHL produced by generator cell would be degraded by block cells at the very beginning resulting in no amplification of the signal, thus, hardly AHL could be detected in the outflow culture, a condition called ‘block’. By this way, we successfully simulate the two most important features of diode and our project could be connected with other devices to construct more complex circuit system in the future.
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<table width="99%" border="10"  cellpadding="0" cellspacing="0" style="padding: 10px; background-color: #0099FF">
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[[Image:TJU1DD001.jpg|500px]]<br>
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<tr>
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<td><center>[[Image:diodedh.gif|500px]]            <font size="8" color="#0000CC">OK!</font> [[Image:happy.gif|150px]]</center></td>
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</tr></table>
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<br><br>
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<font size="3" color="#ffff00" rowspan=2 >On the contrary, if the positions of amplifier cells and block cells in our equipment are reversed, the AHL produced by generator cells would be degraded by block cells at the very beginning resulting in no amplification of the signal. Thus, hardly AHL could be detected in the outflow culture, a condition called ‘negative-biased block’. </font>
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<table width="99%" border="10"  cellpadding="0" cellspacing="0" style="padding: 10px; background-color: #0099FF">
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<td><center>[[Image:diodeblock.gif|500px]]            <font size="8" color="#0000CC">Blocked!</font>[[Image:sad.gif]]</center></td>
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</tr></table>
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<br><br>
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<font size="3" color="#ffff00" rowspan=2 >When the input AHL signal produced by the generator cells is stronger than digesting capacity of the block cells , the amplifier cells will still be activated by the left AHL signal, which is what we called "breakdown".</font>
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<table width="99%" border="10" cellpadding="0" cellspacing="0" style="padding: 10px; background-color: #0099FF">
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<td><center>[[Image:breakdown.gif|500px]]           <font size="5" color="#0000CC">Broken down!</font>[[Image:cry.gif]]</center></td>
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</tr></table>
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<br><br>
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==Model==
==Model==
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Figure 1: the production of AHL could be accelerated both by the addition of IPTG and increasement if living cells.
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We construct a mathematical model to simulate the features of our bio-diode mainly by Ordinary Differential Equations. With the help of this model,we show the ideal performance of each immobilized cells ,especially the amplifier cells, under different cell densities and flux rates.We also examine the features of different cells under the conditions of positive-biased through, negative-biased block and  negative-biased breakdown. Thus the data and conclusion derived from the model are quite helpful for our further wet experiment.
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[[Image:TJU4L1.jpg|500px]]<br>
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<br>
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Figure 2: the concentration of AHL decreases nearly proportionally to the flux rate of liquid culture since the rate of AHL production maintains stable.  
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<font size="3" color="#0000CC" bgcolor="#FF9933">1. </font>[[Tianjin/DIODE/Model0|<font size="3" color="#0000CC">Model design and construction</font>]]
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[[Image:TJU4L2.jpg|500px]]<br>
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Figure 3: concentration of AHL increases expotentially during the first 40 minutes after the input of liquid culture, but keep constant from then on.
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[[Image:TJU4L.jpg|500px]]<br>
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Figure 4: Rough description of relationship between the input of AHL signal and the output of AHL signal when the flux passes through the amplified cells. Low concentration input failed to initialize the amplication process.
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[[Image:TJU8P1.jpg|500px]]<br>
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Figure 5: With the rising of flux and input signal, the output signal tend to rocketrise at a particular value,which indicates the value of threshold.
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[[Image:TJU8P2.jpg|500px]]<br>
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==Experiment==
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1.RFP fluorescence of the decter at different cell cencertration<br>
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[[Image:TJU1DE002.jpg|500px]]<br>
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<font size="3" color="#0000CC">2. <font size="3" color="#0000CC">Model result</font>
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[[Image:TJU1DE003.jpg|500px]]<br>
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<font size="3" color="#0000CC">2.1 </font>[[Tianjin/DIODE/Model|<font size="3" color="#0000CC">Model of Generator Cells</font>]]<br>[[Image:TJU4L1.jpg|80px]][[Image:TJU4L2.jpg|80px]][[Image:TJU4L.jpg|80px]][[Image:TJU8P1.jpg|80px]][[Image:TJU8P2.jpg|80px]]
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<font size="3" color="#0000CC">2.2 </font>[[Tianjin/DIODE/Model1|<font size="3" color="#0000CC">Model of Block Cells</font>]]
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<br>[[Image:TJU4j.jpg|80px]][[Image:TJU4j2.jpg|80px]]
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2.GFP fluorescence of the decter at different cell cencertration<br>
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<font size="3" color="#0000CC">2.3 </font>[[Tianjin/DIODE/Model2|<font size="3" color="#0000CC">Model of Amplifier Cells</font>]]
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[[Image:TJU1DE004.jpg|500px]]<br>
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<br>[[Image:TJU8P1.jpg|80px]][[Image:TJU8P2.jpg|80px]]
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<font size="3" color="#0000CC">2.4 </font>[[Tianjin/DIODE/Model3|<font size="3" color="#0000CC">Model of whole system</font>]]
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<br>[[Image:TJUI.jpg|80px]][[Image:TJUI1.jpg|80px]][[Image:TJUI2.jpg|80px]][[Image:TJUIJ.jpg|80px]][[Image:TJUIJP.jpg|80px]][[Image:TJUzf.jpg|80px]]
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3.GFP fluorescence of the decter at different cencertration of AHL<br>
 
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[[Image:TJU1DE001.jpg|500px]]<br>
 
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|width="960px" style="padding: 10px; background-color: #0099FF" |
|width="960px" style="padding: 10px; background-color: #0099FF" |
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==Modeling==
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==Experiment==
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We use ODEs to simulate the system. Datas used in the model are based on the literature and would be adjusted to match the experimental results.In the model we assume that the flux through each pipeline is identical and the block maintains a stable state.
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The function of detector cells has to be defined first to build a relationship between the concentration of AHL and the fluorescence intensity. The experimental results show that at a range of AHL concentrations the concentration of AHL is proportional to the fluorescence intensity, so within this extent, the fluorescence intensity could be regarded as the value of output signal which is convenient to detect and quantificationally defined. Then we test the functions of other three types of cells in immobilized beads by using the detector cells, proving that the generator and block cells work perfectly but the amplified cells should be improved due to the self-induction of its Lux Promoter. Finally, the three types of cells are fulfilled into our particular designed equipment whose functions are also researched carefully, and in order to solve the problem of amplifier cells, we transfer this part into another plasmid whose replication is controlled by IPTG. Although the data are not the same as our in silico model, it is substantiated that every part of our design has worked well and the main characters of diode including the 'positive-biased through', 'negative-biased block', 'negative-biased breakdown' are realized by our equipment.
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The simulation result is as follows:<br>
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<font size="3" color="#0000CC">1.</font>[[Tianjin/DIODE/Experiment|<font size="3" color="#0000CC">Data and Graphs of Detector cells</font>]]
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<font size="6" >Amplifier</font> <br>
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<font size="3" color="#0000CC">2.</font>[[Tianjin/DIODE/Experiment1|<font size="3" color="#0000CC">Function of immobilized cells</font>]]
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1.relation between the input concentration of the amplifier and the output concentration of the amplifier <br>
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<center>[[Image:TJU1DM001.jpg|370px]]<br></center>
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2.relation between the flux of the amplifier and the output concentration of the amplifier<br>
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<center>[[Image:TJU1DM002.jpg|370px]]<br></center>
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<font size="6" >Block</font> <br>
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<font size="3" color="#0000CC">3.</font>[[Tianjin/DIODE/Experiment2|<font size="3" color="#0000CC">Assembly of equipment</font>]]
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1.relation between the input concentration of the block and the output concentration of the block <br>
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<center>[[Image:TJU1DM003.jpg|370px]]<br></center>
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2.relation between the flux of the block and the output concentration of the block<br>
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<center>[[Image:TJU1DM004.jpg|370px]]<br></center>
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3.relation between the concentration of AIIA in the block and the output concentration of the block <br>
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<center>[[Image:TJU1DM005.jpg|370px]]<br></center>
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4.relation between the flux of the block and the output concentration of the block in different concentration of AIIA<br>
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<center>[[Image:TJU1DM006.jpg|370px]]<br></center>
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<font size="6" >Flow direction from amplifier to block</font> <br>
 
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1.relation between the concentration and the flux at different proportion between the volume of the ampilier and the volume of the block.(the left image is a part of the left image)<br>
 
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<center>[[Image:TJU1DM007.jpg|370px]][[Image:TJU1DM008.jpg|370px]]<br></center>
 
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<font size="6" >Flow direction from block to amplifier(breakdown)</font> <br>
 
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1.relation between the concentration and the input concentration at different proportion betweeen the volume of the amplifier and the volume of the block.(the concentretion contains two curves,the amplifier(the output concentration) and the block)<br>
 
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<center>[[Image:TJU1DM009.jpg|370px]]<br></center>
 
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2.relation between the concentration and the input concentration at different concentration of AIIA.(the concentretion contains two curves,the amplifier(the output concentration) and the block)<br>
 
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<center>[[Image:TJU1DM010.jpg|370px]]<br></center>
 
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3.relation between the concentration and the input concentration at different flux.(the concentretion contains two curves,the amplifier(the output concentration) and the block)<br>
 
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<center>[[Image:TJU1DM011.jpg|370px]]<br></center>
 
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Latest revision as of 03:52, 27 October 2007

Design

Brief Introduction of Bio-diode

The diode, one of the most significant parts in the electric integrate circuit and electronic equipment, functions as allowing the current get through when the diode is positive-biased, and cut off while it is negative-biased. When the negative voltage increases to a threshold, the diode tends to be negatively broken down. In this project, the molecular signal AHL is considered as the current of electric circuit.Three types of cells: Generator cellsAmplifier cells and Block cells are immobilized into small beads with a diameter of 0.5 millimeter. Then different beads are settled into particular parts of our specially designed equipment sequentially and respectively.The diode is built up with the amplifier cells and the block cells.The generator cells are thought as the electrical source and there are detector cells,which are considered as the lamp ,to detect the AHL.


The liquid culture flows through the generator cells and carries the AHL signal into the amplifier cells which are activated to make more AHL signals.
Then the concentration of AHL accumulates to such a level that block cells could not eliminate them completely, and the rest could be detected by Detector cells, a condition called ‘positive-biased pass’.


Diodedh.gif OK! Happy.gif




On the contrary, if the positions of amplifier cells and block cells in our equipment are reversed, the AHL produced by generator cells would be degraded by block cells at the very beginning resulting in no amplification of the signal. Thus, hardly AHL could be detected in the outflow culture, a condition called ‘negative-biased block’.


Diodeblock.gif Blocked!Sad.gif




When the input AHL signal produced by the generator cells is stronger than digesting capacity of the block cells , the amplifier cells will still be activated by the left AHL signal, which is what we called "breakdown".

Breakdown.gif Broken down!Cry.gif



Model

We construct a mathematical model to simulate the features of our bio-diode mainly by Ordinary Differential Equations. With the help of this model,we show the ideal performance of each immobilized cells ,especially the amplifier cells, under different cell densities and flux rates.We also examine the features of different cells under the conditions of positive-biased through, negative-biased block and negative-biased breakdown. Thus the data and conclusion derived from the model are quite helpful for our further wet experiment.
1. Model design and construction


2. Model result

2.1 Model of Generator Cells
TJU4L1.jpgTJU4L2.jpgTJU4L.jpgTJU8P1.jpgTJU8P2.jpg
2.2 Model of Block Cells
TJU4j.jpgTJU4j2.jpg
2.3 Model of Amplifier Cells
TJU8P1.jpgTJU8P2.jpg
2.4 Model of whole system
TJUI.jpgTJUI1.jpgTJUI2.jpgTJUIJ.jpgTJUIJP.jpgTJUzf.jpg

Experiment

The function of detector cells has to be defined first to build a relationship between the concentration of AHL and the fluorescence intensity. The experimental results show that at a range of AHL concentrations the concentration of AHL is proportional to the fluorescence intensity, so within this extent, the fluorescence intensity could be regarded as the value of output signal which is convenient to detect and quantificationally defined. Then we test the functions of other three types of cells in immobilized beads by using the detector cells, proving that the generator and block cells work perfectly but the amplified cells should be improved due to the self-induction of its Lux Promoter. Finally, the three types of cells are fulfilled into our particular designed equipment whose functions are also researched carefully, and in order to solve the problem of amplifier cells, we transfer this part into another plasmid whose replication is controlled by IPTG. Although the data are not the same as our in silico model, it is substantiated that every part of our design has worked well and the main characters of diode including the 'positive-biased through', 'negative-biased block', 'negative-biased breakdown' are realized by our equipment.

1.Data and Graphs of Detector cells

2.Function of immobilized cells

3.Assembly of equipment