Team:Peking/Project/Devices

From 2013.igem.org

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</li>
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<li><a href="https://2013.igem.org/Team:Peking/Project/Plugins">Adaptors</a></li>
<li><a href="https://2013.igem.org/Team:Peking/Project/Plugins">Adaptors</a></li>
<li><a href="https://2013.igem.org/Team:Peking/Project/BandpassFilter">Band-pass Filter</a></li>
<li><a href="https://2013.igem.org/Team:Peking/Project/BandpassFilter">Band-pass Filter</a></li>
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                                <li><a href="https://2013.igem.org/Team:Peking/Project/Devices">Devices</a></li>
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                                <li><a href="https://2013.igem.org/Team:Peking/Model">Band-pass Filter</a></li>
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                                <li><a href="https://2013.igem.org/Team:Peking/ModelforFinetuning">Biosensor Fine-tuning</a></li>
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<a >Data page</a>
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<a href="">Data page</a>
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                                 <li><a href="https://2013.igem.org/Team:Peking/HumanPractice/Questionnaire">Questionnaire</a></li>
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                                 <li><a href="https://2013.igem.org/Team:Peking/HumanPractice/Questionnaire">Questionnaire Survey</a></li>
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<li><a href="https://2013.igem.org/Team:Peking/HumanPractice/FactoryVisit">Factory Visit</a></li>
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<li><a href="https://2013.igem.org/Team:Peking/HumanPractice/FactoryVisit">Visit and Interview</a></li>
                                 <li><a href="https://2013.igem.org/Team:Peking/HumanPractice/ModeliGEM">Practical Analysis</a></li>
                                 <li><a href="https://2013.igem.org/Team:Peking/HumanPractice/ModeliGEM">Practical Analysis</a></li>
<li><a href="https://2013.igem.org/Team:Peking/HumanPractice/iGEMWorkshop">Team Communication</a></li>
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         <a href="https://2013.igem.org/Main_Page"><img id="iGEM_logo" src="https://static.igem.org/mediawiki/igem.org/4/48/Peking_igemlogo.jpg"/></a>
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<div id="MajorBody">   
<div id="MajorBody">   
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          <h1 id="SensorsListTitle">Purpose-Built Device</h1>
           <ul id="ProjectList">
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                 <li class="SensorsListItem"><a href="#Mileston1">Introduction</a><li>
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                 <li class="SensorsListItem"><a href="#Milestone1">Purposes</a><li>
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                 <li class="SensorsListItem"><a href="#Milestone2">Alginate Encapsulation</a><li>
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                 <li class="SensorsListItem"><a href="#Mileston3">Construction </a><li>
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                 <li class="SensorsListItem"><a href="#Milestone4">Advanced Design</a><li>
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                 </ul>
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           <h1 id="ModelOverviewTitle">Band-pass Filter</h1>
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           <h1 id="ModelOverviewTitle">Purpose-Built Device</h1>
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<p id="PageSubtitle1">Introduction</p>
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      <h1 id="Purposes";>Purposes</h1>
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      <p style="position:relative; top:-10px;">In-field detection of aromatic compounds in environments has always been desirable, and convenience has always been an important requirement for in-field detection. To meet this requirement, the detection process should be fast and the result should be easily read by naked eyes. Furthermore, the device we design to realize all these should be readily portable.  
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    <p id="Content1">Hitherto we have constructed a biosensor toolkit for aromatic compounds with wide sensing coverage and high orthogonality between different sensing modules. However, in order to cope with the need of in-field detection, we should further develop advanced equipment for our toolkit to implement fast, economical and convenient measurement of aromatic compounds in various environments.  
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When designing such a portable user-friendly device, the most challenging part would be developing the preservation method. As we choose <i>Escherichia coli</i>, which is unable to germinate spores, as our host strain, special method should be developed to protect the bacteria from temperature changes and physical stress while keeping them alive. We used alginate encapsulation as a basic solution to this problem. Based on such preliminary design, we further built an advanced device to realize convenient quantitative measuring by pattern formation.<a href="#Milestone6"><sup>[1]</sup></a>
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Unfortunately, common reporting systems often failed to meet these requirements. This is because they often possess a Hill-function type dose-response curve. As can be observed from the dose-response curve of a typical Hill function (<B>Fig. 1a</B>), the linear range of a Hill function could be rather narrow, and the transition from low-output to high-output may be quite obscure to naked eyes. Thus appropriate equipment would be required to accurately measure output that follows Hill function type dose-response curve, making the measurement expensive and time consuming. </p>
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<B>Figure 1.</B> Dose-response curves and typical measurement results for a canonical reporting system <b>a</b>, and Band-pass Filter <b>b</b>. <b>a</b>, A general reporting systems typically possesses a Hill function type dose-response curve, and it's quite difficult to determine the absolute intensity of a particular signal among its gradually increasing outputs. <b>b</b>, dose-response curve of a Band-pass Filter possesses a single peak, and it's relatively easy to determine the position of the peak in its output series.
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</p>
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    <div id="Milestone2"></div>
 +
<h2 id="Alginate Encapsulation" style="position: reative; top:20px;">Alginate Encapsulation</h2>
 +
        <img src="https://static.igem.org/mediawiki/2013/a/a1/Peking2013_Device_Fig1.png" style="position:relative; top:20px; width:700px; left:150px;" />
 +
      <legend>
 +
<b>Figure 1.</b> (<b>a</b>) The structure of alginate chelating calcium ion.(<b>b</b>) the beads formed after alginate was exposed to calcium ion.
 +
      </legend>
 +
      <p>Alginate is a polysaccharide consisting of β-D-mannuronic acid (M) and α-L-guluronic acid (G) residues. When exposed to calcium ions, G residues will immediately cross-link to form a gel-like material that may serve as a matrix to safely contain our biosensors(<b>Fig.1</b>).
 +
</br>Alginate has been frequently used as the biological encapsulation material for various organisms such as <i>Saccharomyces cerevisiae, Escherichia coli</i> and mammalian cells<a href="#Milestone6"><sup>[2][3]</sup></a>. It stands out because of several distinct characteristics<a href="#Milestone6"><sup>[4]</sup></a>:</br>
 +
(1) Stable and inexpensive, even edible.</br>
 +
(2) Does not interfere with biosensors we constructed.</br>
 +
(3) Ease to shape and manipulate.</br>
 +
(4) Provide reliable protection against environmental stresses.</br>
 +
The alginate encapsulation successfully solve the problem of lethal dehydration and oxidation stress upon our biosensor strains, so no recovering process is required. </br>
 +
    </p>
 +
    <p style="position:relative; top:30px; width:800px;left:100px;"><b>Experimental protocol:</b></br>
 +
1.5% Alginate solution was boiled and kept warm at 40°C.</br>
 +
<i>E.coli</i> was grown overnight in LB medium at 37 °C in 15 ml Falcon tube, then were harvested by centrifugation at 4000 r.p.m. for 10 minutes and then resuspended in 500 μl of fresh LB media. mixed with 3 ml alginate solution, and dropped into 0.2M calcium chloride in room temperature(20 to 24℃) to form bead-like alginate encapsulation.</br>
 +
Alginate beads were washed in PBS to eliminate calcium ions and then stored in the solution with protective agents or drilled-water.
 +
    </p>
 +
</br>
 +
</br>
-
<p id="Content3">
 
-
Although unaided eyes can barely determine the absolute intensity value of a particular signal among a series of signals with various intensities (<B>Fig. 1a</B>), humans are pretty competent at determining which signal is the strongest one, especially when there is a single peak among the signals (<B>Fig. 1b</B>). Thus it can be reasoned that if we are capable of transforming a series of signals with intensities changing monotonously into a series of signals with an unique intensity peak, reading and interpreting of the output signals will become much more intuitive and convenient. Fortunately, a Band-pass Filter is exactly the equipment that can turn a series of gradually increasing input signals into a series of output signals with a single peak.
 
-
<br/><br/>It can be expected that when a Band-pass Filter is successful constructed, we may serially dilute our sample into a concentration gradient and put our biosensor into the sample. The analyte concentration can be easily determined by the serial number of the test tube exhibiting highest output intensity (<B>Fig. 2</B>). We hoped that by implementing a Band-pass Filter circuit in our bacterial host cells, we might realize fast, economical and convenient detection of aromatic compounds in environment.
 
-
</p>
 
-
<img id="FigurePic3" src="https://static.igem.org/mediawiki/igem.org/7/7f/Peking2013_Bpfigure3.png" />
 
-
<p id="Figure2">
 
-
<B>Figure 2.</B> Graph illustration of proposed Band-pass Filter testing method. First a sample series need to be created by serially diluting the original sample. Then bacterial cells expressing Band-pass Filter circuits will be exposed to the sample series and the concentration of original sample will be determined based on serial number of the sample inducing highest output.
 
-
</p>
 
-
<p id="PageSubtitle2">Concept of Band-Pass Filter
+
<p id="Results"; style="font-size:32px">Results</p>
 +
<p style="position: reative; top:20px;">Demonstration of our convenient in-field detection device was performed by soaking the alginate capsules containing bacteria with specific biosensors into solutions with corresponding aromatics and incubate for a certain time. </p>
 +
<p style="position: reative; top:20px;"> 
 +
We adopted NahR biosensor for the low basal level and high induction ratio. Based on previous works determining the most suitable concentration range for induction, our device was exposed to inducers below 100 μM, because it is found that higher concentration of inducer may inhibit the bacteria's growth.
</p>
</p>
-
 
+
<p>
-
<p id="Content4">
+
<p style="position: reative; top:20px;">
-
Band-pass Filter is a term used in electric engineering. It describes a device that passes signals with frequencies confined to a certain range and blocks signals with frequencies outside that range. The Band-pass Filter is constructed by combining a high-pass filter, which only pass signals with high frequencies, and a low-pass filter, which only pass signals with low frequencies (<B>Fig. 3</B>).</p>
+
4 different inducer concentrations were tested which are selected according to previous works and the National Standards for Drinking Water Quality of China. We tracked the change of fluorescence intensity in six hours. Every hour the photographs of 4 concentrations were taken respectively. It is obvious that this device is capable enough to test these aromatic compounds mentioned in the national standards, as well as it is user friendly and efficiency.<b>(Fig.2)</b>
-
 
+
<img src="https://static.igem.org/mediawiki/2013/thumb/1/13/Peking2013_Device_result.png/800px-Peking2013_Device_result.png" style="position:relative; top:20px; width:800px; left:10px" />
-
<img id="FigurePic4" src="https://static.igem.org/mediawiki/igem.org/5/52/Peking2013_Bpfigure4.png" />
+
<legend><b>Figure 2.</b> Tests for alginate encapsulation beads with NahR biosensor. Vertical line represents concentrations of inducer 4-MeSaA at 0μM, 1μM, 10μM and 100μM respectively. Horizontal line stands for time points in six hours. As is illustrated, 5 hours is sufficient for our device to detect 1μM 4-MeSaA, which is lower than the requirements by Chinese government, with naked eyes.</br>
-
<p id="Figure3">
+
<a href="http://www.steriq.cn/pdf/34.pdf">(Link to the Chinese Government’s Requirements)</a></legend>
-
<B>Figure 3.</B> Sketch diagram of a typical Band-pass Filter in electric engineering. Vertical arrows show the input-output relationships of individual high-pass filters (<b>left circle</b>), and individual low-pass filters (<b>right circle</b>). The horizontal arrows show input-output relationship of a Band-pass Filter constructed by concatenating a high-pass filter and a low-pass filter. In an electric Band-pass Filter, the input signal is first processed by the high-pass filter to filter out low-frequency signals and then processed by the low-pass filter to filter out high-frequency signals, leaving only medium-frequency signals.
+
</p>
</p>
-
 
+
<p>
-
 
+
<p style="position: reative; top:20px;">
-
 
+
To further combine with Adptors to expand detection profile, this device coating NahR was cultured in M9 medium in which Adptor NahF had been treated previously. Evident fluorescence could also be observed, indicating that it is possible to connect this device with adaptors. But it still needs further research to confirm.  
-
 
+
-
<p id="Content5">
+
-
In analogy to an electric Band-pass Filter, a biological Band-pass Filter is a device that can be activated only by an input signal with medium intensity. Neither signal with low nor high intensity will generate an output signal (<B>Fig. 4</B>).
+
-
<br/><br/>
+
-
Quite similar to an electric Band-pass Filter, a biological Band-pass Filter can also be separated into two components, namely the two types of regulation the input node exerts on the output node in the network topology. In one way, the input node activates the output node through a positive feed-forward loop; in another way, the input node inhibits the output node through a negative feed-forward loop. Such a network topology, with two counteracting regulatory feed-forward loops connecting input node and output node, is called an incoherent feed-forward loop topology (<B>Fig. 4a</B>). The positive feed-forward loop will respond only to high intensity input signal (<B>Fig. 4b</B>),serving as a 'high-pass filter'. The negative feed-forward loop will respond only to the low intensity input signal (<B>Fig. 4c</B>), serving as the 'low-pass filter'. By fine-tuning transition points of the dose-response curves of the two counteracting feed-forward loops so that the transition point of the negative loop is higher than that of the positive loop, the biological Band-pass Filter, constructed by combining these two loops together, will respond only to a medium intensity input signal and generate an output peak at a specific concentration (<B>Fig. 4d</B>).  
+
</p>
</p>
-
<img id="FigurePic5" src="https://static.igem.org/mediawiki/igem.org/e/e3/Peking2013_Bpfigure5x.png" />
+
<p>All the demonstration above has verified that our products are usable. Comparing with traditional chemical methods which need laboratory work, it is convenient to operate. As for sensitivity, though this device couldn't compare with chemical methods, it is capable enough to test these aromatic compounds mentioned in the national standards. So it could be used in field to achieve a rough detection of aromatics.  
-
<p id="Figure4">
+
-
<B>Figure 4.</B> Sketch diagram of a possible topology (<b>a</b>) and functioning mechanism (<b>b</b>, <b>c</b> and <b>d</b>) of a biological Band-pass Filter. <b>a</b>, A network topology containing an Incoherent feed-forward loop, capable of generating a Band-pass Filter. The input node A directly represses output node C, creating a negative feed-forward loop, while indirectly activating output node C through repressing internode B which represses node C, creating a positive feed-forward loop. <b>b</b>, dose-response curve of positive feed-forward loop when characterized independently. The positive loop will respond only to high intensity input. <b>c</b>, Dose-response curve of negative feed-forward loop when characterized independently. The negative loop will respond only to low intensity input. <b>d</b>, The integrated dose-response curve of the incoherent feed-forward loop. High intensity input is filtered out by negative loop and low intensity input is filtered out by positive loop, only medium intensity input will induce a significant response.
+
</p>
</p>
-
<p id="PageSubtitle3">Constructing Band-pass Filter
 
-
</p>
 
-
<p id="Content6">
 
-
Having illustrated the basic principles of a Band-pass Filter, we set out to rationally design its genetic circuit.
 
-
<br/><br/>
 
-
First we selected three potential circuit networks (<b>Fig. 5</b>) with incoherent feed-forward loop as their core topology and then used Ordinary Differential Equations (ODEs) to analyze these circuit networks to identify the most robust circuit network. We chose to follow the four-node network because its performance remained more satisfactory than the others when the parameters varied randomly.
 
-
<br/><br/><br/><br/><br/></p>
 
-
<img id="realfigure5" src="https://static.igem.org/mediawiki/2013/8/81/Peking_2013_Project_band-pass_filter_Fig_5.png" />
 
-
<p id="realfigurelegend5"><b>Figure 5.</b> Graphs of three circuit networks we analyzed in our modeling. Each node represent a regulatory protein, either an activator or and repressor. All four networks possess incoherent feed-forward loops as core topology. Components of the activating half of an incoherent feed-forward loop are marked as green while components of repressing half are marked as black. <b>a</b>, <b>b</b>, Three-node networks taken into consideration. The principal difference between these two networks is the regulatory function of input node A. <b>a</b>, Three-node network where input node A functions as repressor. A directly repress output node C while indirectly activating it by inhibiting B, which represses C directly. <b>b</b>, Three-node network where input node A functions as activator. A directly repress output node C while indirectly activating it by activating B, which represses C directly. <b>c</b>, Four-node network taken into consideration. A indirectly activates output node D by activating C which activates D, while indirectly represses output by activating B that inhibits D.
 
</br>
</br>
 +
<div id="Milestone4"></div>
 +
      <h1 id="Advanced Design"; style="position: reative; top:20px; width:300px;">Advanced Design</h1>
 +
      <p style="position: reative; top:20px;">Based on previous test results on the alginate encapsulation method, we reasoned that a hydrogel patterning could serve multi-purposes, including implementing adaptors through cell communication, and realization of bandpass filter by constructing a inducer concentration gradient.
 +
</p>
 +
      <p style="position: reative; top:20px;left:100px;">
</br>
</br>
 +
<b>PDMS Template Design</b>
 +
PDMS (polydimethylsiloxane), particularly known for its unusual rheological (or flow) properties, is optically clear and, in general, inert, non-toxic, and non-flammable. It is a material with no marked harmful effects on organisms in the environment and is frequently used in the microfluridic chips.</br>
 +
Parallel square wells which are 500μm wide, 500μm long and 170μm deep were etched on to a PDMS template, with a distance of 500 μm between them. This design was aiming at preventing interaction of E.coli between different wells. The pattern of our design could be easily adjusted according to customers' need.<a href="#Milestone6"><sup>[1]</sup></a>.
 +
</br>
 +
<b>Alginate Pattern formation</b>
 +
Alginate is still adopted as encapsulation. The mixture of alginate and bacterial culture was added into the wells of PDMS. After treating with calcium ions, the mixture solidify. Then PDMS with the mixture was adhered upside-down to an agarose layer whose concentration is of 1.5% or 2%. After 5 minutes incubation in 40℃, the PDMS template was peeled off with the solidified alginate mixture left on the agarose layer, forming corresponding pattern.
</br>
</br>
</p>
</p>
-
<p id="Content6_2">
+
      <img src="https://static.igem.org/mediawiki/2013/5/52/Peking2013_Device_Fig6.png" style="position:relative; top:20px; width:500px; left:250px;" />
-
Our next step is to select appropriate proteins to serve as individual nodes in the chosen circuit network. First we figured out the most crucial parameters in the ODE model through a parameter sensitivity analysis and determined the most desirable value for these parameters. Then we chose regulatory proteins whose kinetic parameter values are close to the desirable values, based on the reasoning that they would work much more efficiently than casually chosen ones.
+
      <legend><b>Figure 3.</b> The design and experiment protocol of hydrogel patterning and transferring method. This method can be applied to cell communication and semi-quantitative detection.  
-
<br/><br/>
+
</legend>
-
Based on the analysis above, we selected phage transcription activator ϕR73&delta; as internode activator and phage transcription inhibitor cI as internode inhibitor while choosing NahR as the input sensor and sfGFP as reporter. The final construct is shown in <b>Figure 6</b>.</p>
+
<p>
-
<img id="model15" src="https://static.igem.org/mediawiki/igem.org/d/df/Peking2013_model15_.png" />
+
-
<p id="Legend7"><b>Figure 6.</b>The final construct of our Band-pass Filter. The aromatic sensor (input node) will activate transcription of &phi;R73&delta; and cI gene. The &phi;R73&delta; will activate transcription of sfGFP reporter gene while cI represses transcription of the reporter gene, creating an incoherent loop. With proper parameter sets, such a genetic circuit will serve the function as a Band-pass Filter.</p>  
+
<p id="Results"; style="font-size:32px">Results</p>
-
<p id ="Content6_1">
+
<img src="https://static.igem.org/mediawiki/2013/4/44/Peking2013_Device_Fig8.png" style="position:relative; top:20px; width:700px; left:150px;" />
-
Details of constructing process can be viewed at <a href="https://2013.igem.org/Team:Peking/Model">Model</a> page.
+
<legend><b>Figure 4.</b> The primary attempt to construct the advanced device. 100μM 4-MeSaA was dropped on the left side of PDMS, then the plate was incubated in 37℃ for 6 hours. It's shown that a concentration was constructed and the response could be detected by blue LED. For the transferred patterning, This experiment also indicated that even with the PMDS adhered, the biosensor could be induced within a relative short time.</br>  </legend>
-
</p>
+
-
<p id="PageSubtitle4">Building Our Hybrid Promoter
+
<p>
 +
<p style="position: reative; top:20px;"> For further improvement, this device could be combined with adaptors,and the adaptor <i>E.coli</i> cells could be cultured in agarose layer. When exposed to suitable substrates, adaptors will convert the substrates into compounds which can be detected by corresponding biosensor encapsulated in alginate.</br>
 +
For combination with the Band-pass filter, the bacterial strains possessing different detecting profiles as well as different concentration ranges could be encapsulated into alginate and located in distinct units on agarose. When treated with unknown samples, the biosensor shows different fluorescence intensity in each strain, thus it is possible to deduce types and concentrations of certain aromatics in the samples.
</p>
</p>
 +
</br>
-
<p id="Content7">
 
-
After determining the circuit network and protein candidates, we still need to address an important issue: we need to find a way to enable co-regulation of the reporter gene by two different transcription regulators. So we modified a bacteriophage ϕR73’s <em>P<sub>2</sub></em> promoter into a hybrid promoter that can be activated by the ϕR73&delta; activator and repressed by the repressor cI simultaneously and put reporter sfGFP under its regulation. We constructed the hybrid promoter by replacing the sequence between position -1 and -25  of <em>P<sub>2</sub></em> promoter with the cI binding site <em>OR1</em> from Phage &lambda; <em>P<sub>R</sub></em> promoter. When ϕR73&delta; activator binds to its target sequence upstream of -35 element of the hybrid promoter, the transcription will start. The binding of cI dimers downstream of -35 element will block the binding of &sigma;<sup>70</sup> factors and thus repress the transcription even when ϕR73&delta; is bound. (<B>Fig. 7</B>). </p>
 
-
 
-
 
-
<img id="FigurePic6" src="https://static.igem.org/mediawiki/2013/b/bf/Peking2013_hybrid_promoter_2.2.png" />
 
-
 
-
<p id="Figure5">
 
-
<B>Figure 7.</B> Construction of Our Hybrid Promoter. Sequence information of phage &phi;R73 <em>P<sub>2</sub></em> promoter (<b>a</b>), phage &lambda; <em>P<sub>R</sub></em> promoter (<b>b</b>) and our hybrid promoter (<b>c</b>) are shown. <b>a</b>, In the <em>P<sub>2</sub></em> promoter, &phi;R73&delta; binds to a region between position -42 and -71 and activates transcription. <b>b</b>, In <em>P<sub>R</sub></em> promoter, cI dimer binds to <em>OR1</em> site (marked as blue) between position -9 and -25, blocking binding of  &sigma;<sup>70</sup> factors and inhibiting transcription. cI binding region indicates the sequence we used to replace the corresponding region in <em>P<sub>2</sub></em> promoter.<b>c</b>, The hybrid promoter is constructed by replacing sequence between position -1 and -25 of &phi;R73 <em>P<sub>2</sub></em> promoter with sequence at the same position in phage &lambda; <em>P<sub>R</sub></em> promoter that contains an <em>OR1</em> site. The hybrid promoter is co-regulated by &phi;R73&delta; and cI, with &phi;R73&delta; activating and cI repressing. The repression of cI dominates over the activation of  &phi;R73&delta;, since the steric hindrance created by cI dimer prevents formation of  transcription initiation complex even when RNA polymerases are recruited through the help of  &phi;R73&delta;.
 
-
</p>
 
-
 
-
 
-
<p id="Content8">
 
-
In the Band-pass Filter circuit we constructed above (<b>Fig. 6</b>), the promoter will function in the following way as input intensity gradually increase: when the input intensity is weak, the concentration of ϕR73&delta; is too low to generate a strong output; when the input intensity is medium, despite a portion of promoters occupied by cI dimmers, the rest still can be activated by ϕR73&delta; and bring about a visible output; when the input intensity is strong, almost all of the promoters are blocked by cI dimers and the output is shut down. Hence only medium input signal can induce a significant output and the an single peak of output signal would be generated.</p>
 
-
 
-
<p id="PageSubtitle5">Characterizing Hybrid Promoter
 
-
</p>
 
-
 
-
<p id ="Content9">
 
-
As a key component of our Band-pass Filter circuit, the hybrid promoter must be carefully characterized in order to evaluate the feasibility of our Band-pass Filter circuit. To comprehensively characterize the dynamic performance of the hybrid promoter, we put two regulators of the hybrid promoter,  ϕR73&delta; and cI, under the control of two different inducible promoters, <em>P<sub>sal</sub></em> promoter and <em>P<sub>tac</sub></em> promoter. (<b>Fig. 8</b>). This enables us to manipulate separately the expression levels of two regulatory proteins through tuning <em>P<sub>sal</sub></em> and <em>P<sub>tac</sub></em> promoter by adding different concentration combinations of inducers (salicylic acid for <em>P<sub>sal</sub></em> promoter and IPTG for <em>P<sub>tac</sub></em> promoter).
 
-
</p>
 
-
<img id="FigurePic8" src="https://static.igem.org/mediawiki/2013/d/d2/Peking2013_bandpass_figure1_better.png" />
 
-
<p id = "Figure8">
 
-
<b>Figure 8.</b> Testing construct for hybrid promoter. &phi;R73&delta; was put under the regulation of <em>P<sub>sal</sub></em> promoter and cI was put under the control of <em>P<sub>tac</sub></em> promoter. Salicylic acid (SaA) will induce &phi;R73&delta; expression and activate the hybrid promoter. Isopropyl β-D-1-thiogalactopyranoside (IPTG) will induce cI expression and repress the hybrid promoter. Expression level of the two regulatory proteins can be manipulated separately by adding different concentration combinations of SaA and IPTG.
 
-
</p>
 
-
<p id = "Content10">
 
-
To comprehensively characterized the hybrid promoter's transcription activity,  we exposed the characterization circuit (<b>Fig. 8</b>) to a 8x8 two-dimensional induction assay established by combining 8 different concentrations of salicylic acid and 8 different concentrations of IPTG and measured the fluorescence intensity of sfGFP reporter using Flow Cytometry. (<b>Fig. 9</b>)
 
-
</p>
 
-
<img id="FigurePic9" src="https://static.igem.org/mediawiki/2013/3/3f/Peking2013_bandpass_figure_2Dassay.png" />
 
-
<p id = "Figure9">
 
-
<b>Figure 9</b>. Characterization of hybrid promoter's dynamic performance. A two-dimensional inducer concentration assay was established by combining 8 different SaA concentrations (0, 0.1, 0.5, 1, 5, 10, 50 and 100&micro;M) with 8 different IPTG concentrations (0, 1, 10, 50, 100, 150, 200 and 300&micro;M). Bacteria cells expressing the testing construct were exposed to the assay and sfGFP fluorescence intensity was measured using Flow Cytometry. For a fixed IPTG concentration,  fluorescence intensity gradually increased as SaA concentration increased. For a fixed SaA concentration , fluorescence intensity gradually decreased as IPTG concentration increased. These features indicated that the promoter functioned as expected.
 
-
</p>
 
-
<p id = "Content10">
 
-
The hybrid promoter worked as expected. For a fixed IPTG concentration, the sfGFP fluorescence gradually increased as the salicylic acid concentration increased, exhibiting a Hill-function type dose-response curve. For a fixed high salicylic acid concentration under which sfGFP expression is visibly induced, the fluorescence gradually decreased as the IPTG concentration increased, also exhibiting a Hill-function type dose-response curve. These data prove that the hybrid promoter can indeed be activated by ϕR73&delta; and repressed by cI, and the repressing effect of cI protein dominates over the activating effect of  ϕR73&delta; protein, because transcription of the hybrid promoter can still be repressed to a very low level by cI even when  ϕR73&delta; is expressed at a very high level.
 
-
</br>
 
-
</br>
 
-
Simply characterizing the hybrid promoter won't satisfy us. We want to glean more information from this experiment in order to assess whether our Band-pass Filter design is really feasible or, in another word, whether the kinetic/dynamic parameter values of our genetic circuit actually fall within the range where a single output peak can be generated. However, there is an important feature in this testing construct that is radically different from our Band-pass Filter construct: the promoters driving the expression of ϕR73&delta; and cI are not the same, one is <em>P<sub>sal</sub></em>, the other is <em>P<sub>tac</sub></em>.
 
-
</br>
 
-
</br>
 
-
But this difference doesn't preclude the possibility of using data from this testing construct to give us insight on our original design. If the regulation mechanisms of the two promoters are close enough, we may reason that the Hill-functions describing the dynamic performance of the two promoters would also be similar (in the sense that their graphs can be overlapped by linearly stretching or compressing both axises). It is indeed the case. The <em>P<sub>sal</sub></em> promoter is repressed by NahR tetramer through bending of DNA when salicylic acid is absent, and when salicylic acid is present, NahR will undergo a conformation change and transcription will start. (See Project, biosensors, NahR) Mechanism for <em>P<sub>tac</sub></em> promoter is rather similar: LacI inhibits transcription through tetramerization and DNA bending when lactose is absent and the inhibition is eliminated through conformational change.
 
-
</br>
 
-
</br>
 
-
Following the reasoning above, we hypothesized that the negative feed-forward loop in the testing construct may actually represent a transformed version of the negative loop in the original Band-pass Filter construct. So we fit our model to the data from the testing construct in order to get real parameters for the Band-pass Filter circuit. (<b>Fig. 10</b>)
 
-
</p>
 
-
<img id="FigurePic10" src="https://static.igem.org/mediawiki/2013/8/82/Peking2013_bandpass_figure3_data_fitting.png" />
 
-
<p id = "Figure10">
 
-
<b>Figure 10.</b> Model based data fitting for &phi;R73&delta; activator (<b>a</b>) and cI repressor (<b>b</b>). <b>a</b>, Experimental points are sfGFP fluorescence intensities under different SaA concentrations without IPTG. The model based fitting curve provided parameter values for n<sub>A'</sub> and K<sub>AG</sub>. <b>b</b>, Experimental points are sfGFP fluorescence intensities under different IPTG concentrations along with 100&micro;M SaA. Model based data fitting curve provided parameter values for n<sub>B'</sub>, K<sub>BG</sub> and k<sub>AG</sub>&bull;k<sub>BG</sub>. Fitting results: n<sub>A'</sub>=0.72705; K<sub>AG</sub>=62.99928; n<sub>B'</sub>=1.15498; K<sub>BG</sub>=11.53988; k<sub>AG</sub>&bull;k<sub>BG</sub>=24671.78415. Definitions for the parameters can be viewed in equations written in <a href="https://2013.igem.org/Team:Peking/Model">Model</a> page.
 
-
</p>
 
-
<p id = "Content12">
 
-
We substituted the parameters obtained from data fitting into the original Band-pass Filter to observe whether a peak is generated. (<b>Fig. 11</b>) Result showed that provided that our hypothesis is correct, our Band-pass Filter could indeed function as we expected.
 
-
</p>
 
-
<img id="FigurePic11" src="https://static.igem.org/mediawiki/2013/e/e2/Peking2013_bandpassfilter_finalfit.png" />
 
-
<p id = "Figure11">
 
-
<b>Figure 11.</b> Result of modeling based on parameters obtained from data fitting mentioned in <b>Figure 10</b>. Clearly a unique output peak is formed. This indicates that our band-pass filter circuit is feasible.
 
-
</p>
 
-
<p id="ReferenceBPF">
+
<div id="Milestone6"></div>
-
<B>Reference:</B></br>
+
<p style="position:relative;top:35px;">
-
[1] SOHKA, Takayuki, et al. An externally tunable bacterial band-pass filter.<I>Proceedings of the National Academy of Sciences</I>, 2009, 106.25: 10135-10140.<br/>
+
<b>References</b></br>
-
[2] MA, Wenzhe, et al. Defining network topologies that can achieve biochemical adaptation. <I>Cell</I>, 2009, 138.4: 760-773.<br/>
+
[1] Choi, W. S., Kim, M., Park, S., Lee, S. K., & Kim, T. (2012). Patterning and transferring hydrogel-encapsulated bacterial cells for quantitative analysis of synthetically engineered genetic circuits. Biomaterials, 33(2), 624-633.</br>
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[3] BASU, Subhayu, et al. A synthetic multicellular system for programmed pattern formation. <I>Nature</I>, 2005, 434.7037: 1130-1134.<br/>
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[2] Koch, S., Schwinger, C., Kressler, J., Heinzen, C. H., & Rainov, N. G. (2003). Alginate encapsulation of genetically engineered mammalian cells: comparison of production devices, methods and microcapsule characteristics. Journal of microencapsulation, 20(3), 303-316.</br>
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[3] Wang, N., Adams, G., Buttery, L., Falcone, F. H., & Stolnik, S. (2009). Alginate encapsulation technology supports embryonic stem cells differentiation into insulin-producing cells. Journal of biotechnology, 144(4), 304-312.</br>
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[4] <a href="https://2009.igem.org/Team:Imperial_College_London/M2">iGEM: Imperial Collage/Encapsulation, 2009</a></br>
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Latest revision as of 03:51, 29 October 2013

Purpose-Built Device

Purposes

In-field detection of aromatic compounds in environments has always been desirable, and convenience has always been an important requirement for in-field detection. To meet this requirement, the detection process should be fast and the result should be easily read by naked eyes. Furthermore, the device we design to realize all these should be readily portable.
When designing such a portable user-friendly device, the most challenging part would be developing the preservation method. As we choose Escherichia coli, which is unable to germinate spores, as our host strain, special method should be developed to protect the bacteria from temperature changes and physical stress while keeping them alive. We used alginate encapsulation as a basic solution to this problem. Based on such preliminary design, we further built an advanced device to realize convenient quantitative measuring by pattern formation.[1]

Alginate Encapsulation

Figure 1. (a) The structure of alginate chelating calcium ion.(b) the beads formed after alginate was exposed to calcium ion.

Alginate is a polysaccharide consisting of β-D-mannuronic acid (M) and α-L-guluronic acid (G) residues. When exposed to calcium ions, G residues will immediately cross-link to form a gel-like material that may serve as a matrix to safely contain our biosensors(Fig.1).
Alginate has been frequently used as the biological encapsulation material for various organisms such as Saccharomyces cerevisiae, Escherichia coli and mammalian cells[2][3]. It stands out because of several distinct characteristics[4]:
(1) Stable and inexpensive, even edible.
(2) Does not interfere with biosensors we constructed.
(3) Ease to shape and manipulate.
(4) Provide reliable protection against environmental stresses.
The alginate encapsulation successfully solve the problem of lethal dehydration and oxidation stress upon our biosensor strains, so no recovering process is required.

Experimental protocol:
1.5% Alginate solution was boiled and kept warm at 40°C.
E.coli was grown overnight in LB medium at 37 °C in 15 ml Falcon tube, then were harvested by centrifugation at 4000 r.p.m. for 10 minutes and then resuspended in 500 μl of fresh LB media. mixed with 3 ml alginate solution, and dropped into 0.2M calcium chloride in room temperature(20 to 24℃) to form bead-like alginate encapsulation.
Alginate beads were washed in PBS to eliminate calcium ions and then stored in the solution with protective agents or drilled-water.



Results

Demonstration of our convenient in-field detection device was performed by soaking the alginate capsules containing bacteria with specific biosensors into solutions with corresponding aromatics and incubate for a certain time.

We adopted NahR biosensor for the low basal level and high induction ratio. Based on previous works determining the most suitable concentration range for induction, our device was exposed to inducers below 100 μM, because it is found that higher concentration of inducer may inhibit the bacteria's growth.

4 different inducer concentrations were tested which are selected according to previous works and the National Standards for Drinking Water Quality of China. We tracked the change of fluorescence intensity in six hours. Every hour the photographs of 4 concentrations were taken respectively. It is obvious that this device is capable enough to test these aromatic compounds mentioned in the national standards, as well as it is user friendly and efficiency.(Fig.2) Figure 2. Tests for alginate encapsulation beads with NahR biosensor. Vertical line represents concentrations of inducer 4-MeSaA at 0μM, 1μM, 10μM and 100μM respectively. Horizontal line stands for time points in six hours. As is illustrated, 5 hours is sufficient for our device to detect 1μM 4-MeSaA, which is lower than the requirements by Chinese government, with naked eyes.
(Link to the Chinese Government’s Requirements)

To further combine with Adptors to expand detection profile, this device coating NahR was cultured in M9 medium in which Adptor NahF had been treated previously. Evident fluorescence could also be observed, indicating that it is possible to connect this device with adaptors. But it still needs further research to confirm.

All the demonstration above has verified that our products are usable. Comparing with traditional chemical methods which need laboratory work, it is convenient to operate. As for sensitivity, though this device couldn't compare with chemical methods, it is capable enough to test these aromatic compounds mentioned in the national standards. So it could be used in field to achieve a rough detection of aromatics.


Advanced Design

Based on previous test results on the alginate encapsulation method, we reasoned that a hydrogel patterning could serve multi-purposes, including implementing adaptors through cell communication, and realization of bandpass filter by constructing a inducer concentration gradient.


PDMS Template Design PDMS (polydimethylsiloxane), particularly known for its unusual rheological (or flow) properties, is optically clear and, in general, inert, non-toxic, and non-flammable. It is a material with no marked harmful effects on organisms in the environment and is frequently used in the microfluridic chips.
Parallel square wells which are 500μm wide, 500μm long and 170μm deep were etched on to a PDMS template, with a distance of 500 μm between them. This design was aiming at preventing interaction of E.coli between different wells. The pattern of our design could be easily adjusted according to customers' need.[1].
Alginate Pattern formation Alginate is still adopted as encapsulation. The mixture of alginate and bacterial culture was added into the wells of PDMS. After treating with calcium ions, the mixture solidify. Then PDMS with the mixture was adhered upside-down to an agarose layer whose concentration is of 1.5% or 2%. After 5 minutes incubation in 40℃, the PDMS template was peeled off with the solidified alginate mixture left on the agarose layer, forming corresponding pattern.

Figure 3. The design and experiment protocol of hydrogel patterning and transferring method. This method can be applied to cell communication and semi-quantitative detection.

Results

Figure 4. The primary attempt to construct the advanced device. 100μM 4-MeSaA was dropped on the left side of PDMS, then the plate was incubated in 37℃ for 6 hours. It's shown that a concentration was constructed and the response could be detected by blue LED. For the transferred patterning, This experiment also indicated that even with the PMDS adhered, the biosensor could be induced within a relative short time.

For further improvement, this device could be combined with adaptors,and the adaptor E.coli cells could be cultured in agarose layer. When exposed to suitable substrates, adaptors will convert the substrates into compounds which can be detected by corresponding biosensor encapsulated in alginate.
For combination with the Band-pass filter, the bacterial strains possessing different detecting profiles as well as different concentration ranges could be encapsulated into alginate and located in distinct units on agarose. When treated with unknown samples, the biosensor shows different fluorescence intensity in each strain, thus it is possible to deduce types and concentrations of certain aromatics in the samples.


References
[1] Choi, W. S., Kim, M., Park, S., Lee, S. K., & Kim, T. (2012). Patterning and transferring hydrogel-encapsulated bacterial cells for quantitative analysis of synthetically engineered genetic circuits. Biomaterials, 33(2), 624-633.
[2] Koch, S., Schwinger, C., Kressler, J., Heinzen, C. H., & Rainov, N. G. (2003). Alginate encapsulation of genetically engineered mammalian cells: comparison of production devices, methods and microcapsule characteristics. Journal of microencapsulation, 20(3), 303-316.
[3] Wang, N., Adams, G., Buttery, L., Falcone, F. H., & Stolnik, S. (2009). Alginate encapsulation technology supports embryonic stem cells differentiation into insulin-producing cells. Journal of biotechnology, 144(4), 304-312.
[4] iGEM: Imperial Collage/Encapsulation, 2009