Team:Peking/Project/Devices

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                 <li class="SensorsListItem"><a href="#Milestone1">Purposes</a><li>
                 <li class="SensorsListItem"><a href="#Milestone1">Purposes</a><li>
                 <li class="SensorsListItem"><a href="#Milestone2">Alginate Encapsulation</a><li>
                 <li class="SensorsListItem"><a href="#Milestone2">Alginate Encapsulation</a><li>
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                <li class="SensorsListItem"><a href="#Milestone3">Protective Agents</a><li>
 
                 <li class="SensorsListItem"><a href="#Milestone4">Advanced Design</a><li>
                 <li class="SensorsListItem"><a href="#Milestone4">Advanced Design</a><li>
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                <li class="SensorsListItem"><a href="#Milestone5">Potentials</a><li>
 
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       <h1 id="Purposes";>Purposes</h1>
       <h1 id="Purposes";>Purposes</h1>
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       <p>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 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.  
</br>
</br>
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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 and built advanced device based on such preliminary design to measure the concentration of aromatics in samples.<sup>[5]</sup>
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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>
</p>
</p>
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         <img src="http://2013.igem.org/wiki/images/a/a1/Peking2013_Device_Fig1.png" style="position:relative; top:20px; width:700px; left:150px;" />
         <img src="http://2013.igem.org/wiki/images/a/a1/Peking2013_Device_Fig1.png" style="position:relative; top:20px; width:700px; left:150px;" />
       <legend>
       <legend>
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<b>Figure 1.</b> The structure of alginate chelating calcium ion (<b>a</b>) and the beads formed after alginate was exposed to calcium ion (<b>b</b>).
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<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>
       </legend>
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       <p>Alginate is a polysaccharide consisting of β-D-mannuronic acid (M) and α-L-guluronic acid (G) residues. When exposed to calcium irons, G residues will immediately cross-link to form a gel-like material that may serve as a matrix to safely contain our biosensors.
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       <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>).
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  </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<sup>[1][2]</sup>. It stands out because of several distinct characteristics<sup>[8]</sup>:</br>
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  </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>
(1) Stable and inexpensive, even edible.</br>
(2) Does not interfere with biosensors we constructed.</br>
(2) Does not interfere with biosensors we constructed.</br>
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     <p style="position:relative; top:30px; width:800px;left:100px;"><b>Experimental protocol:</b></br>
     <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>
1.5% Alginate solution was boiled and kept warm at 40°C.</br>
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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.</br>
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<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>
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Alginate beads were washed in PBS to eliminate calcium irons and then stored in the solution with protective agents or drilled-water.
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Alginate beads were washed in PBS to eliminate calcium ions and then stored in the solution with protective agents or drilled-water.
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     </p>
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<div id="Milestone3"></div>
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</br>
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    <h2 id="Protective Agents";>Protective Agents</h2>
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</br>
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    <p style="position: reative; top:20px;"><b>Inositol</b></p>
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      <img src="http://2013.igem.org/wiki/images/b/bc/Peking2013_Device_Fig2.png" style="position:relative; width:250px; left:150px;" />
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      <legend style="position:relative; top:-130px;left:450px;width:300px;">
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<b>Figure 2.</b> The structure of myo-inositol. It is optically active and plays a crucial role in eukaryotic cells.
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<p style="position: reative;top:-80px;">
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So far alginate capsule serves our purpose well. However, if additional protective agents were applied, the biosensor strains may be preserved for a longer time at 4℃, even exceeding a month. </br>
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Bacterial under dehydration may face an increasing osmotic stress as the water activity decrease. A possible way to counteract the osmotic stress is to accumulate non-toxic solutes in cytoplasm to maintain a high osmotic pressure, stablize proteins and balance the dehydration in the environment<sup>[4]</sup>. Inositol suits this purpose well due to its stability and hydrophilicity. And this was corroborated by work done by Aitor. et al. </br>
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The link to the original paper:
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<a href="http://link.springer.com/article/10.1007/s00216-010-4558-y">http://link.springer.com/article/10.1007/s00216-010-4558-y</a>
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</p>
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    <p style="position: reative; top:20px;"><b>Trehalose</b></p>
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<img src="http://2013.igem.org/wiki/images/f/fe/Peking2013_Device_Fig4.png" style="position:relative; top:20px; width:350px; left:100px;" />
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<legend style="position:relative; top:-90px;left:510px;width:350px;"><b>Figure 3.</b> The structure of trehalose. It is efficient in preventing dehydration.  </legend>
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    <p style="position: reative; top:-50px;">Trehalose has an advantage over other kinds of sugars for it has a relatively high glass transition, so the bacteria may maintain a glassy form with the temperature and humidity change.Additionally, high concentration of NaCl solution induces the expression of intracellular trehalose. <sup>[7]</sup></br></br>
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To maintain the bacteria encapsulated alive for as long as possible, the protective agents were used in high concentration. Thus, testing whether the induction process will be interfered by the protecting agents would be necessary. A series inositol and trehalose solutions with different concentration were used as medium for induction test. Results showed that the presence of protective agents does not significantly influence the behavior of our biosensors.
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  </p>
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<img src="http://2013.igem.org/wiki/images/d/d3/Peking2013_Device_Fig5.png" style="position:relative; top:-20px; width:700px; left:150px;" />
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<legend><b>Figure 4.</b> Influence of protection agents (inositol and trehalose) on the induction response of NahR biosensor. Induction ratios were measured for NahR's inducer 4-methyl-salicylic acid at 100 μM in presence of protective agents at different concentrations and compared with the induction response in presence of no protective agents (shown as the thin horizontal solid line). Results showed that these two protective agents did not interfere the inducing process of NahR biosensor even when they were in fairly high concentrations. </legend>
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<p id="Results"; style="font-size:32px">Results</p>
<p id="Results"; style="font-size:32px">Results</p>
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<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 amount of time. </p>
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<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>
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<img src="http://2013.igem.org/wiki/images/b/b9/Peking2013_Device_Fig7.png" style="position:relative; top:20px; width:700px; left:150px;" />
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<legend><b>Figure 5.</b>The test results of alginate encapsulation beads with XylS and NahR biosensors in it. <b>a:</b> After 10h incubation, the fluorescence intensity of XylS is sufficiently high to be discriminated by blue LED and after 12 hours, by naked eyes.
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<b>b:</b>The induction experiment conducted after 12 days preservation. Left: blank. Right: response of XylS with 100μM 3-MeBzO.
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</legend>
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<p style="position: reative; top:20px;">   
<p style="position: reative; top:20px;">   
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For NahR biosensor, after 3 hours of incubation, the fluorescence generated by capsules soaked in solution with 100μM 4-MeBzO can be easily discerned by naked eyes using a simple blue light source, while the capsules soaked in blank solution has no visible fluorescence. The concentration of 100μM was chosen according to previous tests because under such a concentration, high induction ratio can be obtained without noticable toxicity to bacteria. <b>(fig. 5b)</b>
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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.
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</br>
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<img src="Fig10.png" style="position:relative; top:20px; width:800px; left:100px;" />
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<legend><b>Figure 6.</b> Tests for alginate encapsulation beads with NahR biosensor. The concentrations of inducer 4-MeSaA are: 100μM, 10μM, 1μM and blank.(Each photograph, from left to right.) and photographs were taken every hour. As is illustrated, different inducer concentration requires different induction time, and it’s possible to detect 1μM 4-MeSaA in 6 hours, this concentration is lower than the requirements by Chinese government, and the higher the concentration of inducer is, the faster detection may conduct. </br>
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<p>
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<p style="position: reative; top:20px;">
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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>
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<img src="http://2013.igem.org/wiki/images/thumb/1/13/Peking2013_Device_result.png/800px-Peking2013_Device_result.png" style="position:relative; top:20px; width:800px; left:10px" />
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<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>
<a href="http://www.steriq.cn/pdf/34.pdf">(Link to the Chinese Government’s Requirements)</a></legend>
<a href="http://www.steriq.cn/pdf/34.pdf">(Link to the Chinese Government’s Requirements)</a></legend>
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</p>
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<p>
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<p style="position: reative; top:20px;">
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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. 
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</p>
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<img src="http://2013.igem.org/wiki/images/3/32/Peking2013_Device_Fig9.png" style="position:relative; top:20px; width:440px; left:280px;" />
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<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.  
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<legend><b>Figure 7.</b> The response of NahR to the sample with 4-MeSaD treated by NahF. From left to right: 1,2: not been treated by the adaptor, NahF; 3.4: treated by NahF for 4 hours. 5,6: treated by NahF for 12 hours. 1,3,5 contain  10μM 4-MeSaD and 2,4,6 contain  100μM 4-MeSaD.
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</legend>
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<p>Although such demonstration was not sufficiently rigorous, it verifies the possibility that our products could be used in field to achieve a rough detection. </br>
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</p>
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  <div id="Milestone4"></div>
  <div id="Milestone4"></div>
       <h1 id="Advanced Design"; style="position: reative; top:20px; width:300px;">Advanced Design</h1>
       <h1 id="Advanced Design"; style="position: reative; top:20px; width:300px;">Advanced Design</h1>
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       <p style="position: reative; top:20px;">Based previous test results on the alginate encapsulation method, we reasoned that a hydrogel patterning and transferring method could serve multi-purposes, including quantitative detection, implementing adaptors through cell communication, and realization of bandpass filter by constructing a inducer concentration gradient.
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       <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>
       <p style="position: reative; top:20px;left:100px;">
       <p style="position: reative; top:20px;left:100px;">
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</br>
<b>PDMS Template Design</b>
<b>PDMS Template Design</b>
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PDMS (polydimethylsiloxane) is particularly known for its unusual rheological (or flow) properties. PDMS is optically clear and, in general, inert, non-toxic, and non-flammable. PDMS is a material with no marked harmful effects on organisms in the environment and is frequently used in the microfluridic chips.</br>
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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>
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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 shape of our design could be easily adjusted according to customers' need.<sup>[5]</sup>. </br>
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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>.  
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<b>Pattern Transferring</b>
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</br>
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The alginate solution and bacterial culture mixture (described in the protocol of alginate encapsulation) was transferred into the wells. After treatment with calcium irons, the mixture solidify. The PDMS with the mixture was then transferred to a agarose layer. (Its concentration is of 1.5% or 2%) In 5 minutes, the PDMS template was peeled off with the cell patterns left on the agarose layer.</br>
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<b>Alginate Pattern formation</b>
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<b>Improvements</b>
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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.
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For adaptors, the adaptor E.coli cells could be cultured in the agarose layer. If the substrate of adaptor exists, it would be converted into the compound which could be dected by corresponding biosensor.</br>
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</br>
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For the bandpass filter, the agarose layer could be pre-treated so that the sample and water was aliquoted on two sides of the agarose layer. After 6 to 12 hours treatment, a concentration gradient would be constructed by diffusion.
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</p>
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(The diffusion time could be calculated according to the mass of inducers and the concentration of agarose layer.)
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       <img src="http://2013.igem.org/wiki/images/5/52/Peking2013_Device_Fig6.png" style="position:relative; top:20px; width:500px; left:250px;" />
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  </p>
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       <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.  
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       <img src="http://2013.igem.org/wiki/images/5/52/Peking2013_Device_Fig6.png" style="position:relative; top:20px; width:500px; left:100px;" />
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       <legend><b>Figure 8.</b> The design and experiment protocol of hydrogel patterning and transferring method. This method is potential for conducting cell communication and semi-quantitative detection.  
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<p id="Results"; style="font-size:32px">Results</p>
<p id="Results"; style="font-size:32px">Results</p>
<img src="http://2013.igem.org/wiki/images/4/44/Peking2013_Device_Fig8.png" style="position:relative; top:20px; width:700px; left:150px;" />
<img src="http://2013.igem.org/wiki/images/4/44/Peking2013_Device_Fig8.png" style="position:relative; top:20px; width:700px; left:150px;" />
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<legend><b>Figure 9.</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>
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<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>
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<p>  
<p>  
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<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>
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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.
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<p style="position:relative;top:35px;">
<p style="position:relative;top:35px;">
<b>References</b></br>
<b>References</b></br>
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[1] 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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[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>
-
[2] 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>
+
[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] Morgan, C. A., Herman, N., White, P. A., & Vesey, G. (2006). Preservation of micro-organisms by drying; a review. Journal of Microbiological Methods,66(2), 183-193.</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] de las Heras, A., & de Lorenzo, V. (2011). In situ detection of aromatic compounds with biosensor Pseudomonas putida cells preserved and delivered to soil in water-soluble gelatin capsules. Analytical and bioanalytical chemistry, 400(4), 1093-1104.</br>
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[4] <a href="http://2009.igem.org/Team:Imperial_College_London/M2">iGEM: Imperial Collage/Encapsulation, 2009</a></br>
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[5] 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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[6] Leslie, S. B., Israeli, E., Lighthart, B., Crowe, J. H., & Crowe, L. M. (1995). Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying. Applied and environmental microbiology, 61(10), 3592-3597.</br>
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[7] Peking iGEM 2010 wiki</br>
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[8] iGEM: Imperial Collage/Encapsulation, 2009</br>
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[9] van der Meer, J. R., & Belkin, S. (2010). Where microbiology meets microengineering: design and applications of reporter bacteria. Nature Reviews Microbiology, 8(7), 511-522.</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