Team:Peking/Project/Devices

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<li><a href="https://2013.igem.org/Team:Peking/Project/Plugins">Adaptors</a></li>
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           <ul id="ProjectList">
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                 <li class="SensorsListItem"><a href="#Milestone1">Purposes</a><li>
                 <li class="SensorsListItem"><a href="#Milestone1">Purposes</a><li>
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                 <li class="SensorsListItem"><a href="#Alginate Encapsulation">Alginate Encapsulation</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="#Protective Agents">Protective Agents</a><li>
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                 <li class="SensorsListItem"><a href="#Milestone4">Advanced Design</a><li>
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                <li class="SensorsListItem"><a href="#Advanced Design">Advanced Design</a><li>
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                <li class="SensorsListItem"><a href="#Results">Results</a><li>
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                <li class="SensorsListItem"><a href="#Potentials">Potentials</a><li>
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       <h1 id="Purposes";>Purposes</h1>
       <h1 id="Purposes";>Purposes</h1>
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      <p>To realize the idea of in-field detection, a device with remarkable convenience for monitoring environmental water pollution should be proposed. This device must be capable of determining if the specific kind of aromatic compound exists in a water sample, and perhaps more significantly, measuring specific aromatic compound’s concentration semi-quantitatively. To meet the requirement of convenience, the detection process must be fast and the result must be read with naked eyes or with user-friendly devices. As for measuring the concentration, a concentration gradient could be constructed by the pre-treating method of the device, so different response patterns may roughly reflect the concentration. All this requirements must be carefully designed with biosafety concerns.
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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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The most challenging part of a biological detection device is the preservation method. The sensor strain we use, Escherichia coli, failed to germinate spores or gemma to resist general preservation conditions of dehydration, temperature changes and physical interference. Several approaches were designed to achieve valid maintainance. Based on the fundamental designs, an advanced device with multifunctions was developed to measure the concentration carrying potential for further improvements<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>
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     <div id="Milestone2"></div>
<h2 id="Alginate Encapsulation" style="position: reative; top:20px;">Alginate Encapsulation</h2>
<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;" />
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       <legend>
       <legend>
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<b>Figure 1.</b> The structure of alginate and the cross-link between encapsulation product.
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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 consists of β-D-mannuronic acid (M) and α-L-guluronic acid (G) residues. If treated by calcium irons, G residues are cross-linked and a coat would be formed within a rather short period of time.
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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 is frequently used as the biological encapsulation material for various organisms such as Saccharomyces cerevisiae, Escherichia coli and mammalian cells<sup>[1][2]</sup>. It came into our consideration as the encapsulating material for its outstanding specifications, which include<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>
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(1) Food grade materials which are stable and inexpensive</br>
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(1) Stable and inexpensive, even edible.</br>
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(2) Possibility to use every biosensor we constructed</br>
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(2) Does not interfere with biosensors we constructed.</br>
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(3) Ease to shape and manipulate</br>
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(3) Ease to shape and manipulate.</br>
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(4) Protection against environmental stresses</br>
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(4) Provide reliable protection against environmental stresses.</br>
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The alginate encapsulation successfully solve the problem of dehydration and oxidation stress upon our biosensor strains, so no reviving process is required. With the support of protective agents, the period of validity could be longer than a month in 4℃.
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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>
     <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>
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1.5% Alginate solution was boiled and kept warm in 40°C.</br>
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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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    <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="https://static.igem.org/mediawiki/2013/b/bc/Peking2013_Device_Fig2.png" style="position:relative; width:400px; left:300px;" />
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      <legend style="position:relative; top:-10px;">
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<b>Figure 2.</b> The structure of myo-inositol, which is the active steroisomer, having crucial function in eukaryotic cells.
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      </legend>
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<p style="position: reative; top:20px;"> 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 compatible solutes to maintain a high osmotic pressure in cytoplasm to stablize proteins and balance the dehydration in the environment<sup>[4]</sup>. Inositol is highly compatible for this usage for its stability and hydrophilicity. </br>
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Previous work done by Aitor. et al. showed that inositol is highly efficient for preserving bacterial cells. (In that case, P. putida, which is the host of the transcriptional factors we used in our biosensors. )
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<legend><b>Figure 3.</b> The results indicated that inositol was a protective agent of maintained the viability of bacterial cells. With the existance of maltodextrins(MD), the optimal validity maintaining could be achieved. (figure from: A. Heras and V. Lorenzo, 2011)</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 style="position: reative; top:20px;"><b>Trehalose</b></p>
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<legend><b>Figure 4.</b>The structure of trehalose, which is a disarrcharide that is a major constituent of insects surving as energy storage compound. It has also been found efficient in preventing dehydration.  </legend>
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    <p style="position: reative; top:20px;">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. It is also important to produce in cell trehalose by the bacteria spontaneously, which involves certain gene expression and an osmotic press induction. In E. coli, the otsA and otsB genes are responsible for trehalose biosynthesis from UDP glucose. These genes encode trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase, respectively. And high concentration of NaCl solution induces the expression of intracellular trehalose. <sup>[7]</sup></br>
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To obtain a high validity maintainance, the protective agents’ we use were of ralatively high concentration, so we should test that the induction process was not interfered by the protecting agents. A series inositol and trehalose solutions of different concentration were tested.
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  </p>
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<img src="https://static.igem.org/mediawiki/2013/d/d3/Peking2013_Device_Fig5.png" style="position:relative; top:20px; width:700px; left:150px;" />
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<legend><b>Figure 5.</b>The tests of induction with the existance of different concentration of protective agents, inositol and trehalose. The NahR was induced by 100μM of inducers. The basal line of none protective agent is shown. As is illustrated, these two protective agents didn’t interfere the inducing process within a fairly high concentration. </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;">There is visible difference between the beads with and without induction. and it would be clearer under blue light.  </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="https://static.igem.org/mediawiki/2013/b/b9/Peking2013_Device_Fig7.png" style="position:relative; top:20px; width:700px; left:150px;" />
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<legend><b>Figure 7.</b>The test results of alginate encapsulation beads with XylS and NahR biosensors in it. <b>a and b:</b> After 10h incubation, the fluorescence intensity is sufficiently high to be discriminated by blue LED and after 12 hours, by naked eyes.<b> c: </b> Same inducer(4-MeSaA,100μM) with different incubation time. (from left to right: 0h, 3h, 6h.) showed that the fluorenscent intensity increase during the 3 to 6 hours after induction, but 3 hour is sufficient to create visible difference. <b>d: </b> results for different inducer’s concentration, incubated for 4 hours. (from left to right, 100μM, 10μM and 0μM of 4-MeSaA.)  </legend>
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<p style="position: reative; top:20px;">   
<p style="position: reative; top:20px;">   
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For NahR, 3 hour induction was sufficient to discriminate the 100μM 4-MeBzO’s induction with the blank by naked eyes. The concentration of 100μM was chosen according to previous tests, indicating this concentration was to obtain highest induction ratio without noticable toxicity. (fig. 7b)
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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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3 hours treatment would be possible to obtain noticable difference of induction, and 6 hours would be appropriate to detect aromatics to determine if the water sample can meet the Chinese government’s requirements for water sample in the environment.<b>(fig. 7c)</b>
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<a href="http://www.steriq.cn/pdf/34.pdf">(Link to the Chinese Government’s Requirements)</a>
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</br>
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After 4 hours incubation, the fluoresence of NahR biosensor worked as expected, and 10μM of 4-MeSaA can be detected by this device. <b>(fig. 7d)</b></br>
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Additionally, This experiment was done after 10-day-storage in the 4℃, so the bacteria were likely to survive for more than 10 days. According to the paper’s report, the maintenance of validity of the bacteria could be sure to meet the detection requirements in more than a month<sup>[4]</sup>. </br>
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Although the evidence of this device was inadequately rigorous, the actual products could be used in field to achieve a rough detection. </br>
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</p>
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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="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" />
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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>
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<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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<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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</p>
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</br>
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  <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 on the alginate encapsulation method and previous test results, a hydrogel patterning and transferring method could surve our purposes. This method would be multi-purposes including aromatic detection in a quantitative view, possibility of the cell communication serving the idea of adaptor, and potential for the application 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, which 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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The parallel wells were etched on PDMS, using a PDMS template in which 500 μm×500 μm square patterns are microfabricated and equidistant from each other by 500 μm. The depth of the wells was 170μm. This design was aiming at prevent interaction of E.coli between different suqares, because the interaction may influence the diffusion process for constucting a concentration gradient. Addtionally, the shape could be altered according to the basal level and detection convenience<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 in the wells. After treated with Calcium irons, the PDMS with the mixture was 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 substrate.</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(Link:), the adaptor E.coli cells could be encapsulated in the agarose layer. Then if the substrate of adaptor exist, it would be tranferred into the inducer which could be dected by corresponding biosensor.
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</br>
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For the bandpass filter(Link:), the agarose layer could be pre-treated, that sample and water was drilled on each side 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="https://static.igem.org/mediawiki/2013/5/52/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="https://static.igem.org/mediawiki/2013/5/52/Peking2013_Device_Fig6.png" style="position:relative; top:20px; width:500px; left:100px;" />
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       <legend><b>Figure 6.</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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</legend>
</legend>
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<p>
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<p id="Results"; style="font-size:32px">Results</p>
<p id="Results"; style="font-size:32px">Results</p>
<img src="https://static.igem.org/mediawiki/2013/4/44/Peking2013_Device_Fig8.png" style="position:relative; top:20px; width:700px; left:150px;" />
<img src="https://static.igem.org/mediawiki/2013/4/44/Peking2013_Device_Fig8.png" style="position:relative; top:20px; width:700px; left:150px;" />
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<legend><b>Figure 8.</b> The primary attempt to construct the advanced device. The agarose plate was pre-treated with 100μM 4-MeSaA for 12 hours, and the Photograph was took 4 hours after the induction.The lecuna in the left was designed for the inducer’s loading. For the transferred patterning, some alginate out of wells wasn’t removed. The one on the bottom indicating 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>  
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<h1 id="Potentials"; style="position: reative; top:20px;">Potentials</h1>
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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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<p style="position: reative; top:20px;"> The advanced device design has potentials for the entire aroamtic toolkit’s application. Various biosensors could be encapsulated by the same method, and the agarose layer may also encapsulate another kind of biosensor, or the adaptor bacterial cells. As the fact that the communication between the agarose layer and alginate is fast, it’s possible to conduct multi-sensor assay by this device. It’s also applicable for the adaptors.
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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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</br></br>
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</p>
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Different concentration gradient could be constructed on the agarose layers. More tests are required to obtain the diffusion constant and this parameter could be used to calculate the diffusion time. After determine the pre-treating time, the alginate encapsulation of bandpass filter would be applied to this device. If the concentration of each square pattern could be calculated, then the bandpass filter would show response in different position. Then we may roughly read out the concentration by the response position. This approach would provide an opportunity for in field aromatic detection. </br>
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</br>
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  </p>
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<div id="Milestone6"></div>
<p style="position:relative;top:35px;">
<p style="position:relative;top:35px;">
<b>References</b></br>
<b>References</b></br>
-
[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>
-
[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>
+
[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>
-
[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>
+
[4] <a href="https://2009.igem.org/Team:Imperial_College_London/M2">iGEM: Imperial Collage/Encapsulation, 2009</a></br>
-
[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>
+
-
[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>
+
-
[7] Peking iGEM 2010 wiki</br>
+
-
[8] iGEM: Imperial Collage/Encapsulation, 2009</br>
+
-
[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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-
 
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</p>
</p>

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