Difference between revisions of "Team:ZJU-China/Demonstrate"

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     <div align="left" style="font-family: 'spr';font-size:40px;border-bottom:2px solid #584b4f;">Light Control</div>
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     <div align="left" style="font-family: 'spr';font-size:40px;border-bottom:2px solid #584b4f;">Mechanical Device</div>
 
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&nbsp;&nbsp;&nbsp;&nbsp;As a cipher machine, it must have the ability to input information conveniently. Therefore, it is crucial to have a rapid and reliable input system. After research and discussion, we finally find the ideal solution: Light-switchable two-component systems (TCSs). Its two components are green-light system (CcaS-CcaR system) and red-light system (Cph8-OmpR system). Besides, our encrypted information is in binary form, this system could satisfy our input requirement. Following is a detailed description.
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&nbsp;&nbsp;&nbsp;&nbsp;In addition to the microfluidics device as the core part, we have designed the mechanical device for the project. The whole system consists of four parts: the main body, lighting board, shading baffle and microfluidics chip. The details for each part are described in the Hardware page.
 
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&nbsp;&nbsp;&nbsp;&nbsp;When assembling, four LED lights are inserted into the corresponding four holes. Four single-pole double-throw switches on the external circuit board can switch the light color (red or green). The power switch is on the battery box. Microfluidics chip is inserted into the upper slot. The shading baffle, which has the same size as the microfluidics chip, is inserted into the lower slot, to avoid the interference from unrelated light.
 
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        <img src="https://static.igem.org/mediawiki/2016/f/f1/ZJUfig1.jpg" height="60%" width="75%">
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</br>Fig.1 Operation Demonstration
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&nbsp;&nbsp;&nbsp;&nbsp;In the green-light system, ccaS protein is fixed on the cell membrane. After combined with chromophore ho1 and pcyA, ccaS could absorb green light around 520nm wavelength long, and thus, its own phosphate group will transfer, resulting in the phosphorylation of ccaR, an intracellular protein. The ccaR then could activate the expression of PcpcG2 promoter. Using this system, optical signal can be used as input, and can be received by engineered E.coli. However, after being irradiated by red light around 650nm wavelength long, the phosphorylation transfer process (from ccaS to ccaR) will be inhibited.
 
 
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  <div align="left" style="font-family: 'spr';font-size:40px;border-bottom:2px solid #584b4f;">Enigma</div>
&nbsp;&nbsp;&nbsp;&nbsp;After a discussion about our literature, we settled the size of each trap and the length, width, and height are 100 um, 85um and 5um, respectively, which is the most favorable one for oscillation experiment and dynamic response model. Two traps stand 150um apart at the same side, while for the opposite side, the groove is 200um wide and 100um deep which is convenient for fluid to pass. Also, according to our calculation, we are quite sure that this design can increase the flow of AHL quorum sensing substance, and can let the cells at the edge of expansion colonies be watered away, making it possible for bacteria in the traps maintaining a continuous exponential growth.For observing convenience, we divide the traps into 4 groups, and each has 30 traps. The distance between two groups is sixty thousands um.
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&nbsp;&nbsp;&nbsp;&nbsp;When using, the overall device will be placed in incubator of 37 ° . Pressing the four switches can adjust the LED light into the appropriate color, waiting for bacteria to respond to light signals. Later, take out the baffle and observe the color of each chamber, and that is the "cipher text" we get.
 
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&nbsp;&nbsp;&nbsp;&nbsp;When working, there are five kinds of bacteria in our second generation of cipher machine: the core bacteria with the oscillation circuit and four affiliated bacteria (which are actually the first generation of cipher machine). And as for these four bacteria, they are controlled by the oscillation system and contain the signal transformation circuit (which is composed of the light-induced circuit and the logic gate circuit). Before using the cipher machine, we should first mix these five bacteria together according to a certain proportion and insert them into the microfluidics chip. And then, insert the chip in our device and put the whole device in the thermostated container.
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</br></br>
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<div align="center"><img src="https://static.igem.org/mediawiki/2016/3/35/ZJUfig2a.jpg" height="60%" width="75%">
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</br>Fig.2
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</div>
  
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&nbsp;&nbsp;&nbsp;&nbsp;When encrypting, we should select a specific time (for example, to begin counting when the bacteria solution is poured into the microfluidics chip) to press the LED switch on the top of the device according to the input information (for example, if the input is 0101, then the LED light should be red, green, red, green). Using corresponding light to irradiate different areas on the chip (we regard each area as an encryption unit. In our initial device, we select 4 areas as encryption units). After a period of time, we can observe fluorescence which is the cipher text (for example, if we observe gfp, rfp, rfp, gfp, then the output is 1001).
  
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        <img src="https://static.igem.org/mediawiki/2016/b/b8/Hardware-2.jpg" height="60%" width="75%">
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</br>Fig.3
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&nbsp;&nbsp;&nbsp;&nbsp;When decrypting, input the cipher text by pressing the LED switch at the same time, or at the same phase in the oscillation period (for example, if the cipher text is 1001, then the LED light should be green, red, red, green). After a period of time, we can observe the original text (for example, if we observe rfp, gfp, rfp, gfp, then the output is 0101).
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    <div align="center" style="font-family: 'spr';font-size:40px;">Others</div>
 
&nbsp;&nbsp;&nbsp;&nbsp;For overall experiment, we also designed a device to help us using the microfludic chip. As for the body part, the upper platform has four through-holes, for putting in the LEDs. The lower part has the two slots, size of each slot are the same as the microfluidic chip. And we have a shading baffle whose size is appropriate with slots. Devices and shading baffle are produced by 3D printing, the material we chose is resin. It has smooth surface, high precision, and good hardness. Taking into account that the device will be placed in the thermostat, we used a kind of resin that has small thermal deformation coefficient, this will ensure that assembling won’t be a problem. In order to ensure that the experiment will not be interfered by the stray light, all surfaces are sprayed with black matte paint.
 
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&nbsp;&nbsp;&nbsp;&nbsp;However, if we input the cipher text at a wrong time (which means the user does not know the encryption time), even if the cipher text is true (1001), the output is not convincing, sometimes even opposite from the real information (for example, 0110) because at this phase, the mechanism of cipher machine is not the same as the one when encrypting. So, compared with the first generation, this second generation of cipher machine is more reliable.
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  The LED we used is a common cathode four-pin light-emitting diodes, we only use 1 (red), 2 (negative), 3 (green) three pins. The wavelengths were 630 ~ 640 nm and 515 ~ 525 nm, respectively. The brightness of the lights is around gigahertz candela. All of them can meet our experimental requirements
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&nbsp;&nbsp;&nbsp;&nbsp;We have got the double-period oscillation circuit already. But when doing an experiment, we could not realize a stable oscillation because of the continuous split of bacteria and the accumulation of autoinducers. In order to solve this problem, we use micro-fluid technology (Fig.5a). Microfluidics is science and technology using micro-channel to operate micro fluid. Using this technology, we can keep the quantity of bacteria at a certain level and take the accumulated autoinducers away, making the system a better coupling and could operate steadily. More details about this device could be found in Hardware page.
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<address>
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  <strong>Contact Us</strong></br>
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  Room 413,Biology lab center, Zijingang Campus</br>
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  Zhejiang University, YuHangTang Road NO.866</br>
 +
  Hangzhou, China</br>
 +
</address>
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 +
<address>
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  <strong>iGEM ZJU-China 2016 Team</strong><br>
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  <a href="mailto:#">igem_zjuchina_2016@outlook.com</a>
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</address>
  
  
 
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Revision as of 13:30, 19 October 2016

Design

Mechanical Device

    In addition to the microfluidics device as the core part, we have designed the mechanical device for the project. The whole system consists of four parts: the main body, lighting board, shading baffle and microfluidics chip. The details for each part are described in the Hardware page.
    When assembling, four LED lights are inserted into the corresponding four holes. Four single-pole double-throw switches on the external circuit board can switch the light color (red or green). The power switch is on the battery box. Microfluidics chip is inserted into the upper slot. The shading baffle, which has the same size as the microfluidics chip, is inserted into the lower slot, to avoid the interference from unrelated light.

Fig.1 Operation Demonstration




Enigma

    When using, the overall device will be placed in incubator of 37 ° . Pressing the four switches can adjust the LED light into the appropriate color, waiting for bacteria to respond to light signals. Later, take out the baffle and observe the color of each chamber, and that is the "cipher text" we get.
    When working, there are five kinds of bacteria in our second generation of cipher machine: the core bacteria with the oscillation circuit and four affiliated bacteria (which are actually the first generation of cipher machine). And as for these four bacteria, they are controlled by the oscillation system and contain the signal transformation circuit (which is composed of the light-induced circuit and the logic gate circuit). Before using the cipher machine, we should first mix these five bacteria together according to a certain proportion and insert them into the microfluidics chip. And then, insert the chip in our device and put the whole device in the thermostated container.


Fig.2

    When encrypting, we should select a specific time (for example, to begin counting when the bacteria solution is poured into the microfluidics chip) to press the LED switch on the top of the device according to the input information (for example, if the input is 0101, then the LED light should be red, green, red, green). Using corresponding light to irradiate different areas on the chip (we regard each area as an encryption unit. In our initial device, we select 4 areas as encryption units). After a period of time, we can observe fluorescence which is the cipher text (for example, if we observe gfp, rfp, rfp, gfp, then the output is 1001).


Fig.3

    When decrypting, input the cipher text by pressing the LED switch at the same time, or at the same phase in the oscillation period (for example, if the cipher text is 1001, then the LED light should be green, red, red, green). After a period of time, we can observe the original text (for example, if we observe rfp, gfp, rfp, gfp, then the output is 0101).


Fig.4

    However, if we input the cipher text at a wrong time (which means the user does not know the encryption time), even if the cipher text is true (1001), the output is not convincing, sometimes even opposite from the real information (for example, 0110) because at this phase, the mechanism of cipher machine is not the same as the one when encrypting. So, compared with the first generation, this second generation of cipher machine is more reliable.
    We have got the double-period oscillation circuit already. But when doing an experiment, we could not realize a stable oscillation because of the continuous split of bacteria and the accumulation of autoinducers. In order to solve this problem, we use micro-fluid technology (Fig.5a). Microfluidics is science and technology using micro-channel to operate micro fluid. Using this technology, we can keep the quantity of bacteria at a certain level and take the accumulated autoinducers away, making the system a better coupling and could operate steadily. More details about this device could be found in Hardware page.

Contact Us
Room 413,Biology lab center, Zijingang Campus
Zhejiang University, YuHangTang Road NO.866
Hangzhou, China
iGEM ZJU-China 2016 Team
igem_zjuchina_2016@outlook.com