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Team:BIT/project AC
From 2013.igem.org
| Amplifier and Controller |
| Amplifier |
| If we give the biosensor an input signal, we will get an output signal, which, however, may not be strong enough for us to detect. Therefore, we have designed an amplifier, which is based on the high activity of T7 promoter, to increase the intensity of the output signal to a specific magnification. We replaced the sequence of the green fluorescent protein of the sensor with the DNA of T7 RNA polymerase to promote the expression of the downstream DNA. Thus we can get stronger fluorescent intensity as expected. |
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| As we all know, if the sample of material which the concentration is high enough to be detected is inserted as the input signal, the sensor will be able to “feel” it and produce an output signal. |
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| However, sometimes, the output is not strong enough for us to detect. To magnify the output signal, we took advantage of the high activity of the T7 promoter. Because the T7 promoter can only be activated by the T7 RNA polymerase, a gene of T7 RNA polymerase and a T7 promoter were inserted at the downstream site of the sensor to get a stronger output. This part is what we call an “amplifier”. |
| Controller |
| Sometimes we need to enhance the output signal to different degrees. In other words, we want to control the magnification. A “controller” is designed to solve this problem. We inserted a lacO operator between the DNA of T7 RNA polymerase and green fluorescent protein, and added a lacI biobrick in the system. When there is low concentration of IPTG, the lacI will close the lacO to inhibit the expression of gfp DNA. When we add IPTG to the sample, the lacI will be combined with IPTG, and the inhibition of the expression of the downstream DNA will be inhibited. Thus we can control the magnification by controlling the concentration of IPTG. |
| Here we introduced a new part which contains lacI and lacO in the system. The gene of lacI is always expressing, which inhibits the expression of lacO. In this case, even if there is an input signal, no egfp will be expressed. |
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| If we put IPTG in the environment as an inducer, the lacI protein will combine to the IPTG molecules and thus the inhibition will be ceased. As a result, the lacO will be activated, which will lead to the expression of downstream egfp. |
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| With a stable concentration of IPTG, the system will work as expected. When a weak input is given, a weak green fluorescence will be detected, while if the input gets stronger, the intensity of the green fluorescence will increase simultaneously. |
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| Sometimes we would like to control the magnification. This could be realized by regulating the concentration of IPTG. The higher the concentration of IPTG is, the more lacI will be combined. As a result, the expression of downstream egfp will be enhanced. |
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| Similarly, we can decrease the magnification by lowering the concentration of IPTG. This is the part what we call a “controller”. |
| Result |
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