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Revision as of 18:30, 1 November 2017
Abstract
To transform the lysine signal to the kind of signal which is easy to be detected, we chose to use a lysine deficient E.coli. It can hardly grow without lysine. But when the medium contains lysine released from the complementary chain, the E.coli ΔLysA begins to grow. Meanwhile, we transformed a plasmid with fluorescent protein gene into the E.coli ΔLysA. So with the growth of the E.coli ΔLysA, there will be fluorescence which is easy to be detected. In this way, we can transform the lysine signal to the fluorescence signal. Consider that the lysine concentration could be weak, we decided to use a strong promoter(BBa_J23100) and a cyclic amplifier to improve the signal, and a dual fluorescence system to improve the sensitivity.
Introduction
1
After the lysine has been dissociated from complementary chain, it will induce the growth of E.coli ΔLysA, which can hardly grow without the addition of lysine. Thus we have transformed our own-designed circuit to the E.coli ΔLysA, which can express fluorescent protein as it grows. In this way, we can transform the concentration of lysine to the fluorescent intensity which can be detected through the machine.
4.
Besides the luxR and luxI generator, there are also lacI generator and GFP reporter downstream the plux promoter. LacI represses plac promoter, leading to an inhibition to the RFP expression. So with the induction of plux promoter, there will be a higher GFP fluorescence and a lower RFP fluorescence.
2.
But the concentration of the lysine is very low, resulting in a low fluorescent intensity. To solve this problem, we designed a strong promoter, cyclic amplifier system and a dual fluorescence system in our genetic circuit to ensure a much higher signal strength and sensitivity, which is definitely beneficial to our detection of AFP.
3.
As shown, pcat is a strong constitutive promoter, which can promote the induced promoter plux. Promoter plux can be induced by the compound of LuxR and AHL. Downstream the plux, we designed the a cyclic amplifier system: luxR generator codes LuxR, and LuxI generator codes luxI, which can catalyze the synthesis of AHL. When the concentration of AHL has reached the threshold value, AHL combines LuxR to form a compound, strongly inducing the promoter plux. This induction will continue to induce the expression of the luxR and luxI generator downstream, forming a positive feedback. That’s how our cyclic amplifier works.
5.
Thus, the whole process is as follows: When the AFP concentration has reached the LOD, it will combine the aptamer and release the complementary chain and lysine, which will express the fluorescent protein. With the help of the strong promoter and the cyclic amplifier, induced promoter will lead to a higher GFP fluorescence and a lower RFP fluorescence. Finally, we use the GFP and RFP fluorescence ratio, with a higher sensitivity, to be the output signal of our detecting machine.
