Difference between revisions of "Team:BIT/BA1"

 
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<h2>Abstract</h2>
 
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<p><font size=3>Cancer is one of the three major killers that threatened mankind since the 21st century. The reason is that it is not taken seriously at the beginning of cancer until it is detected in the middle and late stages, resulting in a very low cure rate. Therefore, early detection becomes a decisive factor to improve the cure rate of cancer. In this project, we use alpha-fetoprotein (AFP)as an example, using the factor that AFP can specifically bind to the aptamer, to achieve a signal conversion that using a small molecule (lysine) to replace AFP content in serum, just like equivalent substitutions in mathematics, to achieve the early detection of liver cancer. This system can be extended to all of the disease detection, which use protein as biomarkers.</font> </div>
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<p><font size=3>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.      
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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. </font> </div>
 
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<p>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.</span><br>
 
<p>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.</span><br>
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<p>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.</span><br>
 
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<p>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.</p>
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<p>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. </p>
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<p>The conventional methodAs 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. is ELISA and other antibody-based techniques to enrich low-abundance molecular markers. ELISA which is based on "antigen-antibody reaction" of the detection method is a little bit expensive. Time and temperature are having a greater impact for its detection. </p>
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<center><font size=1>Figure 1：BIT_Figure_About genetic circiut.</font></center><br>
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                                      <h3>3.</h3>
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<p><br>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.<br>
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                                      <h3>5.</h3>
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<p><br>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.
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<p>The purpose of this project is to establish a detection platform suitable for most molecular markers. The platform is low cost, simple to operate and has strong anti - interference ability. </p>
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<p><font size=3>This project uses the AP273 with the highest binding to AFP and the complementary chain paired with the aptamer.
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Aptamer is an oligonucleotide sequence or short polypeptide obtained by in vitro screening. Aptamer, as an alternative to the antibody, can bind to the corresponding ligand with high affinity and strong specificity. Its low price, easy to screen synthesis, the nature of stability and other advantages are widely used in the detection of molecular markers of disease. Its presence provides a new way to quickly and efficiently identify bio-makers.</font></span><br>
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  Figure 4 ：The 3D Structure of AP273<br>
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<p><font size=3>First of all, we select an aptamers AP273, which could best affinity for AFP. We modified a biotin at the end of its 5’ end, which could affinity for streptavidin. Because of the presence of streptavidin-modified beads, aptamer has been locked firmly on beads. At the same time, a complementary chain was synthesized in its protein binding site. We modified an amino at the 3’ end of the complementary chain which can be combined with lysine protected by BOC anhydride to link lysine to the complementary chain. Because Van der Waals forces between AFP and aptamers is stronger than hydrogen bond, due to the action of magnet lying at the bottom of the reaction system, aptamers are separated from complementary chain, one at the bottom, the other in the supernatant. At this point, with the action of trypsin, lysine can be separated from the complementary chain, and start his adventure.</font>
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Latest revision as of 17:23, 31 October 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.

Free HTML5 Bootstrap Template
Figure 1：BIT_Figure_About genetic circiut.

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.