http://2013.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=500&target=GaoJian+NJU&year=&month=2013.igem.org - User contributions [en]2024-03-29T05:17:39ZFrom 2013.igem.orgMediaWiki 1.16.5http://2013.igem.org/Team:Nanjing-China/qsTeam:Nanjing-China/qs2013-10-27T12:44:29Z<p>GaoJian NJU: </p>
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<dt><a href="###">Aggregation</a></dt><br />
<dd class="dd_1"><br />
In our project, we considered devising our bacteria into some kind of automatic machinery that would be able to sense a target molecule and then assemble to degrade it. To fulfill all these functions, we were bound to design some gene circuits to guide our bacteria.<br><br><br />
<br />
First of all, we should have an understanding of how bacteria move<sup>[1]</sup>. For E.coli, they have flagellar motors to control their movement (Fig. 3-5-1). The direction of rotation of their flagellar motors is controlled by the protein CheY. When CheY is not phosphorylated, the flagellar motors rotate counterclockwise (CCW) and bacteria can migrate. When CheY is phosphorylated (CheY-P), it can bind to the flagellar motorprotein FliM, causing the cell to tumble and thus nonmotile. Phosphatase CheZ dephosphorylates CheY-P and causes the flagellum to rotate CCW. E. coli lacking the cheZ gene (ΔcheZ) cannot dephosphorylate CheY-P, and thus tumble incessantly, being nonmotile. So we only need to take control of gene cheZ to control the motility of our bacteria. <br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/0/04/Fig.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-1</strong> Illustration of how bacteria move. CheZ is the key of controlling the motility of bacteria. For wild type, CheZ can be expressed normally, so the flagellar motor alternates between CCW and CW rotation, thus the cell can run or tumble. On the contrary, if there is no CheZ, CheY will remain phosphorylated, causing the flagellar motor rotates CW, thus the cell tumbles exclusively. (Cite by JACS, Shana Toop et al, 2007.)</div><br><br><br />
We would like our bacteria to seek and destroy atrazine in the soil. To achieve this goal, a classic system can do the job (Fig. 3-5-2A). In this system, bacteria can move around randomly but fix when sensing atrazine as the ribosome switch turns on and protein CI is expressed to repress the gene cheZ. As a result, our bacteria would just assemble around the target molecules after a period of time.<br><br><br />
However, we can only wait for the bacteria to hang about until they find their targets. Thus it could take a long time before the bacteria assembly and the system may not be a robust design. So we shall spare no efforts to improve it. For better control over the function of our bacteria, we took the quorum sensing system into consideration. We expect some bacteria which first find the atrazine will recruit other bacteria at a distance through some signal so they move towards the target and then fixed at the source of pollution. Therefore we constructed the following gene circuits to realize it (Fig. 3-5-2B).<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/6/63/Fig_3-5-2.jpg" width="716"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-2</strong> The gene circuits we built to control cheZ. (A) A classic system using CI to repress cheZ. (B) A complex but high-efficiency system using quorum sensing system. (C) A simplified system like the system in (B).</div><br><br><br />
This circuit is composed of three modules: an atrazine-sensing module, a recruiting module, and a brake module. We adopted the quorum-sensing system in Vibrio fischerias the basis for our design. In this system, a small-molecule acyl-homoserine lactone (AHL) synthesized by LuxI excretes as a signal and accumulates intracellularly to activate a constitutively expressed regulator, LuxR<sup>[2]</sup>. Our atrazine-sensing module controls the translation of LuxI through ribosome switch which turns on when combined to atrazine. Meanwhile, the recruiting module is composed of a series of elements. In this system, the LuxR-AHL complex promotes the expression of a repressor TetR, which represses the expression of another repressor CI. The gene cheZ is controlled by promoter Pλ, which is repressed by CI. So for those bacteria which first sense atrazine, they send out AHL signal and let the other bacteria receive it. After that, TetR is expressed to repress CI, leading the transcription of cheZ. These bacteria move towards the sender until they sense atrazine and another repressor CI, which is controlled by ribosome switch, expresses as a brake of the movement<sup>[3]</sup>. <br><br><br />
We also design the third gene circuit to simplify the system (Fig. 3-5-2C). This system also consists of the three modules in Pattern 2 and the atrazine-sensing module, as well as the brake module, is exactly the same composition with those two parts. The only difference is in the recruiting module that we use a double-functional promoter Plux/CI. The atrazine-sensing module works just the same way in Pattern 2. In this system, however, the LuxR-AHL complex promotes cheZ transcription via promoter Plux/CI. At the spot where bacteria sense atrazine, AHL will accumulate. The higher AHL concentration is, the stronger transcription of cheZ which means greater motility is, leading to directional movement towards bacteria which send out the signal. Meanwhile, the brake module also functions via promoter Plux/CI. The repressor CI, controlled by ribosome switch which turns on when combined to atrazine, represses the promoter Plux/CI and results in bacteria fixation at source of pollution.<br><br><br />
<strong>Experiments and Results</strong><br><br />
At the beginning, we verified gene cheZ in genetic level. We had 3 strains, namely CL-1, CL-M, and CL-IM. CL-1 is a strain with deletion of gene cheZ, CL-M is wildtype, and CL-IM is a strain with deletion of the gene that controls the expression of flagella. We extracted the total RNA of these 3 strains and synthesized the 1st cDNA using RT-PCR. Then we did PCR of gene cheZ with the cDNA as template and we also did PCR of 16 sDNA as control. We got the figure through agarose gel electrophoresis (Fig. 3-5-3A). We can see that CL-1 is actually the strain with deletion of gene cheZ, while the other two strains have normal gene cheZ.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/e/e0/Fig_4-3_2.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-3</strong> Verification of gene cheZ. (A) In genetic level, CL-1 is actually the strain with deletion of gene cheZ, while the other two strains have normal gene cheZ. (B) In phenotype level, CL-M and pcheZ is able to migrate on semisolid culture medium, while CL-1 and CL-M is unable to migrate.</div><br><br><br />
Then we needed to verify of gene cheZ from phenotype. We have 3 strains, namely CL-1, CL-M, and CL-IM. CL-1 is a strain with deletion of gene cheZ, CL-M is wild type, and CL-IM is a strain with deletion of the gene that controls the expression of flagella. Then we transformed a plasmid with gene cheZ into CL-1 and got the fourth strain pcheZ.<br><br><br />
We inoculated the suspension of bacteria on semisolid LB plate and incubated for 20h and took the photograph (Fig. 3-5-3B). From the figure, we can see that CL-M and pcheZ is able to migrate on semisolid culture medium, while CL-1 and CL-M is unable to migrate. This indicates that E.coli is nonmotile without gene cheZ or flagella and inducing the expression of gene cheZ can restore motility in a cheZ knockout strain.<br><br><br />
In addition, we testified the classic gene circuit.We mix the suspension of bacteria with pre-heated semisolid culture medium and drop solution of atrazine on the plate. Our bacteria assembled around one of the targets after incubation for a period of time (Fig. 3-5-4). It is highly possible that they assembles around the other target after atrazine in the first area is degraded and they are able to move freely again.<br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/2/26/3-4%EF%BC%88%E5%A4%87%E7%94%A8%EF%BC%89.jpg" width="300"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-4</strong> Our bacteria could just assemble around the target molecules after a period of time.</div><br><br><br />
Finally, we also verified lux system. We constructed a strain with gene circuit as shown in Fig. 3-5-5A from CL-1. This is a lux system without the ribosome switch. AHL synthesized by LuxI excretes as a signal and accumulates intracellularly to activate a constitutively expressed regulator, LuxR. Then the LuxR-AHL complex promotes the expression of a repressor CI, leading to repression of CheZ. So bacteria will assembly at spots where initial density is high in this system. After we inoculated the suspension of bacteria on semisolid LB plate and incubated for 20h, we took the photograph of a stripe pattern (Fig. 3-5-5B), which was formed under the influence of the lux system<sup>[4]</sup>.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/8/8a/Fig_4-4_2.jpg"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-5</strong> Verification of lux system. (A) the gene circuit we constructed. (B) The phenotype of lux system. </div><br/><br />
For more information of our assembly system, please refer to <a href="https://2013.igem.org/Team:Nanjing-China/model"><strong>Model</strong></a>.<br><br />
<br/> <br />
<br />
<strong> Reference</strong><br/><br />
[1] Topp S and Gallivan JP. Guiding bacteria with small molecules and RNA. Journal of the American Chemical Society, 2007, 129 (21): 6807-6811.<br/><br />
[2] Carter KK, et al. Pathway engineering via quorum sensing and sRNA riboregulators-Interconnected networks and controllers. Metabolic Engineering, 2012, 14 (3): 281-288.<br/><br />
[3] Sinha J, et al. Reprogramming bacteria to seek and destroy a herbicide. Nature chemical biology, 2010, 6 (6): 464-470.<br/><br />
[4] Liu C, et al. Sequential establishment of stripe patterns in an expanding cell population. Science, 2011, 334 (6053): 238-241.<br/><br />
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</html></div>GaoJian NJUhttp://2013.igem.org/Team:Nanjing-China/qsTeam:Nanjing-China/qs2013-10-27T12:43:59Z<p>GaoJian NJU: </p>
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<dt><a href="###">Aggregation</a></dt><br />
<dd class="dd_1"><br />
In our project, we considered devising our bacteria into some kind of automatic machinery that would be able to sense a target molecule and then assemble to degrade it. To fulfill all these functions, we were bound to design some gene circuits to guide our bacteria.<br><br><br />
<br />
First of all, we should have an understanding of how bacteria move<sup>[1]</sup>. For E.coli, they have flagellar motors to control their movement (Fig. 3-5-1). The direction of rotation of their flagellar motors is controlled by the protein CheY. When CheY is not phosphorylated, the flagellar motors rotate counterclockwise (CCW) and bacteria can migrate. When CheY is phosphorylated (CheY-P), it can bind to the flagellar motorprotein FliM, causing the cell to tumble and thus nonmotile. Phosphatase CheZ dephosphorylates CheY-P and causes the flagellum to rotate CCW. E. coli lacking the cheZ gene (ΔcheZ) cannot dephosphorylate CheY-P, and thus tumble incessantly, being nonmotile. So we only need to take control of gene cheZ to control the motility of our bacteria. <br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/0/04/Fig.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-1</strong> Illustration of how bacteria move. CheZ is the key of controlling the motility of bacteria. For wild type, CheZ can be expressed normally, so the flagellar motor alternates between CCW and CW rotation, thus the cell can run or tumble. On the contrary, if there is no CheZ, CheY will remain phosphorylated, causing the flagellar motor rotates CW, thus the cell tumbles exclusively. (Cite by JACS, Shana Toop et al, 2007.)</div><br><br><br />
We would like our bacteria to seek and destroy atrazine in the soil. To achieve this goal, a classic system can do the job (Fig. 3-5-2A). In this system, bacteria can move around randomly but fix when sensing atrazine as the ribosome switch turns on and protein CI is expressed to repress the gene cheZ. As a result, our bacteria would just assemble around the target molecules after a period of time.<br><br><br />
However, we can only wait for the bacteria to hang about until they find their targets. Thus it could take a long time before the bacteria assembly and the system may not be a robust design. So we shall spare no efforts to improve it. For better control over the function of our bacteria, we took the quorum sensing system into consideration. We expect some bacteria which first find the atrazine will recruit other bacteria at a distance through some signal so they move towards the target and then fixed at the source of pollution. Therefore we constructed the following gene circuits to realize it (Fig. 3-5-2B).<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/6/63/Fig_3-5-2.jpg" width="716"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-2</strong> The gene circuits we built to control cheZ. (A) A classic system using CI to repress cheZ. (B) A complex but high-efficiency system using quorum sensing system. (C) A simplified system like the system in (B).</div><br><br><br />
This circuit is composed of three modules: an atrazine-sensing module, a recruiting module, and a brake module. We adopted the quorum-sensing system in Vibrio fischerias the basis for our design. In this system, a small-molecule acyl-homoserine lactone (AHL) synthesized by LuxI excretes as a signal and accumulates intracellularly to activate a constitutively expressed regulator, LuxR<sup>[2]</sup>. Our atrazine-sensing module controls the translation of LuxI through ribosome switch which turns on when combined to atrazine. Meanwhile, the recruiting module is composed of a series of elements. In this system, the LuxR-AHL complex promotes the expression of a repressor TetR, which represses the expression of another repressor CI. The gene cheZ is controlled by promoter Pλ, which is repressed by CI. So for those bacteria which first sense atrazine, they send out AHL signal and let the other bacteria receive it. After that, TetR is expressed to repress CI, leading the transcription of cheZ. These bacteria move towards the sender until they sense atrazine and another repressor CI, which is controlled by ribosome switch, expresses as a brake of the movement<sup>[3]</sup>. <br><br><br />
We also design the third gene circuit to simplify the system (Fig. 3-5-2C). This system also consists of the three modules in Pattern 2 and the atrazine-sensing module, as well as the brake module, is exactly the same composition with those two parts. The only difference is in the recruiting module that we use a double-functional promoter Plux/CI. The atrazine-sensing module works just the same way in Pattern 2. In this system, however, the LuxR-AHL complex promotes cheZ transcription via promoter Plux/CI. At the spot where bacteria sense atrazine, AHL will accumulate. The higher AHL concentration is, the stronger transcription of cheZ which means greater motility is, leading to directional movement towards bacteria which send out the signal. Meanwhile, the brake module also functions via promoter Plux/CI. The repressor CI, controlled by ribosome switch which turns on when combined to atrazine, represses the promoter Plux/CI and results in bacteria fixation at source of pollution.<br><br><br />
<strong>Experiments and Results</strong><br><br />
At the beginning, we verified gene cheZ in genetic level. We had 3 strains, namely CL-1, CL-M, and CL-IM. CL-1 is a strain with deletion of gene cheZ, CL-M is wildtype, and CL-IM is a strain with deletion of the gene that controls the expression of flagella. We extracted the total RNA of these 3 strains and synthesized the 1st cDNA using RT-PCR. Then we did PCR of gene cheZ with the cDNA as template and we also did PCR of 16 sDNA as control. We got the figure through agarose gel electrophoresis (Fig. 3-5-3A). We can see that CL-1 is actually the strain with deletion of gene cheZ, while the other two strains have normal gene cheZ.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/e/e0/Fig_4-3_2.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-3</strong> Verification of gene cheZ. (A) In genetic level, CL-1 is actually the strain with deletion of gene cheZ, while the other two strains have normal gene cheZ. (B) In phenotype level, CL-M and pcheZ is able to migrate on semisolid culture medium, while CL-1 and CL-M is unable to migrate.</div><br><br><br />
Then we needed to verify of gene cheZ from phenotype. We have 3 strains, namely CL-1, CL-M, and CL-IM. CL-1 is a strain with deletion of gene cheZ, CL-M is wild type, and CL-IM is a strain with deletion of the gene that controls the expression of flagella. Then we transformed a plasmid with gene cheZ into CL-1 and got the fourth strain pcheZ.<br><br><br />
We inoculated the suspension of bacteria on semisolid LB plate and incubated for 20h and took the photograph (Fig. 3-5-3B). From the figure, we can see that CL-M and pcheZ is able to migrate on semisolid culture medium, while CL-1 and CL-M is unable to migrate. This indicates that E.coli is nonmotile without gene cheZ or flagella and inducing the expression of gene cheZ can restore motility in a cheZ knockout strain.<br><br><br />
In addition, we testified the classic gene circuit.We mix the suspension of bacteria with pre-heated semisolid culture medium and drop solution of atrazine on the plate. Our bacteria assembled around one of the targets after incubation for a period of time (Fig. 3-5-4). It is highly possible that they assembles around the other target after atrazine in the first area is degraded and they are able to move freely again.<br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/2/26/3-4%EF%BC%88%E5%A4%87%E7%94%A8%EF%BC%89.jpg" width="300"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-4</strong> Our bacteria could just assemble around the target molecules after a period of time.</div><br><br><br />
Finally, we also verified lux system. We constructed a strain with gene circuit as shown in Fig. 3-5-5A from CL-1. This is a lux system without the ribosome switch. AHL synthesized by LuxI excretes as a signal and accumulates intracellularly to activate a constitutively expressed regulator, LuxR. Then the LuxR-AHL complex promotes the expression of a repressor CI, leading to repression of CheZ. So bacteria will assembly at spots where initial density is high in this system. After we inoculated the suspension of bacteria on semisolid LB plate and incubated for 20h, we took the photograph of a stripe pattern (Fig. 3-5-5B), which was formed under the influence of the lux system<sup>[4]</sup>.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/8/8a/Fig_4-4_2.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-5</strong> Verification of lux system. (A) the gene circuit we constructed. (B) The phenotype of lux system. </div><br/><br />
For more information of our assembly system, please refer to <a href="https://2013.igem.org/Team:Nanjing-China/model"><strong>Model</strong></a>.<br><br />
<br/> <br />
<br />
<strong> Reference</strong><br/><br />
[1] Topp S and Gallivan JP. Guiding bacteria with small molecules and RNA. Journal of the American Chemical Society, 2007, 129 (21): 6807-6811.<br/><br />
[2] Carter KK, et al. Pathway engineering via quorum sensing and sRNA riboregulators-Interconnected networks and controllers. Metabolic Engineering, 2012, 14 (3): 281-288.<br/><br />
[3] Sinha J, et al. Reprogramming bacteria to seek and destroy a herbicide. Nature chemical biology, 2010, 6 (6): 464-470.<br/><br />
[4] Liu C, et al. Sequential establishment of stripe patterns in an expanding cell population. Science, 2011, 334 (6053): 238-241.<br/><br />
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</html></div>GaoJian NJUhttp://2013.igem.org/File:Fig_4-4_2.jpgFile:Fig 4-4 2.jpg2013-10-27T12:43:30Z<p>GaoJian NJU: </p>
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<div></div>GaoJian NJUhttp://2013.igem.org/Team:Nanjing-China/qsTeam:Nanjing-China/qs2013-10-27T12:39:56Z<p>GaoJian NJU: </p>
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<dt><a href="###">Aggregation</a></dt><br />
<dd class="dd_1"><br />
In our project, we considered devising our bacteria into some kind of automatic machinery that would be able to sense a target molecule and then assemble to degrade it. To fulfill all these functions, we were bound to design some gene circuits to guide our bacteria.<br><br><br />
<br />
First of all, we should have an understanding of how bacteria move<sup>[1]</sup>. For E.coli, they have flagellar motors to control their movement (Fig. 3-5-1). The direction of rotation of their flagellar motors is controlled by the protein CheY. When CheY is not phosphorylated, the flagellar motors rotate counterclockwise (CCW) and bacteria can migrate. When CheY is phosphorylated (CheY-P), it can bind to the flagellar motorprotein FliM, causing the cell to tumble and thus nonmotile. Phosphatase CheZ dephosphorylates CheY-P and causes the flagellum to rotate CCW. E. coli lacking the cheZ gene (ΔcheZ) cannot dephosphorylate CheY-P, and thus tumble incessantly, being nonmotile. So we only need to take control of gene cheZ to control the motility of our bacteria. <br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/0/04/Fig.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-1</strong> Illustration of how bacteria move. CheZ is the key of controlling the motility of bacteria. For wild type, CheZ can be expressed normally, so the flagellar motor alternates between CCW and CW rotation, thus the cell can run or tumble. On the contrary, if there is no CheZ, CheY will remain phosphorylated, causing the flagellar motor rotates CW, thus the cell tumbles exclusively. (Cite by JACS, Shana Toop et al, 2007.)</div><br><br><br />
We would like our bacteria to seek and destroy atrazine in the soil. To achieve this goal, a classic system can do the job (Fig. 3-5-2A). In this system, bacteria can move around randomly but fix when sensing atrazine as the ribosome switch turns on and protein CI is expressed to repress the gene cheZ. As a result, our bacteria would just assemble around the target molecules after a period of time.<br><br><br />
However, we can only wait for the bacteria to hang about until they find their targets. Thus it could take a long time before the bacteria assembly and the system may not be a robust design. So we shall spare no efforts to improve it. For better control over the function of our bacteria, we took the quorum sensing system into consideration. We expect some bacteria which first find the atrazine will recruit other bacteria at a distance through some signal so they move towards the target and then fixed at the source of pollution. Therefore we constructed the following gene circuits to realize it (Fig. 3-5-2B).<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/6/63/Fig_3-5-2.jpg" width="716"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-2</strong> The gene circuits we built to control cheZ. (A) A classic system using CI to repress cheZ. (B) A complex but high-efficiency system using quorum sensing system. (C) A simplified system like the system in (B).</div><br><br><br />
This circuit is composed of three modules: an atrazine-sensing module, a recruiting module, and a brake module. We adopted the quorum-sensing system in Vibrio fischerias the basis for our design. In this system, a small-molecule acyl-homoserine lactone (AHL) synthesized by LuxI excretes as a signal and accumulates intracellularly to activate a constitutively expressed regulator, LuxR<sup>[2]</sup>. Our atrazine-sensing module controls the translation of LuxI through ribosome switch which turns on when combined to atrazine. Meanwhile, the recruiting module is composed of a series of elements. In this system, the LuxR-AHL complex promotes the expression of a repressor TetR, which represses the expression of another repressor CI. The gene cheZ is controlled by promoter Pλ, which is repressed by CI. So for those bacteria which first sense atrazine, they send out AHL signal and let the other bacteria receive it. After that, TetR is expressed to repress CI, leading the transcription of cheZ. These bacteria move towards the sender until they sense atrazine and another repressor CI, which is controlled by ribosome switch, expresses as a brake of the movement<sup>[3]</sup>. <br><br><br />
We also design the third gene circuit to simplify the system (Fig. 3-5-2C). This system also consists of the three modules in Pattern 2 and the atrazine-sensing module, as well as the brake module, is exactly the same composition with those two parts. The only difference is in the recruiting module that we use a double-functional promoter Plux/CI. The atrazine-sensing module works just the same way in Pattern 2. In this system, however, the LuxR-AHL complex promotes cheZ transcription via promoter Plux/CI. At the spot where bacteria sense atrazine, AHL will accumulate. The higher AHL concentration is, the stronger transcription of cheZ which means greater motility is, leading to directional movement towards bacteria which send out the signal. Meanwhile, the brake module also functions via promoter Plux/CI. The repressor CI, controlled by ribosome switch which turns on when combined to atrazine, represses the promoter Plux/CI and results in bacteria fixation at source of pollution.<br><br><br />
<strong>Experiments and Results</strong><br><br />
At the beginning, we verified gene cheZ in genetic level. We had 3 strains, namely CL-1, CL-M, and CL-IM. CL-1 is a strain with deletion of gene cheZ, CL-M is wildtype, and CL-IM is a strain with deletion of the gene that controls the expression of flagella. We extracted the total RNA of these 3 strains and synthesized the 1st cDNA using RT-PCR. Then we did PCR of gene cheZ with the cDNA as template and we also did PCR of 16 sDNA as control. We got the figure through agarose gel electrophoresis (Fig. 3-5-3A). We can see that CL-1 is actually the strain with deletion of gene cheZ, while the other two strains have normal gene cheZ.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/e/e0/Fig_4-3_2.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-3</strong> Verification of gene cheZ. (A) In genetic level, CL-1 is actually the strain with deletion of gene cheZ, while the other two strains have normal gene cheZ. (B) In phenotype level, CL-M and pcheZ is able to migrate on semisolid culture medium, while CL-1 and CL-M is unable to migrate.</div><br><br><br />
Then we needed to verify of gene cheZ from phenotype. We have 3 strains, namely CL-1, CL-M, and CL-IM. CL-1 is a strain with deletion of gene cheZ, CL-M is wild type, and CL-IM is a strain with deletion of the gene that controls the expression of flagella. Then we transformed a plasmid with gene cheZ into CL-1 and got the fourth strain pcheZ.<br><br><br />
We inoculated the suspension of bacteria on semisolid LB plate and incubated for 20h and took the photograph (Fig. 3-5-3B). From the figure, we can see that CL-M and pcheZ is able to migrate on semisolid culture medium, while CL-1 and CL-M is unable to migrate. This indicates that E.coli is nonmotile without gene cheZ or flagella and inducing the expression of gene cheZ can restore motility in a cheZ knockout strain.<br><br><br />
In addition, we testified the classic gene circuit.We mix the suspension of bacteria with pre-heated semisolid culture medium and drop solution of atrazine on the plate. Our bacteria assembled around one of the targets after incubation for a period of time (Fig. 3-5-4). It is highly possible that they assembles around the other target after atrazine in the first area is degraded and they are able to move freely again.<br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/2/26/3-4%EF%BC%88%E5%A4%87%E7%94%A8%EF%BC%89.jpg" width="300"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-4</strong> Our bacteria could just assemble around the target molecules after a period of time.</div><br><br><br />
Finally, we also verified lux system. We constructed a strain with gene circuit as shown in Fig. 3-5-5A from CL-1. This is a lux system without the ribosome switch. AHL synthesized by LuxI excretes as a signal and accumulates intracellularly to activate a constitutively expressed regulator, LuxR. Then the LuxR-AHL complex promotes the expression of a repressor CI, leading to repression of CheZ. So bacteria will assembly at spots where initial density is high in this system. After we inoculated the suspension of bacteria on semisolid LB plate and incubated for 20h, we took the photograph of a stripe pattern (Fig. 3-5-5B), which was formed under the influence of the lux system<sup>[4]</sup>.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/2/27/Fig_4-4.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-5</strong> Verification of lux system. (A) the gene circuit we constructed. (B) The phenotype of lux system. </div><br/><br />
For more information of our assembly system, please refer to <a href="https://2013.igem.org/Team:Nanjing-China/model"><strong>Model</strong></a>.<br><br />
<br/> <br />
<br />
<strong> Reference</strong><br/><br />
[1] Topp S and Gallivan JP. Guiding bacteria with small molecules and RNA. Journal of the American Chemical Society, 2007, 129 (21): 6807-6811.<br/><br />
[2] Carter KK, et al. Pathway engineering via quorum sensing and sRNA riboregulators-Interconnected networks and controllers. Metabolic Engineering, 2012, 14 (3): 281-288.<br/><br />
[3] Sinha J, et al. Reprogramming bacteria to seek and destroy a herbicide. Nature chemical biology, 2010, 6 (6): 464-470.<br/><br />
[4] Liu C, et al. Sequential establishment of stripe patterns in an expanding cell population. Science, 2011, 334 (6053): 238-241.<br/><br />
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<div></div>GaoJian NJUhttp://2013.igem.org/Team:Nanjing-China/qsTeam:Nanjing-China/qs2013-10-27T12:35:42Z<p>GaoJian NJU: </p>
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<dt><a href="###">Aggregation</a></dt><br />
<dd class="dd_1"><br />
In our project, we considered devising our bacteria into some kind of automatic machinery that would be able to sense a target molecule and then assemble to degrade it. To fulfill all these functions, we were bound to design some gene circuits to guide our bacteria.<br><br><br />
<br />
First of all, we should have an understanding of how bacteria move<sup>[1]</sup>. For E.coli, they have flagellar motors to control their movement (Fig. 3-5-1). The direction of rotation of their flagellar motors is controlled by the protein CheY. When CheY is not phosphorylated, the flagellar motors rotate counterclockwise (CCW) and bacteria can migrate. When CheY is phosphorylated (CheY-P), it can bind to the flagellar motorprotein FliM, causing the cell to tumble and thus nonmotile. Phosphatase CheZ dephosphorylates CheY-P and causes the flagellum to rotate CCW. E. coli lacking the cheZ gene (ΔcheZ) cannot dephosphorylate CheY-P, and thus tumble incessantly, being nonmotile. So we only need to take control of gene cheZ to control the motility of our bacteria. <br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/0/04/Fig.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-1</strong> Illustration of how bacteria move. CheZ is the key of controlling the motility of bacteria. For wild type, CheZ can be expressed normally, so the flagellar motor alternates between CCW and CW rotation, thus the cell can run or tumble. On the contrary, if there is no CheZ, CheY will remain phosphorylated, causing the flagellar motor rotates CW, thus the cell tumbles exclusively. (Cite by JACS, Shana Toop et al, 2007.)</div><br><br><br />
We would like our bacteria to seek and destroy atrazine in the soil. To achieve this goal, a classic system can do the job (Fig. 3-5-2A). In this system, bacteria can move around randomly but fix when sensing atrazine as the ribosome switch turns on and protein CI is expressed to repress the gene cheZ. As a result, our bacteria would just assemble around the target molecules after a period of time.<br><br><br />
However, we can only wait for the bacteria to hang about until they find their targets. Thus it could take a long time before the bacteria assembly and the system may not be a robust design. So we shall spare no efforts to improve it. For better control over the function of our bacteria, we took the quorum sensing system into consideration. We expect some bacteria which first find the atrazine will recruit other bacteria at a distance through some signal so they move towards the target and then fixed at the source of pollution. Therefore we constructed the following gene circuits to realize it (Fig. 3-5-2B).<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/6/63/Fig_3-5-2.jpg" width="716"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-2</strong> The gene circuits we built to control cheZ. (A) A classic system using CI to repress cheZ. (B) A complex but high-efficiency system using quorum sensing system. (C) A simplified system like the system in (B).</div><br><br><br />
This circuit is composed of three modules: an atrazine-sensing module, a recruiting module, and a brake module. We adopted the quorum-sensing system in Vibrio fischerias the basis for our design. In this system, a small-molecule acyl-homoserine lactone (AHL) synthesized by LuxI excretes as a signal and accumulates intracellularly to activate a constitutively expressed regulator, LuxR<sup>[2]</sup>. Our atrazine-sensing module controls the translation of LuxI through ribosome switch which turns on when combined to atrazine. Meanwhile, the recruiting module is composed of a series of elements. In this system, the LuxR-AHL complex promotes the expression of a repressor TetR, which represses the expression of another repressor CI. The gene cheZ is controlled by promoter Pλ, which is repressed by CI. So for those bacteria which first sense atrazine, they send out AHL signal and let the other bacteria receive it. After that, TetR is expressed to repress CI, leading the transcription of cheZ. These bacteria move towards the sender until they sense atrazine and another repressor CI, which is controlled by ribosome switch, expresses as a brake of the movement<sup>[3]</sup>. <br><br><br />
We also design the third gene circuit to simplify the system (Fig. 3-5-2C). This system also consists of the three modules in Pattern 2 and the atrazine-sensing module, as well as the brake module, is exactly the same composition with those two parts. The only difference is in the recruiting module that we use a double-functional promoter Plux/CI. The atrazine-sensing module works just the same way in Pattern 2. In this system, however, the LuxR-AHL complex promotes cheZ transcription via promoter Plux/CI. At the spot where bacteria sense atrazine, AHL will accumulate. The higher AHL concentration is, the stronger transcription of cheZ which means greater motility is, leading to directional movement towards bacteria which send out the signal. Meanwhile, the brake module also functions via promoter Plux/CI. The repressor CI, controlled by ribosome switch which turns on when combined to atrazine, represses the promoter Plux/CI and results in bacteria fixation at source of pollution.<br><br><br />
<strong>Experiments and Results</strong><br><br />
At the beginning, we verified gene cheZ in genetic level. We had 3 strains, namely CL-1, CL-M, and CL-IM. CL-1 is a strain with deletion of gene cheZ, CL-M is wildtype, and CL-IM is a strain with deletion of the gene that controls the expression of flagella. We extracted the total RNA of these 3 strains and synthesized the 1st cDNA using RT-PCR. Then we did PCR of gene cheZ with the cDNA as template and we also did PCR of 16 sDNA as control. We got the figure through agarose gel electrophoresis (Fig. 3-5-3A). We can see that CL-1 is actually the strain with deletion of gene cheZ, while the other two strains have normal gene cheZ.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/e/e0/Fig_4-3_2.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-3</strong> Verification of gene cheZ. (A) In genetic level, CL-1 is actually the strain with deletion of gene cheZ, while the other two strains have normal gene cheZ. (B) In phenotype level, CL-M and pcheZ is able to migrate on semisolid culture medium, while CL-1 and CL-M is unable to migrate.</div><br><br><br />
Then we needed to verify of gene cheZ from phenotype. We have 3 strains, namely CL-1, CL-M, and CL-IM. CL-1 is a strain with deletion of gene cheZ, CL-M is wild type, and CL-IM is a strain with deletion of the gene that controls the expression of flagella. Then we transformed a plasmid with gene cheZ into CL-1 and got the fourth strain pcheZ.<br><br><br />
We inoculated the suspension of bacteria on semisolid LB plate and incubated for 20h and took the photograph (Fig. 3-5-3B). From the figure, we can see that CL-M and pcheZ is able to migrate on semisolid culture medium, while CL-1 and CL-M is unable to migrate. This indicates that E.coli is nonmotile without gene cheZ or flagella and inducing the expression of gene cheZ can restore motility in a cheZ knockout strain.<br><br><br />
In addition, we testified the classic gene circuit.We mix the suspension of bacteria with pre-heated semisolid culture medium and drop solution of atrazine on the plate. Our bacteria assembled around one of the targets after incubation for a period of time (Fig. 3-5-4). It is highly possible that they assembles around the other target after atrazine in the first area is degraded and they are able to move freely again.<br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/2/26/3-4%EF%BC%88%E5%A4%87%E7%94%A8%EF%BC%89.jpg" width="300"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-4</strong> Our bacteria could just assemble around the target molecules after a period of time.</div><br><br><br />
Finally, we also verified lux system. We constructed a strain with gene circuit as shown in Fig. 3-5-5A from CL-1. This is a lux system without the ribosome switch. AHL synthesized by LuxI excretes as a signal and accumulates intracellularly to activate a constitutively expressed regulator, LuxR. Then the LuxR-AHL complex promotes the expression of a repressor CI, leading to repression of CheZ. So bacteria will assembly at spots where initial density is high in this system. After we inoculated the suspension of bacteria on semisolid LB plate and incubated for 20h, we took the photograph of a stripe pattern (Fig. 3-5-5B), which was formed under the influence of the lux system<sup>[4]</sup>.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/d/db/Fig_3-5-4_new.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-5</strong> Verification of lux system. (A) the gene circuit we constructed. (B) The phenotype of lux system. </div><br/><br />
For more information of our assembly system, please refer to <a href="https://2013.igem.org/Team:Nanjing-China/model"><strong>Model</strong></a>.<br><br />
<br/> <br />
<br />
<strong> Reference</strong><br/><br />
[1] Topp S and Gallivan JP. Guiding bacteria with small molecules and RNA. Journal of the American Chemical Society, 2007, 129 (21): 6807-6811.<br/><br />
[2] Carter KK, et al. Pathway engineering via quorum sensing and sRNA riboregulators-Interconnected networks and controllers. Metabolic Engineering, 2012, 14 (3): 281-288.<br/><br />
[3] Sinha J, et al. Reprogramming bacteria to seek and destroy a herbicide. Nature chemical biology, 2010, 6 (6): 464-470.<br/><br />
[4] Liu C, et al. Sequential establishment of stripe patterns in an expanding cell population. Science, 2011, 334 (6053): 238-241.<br/><br />
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</html></div>GaoJian NJUhttp://2013.igem.org/File:Fig_4-3_2.jpgFile:Fig 4-3 2.jpg2013-10-27T12:34:48Z<p>GaoJian NJU: </p>
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<dl><br />
<dt><a href="###">Aggregation</a></dt><br />
<dd class="dd_1"><br />
In our project, we considered devising our bacteria into some kind of automatic machinery that would be able to sense a target molecule and then assemble to degrade it. To fulfill all these functions, we were bound to design some gene circuits to guide our bacteria.<br><br><br />
<br />
First of all, we should have an understanding of how bacteria move<sup>[1]</sup>. For E.coli, they have flagellar motors to control their movement (Fig. 3-5-1). The direction of rotation of their flagellar motors is controlled by the protein CheY. When CheY is not phosphorylated, the flagellar motors rotate counterclockwise (CCW) and bacteria can migrate. When CheY is phosphorylated (CheY-P), it can bind to the flagellar motorprotein FliM, causing the cell to tumble and thus nonmotile. Phosphatase CheZ dephosphorylates CheY-P and causes the flagellum to rotate CCW. E. coli lacking the cheZ gene (ΔcheZ) cannot dephosphorylate CheY-P, and thus tumble incessantly, being nonmotile. So we only need to take control of gene cheZ to control the motility of our bacteria. <br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/0/04/Fig.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-1</strong> Illustration of how bacteria move. CheZ is the key of controlling the motility of bacteria. For wild type, CheZ can be expressed normally, so the flagellar motor alternates between CCW and CW rotation, thus the cell can run or tumble. On the contrary, if there is no CheZ, CheY will remain phosphorylated, causing the flagellar motor rotates CW, thus the cell tumbles exclusively. (Cite by JACS, Shana Toop et al, 2007.)</div><br><br><br />
We would like our bacteria to seek and destroy atrazine in the soil. To achieve this goal, a classic system can do the job (Fig. 3-5-2A). In this system, bacteria can move around randomly but fix when sensing atrazine as the ribosome switch turns on and protein CI is expressed to repress the gene cheZ. As a result, our bacteria would just assemble around the target molecules after a period of time.<br><br><br />
However, we can only wait for the bacteria to hang about until they find their targets. Thus it could take a long time before the bacteria assembly and the system may not be a robust design. So we shall spare no efforts to improve it. For better control over the function of our bacteria, we took the quorum sensing system into consideration. We expect some bacteria which first find the atrazine will recruit other bacteria at a distance through some signal so they move towards the target and then fixed at the source of pollution. Therefore we constructed the following gene circuits to realize it (Fig. 3-5-2B).<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/6/63/Fig_3-5-2.jpg" width="716"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-2</strong> The gene circuits we built to control cheZ. (A) A classic system using CI to repress cheZ. (B) A complex but high-efficiency system using quorum sensing system. (C) A simplified system like the system in (B).</div><br><br><br />
This circuit is composed of three modules: an atrazine-sensing module, a recruiting module, and a brake module. We adopted the quorum-sensing system in Vibrio fischerias the basis for our design. In this system, a small-molecule acyl-homoserine lactone (AHL) synthesized by LuxI excretes as a signal and accumulates intracellularly to activate a constitutively expressed regulator, LuxR<sup>[2]</sup>. Our atrazine-sensing module controls the translation of LuxI through ribosome switch which turns on when combined to atrazine. Meanwhile, the recruiting module is composed of a series of elements. In this system, the LuxR-AHL complex promotes the expression of a repressor TetR, which represses the expression of another repressor CI. The gene cheZ is controlled by promoter Pλ, which is repressed by CI. So for those bacteria which first sense atrazine, they send out AHL signal and let the other bacteria receive it. After that, TetR is expressed to repress CI, leading the transcription of cheZ. These bacteria move towards the sender until they sense atrazine and another repressor CI, which is controlled by ribosome switch, expresses as a brake of the movement<sup>[3]</sup>. <br><br><br />
We also design the third gene circuit to simplify the system (Fig. 3-5-2C). This system also consists of the three modules in Pattern 2 and the atrazine-sensing module, as well as the brake module, is exactly the same composition with those two parts. The only difference is in the recruiting module that we use a double-functional promoter Plux/CI. The atrazine-sensing module works just the same way in Pattern 2. In this system, however, the LuxR-AHL complex promotes cheZ transcription via promoter Plux/CI. At the spot where bacteria sense atrazine, AHL will accumulate. The higher AHL concentration is, the stronger transcription of cheZ which means greater motility is, leading to directional movement towards bacteria which send out the signal. Meanwhile, the brake module also functions via promoter Plux/CI. The repressor CI, controlled by ribosome switch which turns on when combined to atrazine, represses the promoter Plux/CI and results in bacteria fixation at source of pollution.<br><br><br />
<strong>Experiments and Results</strong><br><br />
At the beginning, we verified gene cheZ in genetic level. We had 3 strains, namely CL-1, CL-M, and CL-IM. CL-1 is a strain with deletion of gene cheZ, CL-M is wildtype, and CL-IM is a strain with deletion of the gene that controls the expression of flagella. We extracted the total RNA of these 3 strains and synthesized the 1st cDNA using RT-PCR. Then we did PCR of gene cheZ with the cDNA as template and we also did PCR of 16 sDNA as control. We got the figure through agarose gel electrophoresis (Fig. 3-5-3A). We can see that CL-1 is actually the strain with deletion of gene cheZ, while the other two strains have normal gene cheZ.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/f/f3/Fig_3-5-3.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-3</strong> Verification of gene cheZ. (A) In genetic level, CL-1 is actually the strain with deletion of gene cheZ, while the other two strains have normal gene cheZ. (B) In phenotype level, CL-M and pcheZ is able to migrate on semisolid culture medium, while CL-1 and CL-M is unable to migrate.</div><br><br><br />
Then we needed to verify of gene cheZ from phenotype. We have 3 strains, namely CL-1, CL-M, and CL-IM. CL-1 is a strain with deletion of gene cheZ, CL-M is wild type, and CL-IM is a strain with deletion of the gene that controls the expression of flagella. Then we transformed a plasmid with gene cheZ into CL-1 and got the fourth strain pcheZ.<br><br><br />
We inoculated the suspension of bacteria on semisolid LB plate and incubated for 20h and took the photograph (Fig. 3-5-3B). From the figure, we can see that CL-M and pcheZ is able to migrate on semisolid culture medium, while CL-1 and CL-M is unable to migrate. This indicates that E.coli is nonmotile without gene cheZ or flagella and inducing the expression of gene cheZ can restore motility in a cheZ knockout strain.<br><br><br />
In addition, we testified the classic gene circuit.We mix the suspension of bacteria with pre-heated semisolid culture medium and drop solution of atrazine on the plate. Our bacteria assembled around one of the targets after incubation for a period of time (Fig. 3-5-4). It is highly possible that they assembles around the other target after atrazine in the first area is degraded and they are able to move freely again.<br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/2/26/3-4%EF%BC%88%E5%A4%87%E7%94%A8%EF%BC%89.jpg" width="300"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-4</strong> Our bacteria could just assemble around the target molecules after a period of time.</div><br><br><br />
Finally, we also verified lux system. We constructed a strain with gene circuit as shown in Fig. 3-5-5A from CL-1. This is a lux system without the ribosome switch. AHL synthesized by LuxI excretes as a signal and accumulates intracellularly to activate a constitutively expressed regulator, LuxR. Then the LuxR-AHL complex promotes the expression of a repressor CI, leading to repression of CheZ. So bacteria will assembly at spots where initial density is high in this system. After we inoculated the suspension of bacteria on semisolid LB plate and incubated for 20h, we took the photograph of a stripe pattern (Fig. 3-5-5B), which was formed under the influence of the lux system<sup>[4]</sup>.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/d/db/Fig_3-5-4_new.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-5</strong> Verification of lux system. (A) the gene circuit we constructed. (B) The phenotype of lux system. </div><br/><br />
For more information of our assembly system, please refer to <a href="https://2013.igem.org/Team:Nanjing-China/model"><strong>Model</strong></a>.<br><br />
<br/> <br />
<br />
<strong> Reference</strong><br/><br />
[1] Topp S and Gallivan JP. Guiding bacteria with small molecules and RNA. Journal of the American Chemical Society, 2007, 129 (21): 6807-6811.<br/><br />
[2] Carter KK, et al. Pathway engineering via quorum sensing and sRNA riboregulators-Interconnected networks and controllers. Metabolic Engineering, 2012, 14 (3): 281-288.<br/><br />
[3] Sinha J, et al. Reprogramming bacteria to seek and destroy a herbicide. Nature chemical biology, 2010, 6 (6): 464-470.<br/><br />
[4] Liu C, et al. Sequential establishment of stripe patterns in an expanding cell population. Science, 2011, 334 (6053): 238-241.<br/><br />
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</html></div>GaoJian NJUhttp://2013.igem.org/Team:Nanjing-China/qsTeam:Nanjing-China/qs2013-10-27T12:29:56Z<p>GaoJian NJU: </p>
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<dt><a href="###">Aggregation</a></dt><br />
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In our project, we considered devising our bacteria into some kind of automatic machinery that would be able to sense a target molecule and then assemble to degrade it. To fulfill all these functions, we were bound to design some gene circuits to guide our bacteria.<br><br><br />
<br />
First of all, we should have an understanding of how bacteria move<sup>[1]</sup>. For E.coli, they have flagellar motors to control their movement (Fig. 3-5-1). The direction of rotation of their flagellar motors is controlled by the protein CheY. When CheY is not phosphorylated, the flagellar motors rotate counterclockwise (CCW) and bacteria can migrate. When CheY is phosphorylated (CheY-P), it can bind to the flagellar motorprotein FliM, causing the cell to tumble and thus nonmotile. Phosphatase CheZ dephosphorylates CheY-P and causes the flagellum to rotate CCW. E. coli lacking the cheZ gene (ΔcheZ) cannot dephosphorylate CheY-P, and thus tumble incessantly, being nonmotile. So we only need to take control of gene cheZ to control the motility of our bacteria. <br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/0/04/Fig.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-1</strong> Illustration of how bacteria move. CheZ is the key of controlling the motility of bacteria. For wild type, CheZ can be expressed normally, so the flagellar motor alternates between CCW and CW rotation, thus the cell can run or tumble. On the contrary, if there is no CheZ, CheY will remain phosphorylated, causing the flagellar motor rotates CW, thus the cell tumbles exclusively. (Cite by JACS, Shana Toop et al, 2007.)</div><br><br><br />
We would like our bacteria to seek and destroy atrazine in the soil. To achieve this goal, a classic system can do the job (Fig. 3-5-2A). In this system, bacteria can move around randomly but fix when sensing atrazine as the ribosome switch turns on and protein CI is expressed to repress the gene cheZ. As a result, our bacteria would just assemble around the target molecules after a period of time.<br><br><br />
However, we can only wait for the bacteria to hang about until they find their targets. Thus it could take a long time before the bacteria assembly and the system may not be a robust design. So we shall spare no efforts to improve it. For better control over the function of our bacteria, we took the quorum sensing system into consideration. We expect some bacteria which first find the atrazine will recruit other bacteria at a distance through some signal so they move towards the target and then fixed at the source of pollution. Therefore we constructed the following gene circuits to realize it (Fig. 3-5-2B).<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/6/63/Fig_3-5-2.jpg" width="716"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-2</strong> The gene circuits we built to control cheZ. (A) A classic system using CI to repress cheZ. (B) A complex but high-efficiency system using quorum sensing system. (C) A simplified system like the system in (B).</div><br><br><br />
This circuit is composed of three modules: an atrazine-sensing module, a recruiting module, and a brake module. We adopted the quorum-sensing system in Vibrio fischerias the basis for our design. In this system, a small-molecule acyl-homoserine lactone (AHL) synthesized by LuxI excretes as a signal and accumulates intracellularly to activate a constitutively expressed regulator, LuxR<sup>[2]</sup>. Our atrazine-sensing module controls the translation of LuxI through ribosome switch which turns on when combined to atrazine. Meanwhile, the recruiting module is composed of a series of elements. In this system, the LuxR-AHL complex promotes the expression of a repressor TetR, which represses the expression of another repressor CI. The gene cheZ is controlled by promoter Pλ, which is repressed by CI. So for those bacteria which first sense atrazine, they send out AHL signal and let the other bacteria receive it. After that, TetR is expressed to repress CI, leading the transcription of cheZ. These bacteria move towards the sender until they sense atrazine and another repressor CI, which is controlled by ribosome switch, expresses as a brake of the movement<sup>[3]</sup>. <br><br><br />
We also design the third gene circuit to simplify the system (Fig. 3-5-2C). This system also consists of the three modules in Pattern 2 and the atrazine-sensing module, as well as the brake module, is exactly the same composition with those two parts. The only difference is in the recruiting module that we use a double-functional promoter Plux/CI. The atrazine-sensing module works just the same way in Pattern 2. In this system, however, the LuxR-AHL complex promotes cheZ transcription via promoter Plux/CI. At the spot where bacteria sense atrazine, AHL will accumulate. The higher AHL concentration is, the stronger transcription of cheZ which means greater motility is, leading to directional movement towards bacteria which send out the signal. Meanwhile, the brake module also functions via promoter Plux/CI. The repressor CI, controlled by ribosome switch which turns on when combined to atrazine, represses the promoter Plux/CI and results in bacteria fixation at source of pollution.<br><br><br />
<strong>Experiments and Results</strong><br><br />
At the beginning, we verified gene cheZ in genetic level. We had 3 strains, namely CL-1, CL-M, and CL-IM. CL-1 is a strain with deletion of gene cheZ, CL-M is wildtype, and CL-IM is a strain with deletion of the gene that controls the expression of flagella. We extracted the total RNA of these 3 strains and synthesized the 1st cDNA using RT-PCR. Then we did PCR of gene cheZ with the cDNA as template and we also did PCR of 16 sDNA as control. We got the figure through agarose gel electrophoresis (Fig. 3-5-3A). We can see that CL-1 is actually the strain with deletion of gene cheZ, while the other two strains have normal gene cheZ.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/f/f3/Fig_3-5-3.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-3</strong> Verification of gene cheZ. (A) In genetic level, CL-1 is actually the strain with deletion of gene cheZ, while the other two strains have normal gene cheZ. (B) In phenotype level, CL-M and pcheZ is able to migrate on semisolid culture medium, while CL-1 and CL-M is unable to migrate.</div><br><br><br />
Then we needed to verify of gene cheZ from phenotype. We have 3 strains, namely CL-1, CL-M, and CL-IM. CL-1 is a strain with deletion of gene cheZ, CL-M is wild type, and CL-IM is a strain with deletion of the gene that controls the expression of flagella. Then we transformed a plasmid with gene cheZ into CL-1 and got the fourth strain pcheZ.<br><br><br />
We inoculated the suspension of bacteria on semisolid LB plate and incubated for 20h and took the photograph (Fig. 3-5-3B). From the figure, we can see that CL-M and pcheZ is able to migrate on semisolid culture medium, while CL-1 and CL-M is unable to migrate. This indicates that E.coli is nonmotile without gene cheZ or flagella and inducing the expression of gene cheZ can restore motility in a cheZ knockout strain.<br><br><br />
In addition, we testified the classic gene circuit.We mix the suspension of bacteria with pre-heated semisolid culture medium and drop solution of atrazine on the plate. Our bacteria assembled around one of the targets after incubation for a period of time (Fig. 3-5-4). It is highly possible that they assembles around the other target after atrazine in the first area is degraded and they are able to move freely again.<br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/2/26/3-4%EF%BC%88%E5%A4%87%E7%94%A8%EF%BC%89.jpg" width="400"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-4</strong> Our bacteria could just assemble around the target molecules after a period of time.</div><br><br><br />
Finally, we also verified lux system. We constructed a strain with gene circuit as shown in Fig. 3-5-5A from CL-1. This is a lux system without the ribosome switch. AHL synthesized by LuxI excretes as a signal and accumulates intracellularly to activate a constitutively expressed regulator, LuxR. Then the LuxR-AHL complex promotes the expression of a repressor CI, leading to repression of CheZ. So bacteria will assembly at spots where initial density is high in this system. After we inoculated the suspension of bacteria on semisolid LB plate and incubated for 20h, we took the photograph of a stripe pattern (Fig. 3-5-5B), which was formed under the influence of the lux system<sup>[4]</sup>.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/d/db/Fig_3-5-4_new.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-5</strong> Verification of lux system. (A) the gene circuit we constructed. (B) The phenotype of lux system. </div><br/><br />
For more information of our assembly system, please refer to <a href="https://2013.igem.org/Team:Nanjing-China/model"><strong>Model</strong></a>.<br><br />
<br/> <br />
<br />
<strong> Reference</strong><br/><br />
[1] Topp S and Gallivan JP. Guiding bacteria with small molecules and RNA. Journal of the American Chemical Society, 2007, 129 (21): 6807-6811.<br/><br />
[2] Carter KK, et al. Pathway engineering via quorum sensing and sRNA riboregulators-Interconnected networks and controllers. Metabolic Engineering, 2012, 14 (3): 281-288.<br/><br />
[3] Sinha J, et al. Reprogramming bacteria to seek and destroy a herbicide. Nature chemical biology, 2010, 6 (6): 464-470.<br/><br />
[4] Liu C, et al. Sequential establishment of stripe patterns in an expanding cell population. Science, 2011, 334 (6053): 238-241.<br/><br />
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<dt><a href="###">Aggregation</a></dt><br />
<dd class="dd_1"><br />
In our project, we considered devising our bacteria into some kind of automatic machinery that would be able to sense a target molecule and then assemble to degrade it. To fulfill all these functions, we were bound to design some gene circuits to guide our bacteria.<br><br><br />
<br />
First of all, we should have an understanding of how bacteria move<sup>[1]</sup>. For E.coli, they have flagellar motors to control their movement (Fig. 3-5-1). The direction of rotation of their flagellar motors is controlled by the protein CheY. When CheY is not phosphorylated, the flagellar motors rotate counterclockwise (CCW) and bacteria can migrate. When CheY is phosphorylated (CheY-P), it can bind to the flagellar motorprotein FliM, causing the cell to tumble and thus nonmotile. Phosphatase CheZ dephosphorylates CheY-P and causes the flagellum to rotate CCW. E. coli lacking the cheZ gene (ΔcheZ) cannot dephosphorylate CheY-P, and thus tumble incessantly, being nonmotile. So we only need to take control of gene cheZ to control the motility of our bacteria. <br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/0/04/Fig.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-1</strong> Illustration of how bacteria move. CheZ is the key of controlling the motility of bacteria. For wild type, CheZ can be expressed normally, so the flagellar motor alternates between CCW and CW rotation, thus the cell can run or tumble. On the contrary, if there is no CheZ, CheY will remain phosphorylated, causing the flagellar motor rotates CW, thus the cell tumbles exclusively. (Cite by JACS, Shana Toop et al, 2007.)</div><br><br><br />
We would like our bacteria to seek and destroy atrazine in the soil. To achieve this goal, a classic system can do the job (Fig. 3-5-2A). In this system, bacteria can move around randomly but fix when sensing atrazine as the ribosome switch turns on and protein CI is expressed to repress the gene cheZ. As a result, our bacteria would just assemble around the target molecules after a period of time.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/2/26/3-4%EF%BC%88%E5%A4%87%E7%94%A8%EF%BC%89.jpg" width="400"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-3</strong> Our bacteria could just assemble around the target molecules after a period of time.</div><br><br><br />
However, we can only wait for the bacteria to hang about until they find their targets. Thus it could take a long time before the bacteria assembly and the system may not be a robust design. So we shall spare no efforts to improve it. For better control over the function of our bacteria, we took the quorum sensing system into consideration. We expect some bacteria which first find the atrazine will recruit other bacteria at a distance through some signal so they move towards the target and then fixed at the source of pollution. Therefore we constructed the following gene circuits to realize it (Fig. 3-5-2B).<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/6/63/Fig_3-5-2.jpg" width="716"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-2</strong> The gene circuits we built to control cheZ. (A) A classic system using CI to repress cheZ. (B) A complex but high-efficiency system using quorum sensing system. (C) A simplified system like the system in (B).</div><br><br><br />
This circuit is composed of three modules: an atrazine-sensing module, a recruiting module, and a brake module. We adopted the quorum-sensing system in Vibrio fischerias the basis for our design. In this system, a small-molecule acyl-homoserine lactone (AHL) synthesized by LuxI excretes as a signal and accumulates intracellularly to activate a constitutively expressed regulator, LuxR<sup>[2]</sup>. Our atrazine-sensing module controls the translation of LuxI through ribosome switch which turns on when combined to atrazine. Meanwhile, the recruiting module is composed of a series of elements. In this system, the LuxR-AHL complex promotes the expression of a repressor TetR, which represses the expression of another repressor CI. The gene cheZ is controlled by promoter Pλ, which is repressed by CI. So for those bacteria which first sense atrazine, they send out AHL signal and let the other bacteria receive it. After that, TetR is expressed to repress CI, leading the transcription of cheZ. These bacteria move towards the sender until they sense atrazine and another repressor CI, which is controlled by ribosome switch, expresses as a brake of the movement<sup>[3]</sup>. <br><br><br />
We also design the third gene circuit to simplify the system (Fig. 3-5-2C). This system also consists of the three modules in Pattern 2 and the atrazine-sensing module, as well as the brake module, is exactly the same composition with those two parts. The only difference is in the recruiting module that we use a double-functional promoter Plux/CI. The atrazine-sensing module works just the same way in Pattern 2. In this system, however, the LuxR-AHL complex promotes cheZ transcription via promoter Plux/CI. At the spot where bacteria sense atrazine, AHL will accumulate. The higher AHL concentration is, the stronger transcription of cheZ which means greater motility is, leading to directional movement towards bacteria which send out the signal. Meanwhile, the brake module also functions via promoter Plux/CI. The repressor CI, controlled by ribosome switch which turns on when combined to atrazine, represses the promoter Plux/CI and results in bacteria fixation at source of pollution.<br><br><br />
<strong>Experiments and Results</strong><br><br />
At the beginning, we verified gene cheZ in genetic level. We had 3 strains, namely CL-1, CL-M, and CL-IM. CL-1 is a strain with deletion of gene cheZ, CL-M is wildtype, and CL-IM is a strain with deletion of the gene that controls the expression of flagella. We extracted the total RNA of these 3 strains and synthesized the 1st cDNA using RT-PCR. Then we did PCR of gene cheZ with the cDNA as template and we also did PCR of 16 sDNA as control. We got the figure through agarose gel electrophoresis (Fig. 3-5-3A). We can see that CL-1 is actually the strain with deletion of gene cheZ, while the other two strains have normal gene cheZ.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/f/f3/Fig_3-5-3.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-3</strong> Verification of gene cheZ. (A) In genetic level, CL-1 is actually the strain with deletion of gene cheZ, while the other two strains have normal gene cheZ. (B) In phenotype level, CL-M and pcheZ is able to migrate on semisolid culture medium, while CL-1 and CL-M is unable to migrate.</div><br><br><br />
Then we needed to verify of gene cheZ from phenotype. We have 3 strains, namely CL-1, CL-M, and CL-IM. CL-1 is a strain with deletion of gene cheZ, CL-M is wild type, and CL-IM is a strain with deletion of the gene that controls the expression of flagella. Then we transformed a plasmid with gene cheZ into CL-1 and got the fourth strain pcheZ.<br><br><br />
We inoculated the suspension of bacteria on semisolid LB plate and incubated for 20h and took the photograph (Fig. 3-5-3B). From the figure, we can see that CL-M and pcheZ is able to migrate on semisolid culture medium, while CL-1 and CL-M is unable to migrate. This indicates that E.coli is nonmotile without gene cheZ or flagella and inducing the expression of gene cheZ can restore motility in a cheZ knockout strain.<br><br><br />
Finally, we also verified lux system. We constructed a strain with gene circuit as shown in Fig. 3-5-4A from CL-1. This is a lux system without the ribosome switch. AHL synthesized by LuxI excretes as a signal and accumulates intracellularly to activate a constitutively expressed regulator, LuxR. Then the LuxR-AHL complex promotes the expression of a repressor CI, leading to repression of CheZ. So bacteria will assembly at spots where initial density is high in this system. After we inoculated the suspension of bacteria on semisolid LB plate and incubated for 20h, we took the photograph of a stripe pattern (Fig. 3-5-4B), which was formed under the influence of the lux system<sup>[4]</sup>.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/d/db/Fig_3-5-4_new.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-4</strong> Verification of lux system. (A) the gene circuit we constructed. (B) The phenotype of lux system. </div><br/><br />
For more information of our assembly system, please refer to <a href="https://2013.igem.org/Team:Nanjing-China/model"><strong>Model</strong></a>.<br><br />
<br/> <br />
<br />
<strong> Reference</strong><br/><br />
[1] Topp S and Gallivan JP. Guiding bacteria with small molecules and RNA. Journal of the American Chemical Society, 2007, 129 (21): 6807-6811.<br/><br />
[2] Carter KK, et al. Pathway engineering via quorum sensing and sRNA riboregulators-Interconnected networks and controllers. Metabolic Engineering, 2012, 14 (3): 281-288.<br/><br />
[3] Sinha J, et al. Reprogramming bacteria to seek and destroy a herbicide. Nature chemical biology, 2010, 6 (6): 464-470.<br/><br />
[4] Liu C, et al. Sequential establishment of stripe patterns in an expanding cell population. Science, 2011, 334 (6053): 238-241.<br/><br />
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<dt><a name="Interest">Interest Group of Synthetic Biology of Nanjing University</a></dt><br />
<dd class="dd_1"><br />
In order to unite the students who embrace huge passion in the field of synthetic biology and propagandize iGEM, we established this group in March, 2013. We aim to combine amusement and science together to live a wonderful lab life. Meanwhile, we also communicated with other iGEM teams as a group. This summer, most students of our group devoted themselves into working on the idea we came up with independently half a year ago. Moreover, we hope that through iGEM, we can not only increase our quality of research, but also strengthen own team cooperation. After iGEM 2013, this group will still exist and we are going to make it more influential.<br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/a/a4/Renhaibo_1.png"></p><br />
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<dt><a name="Visit">Visit iGEM Teams of Tsinghua University</a></dt><br />
<dd class="dd_1"><br />
Being fresh in iGEM, it's important for us to communicate with a sophisticated team who can help us grow more quickly. In this way, we can also establish friendship with people who have the same dream with us. Under this circumstance, we went to Beijing and visited Tsinghua University in November, 2012.<br><br><br />
During this trip, we visited 3 teams, Tsinghua-A, Tsinghua-C and Tsinghua-D, and communicated with them well. As experienced iGEM competitors, they successfully showed a more clear scenery of iGEM and synthetic biology to us, who haven't stepped into this field before. Besides, Tsinghua-A have introduced the model part in iGEM to us and promised to help us for the lack of modeler in our team. Of course, all of them sincerely remind us of the hardship in this process and we have also talked over the questions like how to come up with a good idea, how to assess and realize them. When we left THU, we had learned so much and both Tsinghua and our team members thought that we should keep contact with each other in the future and step forward together.<br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/igem.org/b/b3/Pic-8-2-1.jpg"width="600"></p><br />
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<dt><a name="iGEM">iGEM Communication with Shanghai Jiao Tong University And Tsinghua University</a></dt><br />
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This is the 2nd year since for Nanjing University to participate in the IGEM, so it is necessary to spread the basic principles of synthetic biology and introduce the iGEM to more students in NJU.<br><br />
<br><br />
To achieve this goal, in April, we invited 2 leaders of other iGEM teams in China to give lectures on synthetic biology for students from different majors. XiRui, the leader of SJTU-BioX-Shanghai, 2012, introduced the iGEM from a new perspective. She shared their experience on the IGEM project, which have had profound impact on our project. Meanwhile, Wei Lei, the leader of Tsinghua-A, 2012, showed us how they combine synthetic biology with automation technology, promoting our understanding of the concept and methods of interdisciplinary. Audiences attending the lecture were all deeply impressed and attracted by the iGEM and synthetic biology.<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/3/32/Pic-8-3-1.jpg"width="600"></p><br />
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<dt><a name="Leaflets">Leaflets on China Adolescents Science and Technology Innovation Contest</a></dt><br />
<dd class="dd_1"><br />
The 28th China Adolescents Science and Technology Innovation Contest (CASTIC) was held on August 4th in Nanjing. This annual contest covering science, engineering and social science, is held by the China Association for Science and Technology and the ministries of education, science and technology. Every year, more than 15 million young people take part in the contest.China's future stars of science and technology from primary and secondary schools nationwide gathered in Nanjing to show the fruit of their scientific research.<br><br><br />
<br />
Obviously this was a great chance to introduce synthetic biology and iGEM to youth in China, so we propagated the basic theory of synthetic biology and the idea of our team to them by handing out leaflets and communicating face to face.Many of them showed great interest in IGEM. We also hope to grab this opportunity to allow them a greater interest in synthetic biology.<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/a/a1/Renhaibo_2.JPG"width="600"></p><br />
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<dt><a name="help">Help Another iGEM Team, ZJU-China</a></dt><br />
<dd class="dd_1"> <br />
Throughout our project, we took notice of the fact that ZJU-China team and our team both focus on the degradation of atrazine.<br/> <br />
Under this circumstance, we collaborated a lot with them, such as sharing experience, exchanging ideas about environmental protection, and encouraging each other when got stricken. Especially,we offered TrzN, a gene can degrade atrazine to them, which may be of great help for their projects.<br/> <br />
Both of the two outstanding teams learnt a lot from each other's experience, and improved our ideas and designs through the process of free discussion.<br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/4/4d/Renhaibo_3.jpg"width="600"></p><br />
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<dt><a name="Lecture">Lecture in Nanjing Jinling High School</a></dt><br />
<dd class="dd_1"><br />
Although iGEM also has High School Division, few local high school students know about iGEM and synthetic biology. In order to let them have a rational understanding and encourage more students to participate in the IGEM competition, we decided to introduce synthetic biology, IGEM and our project in the form of lectures. <br><br><br />
In the lecture, we used the third biological revolution to give way to synthetic biology. And we introduced synthetic biology and iGEM in a vivid way.When we talked about synthetic biology, we delivered the idea of synthesis. When we speak of iGEM, we delivered several key points that comprise a whole project in iGEM: a practical idea, construction of gene circuits, verification, modification, safety and human practice. What's more, we emphasized the idea of engineer and standardization. At last, we introduced our project.<br><br><br />
After the lecture, many students showed great interest in iGEMHigh School Division. We hope that we did could influence more and more people to participate in this competition and enjoy it.<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/5/53/Pic-8-5-1.jpg"width="600"></p><br />
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<dt><a name="Apply">Apply Our Project to Time Farm</a></dt><br />
<dd class="dd_1"><br />
We have always been considering to apply our project tothe field of farming industry and environmental governance. Guided by this belief, we visited the Time Farm, a newly built organic farm in Nanjing.<br><br />
<br><br />
The technology of this farm is based on permaculture. They want to cultivate healthy products by constructing harmonized eco-environment. In the process of communication, we learned that they purify water mainly by constructing artificial wetlands and spraying organic fertilizer which contains a lot of microorganisms. These strategies indeed obtains satisfying results in water purification, and the products are much healthier than those cultivated in the original farms. However, some refractory deleterious organic compounds used in other areas around the organic farm, for example, atrazine, may still remain in the water. In this case, bacteria with biological function may benefit the farmers a lot. They also realized that synthetic biology could be an effective and environmental friendly tool in the near future.(Friendly link: Time Farm <a href="http://www.timefarm.com.cn">http://www.timefarm.com.cn</a>)<br />
<br/><br />
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<li class=trunk onmouseover=listTrigger(0);><a href="https://2013.igem.org/Team:Nanjing-China/practice#Apply">Apply Our Project to Time Farm</a></li><br />
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<dt><a name="Interest">Interest Group of Synthetic Biology of Nanjing University</a></dt><br />
<dd class="dd_1"><br />
In order to unite the students who embrace huge passion in the field of synthetic biology and propagandize iGEM, we established this group in March, 2013. We aim to combine amusement and science together to live a wonderful lab life. Meanwhile, we also communicated with other iGEM teams as a group. This summer, most students of our group devoted themselves into working on the idea we came up with independently half a year ago. Moreover, we hope that through iGEM, we can not only increase our quality of research, but also strengthen own team cooperation. After iGEM 2013, this group will still exist and we are going to make it more influential.<br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/a/a4/Renhaibo_1.png"></p><br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a name="Visit">Visit iGEM Teams of Tsinghua University</a></dt><br />
<dd class="dd_1"><br />
Being fresh in iGEM, it's important for us to communicate with a sophisticated team who can help us grow more quickly. In this way, we can also establish friendship with people who have the same dream with us. Under this circumstance, we went to Beijing and visited Tsinghua University in November, 2012.<br><br><br />
During this trip, we visited 3 teams, Tsinghua-A, Tsinghua-C and Tsinghua-D, and communicated with them well. As experienced iGEM competitors, they successfully showed a more clear scenery of iGEM and synthetic biology to us, who haven't stepped into this field before. Besides, Tsinghua-A have introduced the model part in iGEM to us and promised to help us for the lack of modeler in our team. Of course, all of them sincerely remind us of the hardship in this process and we have also talked over the questions like how to come up with a good idea, how to assess and realize them. When we left THU, we had learned so much and both Tsinghua and our team members thought that we should keep contact with each other in the future and step forward together.<br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/igem.org/b/b3/Pic-8-2-1.jpg"width="600"></p><br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a name="iGEM">iGEM Communication with Shanghai Jiao Tong University And Tsinghua University</a></dt><br />
<dd class="dd_1"><br />
This is the 2nd year since for Nanjing University to participate in the IGEM, so it is necessary to spread the basic principles of synthetic biology and introduce the iGEM to more students in NJU.<br><br />
<br><br />
To achieve this goal, in April, we invited 2 leaders of other iGEM teams in China to give lectures on synthetic biology for students from different majors. XiRui, the leader of SJTU-BioX-Shanghai, 2012, introduced the iGEM from a new perspective. She shared their experience on the IGEM project, which have had profound impact on our project. Meanwhile, Wei Lei, the leader of Tsinghua-A, 2012, showed us how they combine synthetic biology with automation technology, promoting our understanding of the concept and methods of interdisciplinary. Audiences attending the lecture were all deeply impressed and attracted by the iGEM and synthetic biology.<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/3/32/Pic-8-3-1.jpg"width="600"></p><br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a name="Leaflets">Leaflets on China Adolescents Science and Technology Innovation Contest</a></dt><br />
<dd class="dd_1"><br />
The 28th China Adolescents Science and Technology Innovation Contest (CASTIC) was held on August 4th in Nanjing. This annual contest covering science, engineering and social science, is held by the China Association for Science and Technology and the ministries of education, science and technology. Every year, more than 15 million young people take part in the contest.China's future stars of science and technology from primary and secondary schools nationwide gathered in Nanjing to show the fruit of their scientific research.<br><br><br />
<br />
Obviously this was a great chance to introduce synthetic biology and iGEM to youth in China, so we propagated the basic theory of synthetic biology and the idea of our team to them by handing out leaflets and communicating face to face.Many of them showed great interest in IGEM. We also hope to grab this opportunity to allow them a greater interest in synthetic biology.<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/a/a1/Renhaibo_2.JPG"width="600"></p><br />
<br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a name="help">Help Another iGEM Team, ZJU-China</a></dt><br />
<dd class="dd_1"> <br />
Throughout our project, we took notice of the fact that ZJU-China team and our team both focus on the degradation of atrazine.<br/> <br />
Under this circumstance, we collaborated a lot with them, such as sharing experience, exchanging ideas about environmental protection, and encouraging each other when got stricken. Especially,we offered TrzN, a gene can degrade atrazine to them, which may be of great help for their projects.<br/> <br />
Both of the two outstanding teams learnt a lot from each other's experience, and improved our ideas and designs through the process of free discussion.<br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/4/4d/Renhaibo_3.jpg"width="600"></p><br />
</dd><br />
</dl><br />
<br />
<br />
<dl><br />
<dt><a name="Lecture">Lecture in Nanjing Jinling High School</a></dt><br />
<dd class="dd_1"><br />
Although iGEM also has High School Division, few local high school students know about iGEM and synthetic biology. In order to let them have a rational understanding and encourage more students to participate in the IGEM competition, we decided to introduce synthetic biology, IGEM and our project in the form of lectures. <br><br><br />
In the lecture, we used the third biological revolution to give way to synthetic biology. And we introduced synthetic biology and iGEM in a vivid way.When we talked about synthetic biology, we delivered the idea of synthesis. When we speak of iGEM, we delivered several key points that comprise a whole project in iGEM: a practical idea, construction of gene circuits, verification, modification, safety and human practice. What's more, we emphasized the idea of engineer and standardization. At last, we introduced our project.<br><br><br />
After the lecture, many students showed great interest in iGEMHigh School Division. We hope that we did could influence more and more people to participate in this competition and enjoy it.<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/5/53/Pic-8-5-1.jpg"width="600"></p><br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a name="Apply">Apply Our Project to Time Farm</a></dt><br />
<dd class="dd_1"><br />
We have always been considering to apply our project tothe field of farming industry and environmental governance. Guided by this belief, we visited the Time Farm, a newly built organic farm in Nanjing.<br><br />
<br><br />
The technology of this farm is based on permaculture. They want to cultivate healthy products by constructing harmonized eco-environment. In the process of communication, we learned that they purify water mainly by constructing artificial wetlands and spraying organic fertilizer which contains a lot of microorganisms. These strategies indeed obtains satisfying results in water purification, and the products are much healthier than those cultivated in the original farms. However, some refractory deleterious organic compounds used in other areas around the organic farm, for example, atrazine, may still remain in the water. In this case, bacteria with biological function may benefit the farmers a lot. They also realized that synthetic biology could be an effective and environmental friendly tool in the near future.(Friendly link: Time Farm <a href="http://www.timefarm.com.cn">http://www.timefarm.com.cn</a>)<br />
<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/3/32/Pic-8-3-1.jpg"width="600"></p><br />
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</html></div>GaoJian NJUhttp://2013.igem.org/Team:Nanjing-China/modelTeam:Nanjing-China/model2013-10-27T08:51:29Z<p>GaoJian NJU: </p>
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<dt><a name="Introduction">Introduction</a></dt><br />
<dd class="dd_1"><br />
As you can see in the previous parts, the shining point of our design is that we have introduced the quorum sensing device into our circuit. In order to see what the characteristics of our design are during the process of attracting E. coli to atrazine, we have built three models respectively and compared their possible behaviors.<br><br/><br />
The first circuit, we called circuit one, is the most simple one, which has been constructed by Joy sinha et al in 2010. In this circuit, bacteria will stop walking in the region with high concentration of atrazine, in which way bacteria will finally get together to this region. However, bacteria can't tell each other the information about the destination like ants, which will tell their companies the location of food.<br><br/> <br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/1/11/Circuit1_hah.jpg" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 4-1</strong> Circuit 1. Atrazine will promote the translation of the CI protein. However, the CheZ protein, which represent the motility, will be repressed when the concentration of CI protein is high. So, the consequence will be more and more bacteria stopping in the region with high Atrazine.</div><br/> <br />
The second circuit is our original blueprint with a complex structure and you can see the quorum sensing device here. It is a little complex and has a cascade, which is believed to prolong the respond time of our system. In short, the behavior of bacteria will be affected by the concentration of AHL, which represents the density of bacteria. It means that the bacteria can communicate with each other.<br/> <br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/5/59/Fig_3-5-2-B.jpg" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 4-2</strong> Circuit2. The movement of the bacteria with this circuit will affected by the presence of AHL. In our initiate consideration, the cascade, AHL&LuxR→Plux-tetr---Ptet-CI---Pci-CheZ, will lead to the promotion of the production of CheZ protein.</div><br/> <br />
The third circuit is the ultimate one that we have designed and optimized.We have replaced the cascade mentioned above with a hybridize promoter, which can be repressed by CI protein and activated by AHL-LuxR compound.<br/> <br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/e/ea/Circuit3_a.jpg" width="600"></p> <br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 4-3</strong> Circuit 3. The greatest difference between circuit 2 and circuit 3, as you can see, is that the cascade in circuit 2 have been replaced by the hybridize promoter, Plux/CI. Then, we got a new organism that will respond quickly to the presence of AHL.</div><br/><br />
The most important is that we want to have a preview of the behavior of the three circuits mentioned above. Thus, we can compare them and prove that our final design can attract bacteria efficiently and send out the information of atrazine to bacteria around these regions to make their movement more valuable rather than walking aimless.<br/> <br />
</dd><br />
</dl><br />
<br />
<br />
<dl><br />
<dt><a name="Results">Results</a></dt><br />
<dd class="dd_1"><br />
These three models were all coded in MATLAB (all can be download <a href="https://static.igem.org/mediawiki/2013/6/66/Code_of_Nanjing-China%2C_iGEM_2013.zip"><strong>here</strong></a>). As mentioned above, we have just changed several statements in the second model to construct the first and the third. Finally, we have got how the distribution of bacteria changed with time, which can make us catch the major difference among three circuits more directly. <br><br/><br />
Then, we have made census of the number of bacteria within the atrazine region in different circuits respectively. Finally, we have considered the randomness of their movement. We have introduced the Markov chain into the model. We have also counted out the effective aggregation rate, which will dem-onstrate the value of the movement.<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/8/89/Circuit1_aa.gif" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><p style="text-align:center"><strong>Fig. 4-4</strong> The simulation of circuit 1.</p></div> <br />
<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/9/92/Circuit2.gif" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><p style="text-align:center"><strong>Fig. 4-5</strong> The simulation of circuit 2.</p></div> <br />
<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/5/54/Circuit3.gif" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><p style="text-align:center"><strong>Fig. 4-6</strong> The simulation of circuit 3.</p></div><br/><br />
Let's see the characteristics of each circuit respectively first. Bacteria with circuit 1 move randomly all the time, so thay can aggregate much faster than other two. The results of bacteria with circuit 2, meeting our expectation, demonstrate the dull response of the complex circuit. As to circuit 3, this kind of bacteria gets together in the region of much atrazine gradually. <br><br><br />
Circuit 1 is the classical design, which is proved by so many researchers to have a good result of attracting bacteria. <br><br/><br />
Compared to circuit 1, circuit 2 need too much time to respond.<br><br/><br />
Circuit 3 has speed up the respond time of circuit 2. Besides, circuit 3 can attract the bacteria around the atrazine step by step as circuit 2.<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/e/e4/Cell_num_in_region-time_in_circuit1-census_.jpg" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><p style="text-align:center"><strong>Fig. 4-7</strong> The number of cells in the atrazine region of circuit1.</p></div><br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/7/76/Cell_num_in_region-time_in_circuit2-census_new.jpg" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><p style="text-align:center"><strong>Fig. 4-8</strong> The number of cells in the atrazine region of circuit2.</p></div><br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/9/94/Cell_num_in_region-time_in_circuit3-census_.jpg" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><p style="text-align:center"><strong>Fig. 4-9</strong> The number of cells in the atrazine region of circuit3.</p></div><br/><br />
The Fig. 4-7~9 exhibits some quantitative results of the three circuits. It is easy for us to find that the number of the cells in different circuits changed in different ways. Here, it is clear that, the bacteria with circuit 3 aggregate in specified gradually, which shows a nearly straight-line slope in the fig. 4-9 as the behavior of circuit 2. At last, fig. 4-8, which present circuit 2, exhibits little change about the number of cells in specified region at the beginning. Shortly,the census has further proved the result obtained from figure 4-4~6.<br><br><br />
We have mentioned that we have introduced the QS systems into our design as circuit 2 and circuit 3. However, we can't find out its advantages in figure 4-4~9 and our result is got from limited times of simulations. In order to solve these questions, we have got the figure 4-10~12, which can illustrate this question clearly.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/7/71/Change_number_of_bacteria_in_five_blocks_circuit1.jpg" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><p style="text-align:center"><strong>Fig. 4-10</strong> The aggregation/ the number of moving bacteria ratio of circuit 1.</p></div><br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/7/74/Change_number_of_bacteria_in_five_blocks_circuit2.jpg" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><p style="text-align:center"><strong>Fig. 4-11</strong> The aggregation/ the number of moving bacteria ratio of circuit 2.</p></div><br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/1/15/Change_number_of_bacteria_in_five_blocks_circuit3.jpg" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><p style="text-align:center"><strong>Fig. 4-12</strong> The aggregation/ the number of moving bacteria ratio of circuit 3.</p></div><br/><br />
The figure 4-10~12 have showed the effective aggregation, which is described by the number of cells in regions to the number of moving cells ratio. In this way, we have considered the number of moving bacteria rather than all the bacteria. Because only the moving bacteria are trying to move toward atrazine region.<br><br><br />
As we can see from figure 4-10~12, the value of effective aggregation of circuit 1 is always below 1, which means even though their can aggregate so fast at the beginning, their movement is aimless. The circuit 2 respond too slowly and the value of their effective aggregation is low as well. So it has neither fast aggregation nor effective aggregation. Circuit 3 have a peak in the figure, which reached 3 at the beginning. It proved that their movement is goal-directed and our design is more intelligent than circuit 1.<br><br><br />
Besides, we have introduced the Markov chain into our model in order to takerandomness of movement of bacteria into consideration. As you can see in the figure 4-10~12, they have 3 curves related to the 3 omega values (=1, 3, 5) and these curve are similar, which suggested that the result of our simulation has little variance. It means that our data in figure 4-4~9 are credible and our designed system is robust and their behaviors are stable. <br><br />
<br />
</dd><br />
</dl><br />
<dl><br />
<dt><a name="Equations">Equations</a></dt><br />
<dd class="dd_1"><br />
After drawing a profile of our project, we should make the process of every event more clearly. So, we have constructed the models of our three circuits by the principles of biochemistry.<br><br/><br />
First, we have constructed a model related to the circuit 2, the most complex one, with the help of Tsinghua-A. Then, in order to compare them, we have also constructed the other two based on the model of circuit 2. In our model, we use ODE to describe the process of chemical reaction in organisms.<br><br/><br />
In general, we have divided the chemical events in our bacteria into three parts, the transcription of DNA, the translation of RNA, the production of micromolecule-compounds. The rate of each event can be described by ODE listed below.<br><br/> <br />
<strong>Circuit1:</strong><br/><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Equation1.jpg"><br/><br />
<strong>Circuit2:</strong><br/><br />
<img src="https://static.igem.org/mediawiki/2013/a/a5/Equations2-1.jpg"><img src="https://static.igem.org/mediawiki/2013/0/0b/Equations2-2.jpg"><br/><br />
<strong>Circuit3:</strong><br/><br />
<img src="https://static.igem.org/mediawiki/2013/a/a7/Equation3.jpg"> <br/><br />
m_CI1,m_CI2, m_CheZ, m_TetR, m_TrzN, m_LuxI represent mRNA of different protein.<br/><br />
CI1,CI2, CheZ, TetR, TrzN, LuxI represent proteins.<br/><br />
p_AHL represent the AHL produced in each cell.<br><br/><br />
We have used matrix to describe the state of AHL and the location of every bacteria. By the way, we have also made the density of bacteria related to the distance of bacteria. At last, we have considerate the degradation of atrazine as well, though which proved to have little influence on atrazine latter. And a space lattice of "culture dish" is demonstrated below.<br><br/> <br />
<strong>Space relation:</strong><br/><br />
<img src="https://static.igem.org/mediawiki/2013/8/8e/Space_relation.jpg"><br><br />
e_AHL represent the AHL in the environment.<br/><br />
PXn, Pyn represent the location of bacteria.<br/><br />
Cdn represent the density of bacteria in a lattice.<br/><br />
Atz represent the atrazine. <br/><br/> <br />
<strong>Markov Chain:</strong><br/><br />
<img src="https://static.igem.org/mediawiki/2013/5/5d/Markov_Chain.jpg"><br><br />
The markov chain is used to evaluate the reliability of limited times of simulations and the robustness of our circuit.<br> <br />
<br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a name="Parameter">Parameter</a></dt><br />
<dd class="dd_1"><br />
<img src="https://static.igem.org/mediawiki/2013/4/48/Parametertable.jpg"> <br><br><br/><br />
<strong>Reference</strong><br/><br />
[1] The wiki of iGEM11 TsinghuaA, https://2011.igem.org/Team:Tsinghua-A/Modeling.<br/><br />
[2] The wiki of iGEM11 USTC, https://2011.igem.org/Team:USTC-China/Drylab/modeling.<br/><br />
[3] Basu, S., Gerchman, Y., Collins, C. H., Arnold, F. H. & Weiss, R. A synthetic multicellular system for programmed pattern formation. Nature434, 1130-1134 (2005).<br/><br />
[4] Goryachev, A., Toh, D. & Lee, T. Systems analysis of a quorum sensing network: design constraints imposed by the functional requirements, network topology and kinetic constants. Biosystems83, 178-187 (2006).<br/><br />
[5] Hooshangi, S. & Bentley, W. E. LsrR Quorum Sensing "Switch" Is Revealed by a Bottom-Up Approach. PLoS computational biology7, e1002172 (2011). <br/> <br />
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</html></div>GaoJian NJUhttp://2013.igem.org/File:Change_number_of_bacteria_in_five_blocks_circuit3.jpgFile:Change number of bacteria in five blocks circuit3.jpg2013-10-27T08:51:10Z<p>GaoJian NJU: </p>
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<div></div>GaoJian NJUhttp://2013.igem.org/File:Change_number_of_bacteria_in_five_blocks_circuit2.jpgFile:Change number of bacteria in five blocks circuit2.jpg2013-10-27T08:50:22Z<p>GaoJian NJU: </p>
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<div></div>GaoJian NJUhttp://2013.igem.org/File:Change_number_of_bacteria_in_five_blocks_circuit1.jpgFile:Change number of bacteria in five blocks circuit1.jpg2013-10-27T08:48:14Z<p>GaoJian NJU: </p>
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<div></div>GaoJian NJUhttp://2013.igem.org/Team:Nanjing-China/modelTeam:Nanjing-China/model2013-10-27T08:42:19Z<p>GaoJian NJU: </p>
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<dl><br />
<dt><a name="Introduction">Introduction</a></dt><br />
<dd class="dd_1"><br />
As you can see in the previous parts, the shining point of our design is that we have introduced the quorum sensing device into our circuit. In order to see what the characteristics of our design are during the process of attracting E. coli to atrazine, we have built three models respectively and compared their possible behaviors.<br><br/><br />
The first circuit, we called circuit one, is the most simple one, which has been constructed by Joy sinha et al in 2010. In this circuit, bacteria will stop walking in the region with high concentration of atrazine, in which way bacteria will finally get together to this region. However, bacteria can't tell each other the information about the destination like ants, which will tell their companies the location of food.<br><br/> <br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/1/11/Circuit1_hah.jpg" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 4-1</strong> Circuit 1. Atrazine will promote the translation of the CI protein. However, the CheZ protein, which represent the motility, will be repressed when the concentration of CI protein is high. So, the consequence will be more and more bacteria stopping in the region with high Atrazine.</div><br/> <br />
The second circuit is our original blueprint with a complex structure and you can see the quorum sensing device here. It is a little complex and has a cascade, which is believed to prolong the respond time of our system. In short, the behavior of bacteria will be affected by the concentration of AHL, which represents the density of bacteria. It means that the bacteria can communicate with each other.<br/> <br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/5/59/Fig_3-5-2-B.jpg" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 4-2</strong> Circuit2. The movement of the bacteria with this circuit will affected by the presence of AHL. In our initiate consideration, the cascade, AHL&LuxR→Plux-tetr---Ptet-CI---Pci-CheZ, will lead to the promotion of the production of CheZ protein.</div><br/> <br />
The third circuit is the ultimate one that we have designed and optimized.We have replaced the cascade mentioned above with a hybridize promoter, which can be repressed by CI protein and activated by AHL-LuxR compound.<br/> <br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/e/ea/Circuit3_a.jpg" width="600"></p> <br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 4-3</strong> Circuit 3. The greatest difference between circuit 2 and circuit 3, as you can see, is that the cascade in circuit 2 have been replaced by the hybridize promoter, Plux/CI. Then, we got a new organism that will respond quickly to the presence of AHL.</div><br/><br />
The most important is that we want to have a preview of the behavior of the three circuits mentioned above. Thus, we can compare them and prove that our final design can attract bacteria efficiently and send out the information of atrazine to bacteria around these regions to make their movement more valuable rather than walking aimless.<br/> <br />
</dd><br />
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<br />
<dl><br />
<dt><a name="Results">Results</a></dt><br />
<dd class="dd_1"><br />
These three models were all coded in MATLAB (all can be download <a href="https://static.igem.org/mediawiki/2013/6/66/Code_of_Nanjing-China%2C_iGEM_2013.zip"><strong>here</strong></a>). As mentioned above, we have just changed several statements in the second model to construct the first and the third. Finally, we have got how the distribution of bacteria changed with time, which can make us catch the major difference among three circuits more directly. <br><br/><br />
Then, we have made census of the number of bacteria within the atrazine region in different circuits respectively. Finally, we have considered the randomness of their movement. We have introduced the Markov chain into the model. We have also counted out the effective aggregation rate, which will dem-onstrate the value of the movement.<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/8/89/Circuit1_aa.gif" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><p style="text-align:center"><strong>Fig. 4-4</strong> The simulation of circuit 1.</p></div> <br />
<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/9/92/Circuit2.gif" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><p style="text-align:center"><strong>Fig. 4-5</strong> The simulation of circuit 2.</p></div> <br />
<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/5/54/Circuit3.gif" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><p style="text-align:center"><strong>Fig. 4-6</strong> The simulation of circuit 3.</p></div><br/><br />
Let's see the characteristics of each circuit respectively first. Bacteria with circuit 1 move randomly all the time, so thay can aggregate much faster than other two. The results of bacteria with circuit 2, meeting our expectation, demonstrate the dull response of the complex circuit. As to circuit 3, this kind of bacteria gets together in the region of much atrazine gradually. <br><br><br />
Circuit 1 is the classical design, which is proved by so many researchers to have a good result of attracting bacteria. <br><br/><br />
Compared to circuit 1, circuit 2 need too much time to respond.<br><br/><br />
Circuit 3 has speed up the respond time of circuit 2. Besides, circuit 3 can attract the bacteria around the atrazine step by step as circuit 2.<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/e/e4/Cell_num_in_region-time_in_circuit1-census_.jpg" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><p style="text-align:center"><strong>Fig. 4-7</strong> The number of cells in the atrazine region of circuit1.</p></div><br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/7/76/Cell_num_in_region-time_in_circuit2-census_new.jpg" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><p style="text-align:center"><strong>Fig. 4-8</strong> The number of cells in the atrazine region of circuit2.</p></div><br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/9/94/Cell_num_in_region-time_in_circuit3-census_.jpg" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><p style="text-align:center"><strong>Fig. 4-9</strong> The number of cells in the atrazine region of circuit3.</p></div><br/><br />
The Fig. 4-7~9 exhibits some quantitative results of the three circuits. It is easy for us to find that the number of the cells in different circuits changed in different ways. Here, it is clear that, the bacteria with circuit 3 aggregate in specified gradually, which shows a nearly straight-line slope in the fig. 4-9 as the behavior of circuit 2. At last, fig. 4-8, which present circuit 2, exhibits little change about the number of cells in specified region at the beginning. Shortly,the census has further proved the result obtained from figure 4-4~6.<br />
</dd><br />
</dl><br />
<dl><br />
<dt><a name="Equations">Equations</a></dt><br />
<dd class="dd_1"><br />
After drawing a profile of our project, we should make the process of every event more clearly. So, we have constructed the models of our three circuits by the principles of biochemistry.<br><br/><br />
First, we have constructed a model related to the circuit 2, the most complex one, with the help of Tsinghua-A. Then, in order to compare them, we have also constructed the other two based on the model of circuit 2. In our model, we use ODE to describe the process of chemical reaction in organisms.<br><br/><br />
In general, we have divided the chemical events in our bacteria into three parts, the transcription of DNA, the translation of RNA, the production of micromolecule-compounds. The rate of each event can be described by ODE listed below.<br><br/> <br />
<strong>Circuit1:</strong><br/><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Equation1.jpg"><br/><br />
<strong>Circuit2:</strong><br/><br />
<img src="https://static.igem.org/mediawiki/2013/a/a5/Equations2-1.jpg"><img src="https://static.igem.org/mediawiki/2013/0/0b/Equations2-2.jpg"><br/><br />
<strong>Circuit3:</strong><br/><br />
<img src="https://static.igem.org/mediawiki/2013/a/a7/Equation3.jpg"> <br/><br />
m_CI1,m_CI2, m_CheZ, m_TetR, m_TrzN, m_LuxI represent mRNA of different protein.<br/><br />
CI1,CI2, CheZ, TetR, TrzN, LuxI represent proteins.<br/><br />
p_AHL represent the AHL produced in each cell.<br><br/><br />
We have used matrix to describe the state of AHL and the location of every bacteria. By the way, we have also made the density of bacteria related to the distance of bacteria. At last, we have considerate the degradation of atrazine as well, though which proved to have little influence on atrazine latter. And a space lattice of "culture dish" is demonstrated below.<br><br/> <br />
<strong>Space relation:</strong><br/><br />
<img src="https://static.igem.org/mediawiki/2013/8/8e/Space_relation.jpg"><br><br />
e_AHL represent the AHL in the environment.<br/><br />
PXn, Pyn represent the location of bacteria.<br/><br />
Cdn represent the density of bacteria in a lattice.<br/><br />
Atz represent the atrazine. <br/><br/> <br />
<strong>Markov Chain:</strong><br/><br />
<img src="https://static.igem.org/mediawiki/2013/5/5d/Markov_Chain.jpg"><br><br />
The markov chain is used to evaluate the reliability of limited times of simulations and the robustness of our circuit. <br />
<br />
</dd><br />
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<br />
<dl><br />
<dt><a name="Parameter">Parameter</a></dt><br />
<dd class="dd_1"><br />
<img src="https://static.igem.org/mediawiki/2013/4/48/Parametertable.jpg"> <br><br><br/><br />
<strong>Reference</strong><br/><br />
[1] The wiki of iGEM11 TsinghuaA, https://2011.igem.org/Team:Tsinghua-A/Modeling.<br/><br />
[2] The wiki of iGEM11 USTC, https://2011.igem.org/Team:USTC-China/Drylab/modeling.<br/><br />
[3] Basu, S., Gerchman, Y., Collins, C. H., Arnold, F. H. & Weiss, R. A synthetic multicellular system for programmed pattern formation. Nature434, 1130-1134 (2005).<br/><br />
[4] Goryachev, A., Toh, D. & Lee, T. Systems analysis of a quorum sensing network: design constraints imposed by the functional requirements, network topology and kinetic constants. Biosystems83, 178-187 (2006).<br/><br />
[5] Hooshangi, S. & Bentley, W. E. LsrR Quorum Sensing "Switch" Is Revealed by a Bottom-Up Approach. PLoS computational biology7, e1002172 (2011). <br/> <br />
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</html></div>GaoJian NJUhttp://2013.igem.org/File:Markov_Chain.jpgFile:Markov Chain.jpg2013-10-27T08:42:05Z<p>GaoJian NJU: </p>
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<div class="model"><br />
<dl><br />
<dt><a name="Introduction">Introduction</a></dt><br />
<dd class="dd_1"><br />
As you can see in the previous parts, the shining point of our design is that we have introduced the quorum sensing device into our circuit. In order to see what the characteristics of our design are during the process of attracting E. coli to atrazine, we have built three models respectively and compared their possible behaviors.<br><br/><br />
The first circuit, we called circuit one, is the most simple one, which has been constructed by Joy sinha et al in 2010. In this circuit, bacteria will stop walking in the region with high concentration of atrazine, in which way bacteria will finally get together to this region. However, bacteria can't tell each other the information about the destination like ants, which will tell their companies the location of food.<br><br/> <br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/1/11/Circuit1_hah.jpg" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 4-1</strong> Circuit 1. Atrazine will promote the translation of the CI protein. However, the CheZ protein, which represent the motility, will be repressed when the concentration of CI protein is high. So, the consequence will be more and more bacteria stopping in the region with high Atrazine.</div><br/> <br />
The second circuit is our original blueprint with a complex structure and you can see the quorum sensing device here. It is a little complex and has a cascade, which is believed to prolong the respond time of our system. In short, the behavior of bacteria will be affected by the concentration of AHL, which represents the density of bacteria. It means that the bacteria can communicate with each other.<br/> <br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/5/59/Fig_3-5-2-B.jpg" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 4-2</strong> Circuit2. The movement of the bacteria with this circuit will affected by the presence of AHL. In our initiate consideration, the cascade, AHL&LuxR→Plux-tetr---Ptet-CI---Pci-CheZ, will lead to the promotion of the production of CheZ protein.</div><br/> <br />
The third circuit is the ultimate one that we have designed and optimized.We have replaced the cascade mentioned above with a hybridize promoter, which can be repressed by CI protein and activated by AHL-LuxR compound.<br/> <br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/e/ea/Circuit3_a.jpg" width="600"></p> <br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 4-3</strong> Circuit 3. The greatest difference between circuit 2 and circuit 3, as you can see, is that the cascade in circuit 2 have been replaced by the hybridize promoter, Plux/CI. Then, we got a new organism that will respond quickly to the presence of AHL.</div><br/><br />
The most important is that we want to have a preview of the behavior of the three circuits mentioned above. Thus, we can compare them and prove that our final design can attract bacteria efficiently and send out the information of atrazine to bacteria around these regions to make their movement more valuable rather than walking aimless.<br/> <br />
</dd><br />
</dl><br />
<br />
<br />
<dl><br />
<dt><a name="Results">Results</a></dt><br />
<dd class="dd_1"><br />
These three models were all coded in MATLAB (all can be download <a href="https://static.igem.org/mediawiki/2013/6/66/Code_of_Nanjing-China%2C_iGEM_2013.zip"><strong>here</strong></a>). As mentioned above, we have just changed several statements in the second model to construct the first and the third. Finally, we have got how the distribution of bacteria changed with time, which can make us catch the major difference among three circuits more directly. <br><br/><br />
Then, we have made census of the number of bacteria within the atrazine region in different circuits respectively. Finally, we have considered the randomness of their movement. We have introduced the Markov chain into the model. We have also counted out the effective aggregation rate, which will dem-onstrate the value of the movement.<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/8/89/Circuit1_aa.gif" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><p style="text-align:center"><strong>Fig. 4-4</strong> The simulation of circuit 1.</p></div> <br />
<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/9/92/Circuit2.gif" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><p style="text-align:center"><strong>Fig. 4-5</strong> The simulation of circuit 2.</p></div> <br />
<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/5/54/Circuit3.gif" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><p style="text-align:center"><strong>Fig. 4-6</strong> The simulation of circuit 3.</p></div><br/><br />
Let's see the characteristics of each circuit respectively first. Bacteria with circuit 1 move randomly all the time, so thay can aggregate much faster than other two. The results of bacteria with circuit 2, meeting our expectation, demonstrate the dull response of the complex circuit. As to circuit 3, this kind of bacteria gets together in the region of much atrazine gradually. <br><br><br />
Circuit 1 is the classical design, which is proved by so many researchers to have a good result of attracting bacteria. <br><br/><br />
Compared to circuit 1, circuit 2 need too much time to respond.<br><br/><br />
Circuit 3 has speed up the respond time of circuit 2. Besides, circuit 3 can attract the bacteria around the atrazine step by step as circuit 2.<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/e/e4/Cell_num_in_region-time_in_circuit1-census_.jpg" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><p style="text-align:center"><strong>Fig. 4-7</strong> The number of cells in the atrazine region of circuit1.</p></div><br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/7/76/Cell_num_in_region-time_in_circuit2-census_new.jpg" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><p style="text-align:center"><strong>Fig. 4-8</strong> The number of cells in the atrazine region of circuit2.</p></div><br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/9/94/Cell_num_in_region-time_in_circuit3-census_.jpg" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><p style="text-align:center"><strong>Fig. 4-9</strong> The number of cells in the atrazine region of circuit3.</p></div><br/><br />
The Fig. 4-7~9 exhibits some quantitative results of the three circuits. It is easy for us to find that the number of the cells in different circuits changed in different ways. Here, it is clear that, the bacteria with circuit 3 aggregate in specified gradually, which shows a nearly straight-line slope in the fig. 4-9 as the behavior of circuit 2. At last, fig. 4-8, which present circuit 2, exhibits little change about the number of cells in specified region at the beginning. Shortly,the census has further proved the result obtained from figure 4-4~6.<br />
</dd><br />
</dl><br />
<dl><br />
<dt><a name="Equations">Equations</a></dt><br />
<dd class="dd_1"><br />
After drawing a profile of our project, we should make the process of every event more clearly. So, we have constructed the models of our three circuits by the principles of biochemistry.<br><br/><br />
First, we have constructed a model related to the circuit 2, the most complex one, with the help of Tsinghua-A. Then, in order to compare them, we have also constructed the other two based on the model of circuit 2. In our model, we use ODE to describe the process of chemical reaction in organisms.<br><br/><br />
In general, we have divided the chemical events in our bacteria into three parts, the transcription of DNA, the translation of RNA, the production of micromolecule-compounds. The rate of each event can be described by ODE listed below.<br><br/> <br />
<strong>Circuit1:</strong><br/><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Equation1.jpg"><br/><br />
<strong>Circuit2:</strong><br/><br />
<img src="https://static.igem.org/mediawiki/2013/a/a5/Equations2-1.jpg"><img src="https://static.igem.org/mediawiki/2013/0/0b/Equations2-2.jpg"><br/><br />
<strong>Circuit3:</strong><br/><br />
<img src="https://static.igem.org/mediawiki/2013/a/a7/Equation3.jpg"> <br/><br />
m_CI1,m_CI2, m_CheZ, m_TetR, m_TrzN, m_LuxI represent mRNA of different protein.<br/><br />
CI1,CI2, CheZ, TetR, TrzN, LuxI represent proteins.<br/><br />
p_AHL represent the AHL produced in each cell.<br><br/><br />
We have used matrix to describe the state of AHL and the location of every bacteria. By the way, we have also made the density of bacteria related to the distance of bacteria. At last, we have considerate the degradation of atrazine as well, though which proved to have little influence on atrazine latter. And a space lattice of "culture dish" is demonstrated below.<br><br/> <br />
<strong>Space relation:</strong><br/><br />
<img src="https://static.igem.org/mediawiki/2013/8/8e/Space_relation.jpg"><br><br />
e_AHL represent the AHL in the environment.<br/><br />
PXn, Pyn represent the location of bacteria.<br/><br />
Cdn represent the density of bacteria in a lattice.<br/><br />
Atz represent the atrazine. <br/><br/> <br />
<br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a name="Parameter">Parameter</a></dt><br />
<dd class="dd_1"><br />
<img src="https://static.igem.org/mediawiki/2013/4/48/Parametertable.jpg"> <br><br><br/><br />
<strong>Reference</strong><br/><br />
[1] The wiki of iGEM11 TsinghuaA, https://2011.igem.org/Team:Tsinghua-A/Modeling.<br/><br />
[2] The wiki of iGEM11 USTC, https://2011.igem.org/Team:USTC-China/Drylab/modeling.<br/><br />
[3] Basu, S., Gerchman, Y., Collins, C. H., Arnold, F. H. & Weiss, R. A synthetic multicellular system for programmed pattern formation. Nature434, 1130-1134 (2005).<br/><br />
[4] Goryachev, A., Toh, D. & Lee, T. Systems analysis of a quorum sensing network: design constraints imposed by the functional requirements, network topology and kinetic constants. Biosystems83, 178-187 (2006).<br/><br />
[5] Hooshangi, S. & Bentley, W. E. LsrR Quorum Sensing "Switch" Is Revealed by a Bottom-Up Approach. PLoS computational biology7, e1002172 (2011). <br/> <br />
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<dl><br />
<dt><a name="Introduction">Introduction</a></dt><br />
<dd class="dd_1"><br />
As you can see in the previous parts, the shining point of our design is that we have introduced the quorum sensing device into our circuit. In order to see what the characteristics of our design are during the process of attracting E. coli to atrazine, we have built three models respectively and compared their possible behaviors.<br><br/><br />
The first circuit, we called circuit one, is the most simple one, which has been constructed by Joy sinha et al in 2010. In this circuit, bacteria will stop walking in the region with high concentration of atrazine, in which way bacteria will finally get together to this region. However, bacteria can't tell each other the information about the destination like ants, which will tell their companies the location of food.<br><br/> <br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/1/11/Circuit1_hah.jpg" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 4-1</strong> Circuit 1. Atrazine will promote the translation of the CI protein. However, the CheZ protein, which represent the motility, will be repressed when the concentration of CI protein is high. So, the consequence will be more and more bacteria stopping in the region with high Atrazine.</div><br/> <br />
The second circuit is our original blueprint with a complex structure and you can see the quorum sensing device here. It is a little complex and has a cascade, which is believed to prolong the respond time of our system. In short, the behavior of bacteria will be affected by the concentration of AHL, which represents the density of bacteria. It means that the bacteria can communicate with each other.<br/> <br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/5/59/Fig_3-5-2-B.jpg" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 4-2</strong> Circuit2. The movement of the bacteria with this circuit will affected by the presence of AHL. In our initiate consideration, the cascade, AHL&LuxR→Plux-tetr---Ptet-CI---Pci-CheZ, will lead to the promotion of the production of CheZ protein.</div><br/> <br />
The third circuit is the ultimate one that we have designed and optimized.We have replaced the cascade mentioned above with a hybridize promoter, which can be repressed by CI protein and activated by AHL-LuxR compound.<br/> <br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/e/ea/Circuit3_a.jpg" width="600"></p> <br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 4-3</strong> Circuit 3. The greatest difference between circuit 2 and circuit 3, as you can see, is that the cascade in circuit 2 have been replaced by the hybridize promoter, Plux/CI. Then, we got a new organism that will respond quickly to the presence of AHL.</div><br/><br />
The most important is that we want to have a preview of the behavior of the three circuits mentioned above. Thus, we can compare them and prove that our final design can attract bacteria efficiently and send out the information of atrazine to bacteria around these regions to make their movement more valuable rather than walking aimless.<br/> <br />
</dd><br />
</dl><br />
<br />
<br />
<dl><br />
<dt><a name="Results">Results</a></dt><br />
<dd class="dd_1"><br />
These three models were all coded in MATLAB (all can be download <a href="https://static.igem.org/mediawiki/2013/6/66/Code_of_Nanjing-China%2C_iGEM_2013.zip"><strong>here</strong></a>). As mentioned above, we have just changed several statements in the second model to construct the first and the third. Finally, we have got how the distribution of bacteria changed with time, which can make us catch the major difference among three circuits more directly. <br><br/><br />
Then, we have made census of the number of bacteria within the atrazine region in different circuits respectively. Finally, we have considered the randomness of their movement. We have introduced the Markov chain into the model. We have also counted out the effective aggregation rate, which will dem-onstrate the value of the movement.<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/8/89/Circuit1_aa.gif" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><p style="text-align:center"><strong>Fig. 4-4</strong> The simulation of circuit 1.</p></div> <br />
<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/9/92/Circuit2.gif" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><p style="text-align:center"><strong>Fig. 4-5</strong> The simulation of circuit 2.</p></div> <br />
<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/5/54/Circuit3.gif" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><p style="text-align:center"><strong>Fig. 4-6</strong> The simulation of circuit 3.</p></div><br/><br />
Let's see the characteristics of each circuit respectively first. Bacteria with circuit 1 move randomly all the time, so the result of their aggregation exhibit uncertain scene. The results of bacteria with circuit 2, meeting our expectation, demonstrate the dull response of the complex circuit. As to circuit 3, this kind of bacteria gets together in the region of much atrazine gradually. Circuit 1 is the classical design, which is proved by so many researchers to have a good result of attracting bacteria. <br><br/><br />
Compared to circuit 1, circuit 2 may have a more stable result. However, circuit 2 just need too much time to respond to the signal of cell density.<br><br/><br />
Circuit 3 has speed up the respond time of circuit 2. Besides, circuit 3 can attract the bacteria around the atrazine step by step, which can prevent the situation of too many bacteria aggregate at one point as circuit 1. In another word, circuit 3 works more stably and make it possible to have a mean distribution of bacteria to each atrazine region.<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/e/e4/Cell_num_in_region-time_in_circuit1-census_.jpg" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><p style="text-align:center"><strong>Fig. 4-7</strong> The number of cells in the atrazine region of circuit1.</p></div><br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/7/76/Cell_num_in_region-time_in_circuit2-census_new.jpg" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><p style="text-align:center"><strong>Fig. 4-8</strong> The number of cells in the atrazine region of circuit2.</p></div><br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/9/94/Cell_num_in_region-time_in_circuit3-census_.jpg" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><p style="text-align:center"><strong>Fig. 4-9</strong> The number of cells in the atrazine region of circuit3.</p></div><br/><br />
The Fig. 4-7~9 exhibits some quantitative results of the three circuits. It is easy for us to find that the number of the cells in different circuits changed in different ways. The number of cells with circuit 1 in itself in specified region may have different rate in different situation due to randomness. However, the bacteria with circuit 3 aggregate in specified gradually, which shows a nearly straight-line slope in the fig. 4-9. At last, fig. 4-8, which present circuit 2, exhibits little change about the number of cells in specified region.<br />
</dd><br />
</dl><br />
<dl><br />
<dt><a name="Equations">Equations</a></dt><br />
<dd class="dd_1"><br />
After drawing a profile of our project, we should make the process of every event more clearly. So, we have constructed the models of our three circuits by the principles of biochemistry.<br><br/><br />
First, we have constructed a model related to the circuit 2, the most complex one, with the help of Tsinghua-A. Then, in order to compare them, we have also constructed the other two based on the model of circuit 2. In our model, we use ODE to describe the process of chemical reaction in organisms.<br><br/><br />
In general, we have divided the chemical events in our bacteria into three parts, the transcription of DNA, the translation of RNA, the production of micromolecule-compounds. The rate of each event can be described by ODE listed below.<br><br/> <br />
<strong>Circuit1:</strong><br/><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Equation1.jpg"><br/><br />
<strong>Circuit2:</strong><br/><br />
<img src="https://static.igem.org/mediawiki/2013/a/a5/Equations2-1.jpg"><img src="https://static.igem.org/mediawiki/2013/0/0b/Equations2-2.jpg"><br/><br />
<strong>Circuit3:</strong><br/><br />
<img src="https://static.igem.org/mediawiki/2013/a/a7/Equation3.jpg"> <br/><br />
m_CI1,m_CI2, m_CheZ, m_TetR, m_TrzN, m_LuxI represent mRNA of different protein.<br/><br />
CI1,CI2, CheZ, TetR, TrzN, LuxI represent proteins.<br/><br />
p_AHL represent the AHL produced in each cell.<br><br/><br />
We have used matrix to describe the state of AHL and the location of every bacteria. By the way, we have also made the density of bacteria related to the distance of bacteria. At last, we have considerate the degradation of atrazine as well, though which proved to have little influence on atrazine latter. And a space lattice of "culture dish" is demonstrated below.<br><br/> <br />
<strong>Space relation:</strong><br/><br />
<img src="https://static.igem.org/mediawiki/2013/8/8e/Space_relation.jpg"><br><br />
e_AHL represent the AHL in the environment.<br/><br />
PXn, Pyn represent the location of bacteria.<br/><br />
Cdn represent the density of bacteria in a lattice.<br/><br />
Atz represent the atrazine. <br/><br/> <br />
<br />
</dd><br />
</dl><br />
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<dl><br />
<dt><a name="Parameter">Parameter</a></dt><br />
<dd class="dd_1"><br />
<img src="https://static.igem.org/mediawiki/2013/4/48/Parametertable.jpg"> <br><br><br/><br />
<strong>Reference</strong><br/><br />
[1] The wiki of iGEM11 TsinghuaA, https://2011.igem.org/Team:Tsinghua-A/Modeling.<br/><br />
[2] The wiki of iGEM11 USTC, https://2011.igem.org/Team:USTC-China/Drylab/modeling.<br/><br />
[3] Basu, S., Gerchman, Y., Collins, C. H., Arnold, F. H. & Weiss, R. A synthetic multicellular system for programmed pattern formation. Nature434, 1130-1134 (2005).<br/><br />
[4] Goryachev, A., Toh, D. & Lee, T. Systems analysis of a quorum sensing network: design constraints imposed by the functional requirements, network topology and kinetic constants. Biosystems83, 178-187 (2006).<br/><br />
[5] Hooshangi, S. & Bentley, W. E. LsrR Quorum Sensing "Switch" Is Revealed by a Bottom-Up Approach. PLoS computational biology7, e1002172 (2011). <br/> <br />
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Atrazine is quite hard to degrade naturally in the soil, which makes it persist for a long time in the environment once used. This phenomenon can be considerably severe in that atrazine would cause metabolic disorders both in animals and humankind. For many years, we didn’t have an efficient solution to this problem.<br><br> <br />
However, the discovery of the super power of Arthrobacter aurescens provides us with a light of hope. This amazing species is capable of utilizing atrazine as a sole source of carbon with the help of a series of degrading enzymes which can metabolize atrazine into a kind of nontoxic substance. In our project, we utilize the most useful degrading enzyme TrzN to degrade atrazine. We even mutated the TrzN to make it degrade faster. At the same time, we found a transmembrane transporter TRM, which involves in transporting atrazine from the outside to the inside of the bacteria so that atrazine can be better degraded (more information about <a href="https://2013.igem.org/Team:Nanjing-China/tran"><strong>TRM</strong></a>).<br><br><br />
<strong>Experiments and Results</strong><br />
<br><br />
Firstly, we took 12 15mL-tubes, divided them into 4 groups: Group 0; Group Wild Type; Group Vector and Group TrzN+, 3 tubes each. Then, corresponding materials were added into each group. After 24h of incubation in 37℃ (Fig. 3-6-1), the atrazine concentration in solution of Group TrzN+ was remarkably less than that in solution of other groups.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/7/75/HPLC_TrzN_Col.jpg"width="400"></p><br/><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-6-1</strong> TrzN can distinctively degrade atrazine. This picture shows the atrazine concentration in supernatant of bacterial cultures, which were cultured for 24 hours in 37℃. Group 0 means no bacteria in the cultures; cultures in Group Wild Type contained the wild type K12 bacteria; cultures in Group Vector contained the strain K12 with pGFP; and cultures in Group TrzN+ contained the bacteria which are able to express TrzN. “*” means the difference between two groups which are connected by half square brackets is distinctive. From this chart we can conclude that TrzN is an efficient degrading enzyme in solving the problem of TrzN.</div><br><br><br />
Combining TRM and TrzN together can make the degradation more efficient.<br />
This time, we combined the TRM and TrzN together, under the same method of testing the function of TRM and TrzN, we found that the combination of TRM and TrzN could decrease the atrazine concentration in the bacteria culture to a greater extent (Fig. 3-6-2).<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/e/e8/HPLC_all_Col.jpg"width="550"></p><br/><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-6-2</strong> This picture shows the atrazine concentration in supernatant of bacterial cultures, which were cultured for 24 hours in 37℃. Apart from groups shown above, cultures in Group TRM+TrzN+ contained the bacteria which are able to express TRM as well as TrzN. “*” means the difference between two groups which are connected by half square brackets is distinctive. From this chart we can see that the atrazine concentration in supernatant of Group TRM+TrzN+ was even distinctively less than that in supernatant of TRM+. This means combining TRM and TrzN is indeed a wise choice to polish up our system.</div><br><br><br />
According to these data, we find that TRM can indeed transport atrazine from the outside to the inside of the bacteria, and of course, TrzN can degrade atrazine well and fast.<br/><br/> <br />
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Atrazine is quite hard to degrade naturally in the soil, which makes it persist for a long time in the environment once used. This phenomenon can be considerably severe in that atrazine would cause metabolic disorders both in animals and humankind. For many years, we didn’t have an efficient solution to this problem.<br><br> <br />
However, the discovery of the super power of Arthrobacter aurescens provides us with a light of hope. This amazing species is capable of utilizing atrazine as a sole source of carbon with the help of a series of degrading enzymes which can metabolize atrazine into a kind of nontoxic substance. In our project, we utilize the most useful degrading enzyme TrzN to degrade atrazine. We even mutated the TrzN to make it degrade faster. At the same time, we found a transmembrane transporter TRM, which involves in transporting atrazine from the outside to the inside of the bacteria so that atrazine can be better degraded (more information about <a href="https://2013.igem.org/Team:Nanjing-China/tran"><strong>TRM</strong></a>).<br><br><br />
<strong>Experiments and Results</strong><br />
<br><br />
Firstly, we took 12 15mL-tubes, divided them into 4 groups: Group 0; Group Wild Type; Group Vector and Group TrzN+, 3 tubes each. Then, corresponding materials were added into each group. After 24h of incubation in 37℃ (Fig. 3-6-1), the atrazine concentration in solution of Group TrzN+ was remarkably less than that in solution of other groups.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/7/75/HPLC_TrzN_Col.jpg"width="400"></p><br/><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-6-1</strong> TrzN can distinctively degrade atrazine. This picture shows the atrazine concentration in supernatant of bacterial cultures, which were cultured for 24 hours in 37℃. Group 0 means no bacteria in the cultures; cultures in Group Wild Type contained the wild type K12 bacteria; cultures in Group Vector contained the strain K12 with pGFP; and cultures in Group TrzN+ contained the bacteria which are able to express TrzN. “*” means the difference between two groups which are connected by half square brackets is distinctive. From this chart we can conclude that TrzN is an efficient degrading enzyme in solving the problem of TrzN.</div><br><br><br />
Combining TRM and TrzN together can make the degradation more efficient.<br />
This time, we combined the TRM and TrzN together, under the same method of testing the function of TRM and TrzN, we found that the combination of TRM and TrzN could decrease the atrazine concentration in the bacteria culture to a greater extent (Fig. 3-6-2).<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/e/e8/HPLC_all_Col.jpg"width="550"></p><br/><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-6-2</strong> This picture shows the atrazine concentration in supernatant of bacterial cultures, which were cultured for 24 hours in 37℃. Apart from groups shown above, cultures in Group TRM+TrzN+ contained the bacteria which are able to express TRM as well as TrzN. “*” means the difference between two groups which are connected by half square brackets is distinctive. From this chart we can see that the atrazine concentration in supernatant of Group TRM+TrzN+ was even distinctively less than that in supernatant of TRM+. This means combining TRM and TrzN is indeed a wise choice to polish up our system.</div><br><br><br />
According to these data, we find that TRM can indeed transport atrazine from the outside to the inside of the bacteria, and of course, TrzN can degrade atrazine well and fast.<br/><br/> <br />
<br />
<strong> Reference</strong><br/><br />
[1] 邱玮. 阿特拉津脱氯水解酶 (TrzN) 的定向进化[D]. 南京农业大学, 2011.<br />
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Atrazine, a naughty demon who plays around in the soil, is considerably hard to be eliminated. Its spell causes metabolic disorders in many species.<br><br> <br />
Our elf combined a smart atrazine radar, an amazing transporter, a recruiting system and a degrading enzyme to hunt down this die-hard. With our system, E.coli can recognize atrazine, recruit team members, and eliminate atrazine.<br><br><br />
Our team found the brand new transporter, TRM, from the genome of a bacteria which can utilize atrazine as its only carbon source. With this transporter, the sensitivity of our system and the degrading efficiency are both improved. We also mutated the degrading enzyme, TrzN, to make it better at degrading atrazine than the natural enzyme. We then combined the TRM and the mutated TrzN to achieve a more satisfying result.<br><br> <br />
To take a further step, our team has been devoting ourselves to analyzing and comparing several systems simulated by computer, and we successfully located on the best solution which is equipped with faster moving and quicker degrading.<br><br><br />
Overall, we believe that our system has provided a brilliant solution to make this world a better place for creatures on this planet, hoping it can also become a sincere compensation for the damage resulting from this naughty demon created by ourselves.<br><br />
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</html></div>GaoJian NJUhttp://2013.igem.org/Team:Nanjing-ChinaTeam:Nanjing-China2013-10-27T06:15:51Z<p>GaoJian NJU: </p>
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<p>Atrazine, a naughty demon who plays around in the soil, is considerably hard to be eliminated. Its spell causes metabolic disorders in many species.<br><br />
Our elf combined a smart atrazine radar, an amazing transporter, a recruiting system and a degrading enzyme to hunt down this die-hard. With our system, E.coli can recognize atrazine, recruit team members, and eliminate atrazine.<br><br />
Our team found the brand new transporter, TRM, from the genome of a bacteria which can utilize atrazine as its only carbon source. With this transporter, the sensitivity of our system and the degrading efficiency are both improved. We also mutated the degrading enzyme, TrzN... <a href="https://2013.igem.org/Team:Nanjing-China/abstract">more</a></p><br />
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(Here is an interesting <a href="https://2013.igem.org/Team:Nanjing-China/mp4"><strong>cartoon</strong></a> we made, showing the general idea of our project.)<br><br><br />
Atrazine is one of the most heavily used herbicides and it is used to control the growth of weeds. Atrazine is also a persistent environmental pollutant, it can cause problems like suppressing the growth of some other plants, inducing complete feminization and chemical castration in male frogs, and disrupting the human body's internal systems. Widespread contamination of groundwater has been reported in the United States. As such, there are increasing concerns over the toxicity of atazine in the environment <sup>[1, 2]</sup>.<br/><br/><br />
Considering the fact that atrazine can hardly be degraded naturally in soil and water, we designed and constructed a system in E.coli to make them move towards atrazine simultaneously and absorb it, degrade it into a harmless chemical substance.<br/><br/><br />
For a start, to equip the bacteria with the ability to recognize atrazine, we utilized a smart ribosome switch to recognize the presence of atrazine. In the absence of atrazine, the secondary structure of the ribosome switch forms a pseudoknot, sequestering the ribosome-binding site. In this way, the expression of downstream genes is inhibited. However, in the presence of atrazine, the conformation of the switch changes, the ribosome-binding site is therefore exposed, so the genes downstream can be expressed <sup>[1]</sup>.<br/><br/><br />
When our bacteria can detect the presence of atrazine, we improved their performance by inserting quorum sensing (QS) system into the circuits. When they smell the signal of atrazine, they immediately send out acyl-homoserine lactone (AHL) signal to recruit more team members to gather around the atrazine pollution <sup>[3]</sup>.<br/><br/><br />
Once enough atrazine gather around the atrazine pollution, the density of bacteria pulls the trigger of Plsr, which starts to drive the expression of downstream genes, and the bacteria are therefore able to secrete the super degrading enzyme, TrzN.<br/><br/><br />
However, the low transportation efficiency of atrazine from the outside to the inside of the bacteria posed a problem to us. Luckily, an amazing transporter TRM provided us with an answer. With HPLC, we surprisingly found that TRM can distinctively transport atrazine into our bacteria so the entire system can work more sensitively.<br/><br/><br />
What’s more, in order to acquire a better performance, we modified some of our parts, such as TrzN and CI-tag, to make them function even better.<br/><br/><br />
At the same time, we also used the computer calculation to simulate several systems, and we finally located on the best one which can make the bacteria move, gather and degrade faster.<br/><br/><br />
For the last two months, we have completed some great achievements. A happy and united team was formed and we have successfully constructed 7 brilliant biobrick parts. To take a further step, we improved some parts’ functions with scientific modification. Our human practices spread the concept and notion of synthetic biology as well as our project to many advanced teenagers across the nation.<br/><br/><br />
We know exactly that our work still has a long way to go to get near to industrialization and commercialization, but we do believe that this hope can be eventually realized. Just think about the fascinating progress from the first computer to the PC, iPad and iPhone today, our research perhaps is still lingering in the “old times”, but we have enough confidence that it will shock the world one day, providing us with a final answer to the pollution of atrazine.<br/><br/><br />
<br />
<br />
<strong>Reference</strong><br/><br />
[1] Sinha J, et al. Reprogramming bacteria to seek and destroy a herbicide. Nature chemical biology, 2010, 6 (6): 464-470.<br><br />
<br />
[2] Hayes TB, et al. Atrazine induces complete feminization and chemical castration in male African clawed frogs (Xenopus laevis). Proceedings of the National Academy of Sciences, 2010, 107 (10): 4612-4617.<br><br />
<br />
[3] Carter KK, et al. Pathway engineering via quorum sensing and sRNA riboregulators-Interconnected networks and controllers. Metabolic Engineering, 2012, 14 (3): 281-288.<br/><br />
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<dd class="dd_1"><br />
(Here is an interesting <a href="https://2013.igem.org/Team:Nanjing-China/mp4"><strong>cartoon</strong></a> we made, showing the general idea of our project.)<br><br><br />
Atrazine is one of the most heavily used herbicides and it is used to control the growth of weeds. Atrazine is also a persistent environmental pollutant, it can cause problems like suppressing the growth of some other plants, inducing complete feminization and chemical castration in male frogs, and disrupting the human body's internal systems. Widespread contamination of groundwater has been reported in the United States. As such, there are increasing concerns over the toxicity of atazine in the environment <sup>[1, 2]</sup>.<br/><br/><br />
Considering the fact that atrazine can hardly be degraded naturally in soil and water, we designed and constructed a system in E.coli to make them move towards atrazine simultaneously and absorb it, degrade it into a harmless chemical substance.<br/><br/><br />
For a start, to equip the bacteria with the ability to recognize atrazine, we utilized a smart ribosome switch to recognize the presence of atrazine. In the absence of atrazine, the secondary structure of the ribosome switch forms a pseudoknot, sequestering the ribosome-binding site. In this way, the expression of downstream genes is inhibited. However, in the presence of atrazine, the conformation of the switch changes, the ribosome-binding site is therefore exposed, so the genes downstream can be expressed <sup>[1]</sup>.<br/><br/><br />
When our bacteria could detect the presence of atrazine, we improved their performance by inserting quorum sensing (QS) system into the circuits. When our bacteria smell the signal of atrazine, they send out acyl-homoserine lactone (AHL) to recruit more team members to gather around the atrazine center <sup>[3]</sup>.<br/><br/><br />
Once enough atrazine gathers around the atrazine center, the density of bacteria pulls the trigger of Plsr, and the genes at the downstream of Plsr start to express and the bacteria are therefore able to secrete the super degrading enzyme, TrzN.<br/><br/><br />
However, the low transportation efficiency of atrazine from outside the membrane to the inside of the bacteria posed a problem to us. Luckily, an amazing transporter TRM provided us with an answer. With the help of HPLC (high performance liquid chromatography), we surprisingly found that TRM can distinctively transport atrazine into our bacteria so our entire system can work more sensitively.<br/><br/><br />
What's more, in order to acquire a better performance, we modified some of our parts, such as TrzN and CI, to make them function even better.<br/><br/><br />
At the same time, we also used the computer calculation to simulate the system we constructed, hoping to endow our system with high stability.<br/><br/><br />
For the last two months, we have completed some great achievements. A happy and united team was formed and we have successfully constructed 7 brilliant biobrick parts. Hoping to do better, we improved some of the parts function by scientific modification. Our human practice work has spread the concept and notion of synthetic biology as well as our project to many advanced teenagersacross the nation.<br/><br/><br />
We know exactly that our work still has a long way to go to get near to industrialization and commercialization, but we do believe that this hope can be eventually realized. Just think about the fascinating progress from the first computer to the PC, iPad and iPhone today, our research perhaps is still lingering in the "old times", but we have enough confidence that it will shock the world one day, providing us with a final answer to the pollution of atrazine.<br/><br/><br />
<br />
<br />
<strong>Reference</strong><br/><br />
[1] Sinha J, et al. Reprogramming bacteria to seek and destroy a herbicide. Nature chemical biology, 2010, 6 (6): 464-470.<br><br />
<br />
[2] Hayes TB, et al. Atrazine induces complete feminization and chemical castration in male African clawed frogs (Xenopus laevis). Proceedings of the National Academy of Sciences, 2010, 107 (10): 4612-4617.<br><br />
<br />
[3] Carter KK, et al. Pathway engineering via quorum sensing and sRNA riboregulators-Interconnected networks and controllers. Metabolic Engineering, 2012, 14 (3): 281-288.<br/><br />
</dd><br />
</dl></div>GaoJian NJUhttp://2013.igem.org/Team:Nanjing-China/projectTeam:Nanjing-China/project2013-10-27T06:01:25Z<p>GaoJian NJU: </p>
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(Here is an interesting <a href="https://2013.igem.org/Team:Nanjing-China/mp4"><strong>cartoon</strong></a> we made, showing the general idea of our project.)<br><br><br />
Atrazine is one of the most heavily used herbicides and it is used to control the growth of weeds. Atrazine is also a persistent environmental pollutant, it can cause problems like suppressing the growth of some other plants, inducing complete feminization and chemical castration in male frogs, and disrupting the human body's internal systems. Widespread contamination of groundwater has been reported in the United States. As such, there are increasing concerns over the toxicity of atazine in the environment<sup>[1, 2]</sup>.<br/><br/><br />
Considering the fact that atrazine can hardly be degraded naturally in soil and water, we designed and constructed a system in Escherichia coli to make them move towards atrazine simultaneously up to the gradient and absorb it, degrade it into a harmless chemical substance.<br/><br/><br />
For a start, to equip the bacteria with the ability to recognize atrazine, we utilized a smart ribosome switch to recognize the presence of atrazine. In the absence of atrazine, the secondary structure of the ribosome switch forms a pseudoknot, sequestering the ribosome-binding site. In this way, the expression of downstream genes is inhibited. However, in the presence of atrazine, the conformation of the switch changes, the ribosome-binding site is therefore exposed, so the genes downstream can be expressed.<br/><br/><br />
When our bacteria could detect the presence of atrazine, we improved their performance by inserting quorum sensing (QS) system into the circuits. When our bacteria smell the signal of atrazine, they send out acyl-homoserine lactone (AHL) to recruit more team members to gather around the atrazine center <sup>[3]</sup>.<br/><br/><br />
Once enough atrazine gathers around the atrazine center, the density of bacteria pulls the trigger of Plsr, and the genes at the downstream of Plsr start to express and the bacteria are therefore able to secrete the super degrading enzyme, TrzN.<br/><br/><br />
However, the low transportation efficiency of atrazine from outside the membrane to the inside of the bacteria posed a problem to us. Luckily, an amazing transporter TRM provided us with an answer. With the help of HPLC (high performance liquid chromatography), we surprisingly found that TRM can distinctively transport atrazine into our bacteria so our entire system can work more sensitively.<br/><br/><br />
What's more, in order to acquire a better performance, we modified some of our parts, such as TrzN and CI, to make them function even better.<br/><br/><br />
At the same time, we also used the computer calculation to simulate the system we constructed, hoping to endow our system with high stability.<br/><br/><br />
For the last two months, we have completed some great achievements. A happy and united team was formed and we have successfully constructed 7 brilliant biobrick parts. Hoping to do better, we improved some of the parts function by scientific modification. Our human practice work has spread the concept and notion of synthetic biology as well as our project to many advanced teenagersacross the nation.<br/><br/><br />
We know exactly that our work still has a long way to go to get near to industrialization and commercialization, but we do believe that this hope can be eventually realized. Just think about the fascinating progress from the first computer to the PC, iPad and iPhone today, our research perhaps is still lingering in the "old times", but we have enough confidence that it will shock the world one day, providing us with a final answer to the pollution of atrazine.<br/><br/><br />
<br />
<br />
<strong>Reference</strong><br/><br />
[1] Sinha J, et al. Reprogramming bacteria to seek and destroy a herbicide. Nature chemical biology, 2010, 6 (6): 464-470.<br><br />
<br />
[2] Hayes TB, et al. Atrazine induces complete feminization and chemical castration in male African clawed frogs (Xenopus laevis). Proceedings of the National Academy of Sciences, 2010, 107 (10): 4612-4617.<br><br />
<br />
[3] Carter KK, et al. Pathway engineering via quorum sensing and sRNA riboregulators-Interconnected networks and controllers. Metabolic Engineering, 2012, 14 (3): 281-288.<br/><br />
</dd><br />
</dl></div>GaoJian NJUhttp://2013.igem.org/Team:Nanjing-China/practiceTeam:Nanjing-China/practice2013-10-27T05:53:33Z<p>GaoJian NJU: </p>
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<dt><a name="Interest">Interest Group of Synthetic Biology of Nanjing University</a></dt><br />
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In order to unite the students who embrace huge passion in the field of synthetic biology and propagandize iGEM, we established this group in March, 2013. We aim to combine amusement and science together to live a wonderful lab life. Meanwhile, we also communicated with other iGEM teams as a group. This summer, most students of our group devoted themselves into working on the idea we came up with independently half a year ago. Moreover, we hope that through iGEM, we can not only increase our quality of research, but also strengthen own team cooperation. After iGEM 2013, this group will still exist and we are going to make it more influential.<br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/a/a4/Renhaibo_1.png"></p><br />
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<dd class="dd_1"><br />
Being fresh in iGEM, it's important for us to communicate with a sophisticated team who can help us grow more quickly. In this way, we can also establish friendship with people who have the same dream with us. Under this circumstance, we went to Beijing and visited Tsinghua University in November, 2012.<br><br><br />
During this trip, we visited 3 teams, Tsinghua-A, Tsinghua-C and Tsinghua-D, and communicated with them well. As experienced iGEM competitors, they successfully showed a more clear scenery of iGEM and synthetic biology to us, who haven't stepped into this field before. Besides, Tsinghua-A have introduced the model part in iGEM to us and promised to help us for the lack of modeler in our team. Of course, all of them sincerely remind us of the hardship in this process and we have also talked over the questions like how to come up with a good idea, how to assess and realize them. When we left THU, we had learned so much and both Tsinghua and our team members thought that we should keep contact with each other in the future and step forward together.<br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/igem.org/b/b3/Pic-8-2-1.jpg"width="600"></p><br />
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<dd class="dd_1"><br />
This is the 2nd year since for Nanjing University to participate in the IGEM, so it is necessary to spread the basic principles of synthetic biology and introduce the iGEM to more students in NJU.<br><br />
<br><br />
To achieve this goal, in April, we invited 2 leaders of other iGEM teams in China to give lectures on synthetic biology for students from different majors. XiRui, the leader of SJTU-BioX-Shanghai, 2012, introduced the iGEM from a new perspective. She shared their experience on the IGEM project, which have had profound impact on our project. Meanwhile, Wei Lei, the leader of Tsinghua-A, 2012, showed us how they combine synthetic biology with automation technology, promoting our understanding of the concept and methods of interdisciplinary. Audiences attending the lecture were all deeply impressed and attracted by the iGEM and synthetic biology.<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/3/32/Pic-8-3-1.jpg"width="600"></p><br />
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<dt><a name="Leaflets">Leaflets on China Adolescents Science and Technology Innovation Contest</a></dt><br />
<dd class="dd_1"><br />
The 28th China Adolescents Science and Technology Innovation Contest (CASTIC) was held on August 4th in Nanjing. This annual contest covering science, engineering and social science, is held by the China Association for Science and Technology and the ministries of education, science and technology. Every year, more than 15 million young people take part in the contest.China's future stars of science and technology from primary and secondary schools nationwide gathered in Nanjing to show the fruit of their scientific research.<br><br><br />
<br />
Obviously this was a great chance to introduce synthetic biology and iGEM to youth in China, so we propagated the basic theory of synthetic biology and the idea of our team to them by handing out leaflets and communicating face to face.Many of them showed great interest in IGEM. We also hope to grab this opportunity to allow them a greater interest in synthetic biology.<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/a/a1/Renhaibo_2.JPG"width="600"></p><br />
<br />
</dd><br />
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<dt><a name="help">Help Another iGEM Team, ZJU-China</a></dt><br />
<dd class="dd_1"> <br />
Throughout our project, we took notice of the fact that ZJU-China team and our team both focus on the degradation of atrazine.<br/> <br />
Under this circumstance, we collaborated a lot with them, such as sharing experience, exchanging ideas about environmental protection, and encouraging each other when got stricken. Especially,we offered TrzN, a gene can degrade atrazine to them, which may be of great help for their projects.<br/> <br />
Both of the two outstanding teams learnt a lot from each other's experience, and improved our ideas and designs through the process of free discussion.<br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/4/4d/Renhaibo_3.jpg"width="600"></p><br />
</dd><br />
</dl><br />
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<dt><a name="Lecture">Lecture in Nanjing Jinling Middle School</a></dt><br />
<dd class="dd_1"><br />
Although iGEM also has High School Division, few local high school students know about iGEM and synthetic biology. In order to let them have a rational understanding and encourage more students to participate in the IGEM competition, we decided to introduce synthetic biology, IGEM and our project in the form of lectures. <br><br><br />
In the lecture, we used the third biological revolution to give way to synthetic biology. And we introduced synthetic biology and iGEM in a vivid way.When we talked about synthetic biology, we delivered the idea of synthesis. When we speak of iGEM, we delivered several key points that comprise a whole project in iGEM: a practical idea, construction of gene circuits, verification, modification, safety and human practice. What's more, we emphasized the idea of engineer and standardization. At last, we introduced our project.<br><br><br />
After the lecture, many students showed great interest in iGEMHigh School Division. We hope that we did could influence more and more people to participate in this competition and enjoy it.<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/5/53/Pic-8-5-1.jpg"width="600"></p><br />
</dd><br />
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<br />
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<dt><a name="Apply">Apply Our Project to Time Farm</a></dt><br />
<dd class="dd_1"><br />
We have always been considering to apply our project tothe field of farming industry and environmental governance. Guided by this belief, we visited the Time Farm, a newly built organic farm in Nanjing.<br><br />
<br><br />
The technology of this farm is based on permaculture. They want to cultivate healthy products by constructing harmonized eco-environment. In the process of communication, we learned that they purify water mainly by constructing artificial wetlands and spraying organic fertilizer which contains a lot of microorganisms. These strategies indeed obtains satisfying results in water purification, and the products are much healthier than those cultivated in the original farms. However, some refractory deleterious organic compounds used in other areas around the organic farm, for example, atrazine, may still remain in the water. In this case, bacteria with biological function may benefit the farmers a lot. They also realized that synthetic biology could be an effective and environmental friendly tool in the near future.(Friendly link: Time Farm <a href="http://www.timefarm.com.cn">http://www.timefarm.com.cn</a>)<br />
<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/3/32/Pic-8-3-1.jpg"width="600"></p><br />
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</html></div>GaoJian NJUhttp://2013.igem.org/Team:Nanjing-China/practiceTeam:Nanjing-China/practice2013-10-27T05:51:45Z<p>GaoJian NJU: </p>
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<dt><a name="Interest">Interest Group of Synthetic Biology of Nanjing University</a></dt><br />
<dd class="dd_1"><br />
In order to unite the students who embrace huge passion in the field of synthetic biology and propagandize iGEM, we established this group in March, 2013. We aim to combine amusement and science together to live a wonderful lab life. Meanwhile, we also communicated with other iGEM teams as a group. This summer, most students of our group devoted themselves into working on the idea we came up with independently half a year ago. Moreover, we hope that through iGEM, we can not only increase our quality of research, but also strengthen own team cooperation. After iGEM 2013, this group will still exist and we are going to make it more influential.<br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/a/a4/Renhaibo_1.png"></p><br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a name="Visit">Visit iGEM Teams of Tsinghua University</a></dt><br />
<dd class="dd_1"><br />
Being fresh in iGEM, it's important for us to communicate with a sophisticated team who can help us grow more quickly. In this way, we can also establish friendship with people who have the same dream with us. Under this circumstance, we went to Beijing and visited Tsinghua University in November, 2012.<br><br><br />
During this trip, we visited 3 teams, Tsinghua-A, Tsinghua-C and Tsinghua-D, and communicated with them well. As experienced iGEM competitors, they successfully showed a more clear scenery of iGEM and synthetic biology to us, who haven't stepped into this field before. Besides, Tsinghua-A have introduced the model part in iGEM to us and promised to help us for the lack of modeler in our team. Of course, all of them sincerely remind us of the hardship in this process and we have also talked over the questions like how to come up with a good idea, how to assess and realize them. When we left THU, we had learned so much and both Tsinghua and our team members thought that we should keep contact with each other in the future and step forward together.<br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/igem.org/b/b3/Pic-8-2-1.jpg"width="600"></p><br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a name="iGEM">iGEM Communication with Shanghai Jiao Tong University And Tsinghua University</a></dt><br />
<dd class="dd_1"><br />
This is the 2nd year since for Nanjing University to participate in the IGEM, so it is necessary to spread the basic principles of synthetic biology and introduce the iGEM to more students in NJU.<br><br />
<br><br />
To achieve this goal, in April, we invited 2 leaders of other iGEM teams in China to give lectures on synthetic biology for students from different majors. XiRui, the leader of SJTU-BioX-Shanghai, 2012, introduced the iGEM from a new perspective. She shared their experience on the IGEM project, which have had profound impact on our project. Meanwhile, Wei Lei, the leader of Tsinghua-A, 2012, showed us how they combine synthetic biology with automation technology, promoting our understanding of the concept and methods of interdisciplinary. Audiences attending the lecture were all deeply impressed and attracted by the iGEM and synthetic biology.<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/3/32/Pic-8-3-1.jpg"width="600"></p><br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a name="Leaflets">Leaflets on China Adolescents Science and Technology Innovation Contest</a></dt><br />
<dd class="dd_1"><br />
The 28th China Adolescents Science and Technology Innovation Contest (CASTIC) was held on August 4th in Nanjing. This annual contest covering science, engineering and social science, is held by the China Association for Science and Technology and the ministries of education, science and technology. Every year, more than 15 million young people take part in the contest.China's future stars of science and technology from primary and secondary schools nationwide gathered in Nanjing to show the fruit of their scientific research.<br><br><br />
<br />
Obviously this was a great chance to introduce synthetic biology and iGEM to youth in China, so we propagated the basic theory of synthetic biology and the idea of our team to them by handing out leaflets and communicating face to face.Many of them showed great interest in IGEM. We also hope to grab this opportunity to allow them a greater interest in synthetic biology.<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/a/a1/Renhaibo_2.JPG"width="600"></p><br />
<br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a name="help">Help Another iGEM Team, ZJU-China</a></dt><br />
<dd class="dd_1"> <br />
Throughout our project, we took notice of the fact that ZJU-China team and our team both focus on the degradation of atrazine.<br/> <br />
Under this circumstance, we collaborated a lot with them, such as sharing experience, exchanging ideas about environmental protection, and encouraging each other when got stricken. Especially,we offered TrzN, a gene can degrade atrazine to them, which may be of great help for their projects.<br/> <br />
Both of the two outstanding teams learnt a lot from each other's experience, and improved our ideas and designs through the process of free discussion.<br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/4/4d/Renhaibo_3.jpg"width="600"></p><br />
</dd><br />
</dl><br />
<br />
<br />
<dl><br />
<dt><a name="Lecture">Lecture in Nanjing Jinling Middle School</a></dt><br />
<dd class="dd_1"><br />
Although iGEM also has High School Division, few local high school students know about iGEM and synthetic biology. In order to let them have a rational understanding and encourage more students to participate in the IGEM competition, we decided to introduce synthetic biology, IGEM and our project in the form of lectures. <br><br><br />
In the lecture, we used the third biological revolution to give way to synthetic biology. And we introduced synthetic biology and iGEM in a vivid way.When we talked about synthetic biology, we delivered the idea of synthesis. When we speak of iGEM, we delivered several key points that comprise a whole project in iGEM: a practical idea, construction of gene circuits, verification, modification, safety and human practice. What's more, we emphasized the idea of engineer and standardization. At last, we introduced our project.<br><br><br />
After the lecture, many students showed great interest in iGEMHigh School Division. We hope that we did could influence more and more people to participate in this competition and enjoy it.<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/5/53/Pic-8-5-1.jpg"width="600"></p><br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a name="Apply">Apply Our Project to Time Farm</a></dt><br />
<dd class="dd_1"><br />
We have always been considering to apply our project tothe field of farming industry and environmental governance. Guided by this belief, we visited the Time Farm, a newly built organic farm in Nanjing.<br><br />
<br><br />
The technology of this farm is based on permaculture. They want to cultivate healthy products by constructing harmonized eco-environment. In the process of communication, we learned that they purify water mainly by constructing artificial wetlands and spraying organic fertilizer which contains a lot of microorganisms. These strategies indeed obtains satisfying results in water purification, and the products are much healthier than those cultivated in the original farms. However, some refractory deleterious organic compounds used in other areas around the organic farm, for example, atrazine, may still remain in the water. In this case, bacteria with biological function may benefit the farmers a lot. They also realized that synthetic biology could be an effective and environmental friendly tool in the near future (Friendly link: Time Farm <a href="http://www.timefarm.com.cn">http://www.timefarm.com.cn</a>).<br />
<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/3/32/Pic-8-3-1.jpg"width="600"></p><br />
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</html></div>GaoJian NJUhttp://2013.igem.org/Team:Nanjing-China/practiceTeam:Nanjing-China/practice2013-10-27T03:33:27Z<p>GaoJian NJU: </p>
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<dt><a name="Interest">Interest Group of Synthetic Biology of Nanjing University</a></dt><br />
<dd class="dd_1"><br />
In order to unite the students who embrace huge passion in the field of synthetic biology and propagandize iGEM, we established this group in March, 2013. We aim to combine amusement and science together to live a wonderful lab life. Meanwhile, we also communicated with other iGEM teams as a group. This summer, most students of our group devoted themselves into working on the idea we came up with independently half a year ago. Moreover, we hope that through iGEM, we can not only increase our quality of research, but also strengthen own team cooperation. After iGEM 2013, this group will still exist and we are going to make it more influential.<br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/a/a4/Renhaibo_1.png"></p><br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a name="Visit">Visit iGEM Teams of Tsinghua University</a></dt><br />
<dd class="dd_1"><br />
Being fresh in iGEM, it's important for us to communicate with a sophisticated team who can help us grow more quickly. In this way, we can also establish friendship with people who have the same dream with us. Under this circumstance, we went to Beijing and visited Tsinghua University in November, 2012.<br><br><br />
During this trip, we visited 3 teams, Tsinghua-A, Tsinghua-C and Tsinghua-D, and communicated with them well. As experienced iGEM competitors, they successfully showed a more clear scenery of iGEM and synthetic biology to us, who haven't stepped into this field before. Besides, Tsinghua-A have introduced the model part in iGEM to us and promised to help us for the lack of modeler in our team. Of course, all of them sincerely remind us of the hardship in this process and we have also talked over the questions like how to come up with a good idea, how to assess and realize them. When we left THU, we had learned so much and both Tsinghua and our team members thought that we should keep contact with each other in the future and step forward together.<br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/igem.org/b/b3/Pic-8-2-1.jpg"width="600"></p><br />
</dd><br />
</dl><br />
<br />
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<dt><a name="iGEM">iGEM Communication with Shanghai Jiao Tong University And Tsinghua University</a></dt><br />
<dd class="dd_1"><br />
This is the 2nd year since for Nanjing University to participate in the IGEM, so it is necessary to spread the basic principles of synthetic biology and introduce the iGEM to more students in NJU.<br><br />
<br><br />
To achieve this goal, in April, we invited 2 leaders of other iGEM teams in China to give lectures on synthetic biology for students from different majors. XiRui, the leader of SJTU-BioX-Shanghai, 2012, introduced the iGEM from a new perspective. She shared their experience on the IGEM project, which have had profound impact on our project. Meanwhile, Wei Lei, the leader of Tsinghua-A, 2012, showed us how they combine synthetic biology with automation technology, promoting our understanding of the concept and methods of interdisciplinary. Audiences attending the lecture were all deeply impressed and attracted by the iGEM and synthetic biology.<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/3/32/Pic-8-3-1.jpg"width="600"></p><br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a name="Leaflets">Leaflets on China Adolescents Science and Technology Innovation Contest</a></dt><br />
<dd class="dd_1"><br />
The 28th China Adolescents Science and Technology Innovation Contest (CASTIC) was held on August 4th in Nanjing. This annual contest covering science, engineering and social science, is held by the China Association for Science and Technology and the ministries of education, science and technology. Every year, more than 15 million young people take part in the contest.China's future stars of science and technology from primary and secondary schools nationwide gathered in Nanjing to show the fruit of their scientific research.<br><br><br />
<br />
Obviously this was a great chance to introduce synthetic biology and iGEM to youth in China, so we propagated the basic theory of synthetic biology and the idea of our team to them by handing out leaflets and communicating face to face.Many of them showed great interest in IGEM. We also hope to grab this opportunity to allow them a greater interest in synthetic biology.<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/a/a1/Renhaibo_2.JPG"width="600"></p><br />
<br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a name="help">Help Another iGEM Team, ZJU-China</a></dt><br />
<dd class="dd_1"> <br />
Throughout our project, we took notice of the fact that ZJU-China team and our team both focus on the degradation of atrazine.<br/> <br />
Under this circumstance, we collaborated a lot with them, such as sharing experience, exchanging ideas about environmental protection, and encouraging each other when got stricken. Especially,we offered TrzN, a gene can degrade atrazine to them, which may be of great help for their projects.<br/> <br />
Both of the two outstanding teams learnt a lot from each other's experience, and improved our ideas and designs through the process of free discussion.<br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/4/4d/Renhaibo_3.jpg"width="600"></p><br />
</dd><br />
</dl><br />
<br />
<br />
<dl><br />
<dt><a name="Lecture">Lecture in Nanjing Jinling Middle School</a></dt><br />
<dd class="dd_1"><br />
Although iGEM also has High School Division, few local high school students know about iGEM and synthetic biology. In order to let them have a rational understanding and encourage more students to participate in the IGEM competition, we decided to introduce synthetic biology, IGEM and our project in the form of lectures. <br><br><br />
In the lecture, we used the third biological revolution to give way to synthetic biology. And we introduced synthetic biology and iGEM in a vivid way.When we talked about synthetic biology, we delivered the idea of synthesis. When we speak of iGEM, we delivered several key points that comprise a whole project in iGEM: a practical idea, construction of gene circuits, verification, modification, safety and human practice. What's more, we emphasized the idea of engineer and standardization. At last, we introduced our project.<br><br><br />
After the lecture, many students showed great interest in iGEMHigh School Division. We hope that we did could influence more and more people to participate in this competition and enjoy it.<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/5/53/Pic-8-5-1.jpg"width="600"></p><br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a name="Apply">Apply Our Project to Time Farm</a></dt><br />
<dd class="dd_1"><br />
This is the 2nd year since for Nanjing University to participate in the IGEM, so it is necessary to spread the basic principles of synthetic biology and introduce the iGEM to more students in NJU.<br><br />
<br><br />
To achieve this goal, in April, we invited 2 leaders of other iGEM teams in China to give lectures on synthetic biology for students from different majors. XiRui, the leader of SJTU-BioX-Shanghai, 2012, introduced the iGEM from a new perspective. She shared their experience on the IGEM project, which have had profound impact on our project. Meanwhile, Wei Lei, the leader of Tsinghua-A, 2012, showed us how they combine synthetic biology with automation technology, promoting our understanding of the concept and methods of interdisciplinary. Audiences attending the lecture were all deeply impressed and attracted by the iGEM and synthetic biology.<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/3/32/Pic-8-3-1.jpg"width="600"></p><br />
</dd><br />
</dl><br />
<br />
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</html></div>GaoJian NJUhttp://2013.igem.org/Team:Nanjing-China/practiceTeam:Nanjing-China/practice2013-10-27T03:26:35Z<p>GaoJian NJU: </p>
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<dt><a name="Interest">Interest Group of Synthetic Biology of Nanjing University</a></dt><br />
<dd class="dd_1"><br />
In order to unite the students who embrace huge passion in the field of synthetic biology and propagandize iGEM, we established this group in March, 2013. We aim to combine amusement and science together to live a wonderful lab life. Meanwhile, we also communicated with other iGEM teams as a group. This summer, most students of our group devoted themselves into working on the idea we came up with independently half a year ago. Moreover, we hope that through iGEM, we can not only increase our quality of research, but also strengthen own team cooperation. After iGEM 2013, this group will still exist and we are going to make it more influential.<br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/a/a4/Renhaibo_1.png"></p><br />
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Being fresh in iGEM, it's important for us to communicate with a sophisticated team who can help us grow more quickly. In this way, we can also establish friendship with people who have the same dream with us. Under this circumstance, we went to Beijing and visited Tsinghua University in November, 2012.<br><br><br />
During this trip, we visited 3 teams, Tsinghua-A, Tsinghua-C and Tsinghua-D, and communicated with them well. As experienced iGEM competitors, they successfully showed a more clear scenery of iGEM and synthetic biology to us, who haven't stepped into this field before. Besides, Tsinghua-A have introduced the model part in iGEM to us and promised to help us for the lack of modeler in our team. Of course, all of them sincerely remind us of the hardship in this process and we have also talked over the questions like how to come up with a good idea, how to assess and realize them. When we left THU, we had learned so much and both Tsinghua and our team members thought that we should keep contact with each other in the future and step forward together.<br><br />
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This is the 2nd year since for Nanjing University to participate in the IGEM, so it is necessary to spread the basic principles of synthetic biology and introduce the iGEM to more students in NJU.<br><br />
<br><br />
To achieve this goal, in April, we invited 2 leaders of other iGEM teams in China to give lectures on synthetic biology for students from different majors. XiRui, the leader of SJTU-BioX-Shanghai, 2012, introduced the iGEM from a new perspective. She shared their experience on the IGEM project, which have had profound impact on our project. Meanwhile, Wei Lei, the leader of Tsinghua-A, 2012, showed us how they combine synthetic biology with automation technology, promoting our understanding of the concept and methods of interdisciplinary. Audiences attending the lecture were all deeply impressed and attracted by the iGEM and synthetic biology.<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/3/32/Pic-8-3-1.jpg"width="600"></p><br />
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<dt><a name="Leaflets">Leaflets on China Adolescents Science and Technology Innovation Contest</a></dt><br />
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The 28th China Adolescents Science and Technology Innovation Contest (CASTIC) was held on August 4th in Nanjing. This annual contest covering science, engineering and social science, is held by the China Association for Science and Technology and the ministries of education, science and technology. Every year, more than 15 million young people take part in the contest.China's future stars of science and technology from primary and secondary schools nationwide gathered in Nanjing to show the fruit of their scientific research.<br><br><br />
<br />
Obviously this was a great chance to introduce synthetic biology and iGEM to youth in China, so we propagated the basic theory of synthetic biology and the idea of our team to them by handing out leaflets and communicating face to face.Many of them showed great interest in IGEM. We also hope to grab this opportunity to allow them a greater interest in synthetic biology.<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/a/a1/Renhaibo_2.JPG"width="600"></p><br />
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<dd class="dd_1"> <br />
Throughout our project, we took notice of the fact that ZJU-China team and our team both focus on the degradation of atrazine.<br/> <br />
Under this circumstance, we collaborated a lot with them, such as sharing experience, exchanging ideas about environmental protection, and encouraging each other when got stricken. Especially,we offered TrzN, a gene can degrade atrazine to them, which may be of great help for their projects.<br/> <br />
Both of the two outstanding teams learnt a lot from each other's experience, and improved our ideas and designs through the process of free discussion.<br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/4/4d/Renhaibo_3.jpg"width="600"></p><br />
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<dt><a name="Lecture">Lecture in Nanjing Jinling Middle School</a></dt><br />
<dd class="dd_1"><br />
Although iGEM also has High School Division, few local high school students know about iGEM and synthetic biology. In order to let them have a rational understanding and encourage more students to participate in the IGEM competition, we decided to introduce synthetic biology, IGEM and our project in the form of lectures. <br><br><br />
In the lecture, we used the third biological revolution to give way to synthetic biology. And we introduced synthetic biology and iGEM in a vivid way.When we talked about synthetic biology, we delivered the idea of synthesis. When we speak of iGEM, we delivered several key points that comprise a whole project in iGEM: a practical idea, construction of gene circuits, verification, modification, safety and human practice. What's more, we emphasized the idea of engineer and standardization. At last, we introduced our project.<br><br><br />
After the lecture, many students showed great interest in iGEMHigh School Division. We hope that we did could influence more and more people to participate in this competition and enjoy it.<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/5/53/Pic-8-5-1.jpg"width="600"></p><br />
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</html></div>GaoJian NJUhttp://2013.igem.org/Team:Nanjing-China/qsTeam:Nanjing-China/qs2013-10-27T03:23:03Z<p>GaoJian NJU: </p>
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In our project, we considered devising our bacteria into some kind of automatic machinery that would be able to sense a target molecule and then assemble to degrade it. To fulfill all these functions, we were bound to design some gene circuits to guide our bacteria.<br><br><br />
<br />
First of all, we should have an understanding of how bacteria move<sup>[1]</sup>. For E.coli, they have flagellar motors to control their movement (Fig. 3-5-1). The direction of rotation of their flagellar motors is controlled by the protein CheY. When CheY is not phosphorylated, the flagellar motors rotate counterclockwise (CCW) and bacteria can migrate. When CheY is phosphorylated (CheY-P), it can bind to the flagellar motorprotein FliM, causing the cell to tumble and thus nonmotile. Phosphatase CheZ dephosphorylates CheY-P and causes the flagellum to rotate CCW. E. coli lacking the cheZ gene (ΔcheZ) cannot dephosphorylate CheY-P, and thus tumble incessantly, being nonmotile. So we only need to take control of gene cheZ to control the motility of our bacteria. <br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/0/04/Fig.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-1</strong> Illustration of how bacteria move. CheZ is the key of controlling the motility of bacteria. For wild type, CheZ can be expressed normally, so the flagellar motor alternates between CCW and CW rotation, thus the cell can run or tumble. On the contrary, if there is no CheZ, CheY will remain phosphorylated, causing the flagellar motor rotates CW, thus the cell tumbles exclusively. (Cite by JACS, Shana Toop et al, 2007.)</div><br><br><br />
We would like our bacteria to seek and destroy atrazine in the soil. To achieve this goal, a classic system can do the job (Fig. 3-5-2A). In this system, bacteria can move around randomly but fix when sensing atrazine as the ribosome switch turns on and protein CI is expressed to repress the gene cheZ. As a result, our bacteria would just assemble around the target molecules after a period of time (Fig. 3-5-3).<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/2/26/3-4%EF%BC%88%E5%A4%87%E7%94%A8%EF%BC%89.jpg" width="400"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-3</strong> Our bacteria could just assemble around the target molecules after a period of time.</div><br><br><br />
However, we can only wait for the bacteria to hang about until they find their targets. Thus it could take a long time before the bacteria assembly and the system may not be a robust design. So we shall spare no efforts to improve it. For better control over the function of our bacteria, we took the quorum sensing system into consideration. We expect some bacteria which first find the atrazine will recruit other bacteria at a distance through some signal so they move towards the target and then fixed at the source of pollution. Therefore we constructed the following gene circuits to realize it (Fig. 3-5-2B).<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/6/63/Fig_3-5-2.jpg" width="716"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-2</strong> The gene circuits we built to control cheZ. (A) A classic system using CI to repress cheZ. (B) A complex but high-efficiency system using quorum sensing system. (C) A simplified system like the system in (B).</div><br><br><br />
This circuit is composed of three modules: an atrazine-sensing module, a recruiting module, and a brake module. We adopted the quorum-sensing system in Vibrio fischerias the basis for our design. In this system, a small-molecule acyl-homoserine lactone (AHL) synthesized by LuxI excretes as a signal and accumulates intracellularly to activate a constitutively expressed regulator, LuxR<sup>[2]</sup>. Our atrazine-sensing module controls the translation of LuxI through ribosome switch which turns on when combined to atrazine. Meanwhile, the recruiting module is composed of a series of elements. In this system, the LuxR-AHL complex promotes the expression of a repressor TetR, which represses the expression of another repressor CI. The gene cheZ is controlled by promoter Pλ, which is repressed by CI. So for those bacteria which first sense atrazine, they send out AHL signal and let the other bacteria receive it. After that, TetR is expressed to repress CI, leading the transcription of cheZ. These bacteria move towards the sender until they sense atrazine and another repressor CI, which is controlled by ribosome switch, expresses as a brake of the movement<sup>[3]</sup>. <br><br><br />
We also design the third gene circuit to simplify the system (Fig. 3-5-2C). This system also consists of the three modules in Pattern 2 and the atrazine-sensing module, as well as the brake module, is exactly the same composition with those two parts. The only difference is in the recruiting module that we use a double-functional promoter Plux/CI. The atrazine-sensing module works just the same way in Pattern 2. In this system, however, the LuxR-AHL complex promotes cheZ transcription via promoter Plux/CI. At the spot where bacteria sense atrazine, AHL will accumulate. The higher AHL concentration is, the stronger transcription of cheZ which means greater motility is, leading to directional movement towards bacteria which send out the signal. Meanwhile, the brake module also functions via promoter Plux/CI. The repressor CI, controlled by ribosome switch which turns on when combined to atrazine, represses the promoter Plux/CI and results in bacteria fixation at source of pollution.<br><br><br />
<strong>Experiments and Results</strong><br><br />
At the beginning, we verified gene cheZ in genetic level. We had 3 strains, namely CL-1, CL-M, and CL-IM. CL-1 is a strain with deletion of gene cheZ, CL-M is wildtype, and CL-IM is a strain with deletion of the gene that controls the expression of flagella. We extracted the total RNA of these 3 strains and synthesized the 1st cDNA using RT-PCR. Then we did PCR of gene cheZ with the cDNA as template and we also did PCR of 16 sDNA as control. We got the figure through agarose gel electrophoresis (Fig. 3-5-4A). We can see that CL-1 is actually the strain with deletion of gene cheZ, while the other two strains have normal gene cheZ.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/f/f3/Fig_3-5-3.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-4</strong> Verification of gene cheZ. (A) In genetic level, CL-1 is actually the strain with deletion of gene cheZ, while the other two strains have normal gene cheZ. (B) In phenotype level, CL-M and pcheZ is able to migrate on semisolid culture medium, while CL-1 and CL-M is unable to migrate.</div><br><br><br />
Then we needed to verify of gene cheZ from phenotype. We have 3 strains, namely CL-1, CL-M, and CL-IM. CL-1 is a strain with deletion of gene cheZ, CL-M is wild type, and CL-IM is a strain with deletion of the gene that controls the expression of flagella. Then we transformed a plasmid with gene cheZ into CL-1 and got the fourth strain pcheZ.<br><br><br />
We inoculated the suspension of bacteria on semisolid LB plate and incubated for 20h and took the photograph (Fig. 3-5-4B). From the figure, we can see that CL-M and pcheZ is able to migrate on semisolid culture medium, while CL-1 and CL-M is unable to migrate. This indicates that E.coli is nonmotile without gene cheZ or flagella and inducing the expression of gene cheZ can restore motility in a cheZ knockout strain.<br><br><br />
Finally, we also verified lux system. We constructed a strain with gene circuit as shown in Fig. 3-5-5A from CL-1. This is a lux system without the ribosome switch. AHL synthesized by LuxI excretes as a signal and accumulates intracellularly to activate a constitutively expressed regulator, LuxR. Then the LuxR-AHL complex promotes the expression of a repressor CI, leading to repression of CheZ. So bacteria will assembly at spots where initial density is high in this system. After we inoculated the suspension of bacteria on semisolid LB plate and incubated for 20h, we took the photograph of a stripe pattern (Fig. 3-5-5B), which was formed under the influence of the lux system<sup>[4]</sup>.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/d/db/Fig_3-5-4_new.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-5</strong> Verification of lux system. (A) the gene circuit we constructed. (B) The phenotype of lux system. </div><br/><br />
For more information of our assembly system, please refer to <a href="https://2013.igem.org/Team:Nanjing-China/model"><strong>Model</strong></a>.<br><br />
<br/> <br />
<br />
<strong> Reference</strong><br/><br />
[1] Topp S and Gallivan JP. Guiding bacteria with small molecules and RNA. Journal of the American Chemical Society, 2007, 129 (21): 6807-6811.<br/><br />
[2] Carter KK, et al. Pathway engineering via quorum sensing and sRNA riboregulators-Interconnected networks and controllers. Metabolic Engineering, 2012, 14 (3): 281-288.<br/><br />
[3] Sinha J, et al. Reprogramming bacteria to seek and destroy a herbicide. Nature chemical biology, 2010, 6 (6): 464-470.<br/><br />
[4] Liu C, et al. Sequential establishment of stripe patterns in an expanding cell population. Science, 2011, 334 (6053): 238-241.<br/><br />
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</html></div>GaoJian NJUhttp://2013.igem.org/Team:Nanjing-China/qsTeam:Nanjing-China/qs2013-10-27T03:22:21Z<p>GaoJian NJU: </p>
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<dt><a href="###">Aggregation</a></dt><br />
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In our project, we considered devising our bacteria into some kind of automatic machinery that would be able to sense a target molecule and then assemble to degrade it. To fulfill all these functions, we were bound to design some gene circuits to guide our bacteria.<br><br><br />
<br />
First of all, we should have an understanding of how bacteria move<sup>[1]</sup>. For E.coli, they have flagellar motors to control their movement (Fig. 3-5-1). The direction of rotation of their flagellar motors is controlled by the protein CheY. When CheY is not phosphorylated, the flagellar motors rotate counterclockwise (CCW) and bacteria can migrate. When CheY is phosphorylated (CheY-P), it can bind to the flagellar motorprotein FliM, causing the cell to tumble and thus nonmotile. Phosphatase CheZ dephosphorylates CheY-P and causes the flagellum to rotate CCW. E. coli lacking the cheZ gene (ΔcheZ) cannot dephosphorylate CheY-P, and thus tumble incessantly, being nonmotile. So we only need to take control of gene cheZ to control the motility of our bacteria. <br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/0/04/Fig.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-1</strong> Illustration of how bacteria move. CheZ is the key of controlling the motility of bacteria. For wild type, CheZ can be expressed normally, so the flagellar motor alternates between CCW and CW rotation, thus the cell can run or tumble. On the contrary, if there is no CheZ, CheY will remain phosphorylated, causing the flagellar motor rotates CW, thus the cell tumbles exclusively. (Cite by JACS, Shana Toop et al, 2007.)</div><br><br><br />
We would like our bacteria to seek and destroy atrazine in the soil. To achieve this goal, a classic system can do the job (Fig. 3-5-2A). In this system, bacteria can move around randomly but fix when sensing atrazine as the ribosome switch turns on and protein CI is expressed to repress the gene cheZ. As a result, our bacteria would just assemble around the target molecules after a period of time (Fig. 3-5-3).<br><br><br />
<p style="text-align:center"><img src="https://2013.igem.org/File:3-4%EF%BC%88%E5%A4%87%E7%94%A8%EF%BC%89.jpg" width="400"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-3</strong> Our bacteria could just assemble around the target molecules after a period of time.</div><br><br><br />
However, we can only wait for the bacteria to hang about until they find their targets. Thus it could take a long time before the bacteria assembly and the system may not be a robust design. So we shall spare no efforts to improve it. For better control over the function of our bacteria, we took the quorum sensing system into consideration. We expect some bacteria which first find the atrazine will recruit other bacteria at a distance through some signal so they move towards the target and then fixed at the source of pollution. Therefore we constructed the following gene circuits to realize it (Fig. 3-5-2B).<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/6/63/Fig_3-5-2.jpg" width="716"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-2</strong> The gene circuits we built to control cheZ. (A) A classic system using CI to repress cheZ. (B) A complex but high-efficiency system using quorum sensing system. (C) A simplified system like the system in (B).</div><br><br><br />
This circuit is composed of three modules: an atrazine-sensing module, a recruiting module, and a brake module. We adopted the quorum-sensing system in Vibrio fischerias the basis for our design. In this system, a small-molecule acyl-homoserine lactone (AHL) synthesized by LuxI excretes as a signal and accumulates intracellularly to activate a constitutively expressed regulator, LuxR<sup>[2]</sup>. Our atrazine-sensing module controls the translation of LuxI through ribosome switch which turns on when combined to atrazine. Meanwhile, the recruiting module is composed of a series of elements. In this system, the LuxR-AHL complex promotes the expression of a repressor TetR, which represses the expression of another repressor CI. The gene cheZ is controlled by promoter Pλ, which is repressed by CI. So for those bacteria which first sense atrazine, they send out AHL signal and let the other bacteria receive it. After that, TetR is expressed to repress CI, leading the transcription of cheZ. These bacteria move towards the sender until they sense atrazine and another repressor CI, which is controlled by ribosome switch, expresses as a brake of the movement<sup>[3]</sup>. <br><br><br />
We also design the third gene circuit to simplify the system (Fig. 3-5-2C). This system also consists of the three modules in Pattern 2 and the atrazine-sensing module, as well as the brake module, is exactly the same composition with those two parts. The only difference is in the recruiting module that we use a double-functional promoter Plux/CI. The atrazine-sensing module works just the same way in Pattern 2. In this system, however, the LuxR-AHL complex promotes cheZ transcription via promoter Plux/CI. At the spot where bacteria sense atrazine, AHL will accumulate. The higher AHL concentration is, the stronger transcription of cheZ which means greater motility is, leading to directional movement towards bacteria which send out the signal. Meanwhile, the brake module also functions via promoter Plux/CI. The repressor CI, controlled by ribosome switch which turns on when combined to atrazine, represses the promoter Plux/CI and results in bacteria fixation at source of pollution.<br><br><br />
<strong>Experiments and Results</strong><br><br />
At the beginning, we verified gene cheZ in genetic level. We had 3 strains, namely CL-1, CL-M, and CL-IM. CL-1 is a strain with deletion of gene cheZ, CL-M is wildtype, and CL-IM is a strain with deletion of the gene that controls the expression of flagella. We extracted the total RNA of these 3 strains and synthesized the 1st cDNA using RT-PCR. Then we did PCR of gene cheZ with the cDNA as template and we also did PCR of 16 sDNA as control. We got the figure through agarose gel electrophoresis (Fig. 3-5-4A). We can see that CL-1 is actually the strain with deletion of gene cheZ, while the other two strains have normal gene cheZ.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/f/f3/Fig_3-5-3.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-4</strong> Verification of gene cheZ. (A) In genetic level, CL-1 is actually the strain with deletion of gene cheZ, while the other two strains have normal gene cheZ. (B) In phenotype level, CL-M and pcheZ is able to migrate on semisolid culture medium, while CL-1 and CL-M is unable to migrate.</div><br><br><br />
Then we needed to verify of gene cheZ from phenotype. We have 3 strains, namely CL-1, CL-M, and CL-IM. CL-1 is a strain with deletion of gene cheZ, CL-M is wild type, and CL-IM is a strain with deletion of the gene that controls the expression of flagella. Then we transformed a plasmid with gene cheZ into CL-1 and got the fourth strain pcheZ.<br><br><br />
We inoculated the suspension of bacteria on semisolid LB plate and incubated for 20h and took the photograph (Fig. 3-5-4B). From the figure, we can see that CL-M and pcheZ is able to migrate on semisolid culture medium, while CL-1 and CL-M is unable to migrate. This indicates that E.coli is nonmotile without gene cheZ or flagella and inducing the expression of gene cheZ can restore motility in a cheZ knockout strain.<br><br><br />
Finally, we also verified lux system. We constructed a strain with gene circuit as shown in Fig. 3-5-5A from CL-1. This is a lux system without the ribosome switch. AHL synthesized by LuxI excretes as a signal and accumulates intracellularly to activate a constitutively expressed regulator, LuxR. Then the LuxR-AHL complex promotes the expression of a repressor CI, leading to repression of CheZ. So bacteria will assembly at spots where initial density is high in this system. After we inoculated the suspension of bacteria on semisolid LB plate and incubated for 20h, we took the photograph of a stripe pattern (Fig. 3-5-5B), which was formed under the influence of the lux system<sup>[4]</sup>.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/d/db/Fig_3-5-4_new.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-5</strong> Verification of lux system. (A) the gene circuit we constructed. (B) The phenotype of lux system. </div><br/><br />
For more information of our assembly system, please refer to <a href="https://2013.igem.org/Team:Nanjing-China/model"><strong>Model</strong></a>.<br><br />
<br/> <br />
<br />
<strong> Reference</strong><br/><br />
[1] Topp S and Gallivan JP. Guiding bacteria with small molecules and RNA. Journal of the American Chemical Society, 2007, 129 (21): 6807-6811.<br/><br />
[2] Carter KK, et al. Pathway engineering via quorum sensing and sRNA riboregulators-Interconnected networks and controllers. Metabolic Engineering, 2012, 14 (3): 281-288.<br/><br />
[3] Sinha J, et al. Reprogramming bacteria to seek and destroy a herbicide. Nature chemical biology, 2010, 6 (6): 464-470.<br/><br />
[4] Liu C, et al. Sequential establishment of stripe patterns in an expanding cell population. Science, 2011, 334 (6053): 238-241.<br/><br />
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In our project, we considered devising our bacteria into some kind of automatic machinery that would be able to sense a target molecule and then assemble to degrade it. To fulfill all these functions, we were bound to design some gene circuits to guide our bacteria.<br><br><br />
<br />
First of all, we should have an understanding of how bacteria move<sup>[1]</sup>. For E.coli, they have flagellar motors to control their movement (Fig. 3-5-1). The direction of rotation of their flagellar motors is controlled by the protein CheY. When CheY is not phosphorylated, the flagellar motors rotate counterclockwise (CCW) and bacteria can migrate. When CheY is phosphorylated (CheY-P), it can bind to the flagellar motorprotein FliM, causing the cell to tumble and thus nonmotile. Phosphatase CheZ dephosphorylates CheY-P and causes the flagellum to rotate CCW. E. coli lacking the cheZ gene (ΔcheZ) cannot dephosphorylate CheY-P, and thus tumble incessantly, being nonmotile. So we only need to take control of gene cheZ to control the motility of our bacteria. <br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/0/04/Fig.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-1</strong> Illustration of how bacteria move. CheZ is the key of controlling the motility of bacteria. For wild type, CheZ can be expressed normally, so the flagellar motor alternates between CCW and CW rotation, thus the cell can run or tumble. On the contrary, if there is no CheZ, CheY will remain phosphorylated, causing the flagellar motor rotates CW, thus the cell tumbles exclusively. (Cite by JACS, Shana Toop et al, 2007.)</div><br><br><br />
We would like our bacteria to seek and destroy atrazine in the soil. To achieve this goal, a classic system can do the job (Fig. 3-5-2A). In this system, bacteria can move around randomly but fix when sensing atrazine as the ribosome switch turns on and protein CI is expressed to repress the gene cheZ. As a result, our bacteria would just assemble around the target molecules after a period of time (Fig. 3-5-3).<br><br><br />
<p style="text-align:center"><img src="https://2013.igem.org/File:3-4%EF%BC%88%E5%A4%87%E7%94%A8%EF%BC%89.jpg"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-3</strong> Our bacteria could just assemble around the target molecules after a period of time.</div><br><br><br />
However, we can only wait for the bacteria to hang about until they find their targets. Thus it could take a long time before the bacteria assembly and the system may not be a robust design. So we shall spare no efforts to improve it. For better control over the function of our bacteria, we took the quorum sensing system into consideration. We expect some bacteria which first find the atrazine will recruit other bacteria at a distance through some signal so they move towards the target and then fixed at the source of pollution. Therefore we constructed the following gene circuits to realize it (Fig. 3-5-2B).<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/6/63/Fig_3-5-2.jpg" width="716"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-2</strong> The gene circuits we built to control cheZ. (A) A classic system using CI to repress cheZ. (B) A complex but high-efficiency system using quorum sensing system. (C) A simplified system like the system in (B).</div><br><br><br />
This circuit is composed of three modules: an atrazine-sensing module, a recruiting module, and a brake module. We adopted the quorum-sensing system in Vibrio fischerias the basis for our design. In this system, a small-molecule acyl-homoserine lactone (AHL) synthesized by LuxI excretes as a signal and accumulates intracellularly to activate a constitutively expressed regulator, LuxR<sup>[2]</sup>. Our atrazine-sensing module controls the translation of LuxI through ribosome switch which turns on when combined to atrazine. Meanwhile, the recruiting module is composed of a series of elements. In this system, the LuxR-AHL complex promotes the expression of a repressor TetR, which represses the expression of another repressor CI. The gene cheZ is controlled by promoter Pλ, which is repressed by CI. So for those bacteria which first sense atrazine, they send out AHL signal and let the other bacteria receive it. After that, TetR is expressed to repress CI, leading the transcription of cheZ. These bacteria move towards the sender until they sense atrazine and another repressor CI, which is controlled by ribosome switch, expresses as a brake of the movement<sup>[3]</sup>. <br><br><br />
We also design the third gene circuit to simplify the system (Fig. 3-5-2C). This system also consists of the three modules in Pattern 2 and the atrazine-sensing module, as well as the brake module, is exactly the same composition with those two parts. The only difference is in the recruiting module that we use a double-functional promoter Plux/CI. The atrazine-sensing module works just the same way in Pattern 2. In this system, however, the LuxR-AHL complex promotes cheZ transcription via promoter Plux/CI. At the spot where bacteria sense atrazine, AHL will accumulate. The higher AHL concentration is, the stronger transcription of cheZ which means greater motility is, leading to directional movement towards bacteria which send out the signal. Meanwhile, the brake module also functions via promoter Plux/CI. The repressor CI, controlled by ribosome switch which turns on when combined to atrazine, represses the promoter Plux/CI and results in bacteria fixation at source of pollution.<br><br><br />
<strong>Experiments and Results</strong><br><br />
At the beginning, we verified gene cheZ in genetic level. We had 3 strains, namely CL-1, CL-M, and CL-IM. CL-1 is a strain with deletion of gene cheZ, CL-M is wildtype, and CL-IM is a strain with deletion of the gene that controls the expression of flagella. We extracted the total RNA of these 3 strains and synthesized the 1st cDNA using RT-PCR. Then we did PCR of gene cheZ with the cDNA as template and we also did PCR of 16 sDNA as control. We got the figure through agarose gel electrophoresis (Fig. 3-5-4A). We can see that CL-1 is actually the strain with deletion of gene cheZ, while the other two strains have normal gene cheZ.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/f/f3/Fig_3-5-3.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-4</strong> Verification of gene cheZ. (A) In genetic level, CL-1 is actually the strain with deletion of gene cheZ, while the other two strains have normal gene cheZ. (B) In phenotype level, CL-M and pcheZ is able to migrate on semisolid culture medium, while CL-1 and CL-M is unable to migrate.</div><br><br><br />
Then we needed to verify of gene cheZ from phenotype. We have 3 strains, namely CL-1, CL-M, and CL-IM. CL-1 is a strain with deletion of gene cheZ, CL-M is wild type, and CL-IM is a strain with deletion of the gene that controls the expression of flagella. Then we transformed a plasmid with gene cheZ into CL-1 and got the fourth strain pcheZ.<br><br><br />
We inoculated the suspension of bacteria on semisolid LB plate and incubated for 20h and took the photograph (Fig. 3-5-4B). From the figure, we can see that CL-M and pcheZ is able to migrate on semisolid culture medium, while CL-1 and CL-M is unable to migrate. This indicates that E.coli is nonmotile without gene cheZ or flagella and inducing the expression of gene cheZ can restore motility in a cheZ knockout strain.<br><br><br />
Finally, we also verified lux system. We constructed a strain with gene circuit as shown in Fig. 3-5-5A from CL-1. This is a lux system without the ribosome switch. AHL synthesized by LuxI excretes as a signal and accumulates intracellularly to activate a constitutively expressed regulator, LuxR. Then the LuxR-AHL complex promotes the expression of a repressor CI, leading to repression of CheZ. So bacteria will assembly at spots where initial density is high in this system. After we inoculated the suspension of bacteria on semisolid LB plate and incubated for 20h, we took the photograph of a stripe pattern (Fig. 3-5-5B), which was formed under the influence of the lux system<sup>[4]</sup>.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/d/db/Fig_3-5-4_new.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-5</strong> Verification of lux system. (A) the gene circuit we constructed. (B) The phenotype of lux system. </div><br/><br />
For more information of our assembly system, please refer to <a href="https://2013.igem.org/Team:Nanjing-China/model"><strong>Model</strong></a>.<br><br />
<br/> <br />
<br />
<strong> Reference</strong><br/><br />
[1] Topp S and Gallivan JP. Guiding bacteria with small molecules and RNA. Journal of the American Chemical Society, 2007, 129 (21): 6807-6811.<br/><br />
[2] Carter KK, et al. Pathway engineering via quorum sensing and sRNA riboregulators-Interconnected networks and controllers. Metabolic Engineering, 2012, 14 (3): 281-288.<br/><br />
[3] Sinha J, et al. Reprogramming bacteria to seek and destroy a herbicide. Nature chemical biology, 2010, 6 (6): 464-470.<br/><br />
[4] Liu C, et al. Sequential establishment of stripe patterns in an expanding cell population. Science, 2011, 334 (6053): 238-241.<br/><br />
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</html></div>GaoJian NJUhttp://2013.igem.org/File:3-4%EF%BC%88%E5%A4%87%E7%94%A8%EF%BC%89.jpgFile:3-4(备用).jpg2013-10-27T03:11:38Z<p>GaoJian NJU: </p>
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<dt><a href="###">Recognition</a></dt><br />
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Ribosome switch is used widely in synthetic biology to detect various small molecules. This summer, we chose an efficient ribosome switch created via a new method to detect atrazine <sup>[1]</sup>. And then, our team verified the basic parameters about this amazing ribosome switch, including the lowest detect concentration and the induced time.<br><br><br />
<strong>Experiments and Results</strong><br><br />
Our team used this kind of ribosome switch which can detect atrazine to control the GFP expression in E.coli. E.coli expressed GFP after 20 hours under 500μM of atrazine, but could not express GFP without atrazine (Fig. 3-4-1). This shows the ribosome switch can be turned on under 500μM of atrazine.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/7/7a/Fig_3-4-1.jpg" width="600"></p><br/><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-4-1</strong> The ribosome switch exhibits conformations and function. (A) The principle of how the ribosome switch works. (B) Ribosome switch is induced by atrazine under 500μM of atrazine.</div><br><br />
<br />
Our team used this part to control GFP expression in E.coli. We found all E.coli had expressed GFP under all the concentrations of atrazine, from 1μM to 500μM, in our experiment (Fig. 3-4-2). This data indicates that the ribosome switch can work under 1μM of atrazine.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/0/04/Fig_3-4-2.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-4-2</strong> Various concentrations of atrazine were detected after 20h culturing. We detect the expression of GFP after the strain K-12 is cultured for 20h in the different concentrations from 1μM to 500μM.</div><br/><br/><br />
Our team used this part to control the GFP expression in E.coli. We found E.coli had merely expressed GFP after 12 hours under 200μM of atrazine (Fig. 3-4-3).. This data elucidates that the ribosome switch can work after 12 hours under 200μM of atrazine.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/7/7d/Fig_3-4-3.jpg" width="600"></p><br/><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-4-3</strong> The ribosome switch was induced at different time under 200μM of atrazine. We detect the expression of GFP after that the strain K-12 is cultured under 200μM of atrazine at the different time from 6h to 12h.</div><br><br>According to these data, this ribosome switch is more efficient than the one used before. <br><br><br />
However, for the reason of fluorescence quenching, the fluorescence intensity would descending for too high concentration of GFP. So we detected the RFU changing with time and atrazine’s concentration and identified the range of concentration of atrazine (Fig. 3-4-4). Therefore, we are confident of the fact that we can put this amazing part into detecting atrazine in real life. <br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/2/2f/3-4-4.jpg"></p><br/><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-4-4</strong> The RFU changing with time and atrazine’s concentration.</div><br/><br/><br />
<strong>Reference</strong><br/><br />
[1] Sinha J, et al. Reprogramming bacteria to seek and destroy a herbicide. Nature chemical biology, 2010, 6 (6): 464-470. <br />
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<p>Atrazine, a widely used herbicide, persists for a long period in the environment once used. It causes metabolic disorders in both animals and humankind. Our team utilized the ribosome switch induced by atrazine, a QS system of Plux and a degrading enzyme to control E.coil's motility through regulating its CheZ gene. Therefore, E.coli can recognize atrazine, recruit team workers, and degrade atrazine. Our team found a transporter of atrazine, which we call TRM. We also mutated the degrading enzyme, TrzN, making it better at degradation. We combined TRM and the TrzN to improve atrazine absorbance and degradation. Moreover, our team are trying to analyze ... <a href="https://2013.igem.org/Team:Nanjing-China/abstract">more</a></p><br />
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<p>Atrazine, a widely used herbicide, persists for a long period in the environment once used. It causes metabolic disorders in both animals and humankind. Our team utilized the ribosome switch induced by atrazine, a QS system of Plux and a degrading enzyme to control E.coil's motility through regulating its CheZ gene. Therefore, E.coli can recognize atrazine, recruit team workers, and degrade atrazine. Our team found a transporter of atrazine, which we call TRM. We also mutated the degrading enzyme, TrzN, making it better at degradation. We combined TRM and the TrzN to improve atrazine absorbance and degradation. Moreover, our team are trying to analyze ... <a href="https://2013.igem.org/Team:Nanjing-China/abstract">more</a></p><br />
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<dt><a href="###">Aggregation</a></dt><br />
<dd class="dd_1"><br />
In our project, we considered devising our bacteria into some kind of automatic machinery that would be able to sense a target molecule and then assemble to degrade it. To fulfill all these functions, we were bound to design some gene circuits to guide our bacteria.<br><br><br />
<br />
First of all, we should have an understanding of how bacteria move<sup>[1]</sup>. For E.coli, they have flagellar motors to control their movement (Fig. 3-5-1). The direction of rotation of their flagellar motors is controlled by the protein CheY. When CheY is not phosphorylated, the flagellar motors rotate counterclockwise (CCW) and bacteria can migrate. When CheY is phosphorylated (CheY-P), it can bind to the flagellar motorprotein FliM, causing the cell to tumble and thus nonmotile. Phosphatase CheZ dephosphorylates CheY-P and causes the flagellum to rotate CCW. E. coli lacking the cheZ gene (ΔcheZ) cannot dephosphorylate CheY-P, and thus tumble incessantly, being nonmotile. So we only need to take control of gene cheZ to control the motility of our bacteria. <br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/0/04/Fig.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-1</strong> Illustration of how bacteria move. CheZ is the key of controlling the motility of bacteria. For wild type, CheZ can be expressed normally, so the flagellar motor alternates between CCW and CW rotation, thus the cell can run or tumble. On the contrary, if there is no CheZ, CheY will remain phosphorylated, causing the flagellar motor rotates CW, thus the cell tumbles exclusively. (Cite by JACS, Shana Toop et al, 2007.)</div><br><br><br />
We would like our bacteria to seek and destroy atrazine in the soil. To achieve this goal, a classic system can do the job (Fig. 3-5-2A). In this system, bacteria can move around randomly but fix when sensing atrazine as the ribosome switch turns on and protein CI is expressed to repress the gene cheZ. As a result, our bacteria will just assemble around the target molecules after a period of time.<br><br><br />
However, we can only wait for the bacteria to hang about until they find their targets. Thus it could take a long time before the bacteria assembly and the system may not be a robust design. So we shall spare no efforts to improve it. For better control over the function of our bacteria, we took the quorum sensing system into consideration. We expect some bacteria which first find the atrazine will recruit other bacteria at a distance through some signal so they move towards the target and then fixed at the source of pollution. Therefore we constructed the following gene circuits to realize it (Fig. 3-5-2B).<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/6/63/Fig_3-5-2.jpg" width="716"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-2</strong> The gene circuits we built to control cheZ. (A) A classic system using CI to repress cheZ. (B) A complex but high-efficiency system using quorum sensing system. (C) A simplified system like the system in (B).</div><br><br><br />
This circuit is composed of three modules: an atrazine-sensing module, a recruiting module, and a brake module. We adopted the quorum-sensing system in Vibrio fischerias the basis for our design. In this system, a small-molecule acyl-homoserine lactone (AHL) synthesized by LuxI excretes as a signal and accumulates intracellularly to activate a constitutively expressed regulator, LuxR<sup>[2]</sup>. Our atrazine-sensing module controls the translation of LuxI through ribosome switch which turns on when combined to atrazine. Meanwhile, the recruiting module is composed of a series of elements. In this system, the LuxR-AHL complex promotes the expression of a repressor TetR, which represses the expression of another repressor CI. The gene cheZ is controlled by promoter Pλ, which is repressed by CI. So for those bacteria which first sense atrazine, they send out AHL signal and let the other bacteria receive it. After that, TetR is expressed to repress CI, leading the transcription of cheZ. These bacteria move towards the sender until they sense atrazine and another repressor CI, which is controlled by ribosome switch, expresses as a brake of the movement<sup>[3]</sup>. <br><br><br />
We also design the third gene circuit to simplify the system (Fig. 3-5-2C). This system also consists of the three modules in Pattern 2 and the atrazine-sensing module, as well as the brake module, is exactly the same composition with those two parts. The only difference is in the recruiting module that we use a double-functional promoter Plux/CI. The atrazine-sensing module works just the same way in Pattern 2. In this system, however, the LuxR-AHL complex promotes cheZ transcription via promoter Plux/CI. At the spot where bacteria sense atrazine, AHL will accumulate. The higher AHL concentration is, the stronger transcription of cheZ which means greater motility is, leading to directional movement towards bacteria which send out the signal. Meanwhile, the brake module also functions via promoter Plux/CI. The repressor CI, controlled by ribosome switch which turns on when combined to atrazine, represses the promoter Plux/CI and results in bacteria fixation at source of pollution.<br><br><br />
<strong>Experiments and Results</strong><br><br />
At the beginning, we verified gene cheZ in genetic level. We had 3 strains, namely CL-1, CL-M, and CL-IM. CL-1 is a strain with deletion of gene cheZ, CL-M is wildtype, and CL-IM is a strain with deletion of the gene that controls the expression of flagella. We extracted the total RNA of these 3 strains and synthesized the 1st cDNA using RT-PCR. Then we did PCR of gene cheZ with the cDNA as template and we also did PCR of 16 sDNA as control. We got the figure through agarose gel electrophoresis (Fig. 3-5-3A). We can see that CL-1 is actually the strain with deletion of gene cheZ, while the other two strains have normal gene cheZ.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/f/f3/Fig_3-5-3.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-3</strong> Verification of gene cheZ. (A) In genetic level, CL-1 is actually the strain with deletion of gene cheZ, while the other two strains have normal gene cheZ. (B) In phenotype level, CL-M and pcheZ is able to migrate on semisolid culture medium, while CL-1 and CL-M is unable to migrate.</div><br><br><br />
Then we needed to verify of gene cheZ from phenotype. We have 3 strains, namely CL-1, CL-M, and CL-IM. CL-1 is a strain with deletion of gene cheZ, CL-M is wild type, and CL-IM is a strain with deletion of the gene that controls the expression of flagella. Then we transformed a plasmid with gene cheZ into CL-1 and got the fourth strain pcheZ.<br><br><br />
We inoculated the suspension of bacteria on semisolid LB plate and incubated for 20h and took the photograph (Fig. 3-5-3B). From the figure, we can see that CL-M and pcheZ is able to migrate on semisolid culture medium, while CL-1 and CL-M is unable to migrate. This indicates that E.coli is nonmotile without gene cheZ or flagella and inducing the expression of gene cheZ can restore motility in a cheZ knockout strain.<br><br><br />
Finally, we also verified lux system. We constructed a strain with gene circuit as shown in Fig. 3-5-4A from CL-1. This is a lux system without the ribosome switch. AHL synthesized by LuxI excretes as a signal and accumulates intracellularly to activate a constitutively expressed regulator, LuxR. Then the LuxR-AHL complex promotes the expression of a repressor CI, leading to repression of CheZ. So bacteria will assembly at spots where initial density is high in this system. After we inoculated the suspension of bacteria on semisolid LB plate and incubated for 20h, we took the photograph of a stripe pattern (Fig. 3-5-4B), which was formed under the influence of the lux system<sup>[4]</sup>.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/d/db/Fig_3-5-4_new.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-4</strong> Verification of lux system. (A) the gene circuit we constructed. (B) The phenotype of lux system. </div><br/><br />
For more information of our assembly system, please refer to <a href="https://2013.igem.org/Team:Nanjing-China/model"><strong>Model</strong></a>.<br><br />
<br/> <br />
<br />
<strong> Reference</strong><br/><br />
[1] Topp S and Gallivan JP. Guiding bacteria with small molecules and RNA. Journal of the American Chemical Society, 2007, 129 (21): 6807-6811.<br/><br />
[2] Carter KK, et al. Pathway engineering via quorum sensing and sRNA riboregulators-Interconnected networks and controllers. Metabolic Engineering, 2012, 14 (3): 281-288.<br/><br />
[3] Sinha J, et al. Reprogramming bacteria to seek and destroy a herbicide. Nature chemical biology, 2010, 6 (6): 464-470.<br/><br />
[4] Liu C, et al. Sequential establishment of stripe patterns in an expanding cell population. Science, 2011, 334 (6053): 238-241.<br/><br />
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</html></div>GaoJian NJUhttp://2013.igem.org/Team:Nanjing-China/qsTeam:Nanjing-China/qs2013-09-28T03:57:22Z<p>GaoJian NJU: </p>
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In our project, we considered devising our bacteria into some kind of automatic machinery that would be able to sense a target molecule and then assemble to degrade it. To fulfill all these functions, we were bound to design some gene circuits to guide our bacteria.<br><br><br />
<br />
First of all, we should have an understanding of how bacteria move<sup>[1]</sup>. For E.coli, they have flagellar motors to control their movement (Fig. 3-5-1). The direction of rotation of their flagellar motors is controlled by the protein CheY. When CheY is not phosphorylated, the flagellar motors rotate counterclockwise (CCW) and bacteria can migrate. When CheY is phosphorylated (CheY-P), it can bind to the flagellar motorprotein FliM, causing the cell to tumble and thus nonmotile. Phosphatase CheZ dephosphorylates CheY-P and causes the flagellum to rotate CCW. E. coli lacking the cheZ gene (ΔcheZ) cannot dephosphorylate CheY-P, and thus tumble incessantly, being nonmotile. So we only need to take control of gene cheZ to control the motility of our bacteria. <br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/0/04/Fig.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-1</strong> Illustration of how bacteria move. CheZ is the key of controlling the motility of bacteria. For wild type, CheZ can be expressed normally, so the flagellar motor alternates between CCW and CW rotation, thus the cell can run or tumble. On the contrary, if there is no CheZ, CheY will remain phosphorylated, causing the flagellar motor rotates CW, thus the cell tumbles exclusively.(Cite by JACS, Shana Toop et al, 2007)</div><br><br><br />
We would like our bacteria to seek and destroy atrazine in the soil. To achieve this goal, a classic system can do the job (Fig. 3-5-2A). In this system, bacteria can move around randomly but fix when sensing atrazine as the ribosome switch turns on and protein CI is expressed to repress the gene cheZ. As a result, our bacteria will just assemble around the target molecules after a period of time.<br><br><br />
However, we can only wait for the bacteria to hang about until they find their targets. Thus it could take a long time before the bacteria assembly and the system may not be a robust design. So we shall spare no efforts to improve it. For better control over the function of our bacteria, we took the quorum sensing system into consideration. We expect some bacteria which first find the atrazine will recruit other bacteria at a distance through some signal so they move towards the target and then fixed at the source of pollution. Therefore we constructed the following gene circuits to realize it (Fig. 3-5-2B).<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/6/63/Fig_3-5-2.jpg" width="716"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-2</strong> The gene circuits we built to control cheZ. (A) A classic system using CI to repress cheZ. (B) A complex but high-efficiency system using quorum sensing system. (C) A simplified system like the system in (B).</div><br><br><br />
This circuit is composed of three modules: an atrazine-sensing module, a recruiting module, and a brake module. We adopted the quorum-sensing system in Vibrio fischerias the basis for our design. In this system, a small-molecule acyl-homoserine lactone (AHL) synthesized by LuxI excretes as a signal and accumulates intracellularly to activate a constitutively expressed regulator, LuxR<sup>[2]</sup>. Our atrazine-sensing module controls the translation of LuxI through ribosome switch which turns on when combined to atrazine. Meanwhile, the recruiting module is composed of a series of elements. In this system, the LuxR-AHL complex promotes the expression of a repressor TetR, which represses the expression of another repressor CI. The gene cheZ is controlled by promoter Pλ, which is repressed by CI. So for those bacteria which first sense atrazine, they send out AHL signal and let the other bacteria receive it. After that, TetR is expressed to repress CI, leading the transcription of cheZ. These bacteria move towards the sender until they sense atrazine and another repressor CI, which is controlled by ribosome switch, expresses as a brake of the movement<sup>[3]</sup>. <br><br><br />
We also design the third gene circuit to simplify the system (Fig. 3-5-2C). This system also consists of the three modules in Pattern 2 and the atrazine-sensing module, as well as the brake module, is exactly the same composition with those two parts. The only difference is in the recruiting module that we use a double-functional promoter Plux/CI. The atrazine-sensing module works just the same way in Pattern 2. In this system, however, the LuxR-AHL complex promotes cheZ transcription via promoter Plux/CI. At the spot where bacteria sense atrazine, AHL will accumulate. The higher AHL concentration is, the stronger transcription of cheZ which means greater motility is, leading to directional movement towards bacteria which send out the signal. Meanwhile, the brake module also functions via promoter Plux/CI. The repressor CI, controlled by ribosome switch which turns on when combined to atrazine, represses the promoter Plux/CI and results in bacteria fixation at source of pollution.<br><br><br />
<strong>Experiments and Results</strong><br><br />
At the beginning, we verified gene cheZ in genetic level. We had 3 strains, namely CL-1, CL-M, and CL-IM. CL-1 is a strain with deletion of gene cheZ, CL-M is wildtype, and CL-IM is a strain with deletion of the gene that controls the expression of flagella. We extracted the total RNA of these 3 strains and synthesized the 1st cDNA using RT-PCR. Then we did PCR of gene cheZ with the cDNA as template and we also did PCR of 16 sDNA as control. We got the figure through agarose gel electrophoresis (Fig. 3-5-3A). We can see that CL-1 is actually the strain with deletion of gene cheZ, while the other two strains have normal gene cheZ.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/f/f3/Fig_3-5-3.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-3</strong> Verification of gene cheZ. (A) In genetic level, CL-1 is actually the strain with deletion of gene cheZ, while the other two strains have normal gene cheZ. (B) In phenotype level, CL-M and pcheZ is able to migrate on semisolid culture medium, while CL-1 and CL-M is unable to migrate.</div><br><br><br />
Then we needed to verify of gene cheZ from phenotype. We have 3 strains, namely CL-1, CL-M, and CL-IM. CL-1 is a strain with deletion of gene cheZ, CL-M is wild type, and CL-IM is a strain with deletion of the gene that controls the expression of flagella. Then we transformed a plasmid with gene cheZ into CL-1 and got the fourth strain pcheZ.<br><br><br />
We inoculated the suspension of bacteria on semisolid LB plate and incubated for 20h and took the photograph (Fig. 3-5-3B). From the figure, we can see that CL-M and pcheZ is able to migrate on semisolid culture medium, while CL-1 and CL-M is unable to migrate. This indicates that E.coli is nonmotile without gene cheZ or flagella and inducing the expression of gene cheZ can restore motility in a cheZ knockout strain.<br><br><br />
Finally, we also verified lux system. We constructed a strain with gene circuit as shown in Fig. 3-5-4A from CL-1. This is a lux system without the ribosome switch. AHL synthesized by LuxI excretes as a signal and accumulates intracellularly to activate a constitutively expressed regulator, LuxR. Then the LuxR-AHL complex promotes the expression of a repressor CI, leading to repression of CheZ. So bacteria will assembly at spots where initial density is high in this system. After we inoculated the suspension of bacteria on semisolid LB plate and incubated for 20h, we took the photograph of a stripe pattern (Fig. 3-5-4B), which was formed under the influence of the lux system<sup>[4]</sup>.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/d/db/Fig_3-5-4_new.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-4</strong> Verification of lux system. (A) the gene circuit we constructed. (B) The phenotype of lux system. </div><br/><br />
For more information of our assembly system, please refer to <a href="https://2013.igem.org/Team:Nanjing-China/model"><strong>Model</strong></a>.<br><br />
<br/> <br />
<br />
<strong> Reference</strong><br/><br />
[1] Topp S and Gallivan JP. Guiding bacteria with small molecules and RNA. Journal of the American Chemical Society, 2007, 129 (21): 6807-6811.<br/><br />
[2] Carter KK, et al. Pathway engineering via quorum sensing and sRNA riboregulators-Interconnected networks and controllers. Metabolic Engineering, 2012, 14 (3): 281-288.<br/><br />
[3] Sinha J, et al. Reprogramming bacteria to seek and destroy a herbicide. Nature chemical biology, 2010, 6 (6): 464-470.<br/><br />
[4] Liu C, et al. Sequential establishment of stripe patterns in an expanding cell population. Science, 2011, 334 (6053): 238-241.<br/><br />
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<dt><a href="###">Aggregation</a></dt><br />
<dd class="dd_1"><br />
In our project, we considered devising our bacteria into some kind of automatic machinery that would be able to sense a target molecule and then assemble to degrade it. To fulfill all these functions, we were bound to design some gene circuits to guide our bacteria.<br><br><br />
<br />
First of all, we should have an understanding of how bacteria move<sup>[1]</sup>. For E.coli, they have flagellar motors to control their movement (Fig. 3-5-1). The direction of rotation of their flagellar motors is controlled by the protein CheY. When CheY is not phosphorylated, the flagellar motors rotate counterclockwise (CCW) and bacteria can migrate. When CheY is phosphorylated (CheY-P), it can bind to the flagellar motorprotein FliM, causing the cell to tumble and thus nonmotile. Phosphatase CheZ dephosphorylates CheY-P and causes the flagellum to rotate CCW. E. coli lacking the cheZ gene (ΔcheZ) cannot dephosphorylate CheY-P, and thus tumble incessantly, being nonmotile. So we only need to take control of gene cheZ to control the motility of our bacteria. <br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/0/04/Fig.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-1</strong> Illustration of how bacteria move. CheZ is the key of controlling the motility of bacteria. For wild type, CheZ can be expressed normally, so the flagellar motor alternates between CCW and CW rotation, thus the cell can run or tumble. On the contrary, if there is no CheZ, CheY will remain phosphorylated, causing the flagellar motor rotates CW, thus the cell tumbles exclusively.(Cite by JACS, Shana Toop et al, 2007)</div><br><br><br />
We would like our bacteria to seek and destroy atrazine in the soil. To achieve this goal, a classic system can do the job (Fig. 3-5-2A). In this system, bacteria can move around randomly but fix when sensing atrazine as the ribosome switch turns on and protein CI is expressed to repress the gene cheZ. As a result, our bacteria will just assemble around the target molecules after a period of time.<br><br><br />
However, we can only wait for the bacteria to hang about until they find their targets. Thus it could take a long time before the bacteria assembly and the system may not be a robust design. So we shall spare no efforts to improve it. For better control over the function of our bacteria, we took the quorum sensing system into consideration. We expect some bacteria which first find the atrazine will recruit other bacteria at a distance through some signal so they move towards the target and then fixed at the source of pollution. Therefore we constructed the following gene circuits to realize it (Fig. 3-5-2B).<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/6/63/Fig_3-5-2.jpg" width="716"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-2</strong> The gene circuits we built to control cheZ. (A) A classic system using CI to repress cheZ. (B) A complex but high-efficiency system using quorum sensing system. (C) A simplified system like the system in (B).</div><br><br><br />
This circuit is composed of three modules: an atrazine-sensing module, a recruiting module, and a brake module. We adopted the quorum-sensing system in Vibrio fischerias the basis for our design. In this system, a small-molecule acyl-homoserine lactone (AHL) synthesized by LuxI excretes as a signal and accumulates intracellularly to activate a constitutively expressed regulator, LuxR<sup>[2]</sup>. Our atrazine-sensing module controls the translation of LuxI through ribosome switch which turns on when combined to atrazine. Meanwhile, the recruiting module is composed of a series of elements. In this system, the LuxR-AHL complex promotes the expression of a repressor TetR, which represses the expression of another repressor CI. The gene cheZ is controlled by promoter Pλ, which is repressed by CI. So for those bacteria which first sense atrazine, they send out AHL signal and let the other bacteria receive it. After that, TetR is expressed to repress CI, leading the transcription of cheZ. These bacteria move towards the sender until they sense atrazine and another repressor CI, which is controlled by ribosome switch, expresses as a brake of the movement<sup>[3]</sup>. <br><br><br />
We also design the third gene circuit to simplify the system (Fig. 3-5-2C). This system also consists of the three modules in Pattern 2 and the atrazine-sensing module, as well as the brake module, is exactly the same composition with those two parts. The only difference is in the recruiting module that we use a double-functional promoter Plux/CI. The atrazine-sensing module works just the same way in Pattern 2. In this system, however, the LuxR-AHL complex promotes cheZ transcription via promoter Plux/CI. At the spot where bacteria sense atrazine, AHL will accumulate. The higher AHL concentration is, the stronger transcription of cheZ which means greater motility is, leading to directional movement towards bacteria which send out the signal. Meanwhile, the brake module also functions via promoter Plux/CI. The repressor CI, controlled by ribosome switch which turns on when combined to atrazine, represses the promoter Plux/CI and results in bacteria fixation at source of pollution.<br><br><br />
<strong>Experiments and Results</strong><br><br />
At the beginning, we verified gene cheZ in genetic level. We had 3 strains, namely CL-1, CL-M, and CL-IM. CL-1 is a strain with deletion of gene cheZ, CL-M is wildtype, and CL-IM is a strain with deletion of the gene that controls the expression of flagella. We extracted the total RNA of these 3 strains and synthesized the 1st cDNA using RT-PCR. Then we did PCR of gene cheZ with the cDNA as template and we also did PCR of 16 sDNA as control. We got the figure through agarose gel electrophoresis (Fig. 3-5-3A). We can see that CL-1 is actually the strain with deletion of gene cheZ, while the other two strains have normal gene cheZ.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/f/f3/Fig_3-5-3.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-3</strong> The verification of gene cheZ. (A) In genetic level, CL-1 is actually the strain with deletion of gene cheZ, while the other two strains have normal gene cheZ. (B) In phenotype level, CL-M and pcheZ is able to migrate on semisolid culture medium, while CL-1 and CL-M is unable to migrate.</div><br><br><br />
Then we needed to verify of gene cheZ from phenotype. We have 3 strains, namely CL-1, CL-M, and CL-IM. CL-1 is a strain with deletion of gene cheZ, CL-M is wild type, and CL-IM is a strain with deletion of the gene that controls the expression of flagella. Then we transformed a plasmid with gene cheZ into CL-1 and got the fourth strain pcheZ.<br><br><br />
We inoculated the suspension of bacteria on semisolid LB plate and incubated for 20h and took the photograph (Fig. 3-5-3B). From the figure, we can see that CL-M and pcheZ is able to migrate on semisolid culture medium, while CL-1 and CL-M is unable to migrate. This indicates that E.coli is nonmotile without gene cheZ or flagella and inducing the expression of gene cheZ can restore motility in a cheZ knockout strain.<br><br><br />
Finally, we also verified lux system. We constructed a strain with gene circuit as shown in Fig. 3-5-4A from CL-1. This is a lux system without the ribosome switch. AHL synthesized by LuxI excretes as a signal and accumulates intracellularly to activate a constitutively expressed regulator, LuxR. Then the LuxR-AHL complex promotes the expression of a repressor CI, leading to repression of CheZ. So bacteria will assembly at spots where initial density is high in this system. After we inoculated the suspension of bacteria on semisolid LB plate and incubated for 20h, we took the photograph of a stripe pattern (Fig. 3-5-4B), which was formed under the influence of the lux system<sup>[4]</sup>.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/d/db/Fig_3-5-4_new.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-4</strong> The verification of lux system. (A) the gene circuit we constructed. (B) The phenotype of lux system. </div><br/><br />
For more information of our assembly system, please refer to <a href="https://2013.igem.org/Team:Nanjing-China/model"><strong>Model</strong></a>.<br><br />
<br/> <br />
<br />
<strong> Reference</strong><br/><br />
[1] Topp S and Gallivan JP. Guiding bacteria with small molecules and RNA. Journal of the American Chemical Society, 2007, 129 (21): 6807-6811.<br/><br />
[2] Carter KK, et al. Pathway engineering via quorum sensing and sRNA riboregulators-Interconnected networks and controllers. Metabolic Engineering, 2012, 14 (3): 281-288.<br/><br />
[3] Sinha J, et al. Reprogramming bacteria to seek and destroy a herbicide. Nature chemical biology, 2010, 6 (6): 464-470.<br/><br />
[4] Liu C, et al. Sequential establishment of stripe patterns in an expanding cell population. Science, 2011, 334 (6053): 238-241.<br/><br />
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<dt><img src="https://static.igem.org/mediawiki/2013/d/db/M1.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Mao Yafei</dd><br />
<dd class="dd_2">He is the team leader of Nanjing-China. He is in charge of arranging all team work, making sure that all team members collaborate well with each other. He also participated in all data analysis.</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/c/cd/M2.jpg" width="126" height="129"></dt><br />
<dd class="dd_1">Lv Kun</dd><br />
<dd class="dd_2">He is one of the core members of Nanjing-China. His work mainly lies in designing experiments to test the functions of our parts, TrzN and TRM. And this responsible guy also helped leader complete all the forms.</dd><br />
</dl><br />
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<dt><img src="https://static.igem.org/mediawiki/2013/7/77/M3.jpg" width="126" height="129"></dt><br />
<dd class="dd_1">Yang Jianchen</dd><br />
<dd class="dd_2">He is one of the core members of Nanjing-China.The main idea of this program mainly came from this intellectual guy. He is in charge of constructing our gene circuits and testing the functions of the systems.</dd><br />
</dl><br />
<dl class="member"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/3/3a/CaoZhipeng.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Cao Zhipeng</dd><br />
<dd class="dd_2">He is one of the core members of Nanjing-China. He is a good experimenter whose work mainly lies in constructing our gene circuits. This funnny guy always creates miracles for our team work.</dd><br />
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<dt><img src="https://static.igem.org/mediawiki/2013/4/49/M5.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Zhang Juyan</dd><br />
<dd class="dd_2">He is one of the core members of Nanjing-China. He spent a lot of time helping others, including experiments and website design. This diligent guy contributed a lot to our team. <br />
</dd><br />
</dl><br />
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<dt><img src="https://static.igem.org/mediawiki/2013/9/9e/6_a.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Pu Ruokun</dd><br />
<dd class="dd_2">He is the group leader of the modeling part of Nanjing-China. He completed the main model with help from Tsinghua-A. And he also helped us to collect the basic information of atrazine.</dd><br />
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<dt><img src="https://static.igem.org/mediawiki/2013/6/6c/M7.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Gao Jian</dd><br />
<dd class="dd_2">He is the group leader of the website part of Nanjing-China.He is in charge of working out the idea about website design. And this shutterbug prepared all the photos for our team.</dd><br />
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<dt><img src="https://static.igem.org/mediawiki/2013/0/07/M8.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Wang Yuanyuan</dd><br />
<dd class="dd_2">She is one of the main members of Nanjing-China. She assists others on the experiments, and she independently completed the growth curve drawing.</dd><br />
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<dt><img src="https://static.igem.org/mediawiki/2013/8/8d/M9.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Gao Jiajia</dd><br />
<dd class="dd_2">She is one of the core members of the website part of Nanjing-China. She helped us to complete the wiki and worked out the elegant video for our team.</dd><br />
</dl><br />
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<dt><img src="https://static.igem.org/mediawiki/2013/6/61/M10.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Xiao Shuke</dd><br />
<dd class="dd_2">He is one of the main members of Nanjing-China. He always helps us to complete the basic experiments. This cute guy is good at technology using. He also did some human practices with other team members.</dd><br />
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<dt><img src="https://static.igem.org/mediawiki/2013/5/57/11_a.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Li Yangyang</dd><br />
<dd class="dd_2">He is in charge of the part of human practice. And this clever guy majors in business so that he helped us to collect much information about atrazine, and calculated the value of our program in market.</dd><br />
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<dt><img src="https://static.igem.org/mediawiki/2013/b/b0/12_a.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Li Yezheng</dd><br />
<dd class="dd_2">He is one of the main members of the model part of Nanjing-China. He helped others model from the primary idea.</dd><br />
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<dt><img src="https://static.igem.org/mediawiki/2013/5/5f/13_a.jpg" width="116" height="129"></dt><br />
<dd class="dd_1">Zeng Mingzhi</dd><br />
<dd class="dd_2">He is one of the main members of the wiki design part of Nanjing-China. He worked out the perfect wiki with his brilliant expertise.</dd><br />
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<br />
<br />
<div class="team_bt"><a name="instructors">Instructors</a></div><br />
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<dt><a href="http://114.212.232.25/staff/?p=176"><img src="https://static.igem.org/mediawiki/2013/1/19/Inst_1.jpg" width="140" height="150"></a></dt><br />
<dd class="dd_1">Lan WenZhi</dd><br />
<dd class="dd_2">Professor<br/>Research: the function of iron channel; the mechanism of plant cell signaling</dd><br />
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<dd class="dd_1">Wang Bin</dd><br />
<dd class="dd_2">Associated professor <br/>Research: molecular genetics and evolution of mycorrhizae</dd><br />
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<dl class="instructors"><br />
<dt><a href="http://114.212.232.25/staff/?p=333"><img src="https://static.igem.org/mediawiki/2013/4/45/Inst_3.jpg" width="140" height="150"></a></dt><br />
<dd class="dd_1">Zheng WeiJuan</dd><br />
<dd class="dd_2">Professor<br>Research: the separation and purification of proteins; genetic engineering of peptide drug</dd><br />
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<br />
<br />
<div class="team_bt" style=" margin-top:20px;"><a name="acknowledgement">Acknowledgements</a></div><br />
<dl class="instructors"><br />
<dt><a href="http://114.212.232.25/staff/?p=170" target="_blank"><img src="https://static.igem.org/mediawiki/2013/f/f3/1.jpg" width="140" height="150"></a></dt><br />
<dd class="dd_1">Kong Lingdong</dd><br />
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<dl class="instructors"><br />
<dt><a href="http://gattaca.nju.edu.cn/sihai.html" target="_blank"><img src="https://static.igem.org/mediawiki/2013/b/bd/YangSihai.jpg" width="140" height="150"></a></dt><br />
<dd class="dd_1">Yang Sihai</dd><br />
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<dl class="instructors"><br />
<dt><img src="https://static.igem.org/mediawiki/2013/7/79/3.jpg" width="140" height="150"></dt><br />
<dd class="dd_1">Zeng Shuiyun</dd><br />
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<dl class="instructors"><br />
<dt><a href="https://2013.igem.org/Team:Tsinghua-A" target="_blank"><img src="https://static.igem.org/mediawiki/2013/f/f4/Tsinghua-A.jpg" width="140" height="150"></a></dt><br />
<dd class="dd_1">Tsinghua-A</dd><br />
</dl><br />
<dl class="instructors"><br />
<dt><a href="http://www.biochem.hku.hk/huanglab/Huang_Lab_at_HKU" target="_blank"><img src="https://static.igem.org/mediawiki/2013/a/a5/Huanglab.jpg" width="140" height="150"></a></dt><br />
<dd class="dd_1">Huang Lab</dd><br />
</dl><br />
<div class="miaoshu">Here we express our gratitude to the great support from professor Kong Lingdong and professor Yang Sihai from the School of Life Sciences, Nanjing University, and technical support from technologist Zeng Shuiyun. And especially, we express our heartfelt thanks to Tsinghua-A, another 2013 iGEM team, for their help in our basic model building. Finally, we want to express great gratitude to Dr. Huang Jiandong's lab, University of Hong Kong which provided us with some important plasmids and strains.</div><br />
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(Here is an interesting <a href="https://2013.igem.org/Team:Nanjing-China/mp4"><strong>cartoon</strong></a> we made, showing the general idea of our project.)<br><br><br />
Atrazine is one of the most heavily used herbicides worldwide. It is used to control the growth of weeds. Atrazine is also a persistent environmental pollutant, like suppressing the growth of some other plants, inducing complete feminization and chemical castration in male frogs, and disrupting the human body's internal systems. Widespread contamination of groundwater caused by atrazine has been reported in the United States. As such, there are increasing concerns over the toxicity of atazine in the environment <sup>[1, 2]</sup>.<br/><br/><br />
Considering the fact that atrazine can hardly be degraded naturally in soil and water, we designed and constructed a system in Escherichia coli to make them move towards atrazine simultaneously up to the gradient and absorb it, degrade it into a harmless chemical substance.<br/><br/><br />
For a start, to equip the bacteria with the ability to recognize atrazine, we utilized a smart ribosome switch to recognize the presence of atrazine. In the absence of atrazine, the secondary structure of the ribosome switch forms a pseudoknot, sequestering the ribosome-binding site. In this way, the expression of downstream genes is inhibited. However, in the presence of atrazine, the conformation of the switch changes, the ribosome-binding site is therefore exposed, so the genes downstream can be expressed.<br/><br/><br />
When our bacteria could detect the presence of atrazine, we improved their performance by inserting quorum sensing (QS) system into the circuits. When our bacteria smell the signal of atrazine, they send out acyl-homoserine lactone (AHL) to recruit more team members to gather around the atrazine center <sup>[3]</sup>.<br/><br/><br />
Once enough atrazine gathers around the atrazine center, the density of bacteria pulls the trigger of Plsr, and the genes at the downstream of Plsr start to express and the bacteria are therefore able to secrete the super degrading enzyme, TrzN.<br/><br/><br />
However, the low transportation efficiency of atrazine from outside the membrane to the inside of the bacteria posed a problem to us. Luckily, an amazing transporter TRM provided us with an answer. With the help of HPLC (high performance liquid chromatography), we surprisingly found that TRM can distinctively transport atrazine into our bacteria so our entire system can work more sensitively.<br/><br/><br />
What's more, in order to acquire a better performance, we modified some of our parts, such as TrzN and CI, to make them function even better.<br/><br/><br />
At the same time, we also used the computer calculation to simulate the system we constructed, hoping to endow our system with high stability.<br/><br/><br />
For the last two months, we have completed some great achievements. A happy and united team was formed and we have successfully constructed 7 brilliant biobrick parts. Hoping to do better, we improved some of the parts function by scientific modification. Our human practice work has spread the concept and notion of synthetic biology as well as our project to many advanced teenagersacross the nation.<br/><br/><br />
We know exactly that our work still has a long way to go to get near to industrialization and commercialization, but we do believe that this hope can be eventually realized. Just think about the fascinating progress from the first computer to the PC, iPad and iPhone today, our research perhaps is still lingering in the "old times", but we have enough confidence that it will shock the world one day, providing us with a final answer to the pollution of atrazine.<br/><br/><br />
<br />
<br />
<strong>Reference</strong><br/><br />
[1] Sinha J, et al. Reprogramming bacteria to seek and destroy a herbicide. Nature chemical biology, 2010, 6 (6): 464-470.<br><br />
<br />
[2] Hayes TB, et al. Atrazine induces complete feminization and chemical castration in male African clawed frogs (Xenopus laevis). Proceedings of the National Academy of Sciences, 2010, 107 (10): 4612-4617.<br><br />
<br />
[3] Carter KK, et al. Pathway engineering via quorum sensing and sRNA riboregulators-Interconnected networks and controllers. Metabolic Engineering, 2012, 14 (3): 281-288.<br/><br />
</dd><br />
</dl></div>GaoJian NJUhttp://2013.igem.org/Team:Nanjing-China/qsTeam:Nanjing-China/qs2013-09-28T03:10:55Z<p>GaoJian NJU: </p>
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In our project, we considered devising our bacteria into some kind of automatic machinery that would be able to sense a target molecule and then assemble to degrade it. To fulfill all these functions, we were bound to design some gene circuits to guide our bacteria.<br><br><br />
<br />
First of all, we should have an understanding of how bacteria move<sup>[1]</sup>. For E.coli, they have flagellar motors to control their movement (Fig. 3-5-1). The direction of rotation of their flagellar motors is controlled by the protein CheY. When CheY is not phosphorylated, the flagellar motors rotate counterclockwise (CCW) and bacteria can migrate. When CheY is phosphorylated (CheY-P), it can bind to the flagellar motorprotein FliM, causing the cell to tumble and thus nonmotile. Phosphatase CheZ dephosphorylates CheY-P and causes the flagellum to rotate CCW. E. coli lacking the cheZ gene (ΔcheZ) cannot dephosphorylate CheY-P, and thus tumble incessantly, being nonmotile. So we only need to take control of gene cheZ to control the motility of our bacteria. <br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/0/04/Fig.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-1</strong> Illustration of how bacteria move. CheZ is the key of controlling the motility of bacteria. For wild type, CheZ can be expressed normally, so the flagellar motor alternates between CCW and CW rotation, thus the cell can run or tumble. On the contrary, if there is no CheZ, CheY will remain phosphorylated, causing the flagellar motor rotates CW, thus the cell tumbles exclusively.</div><br><br><br />
We would like our bacteria to seek and destroy atrazine in the soil. To achieve this goal, a classic system can do the job (Fig. 3-5-2A). In this system, bacteria can move around randomly but fix when sensing atrazine as the ribosome switch turns on and protein CI is expressed to repress the gene cheZ. As a result, our bacteria will just assemble around the target molecules after a period of time.<br><br><br />
However, we can only wait for the bacteria to hang about until they find their targets. Thus it could take a long time before the bacteria assembly and the system may not be a robust design. So we shall spare no efforts to improve it. For better control over the function of our bacteria, we took the quorum sensing system into consideration. We expect some bacteria which first find the atrazine will recruit other bacteria at a distance through some signal so they move towards the target and then fixed at the source of pollution. Therefore we constructed the following gene circuits to realize it (Fig. 3-5-2B).<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/6/63/Fig_3-5-2.jpg" width="716"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-2</strong> The gene circuits we built to control cheZ. (A) A classic system using CI to repress cheZ. (B) A complex but high-efficiency system using quorum sensing system. (C) A simplified system like the system in (B).</div><br><br><br />
This circuit is composed of three modules: an atrazine-sensing module, a recruiting module, and a brake module. We adopted the quorum-sensing system in Vibrio fischerias the basis for our design. In this system, a small-molecule acyl-homoserine lactone (AHL) synthesized by LuxI excretes as a signal and accumulates intracellularly to activate a constitutively expressed regulator, LuxR<sup>[2]</sup>. Our atrazine-sensing module controls the translation of LuxI through ribosome switch which turns on when combined to atrazine. Meanwhile, the recruiting module is composed of a series of elements. In this system, the LuxR-AHL complex promotes the expression of a repressor TetR, which represses the expression of another repressor CI. The gene cheZ is controlled by promoter Pλ, which is repressed by CI. So for those bacteria which first sense atrazine, they send out AHL signal and let the other bacteria receive it. After that, TetR is expressed to repress CI, leading the transcription of cheZ. These bacteria move towards the sender until they sense atrazine and another repressor CI, which is controlled by ribosome switch, expresses as a brake of the movement<sup>[3]</sup>. <br><br><br />
We also design the third gene circuit to simplify the system (Fig. 3-5-2C). This system also consists of the three modules in Pattern 2 and the atrazine-sensing module, as well as the brake module, is exactly the same composition with those two parts. The only difference is in the recruiting module that we use a double-functional promoter Plux/CI. The atrazine-sensing module works just the same way in Pattern 2. In this system, however, the LuxR-AHL complex promotes cheZ transcription via promoter Plux/CI. At the spot where bacteria sense atrazine, AHL will accumulate. The higher AHL concentration is, the stronger transcription of cheZ which means greater motility is, leading to directional movement towards bacteria which send out the signal. Meanwhile, the brake module also functions via promoter Plux/CI. The repressor CI, controlled by ribosome switch which turns on when combined to atrazine, represses the promoter Plux/CI and results in bacteria fixation at source of pollution.<br><br><br />
<strong>Experiments and Results</strong><br><br />
At the beginning, we verified gene cheZ in genetic level. We had 3 strains, namely CL-1, CL-M, and CL-IM. CL-1 is a strain with deletion of gene cheZ, CL-M is wildtype, and CL-IM is a strain with deletion of the gene that controls the expression of flagella. We extracted the total RNA of these 3 strains and synthesized the 1st cDNA using RT-PCR. Then we did PCR of gene cheZ with the cDNA as template and we also did PCR of 16 sDNA as control. We got the figure through agarose gel electrophoresis (Fig. 3-5-3A). We can see that CL-1 is actually the strain with deletion of gene cheZ, while the other two strains have normal gene cheZ.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/f/f3/Fig_3-5-3.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-3</strong> The verification of gene cheZ. (A) In genetic level, CL-1 is actually the strain with deletion of gene cheZ, while the other two strains have normal gene cheZ. (B) In phenotype level, CL-M and pcheZ is able to migrate on semisolid culture medium, while CL-1 and CL-M is unable to migrate.</div><br><br><br />
Then we needed to verify of gene cheZ from phenotype. We have 3 strains, namely CL-1, CL-M, and CL-IM. CL-1 is a strain with deletion of gene cheZ, CL-M is wild type, and CL-IM is a strain with deletion of the gene that controls the expression of flagella. Then we transformed a plasmid with gene cheZ into CL-1 and got the fourth strain pcheZ.<br><br><br />
We inoculated the suspension of bacteria on semisolid LB plate and incubated for 20h and took the photograph (Fig. 3-5-3B). From the figure, we can see that CL-M and pcheZ is able to migrate on semisolid culture medium, while CL-1 and CL-M is unable to migrate. This indicates that E.coli is nonmotile without gene cheZ or flagella and inducing the expression of gene cheZ can restore motility in a cheZ knockout strain.<br><br><br />
Finally, we also verified lux system. We constructed a strain with gene circuit as shown in Fig. 3-5-4A from CL-1. This is a lux system without the ribosome switch. AHL synthesized by LuxI excretes as a signal and accumulates intracellularly to activate a constitutively expressed regulator, LuxR. Then the LuxR-AHL complex promotes the expression of a repressor CI, leading to repression of CheZ. So bacteria will assembly at spots where initial density is high in this system. After we inoculated the suspension of bacteria on semisolid LB plate and incubated for 20h, we took the photograph of a stripe pattern (Fig. 3-5-4B), which was formed under the influence of the lux system<sup>[4]</sup>.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/d/db/Fig_3-5-4_new.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-4</strong> The verification of lux system. (A) the gene circuit we constructed. (B) The phenotype of lux system. </div><br/><br />
For more information of our assembly system, please refer to <a href="https://2013.igem.org/Team:Nanjing-China/model"><strong>Model</strong></a>.<br><br />
<br/> <br />
<br />
<strong> Reference</strong><br/><br />
[1] Topp S and Gallivan JP. Guiding bacteria with small molecules and RNA. Journal of the American Chemical Society, 2007, 129 (21): 6807-6811.<br/><br />
[2] Carter KK, et al. Pathway engineering via quorum sensing and sRNA riboregulators-Interconnected networks and controllers. Metabolic Engineering, 2012, 14 (3): 281-288.<br/><br />
[3] Sinha J, et al. Reprogramming bacteria to seek and destroy a herbicide. Nature chemical biology, 2010, 6 (6): 464-470.<br/><br />
[4] Liu C, et al. Sequential establishment of stripe patterns in an expanding cell population. Science, 2011, 334 (6053): 238-241.<br/><br />
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</html></div>GaoJian NJUhttp://2013.igem.org/Team:Nanjing-China/qsTeam:Nanjing-China/qs2013-09-28T03:09:49Z<p>GaoJian NJU: </p>
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<dt><a href="###">Aggregation</a></dt><br />
<dd class="dd_1"><br />
In our project, we considered devising our bacteria into some kind of automatic machinery that would be able to sense a target molecule and then assemble to degrade it. To fulfill all these functions, we were bound to design some gene circuits to guide our bacteria.<br><br><br />
<br />
First of all, we should have an understanding of how bacteria move<sup>[1]</sup>. For E.coli, they have flagellar motors to control their movement (Fig. 3-5-1). The direction of rotation of their flagellar motors is controlled by the protein CheY. When CheY is not phosphorylated, the flagellar motors rotate counterclockwise (CCW) and bacteria can migrate. When CheY is phosphorylated (CheY-P), it can bind to the flagellar motorprotein FliM, causing the cell to tumble and thus nonmotile. Phosphatase CheZ dephosphorylates CheY-P and causes the flagellum to rotate CCW. E. coli lacking the cheZ gene (ΔcheZ) cannot dephosphorylate CheY-P, and thus tumble incessantly, being nonmotile. So we only need to take control of gene cheZ to control the motility of our bacteria. <br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/0/04/Fig.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-1</strong> Illustration of how bacteria move. CheZ is the key of controlling the motility of bacteria. For wild type, CheZ can be expressed normally, so the flagellar motor alternates between CCW and CW rotation, thus the cell can run or tumble. On the contrary, if there is no CheZ, CheY will remain phosphorylated, causing the flagellar motor rotates CW, thus the cell tumbles exclusively.</div><br><br><br />
We would like our bacteria to seek and destroy atrazine in the soil. To achieve this goal, a classic system can do the job (Fig. 3-5-2A). In this system, bacteria can move around randomly but fix when sensing atrazine as the ribosome switch turns on and protein CI is expressed to repress the gene cheZ. As a result, our bacteria will just assemble around the target molecules after a period of time.<br><br><br />
However, we can only wait for the bacteria to hang about until they find their targets. Thus it could take a long time before the bacteria assembly and the system may not be a robust design. So we shall spare no efforts to improve it. For better control over the function of our bacteria, we took the quorum sensing system into consideration. We expect some bacteria which first find the atrazine will recruit other bacteria at a distance through some signal so they move towards the target and then fixed at the source of pollution. Therefore we constructed the following gene circuits to realize it (Fig. 3-5-2B).<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/6/63/Fig_3-5-2.jpg" width="716"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-2</strong> The gene circuits we built to control cheZ. (A) A classic system using CI to repress cheZ. (B) A complex but high-efficiency system using quorum sensing system. (C) A simplified system like the system in (B).</div><br><br><br />
This circuit is composed of three modules: an atrazine-sensing module, a recruiting module, and a brake module. We adopted the quorum-sensing system in Vibrio fischerias the basis for our design. In this system, a small-molecule acyl-homoserine lactone (AHL) synthesized by LuxI excretes as a signal and accumulates intracellularly to activate a constitutively expressed regulator, LuxR<sup>[2]</sup>. Our atrazine-sensing module controls the translation of LuxI through ribosome switch which turns on when combined to atrazine. Meanwhile, the recruiting module is composed of a series of elements. In this system, the LuxR-AHL complex promotes the expression of a repressor TetR, which represses the expression of another repressor CI. The gene cheZ is controlled by promoter Pλ, which is repressed by CI. So for those bacteria which first sense atrazine, they send out AHL signal and let the other bacteria receive it. After that, TetR is expressed to repress CI, leading the transcription of cheZ. These bacteria move towards the sender until they sense atrazine and another repressor CI, which is controlled by ribosome switch, expresses as a brake of the movement<sup>[3]</sup>. <br><br><br />
We also design the third gene circuit to simplify the system (Fig. 3-5-2C). This system also consists of the three modules in Pattern 2 and the atrazine-sensing module, as well as the brake module, is exactly the same composition with those two parts. The only difference is in the recruiting module that we use a double-functional promoter Plux/CI. The atrazine-sensing module works just the same way in Pattern 2. In this system, however, the LuxR-AHL complex promotes cheZ transcription via promoter Plux/CI. At the spot where bacteria sense atrazine, AHL will accumulate. The higher AHL concentration is, the stronger transcription of cheZ which means greater motility is, leading to directional movement towards bacteria which send out the signal. Meanwhile, the brake module also functions via promoter Plux/CI. The repressor CI, controlled by ribosome switch which turns on when combined to atrazine, represses the promoter Plux/CI and results in bacteria fixation at source of pollution.<br><br><br />
<strong>Experiments and Results</strong><br><br />
At the beginning, we verified gene cheZ in genetic level. We had 3 strains, namely CL-1, CL-M, and CL-IM. CL-1 is a strain with deletion of gene cheZ, CL-M is wildtype, and CL-IM is a strain with deletion of the gene that controls the expression of flagella. We extracted the total RNA of these 3 strains and synthesized the 1st cDNA using RT-PCR. Then we did PCR of gene cheZ with the cDNA as template and we also did PCR of 16 sDNA as control. We got the figure through agarose gel electrophoresis (Fig. 3-5-3A). We can see that CL-1 is actually the strain with deletion of gene cheZ, while the other two strains have normal gene cheZ.<br><br><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/f/f3/Fig_3-5-3.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-3</strong> The verification of gene cheZ. (A) In genetic level, CL-1 is actually the strain with deletion of gene cheZ, while the other two strains have normal gene cheZ. (B) In phenotype level, CL-M and pcheZ is able to migrate on semisolid culture medium, while CL-1 and CL-M is unable to migrate.</div><br><br><br />
Then we needed to verify of gene cheZ from phenotype. We have 3 strains, namely CL-1, CL-M, and CL-IM. CL-1 is a strain with deletion of gene cheZ, CL-M is wild type, and CL-IM is a strain with deletion of the gene that controls the expression of flagella. Then we transformed a plasmid with gene cheZ into CL-1 and got the fourth strain pcheZ.<br><br><br />
We inoculated the suspension of bacteria on semisolid LB plate and incubated for 20h and took the photograph (Fig. 3-5-3B). From the figure, we can see that CL-M and pcheZ is able to migrate on semisolid culture medium, while CL-1 and CL-M is unable to migrate. This indicates that E.coli is nonmotile without gene cheZ or flagella and inducing the expression of gene cheZ can restore motility in a cheZ knockout strain.<br><br><br />
Finally, we also verified lux system. We constructed a strain with gene circuit as shown in Fig. 3-5-4A from CL-1. This is a lux system without the ribosome switch. AHL synthesized by LuxI excretes as a signal and accumulates intracellularly to activate a constitutively expressed regulator, LuxR. Then the LuxR-AHL complex promotes the expression of a repressor CI, leading to repression of CheZ. So bacteria will assembly at spots where initial density is high in this system. After we inoculated the suspension of bacteria on semisolid LB plate and incubated for 20h, we took the photograph of a stripe pattern (Fig. 3-5-4B), which was formed under the influence of the lux system<sup>[4]</sup>.<br><br><br />
<p style="text-align:center"><img src="http://https://static.igem.org/mediawiki/2013/d/db/Fig_3-5-4_new.jpg" width="600"></p><br><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 3-5-4</strong> The verification of lux system. (A) the gene circuit we constructed. (B) The phenotype of lux system. </div><br/><br />
For more information of our assembly system, please refer to <a href="https://2013.igem.org/Team:Nanjing-China/model"><strong>Model</strong></a>.<br><br />
<br/> <br />
<br />
<strong> Reference</strong><br/><br />
[1] Topp S and Gallivan JP. Guiding bacteria with small molecules and RNA. Journal of the American Chemical Society, 2007, 129 (21): 6807-6811.<br/><br />
[2] Carter KK, et al. Pathway engineering via quorum sensing and sRNA riboregulators-Interconnected networks and controllers. Metabolic Engineering, 2012, 14 (3): 281-288.<br/><br />
[3] Sinha J, et al. Reprogramming bacteria to seek and destroy a herbicide. Nature chemical biology, 2010, 6 (6): 464-470.<br/><br />
[4] Liu C, et al. Sequential establishment of stripe patterns in an expanding cell population. Science, 2011, 334 (6053): 238-241.<br/><br />
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</html></div>GaoJian NJUhttp://2013.igem.org/File:Fig_3-5-4_new.jpgFile:Fig 3-5-4 new.jpg2013-09-28T03:08:40Z<p>GaoJian NJU: </p>
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<div></div>GaoJian NJUhttp://2013.igem.org/Team:Nanjing-China/modelTeam:Nanjing-China/model2013-09-28T03:02:01Z<p>GaoJian NJU: </p>
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<dl><br />
<dt><a name="Introduction">Introduction</a></dt><br />
<dd class="dd_1"><br />
As you can see in the previous parts, the shining point of our design is that we have introduced the quorum sensing device into our circuit. In order to see what the characteristics of our design are during the process of attracting E. coli to atrazine, we have built three models respectively and compared their possible behaviors.<br><br/><br />
The first circuit, we called circuit one, is the most simple one, which has been constructed by Joy sinha et al in 2010. In this circuit, bacteria will stop walking in the region with high concentration of atrazine, in which way bacteria will finally get together to this region. However, bacteria can't tell each other the information about the destination like ants, which will tell their companies the location of food.<br><br/> <br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/1/11/Circuit1_hah.jpg" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 4-1</strong> Circuit 1. Atrazine will promote the translation of the CI protein. However, the CheZ protein, which represent the motility, will be repressed when the concentration of CI protein is high. So, the consequence will be more and more bacteria stopping in the region with high Atrazine.</div><br/> <br />
The second circuit is our original blueprint with a complex structure and you can see the quorum sensing device here. It is a little complex and has a cascade, which is believed to prolong the respond time of our system. In short, the behavior of bacteria will be affected by the concentration of AHL, which represents the density of bacteria. In this way, the bacteria around the atrazine region are expected to move faster than the bacteria far away from this region.<br/> <br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/5/59/Fig_3-5-2-B.jpg" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 4-2</strong> Circuit2. The movement of the bacteria with this circuit will affected by the presence of AHL. In our initiate consideration, the cascade, AHL&LuxR→Plux-tetr---Ptet-CI---Pci-CheZ, will lead to the promotion of the production of CheZ protein.</div><br/> <br />
The third circuit is the ultimate one that we have designed and optimized.We have replaced the cascade mentioned above with a hybridize promoter, which can be repressed by CI protein and activated by AHL-LuxR compound. As thus, we are able to short the respond time of bacteria, which in turn can make our circuit more efficient.<br/> <br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/e/ea/Circuit3_a.jpg" width="600"></p> <br />
<div style="padding:0 50px; font-size:11px"><strong>Fig. 4-3</strong> Circuit 3. The greatest difference between circuit 2 and circuit 3, as you can see, is that the cascade in circuit 2 have been replaced by the hybridize promoter, Plux/CI. Then, we got a new organism that will respond quickly to the presence of AHL.</div><br/><br />
The most important is that we want to have a preview of the behavior of the three circuits mentioned above. Thus, we can compare them and prove that our final design can attract bacteria efficiently and send out the information of atrazine to bacteria around these regions to make them have a stable movement.<br/> <br />
</dd><br />
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<dl><br />
<dt><a name="Results">Results</a></dt><br />
<dd class="dd_1"><br />
These three models were all coded in MATLAB (all can be download <a href="https://static.igem.org/mediawiki/2013/6/66/Code_of_Nanjing-China%2C_iGEM_2013.zip"><strong>here</strong></a>). As mentioned above, we have just changed several statements in the second model to construct the first and the third. Finally, we have got how the distribution of bacteria changed with time, which can make us catch the major difference among three circuits more directly. <br><br/><br />
Besides, we have made census of the number of bacteria within the atrazine region in different circuits respectively. Then, we compared the outcome to find out the efficiency of different circuit.<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/8/89/Circuit1_aa.gif" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><p style="text-align:center"><strong>Fig. 4-4</strong> The simulation of circuit 1.</p></div> <br />
<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/9/92/Circuit2.gif" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><p style="text-align:center"><strong>Fig. 4-5</strong> The simulation of circuit 2.</p></div> <br />
<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/5/54/Circuit3.gif" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><p style="text-align:center"><strong>Fig. 4-6</strong> The simulation of circuit 3.</p></div><br/><br />
Let's see the characteristics of each circuit respectively first. Bacteria with circuit 1 move randomly all the time, so the result of their aggregation exhibit uncertain scene. The results of bacteria with circuit 2, meeting our expectation, demonstrate the dull response of the complex circuit. As to circuit 3, this kind of bacteria gets together in the region of much atrazine gradually. Circuit 1 is the classical design, which is proved by so many researchers to have a good result of attracting bacteria. <br><br/><br />
Compared to circuit 1, circuit 2 may have a more stable result. However, circuit 2 just need too much time to respond to the signal of cell density.<br><br/><br />
Circuit 3 has speed up the respond time of circuit 2. Besides, circuit 3 can attract the bacteria around the atrazine step by step, which can prevent the situation of too many bacteria aggregate at one point as circuit 1. In another word, circuit 3 works more stably and make it possible to have a mean distribution of bacteria to each atrazine region.<br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/e/e4/Cell_num_in_region-time_in_circuit1-census_.jpg" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><p style="text-align:center"><strong>Fig. 4-7</strong> The number of cells in the atrazine region of circuit1.</p></div><br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/7/76/Cell_num_in_region-time_in_circuit2-census_new.jpg" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><p style="text-align:center"><strong>Fig. 4-8</strong> The number of cells in the atrazine region of circuit2.</p></div><br/><br />
<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2013/9/94/Cell_num_in_region-time_in_circuit3-census_.jpg" width="600"></p><br />
<div style="padding:0 50px; font-size:11px"><p style="text-align:center"><strong>Fig. 4-9</strong> The number of cells in the atrazine region of circuit3.</p></div><br/><br />
The Fig. 4-7~9 exhibits some quantitative results of the three circuits. It is easy for us to find that the number of the cells in different circuits changed in different ways. The number of cells with circuit 1 in itself in specified region may have different rate in different situation due to randomness. However, the bacteria with circuit 3 aggregate in specified gradually, which shows a nearly straight-line slope in the fig. 4-9. At last, fig. 4-8, which present circuit 2, exhibits little change about the number of cells in specified region.<br />
</dd><br />
</dl><br />
<dl><br />
<dt><a name="Equations">Equations</a></dt><br />
<dd class="dd_1"><br />
After drawing a profile of our project, we should make the process of every event more clearly. So, we have constructed the models of our three circuits by the principles of biochemistry.<br><br/><br />
First, we have constructed a model related to the circuit 2, the most complex one, with the help of Tsinghua-A. Then, in order to compare them, we have also constructed the other two based on the model of circuit 2. In our model, we use ODE to describe the process of chemical reaction in organisms.<br><br/><br />
In general, we have divided the chemical events in our bacteria into three parts, the transcription of DNA, the translation of RNA, the production of micromolecule-compounds. The rate of each event can be described by ODE listed below.<br><br/> <br />
<strong>Circuit1:</strong><br/><br />
<img src="https://static.igem.org/mediawiki/2013/4/41/Equation1.jpg"><br/><br />
<strong>Circuit2:</strong><br/><br />
<img src="https://static.igem.org/mediawiki/2013/a/a5/Equations2-1.jpg"><img src="https://static.igem.org/mediawiki/2013/0/0b/Equations2-2.jpg"><br/><br />
<strong>Circuit3:</strong><br/><br />
<img src="https://static.igem.org/mediawiki/2013/a/a7/Equation3.jpg"> <br/><br />
m_CI1,m_CI2, m_CheZ, m_TetR, m_TrzN, m_LuxI represent mRNA of different protein.<br/><br />
CI1,CI2, CheZ, TetR, TrzN, LuxI represent proteins.<br/><br />
p_AHL represent the AHL produced in each cell.<br><br/><br />
We have used matrix to describe the state of AHL and the location of every bacteria. By the way, we have also made the density of bacteria related to the distance of bacteria. At last, we have considerate the degradation of atrazine as well, though which proved to have little influence on atrazine latter. And a space lattice of "culture dish" is demonstrated below.<br><br/> <br />
<strong>Space relation:</strong><br/><br />
<img src="https://static.igem.org/mediawiki/2013/8/8e/Space_relation.jpg"><br><br />
e_AHL represent the AHL in the environment.<br/><br />
PXn, Pyn represent the location of bacteria.<br/><br />
Cdn represent the density of bacteria in a lattice.<br/><br />
Atz represent the atrazine. <br/><br/> <br />
<br />
</dd><br />
</dl><br />
<br />
<dl><br />
<dt><a name="Parameter">Parameter</a></dt><br />
<dd class="dd_1"><br />
<img src="https://static.igem.org/mediawiki/2013/4/48/Parametertable.jpg"> <br><br><br/><br />
<strong>Reference</strong><br/><br />
[1] The wiki of iGEM11 TsinghuaA, https://2011.igem.org/Team:Tsinghua-A/Modeling.<br/><br />
[2] The wiki of iGEM11 USTC, https://2011.igem.org/Team:USTC-China/Drylab/modeling.<br/><br />
[3] Basu, S., Gerchman, Y., Collins, C. H., Arnold, F. H. & Weiss, R. A synthetic multicellular system for programmed pattern formation. Nature434, 1130-1134 (2005).<br/><br />
[4] Goryachev, A., Toh, D. & Lee, T. Systems analysis of a quorum sensing network: design constraints imposed by the functional requirements, network topology and kinetic constants. Biosystems83, 178-187 (2006).<br/><br />
[5] Hooshangi, S. & Bentley, W. E. LsrR Quorum Sensing "Switch" Is Revealed by a Bottom-Up Approach. PLoS computational biology7, e1002172 (2011). <br/> <br />
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