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<div class="HZAU_div_main"> | <div class="HZAU_div_main"> | ||
| − | <a class=" | + | <a class="HZAU_title">Design</a> |
<a class="zhengwen">Our whole plan is to control the cell replication dynamically. After research, we decided to combine the CRISPR and optogenetic to construct our system.</a> | <a class="zhengwen">Our whole plan is to control the cell replication dynamically. After research, we decided to combine the CRISPR and optogenetic to construct our system.</a> | ||
<img src="https://static.igem.org/mediawiki/2017/0/0b/T--HZAU-China--hand.gif" class="tu_1"> | <img src="https://static.igem.org/mediawiki/2017/0/0b/T--HZAU-China--hand.gif" class="tu_1"> | ||
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<a class="zhengwen_disblock">The protein level of light controlled dCas9 system is based on the split protein and light induced dimerization (LID) protein. By infusing split dCas9 and LID protein together dCas9 can be controlled by light. (Figure 2.) The pMag and nMag developed from fungal is chosen to induce the complement of dCas9<a class="yinzhu" href="#yinwen_jiaozheng">$^{[4]}$</a><a class="zhengwen_disblock">. They are engineered VVD protein using FAD as its light sensing molecule. Under the irritation of blue light, the conformation change of FAD influences the structure of pMag and nMag revealing its dimerization domain. We choose this pair of protein due to its low molecular weight and tunable dynamics. (Figue 3. )</a> | <a class="zhengwen_disblock">The protein level of light controlled dCas9 system is based on the split protein and light induced dimerization (LID) protein. By infusing split dCas9 and LID protein together dCas9 can be controlled by light. (Figure 2.) The pMag and nMag developed from fungal is chosen to induce the complement of dCas9<a class="yinzhu" href="#yinwen_jiaozheng">$^{[4]}$</a><a class="zhengwen_disblock">. They are engineered VVD protein using FAD as its light sensing molecule. Under the irritation of blue light, the conformation change of FAD influences the structure of pMag and nMag revealing its dimerization domain. We choose this pair of protein due to its low molecular weight and tunable dynamics. (Figue 3. )</a> | ||
<a><img src="https://static.igem.org/mediawiki/2017/1/18/T--HZAU-China--design_figure3.png" class="tu_1">Figure 3. | <a><img src="https://static.igem.org/mediawiki/2017/1/18/T--HZAU-China--design_figure3.png" class="tu_1">Figure 3. | ||
| − | <a class="zhengwen">These two approaches both can satisfy our need in a certain way, but the former one is simpler but more related to metabolic state of chassis, and the latter one is more difficult to fulfill. We tried two approaches at the same time. For more information please view EXPERIMENT.</a> | + | <a class="zhengwen">These two approaches both can satisfy our need in a certain way, but the former one is simpler but more related to metabolic state of chassis, and the latter one is more difficult to fulfill. We tried two approaches at the same time. For more information please view <a class="zhengwen_disblock" href="https://2017.igem.org/Team:HZAU-China/Experiments">EXPERIMENT.</a></a> |
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| − | <a class="biaoti | + | <a class="biaoti">References</a> |
<a class="yinwen">1. Mott,M.L. and Berger,J.M. (2007) DNA replication initiation: mechanisms and regulation in bacteria. Nat. Rev. Microbiol., 5,343–354.</a> | <a class="yinwen">1. Mott,M.L. and Berger,J.M. (2007) DNA replication initiation: mechanisms and regulation in bacteria. Nat. Rev. Microbiol., 5,343–354.</a> | ||
<a class="yinwen">2. Wiktor, J., Lesterlin, C., Sherratt, D. J., & Dekker, C. (2016). CRISPR-mediated control of the bacterial initiation of replication. Nucleic Acids Res, 44(8), 3801-3810.</a> | <a class="yinwen">2. Wiktor, J., Lesterlin, C., Sherratt, D. J., & Dekker, C. (2016). CRISPR-mediated control of the bacterial initiation of replication. Nucleic Acids Res, 44(8), 3801-3810.</a> | ||
Revision as of 21:38, 1 November 2017
Design
Our whole plan is to control the cell replication dynamically. After research, we decided to combine the CRISPR and optogenetic to construct our system.
Like mentioned in description, DnaA protein is essential for cell cycle control$^{[1]}$, so we decide to interrupt its binding to corresponding DNA using CRISPR/dCas9. Under the guidance of gRNA dCas9 will bind to where DnaA should bind blocking further reaction to form replisome$^{[2]}$. Without replisome, the chromosome can’t replicate block it at phase B. Compared with engineering DnaA protein, this method is more easy and robust, and can be readily integrated into other prokaryotes.
Optogenetic is used to regulate the function of dCas9 due to its specificity in time and space and its easy manipulation, and it is also considered to be a proper connection between organism and computer. So we decided to use light to control the whole system. To accomplish this system, we designed two approaches, one is based on the reaction of transcription level, and the other is based on the protein interaction.
On the transcription level, we used CcaS-CcaR system, which is developed from cyanobacteria and is well used in synthetic biology$^{[3]}$. The CcaS-CcaR system is a two-component system. Under the green light, CcaS protein will be phosphorylated and the CcaR protein accept this phosphate and dimerize into a transcriptor inducing the transcription of gRNA, in which leading to the inhibition of replication. In the red light, the gRNA will stop transcription and degrade in a short time, and the inhibition will decrease in a short time freeing cell from blocking(Figure 1.). Corresponding results please see HERE.
Figure 2. The CcaS-CcaR system is developed from Synechocystis PCC 6803, and engineered into E.coli. It is a two component system(TCS) in which CcaS can sense green light and autophosphorylate as a membrane-binding protein, and CcaR can be phophorylated by CcaS-P and dimerize into a transcription factor.
The protein level of light controlled dCas9 system is based on the split protein and light induced dimerization (LID) protein. By infusing split dCas9 and LID protein together dCas9 can be controlled by light. (Figure 2.) The pMag and nMag developed from fungal is chosen to induce the complement of dCas9$^{[4]}$. They are engineered VVD protein using FAD as its light sensing molecule. Under the irritation of blue light, the conformation change of FAD influences the structure of pMag and nMag revealing its dimerization domain. We choose this pair of protein due to its low molecular weight and tunable dynamics. (Figue 3. )
Figure 3.
These two approaches both can satisfy our need in a certain way, but the former one is simpler but more related to metabolic state of chassis, and the latter one is more difficult to fulfill. We tried two approaches at the same time. For more information please view EXPERIMENT.
References
1. Mott,M.L. and Berger,J.M. (2007) DNA replication initiation: mechanisms and regulation in bacteria. Nat. Rev. Microbiol., 5,343–354.
2. Wiktor, J., Lesterlin, C., Sherratt, D. J., & Dekker, C. (2016). CRISPR-mediated control of the bacterial initiation of replication. Nucleic Acids Res, 44(8), 3801-3810.
3. Fernandez-Rodriguez, J., Moser, F., Song, M., & Voigt, C. A. (2017). Engineering RGB color vision into Escherichia coli. Nature Chemical Biology, 13(7), 706-708.
4. Kawano, F., Suzuki, H., Furuya, A., & Sato, M. (2015). Engineered pairs of distinct photoswitches for optogenetic control of cellular proteins. Nat Commun, 6, 6256.
Like mentioned in description, DnaA protein is essential for cell cycle control$^{[1]}$, so we decide to interrupt its binding to corresponding DNA using CRISPR/dCas9. Under the guidance of gRNA dCas9 will bind to where DnaA should bind blocking further reaction to form replisome$^{[2]}$. Without replisome, the chromosome can’t replicate block it at phase B. Compared with engineering DnaA protein, this method is more easy and robust, and can be readily integrated into other prokaryotes.
Optogenetic is used to regulate the function of dCas9 due to its specificity in time and space and its easy manipulation, and it is also considered to be a proper connection between organism and computer. So we decided to use light to control the whole system. To accomplish this system, we designed two approaches, one is based on the reaction of transcription level, and the other is based on the protein interaction.
On the transcription level, we used CcaS-CcaR system, which is developed from cyanobacteria and is well used in synthetic biology$^{[3]}$. The CcaS-CcaR system is a two-component system. Under the green light, CcaS protein will be phosphorylated and the CcaR protein accept this phosphate and dimerize into a transcriptor inducing the transcription of gRNA, in which leading to the inhibition of replication. In the red light, the gRNA will stop transcription and degrade in a short time, and the inhibition will decrease in a short time freeing cell from blocking(Figure 1.). Corresponding results please see HERE.
Figure 2. The CcaS-CcaR system is developed from Synechocystis PCC 6803, and engineered into E.coli. It is a two component system(TCS) in which CcaS can sense green light and autophosphorylate as a membrane-binding protein, and CcaR can be phophorylated by CcaS-P and dimerize into a transcription factor. The protein level of light controlled dCas9 system is based on the split protein and light induced dimerization (LID) protein. By infusing split dCas9 and LID protein together dCas9 can be controlled by light. (Figure 2.) The pMag and nMag developed from fungal is chosen to induce the complement of dCas9$^{[4]}$. They are engineered VVD protein using FAD as its light sensing molecule. Under the irritation of blue light, the conformation change of FAD influences the structure of pMag and nMag revealing its dimerization domain. We choose this pair of protein due to its low molecular weight and tunable dynamics. (Figue 3. )
Figure 3.
These two approaches both can satisfy our need in a certain way, but the former one is simpler but more related to metabolic state of chassis, and the latter one is more difficult to fulfill. We tried two approaches at the same time. For more information please view EXPERIMENT.
