Team:XHD-Wuhan-B-China/Description
Description
Inspiration
It is well known that the survival and operation of everything, such as the extraction of new branches from trees, the blooming of flowers, the recovery of damaged skin, and so on, are closely related to the process of cell division. But what happens if cell division stopped?
Are all the processes of natural cell division beneficial? First of all, by visiting the fermentation factory, we know that if the cell stops dividing at the stage of accumulating products in the fermentation process, we can obtain more fermentation products, which can not only greatly shorten the product accumulation time, but also reduce the cost. Second, we interviewed a cancer expert, and he told us that if it was possible to stop cell division, it would be a major boon for cancer patients. Therefore, we are committed to exploring the means to stop cell division.
Escherichia coli is an important model species and plays an important role in the field of synthetic biology. E. coli has the advantages of rapid growth, short division interval, and the problems related to E. coli cell division have been widely studied in recent years. Therefore, we chose E. coli as our research object.
Based on above, we hope to be able to control the cell division of Escherichia coli by means of synthetic biology.
Are all the processes of natural cell division beneficial? First of all, by visiting the fermentation factory, we know that if the cell stops dividing at the stage of accumulating products in the fermentation process, we can obtain more fermentation products, which can not only greatly shorten the product accumulation time, but also reduce the cost. Second, we interviewed a cancer expert, and he told us that if it was possible to stop cell division, it would be a major boon for cancer patients. Therefore, we are committed to exploring the means to stop cell division.
Escherichia coli is an important model species and plays an important role in the field of synthetic biology. E. coli has the advantages of rapid growth, short division interval, and the problems related to E. coli cell division have been widely studied in recent years. Therefore, we chose E. coli as our research object.
Based on above, we hope to be able to control the cell division of Escherichia coli by means of synthetic biology.
Background
How to stop the cell from dividing? Through the literature, we have learned about some genes and loci related to cell division, namely dnaA, ftsZ, mreB and oriC. DnaA protein promotes the dissociation of DNA in oriC, and the initiation of bacterial chromosome replication is precisely regulated by DnaA protein. FstZ protein can recruit some proteins needed for cell division for the synthesis of new septum, so fstZ gene plays a key role in cell division. Its deletion mutation blocks the formation of diaphragm and produces filaments. MreB protein is a cytoskeleton element, the rod structure of E. coli depends on MreB protein, dysfunctional MreB inhibits chromosome segregation in E. coli. And oriC is the place where genes begin to replicate.
Can we inhibit the division of E. coli by inhibiting these genes and loci? Yes, of course! That's gene silencing. Gene silencing is a phenomenon in which some genes lose transcriptional activity or decrease expression due to various reasons in the process of expression. Nowadays, the widely used gene silencing techniques are RNA interference (RNAi) and CRISPRi. RNAi technology, which originated about 24 years ago, refers to the phenomenon of efficient and specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA) in the process of evolution. Therefore, RNAi mainly inhibits the translation process of DNA. The CRISPRi system is mainly composed of single-guide RNA (sgRNA) and dCas9 protein with no endonuclease activity. SgRNA guides dCas9 protein to a specific site of the gene. Because of the dCas9 protein is too large, it will block the transcription process. Therefore, through RNAi and CRISPRi techniques, we can inhibit the genes and sites related to cell division, so as to achieve the purpose of inhibiting cell division.
Can we inhibit the division of E. coli by inhibiting these genes and loci? Yes, of course! That's gene silencing. Gene silencing is a phenomenon in which some genes lose transcriptional activity or decrease expression due to various reasons in the process of expression. Nowadays, the widely used gene silencing techniques are RNA interference (RNAi) and CRISPRi. RNAi technology, which originated about 24 years ago, refers to the phenomenon of efficient and specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA) in the process of evolution. Therefore, RNAi mainly inhibits the translation process of DNA. The CRISPRi system is mainly composed of single-guide RNA (sgRNA) and dCas9 protein with no endonuclease activity. SgRNA guides dCas9 protein to a specific site of the gene. Because of the dCas9 protein is too large, it will block the transcription process. Therefore, through RNAi and CRISPRi techniques, we can inhibit the genes and sites related to cell division, so as to achieve the purpose of inhibiting cell division.
Our Project
Our ultimate goal is to be able to switch freely between dividing and ceasing to divide. Although we have found RNAi and CRISPRi techniques that can inhibit cell division genes, we will not be able to achieve our goal without the means to control them. Therefore, we are going to use the optical system to control RNAi and CRISPRi, that is, through different wavelengths of light to control when RNAi and CRISPRi work. We use the two-component optical system CcaR/CcaS, under the green light of 520nm, CcaS is self-phosphorylated, then the phosphate group is transferred to the corresponding regulatory factor CcaR, and the phosphorylated CcaR binds to the cpcG2 promoter to start the expression and transcription of the target gene. The process is reversed at 660nm red light.
We artificially designed the sgRNA sequence of the CRISPRi system. Fortunately, the small RNA (sRNA) DicF, a natural RNAi sequence, was found to inhibit ftsZ mRNA in E. coli.
We combine CcaR/CcaS optical system with RNAi and CRISPRi to target genes and loci related to cell division, so as to achieve the purpose of controlling cell division. Through the optical system, we have realized the process of reversible regulation of cell division.And we have achieved that under the green light of 520nm, the division of MG1655 was inhibited, and the division of MG1655 returned to normal under the red light of 660nm. We hope that the strategy can facilitate some basic research and biological fermentation process.
We artificially designed the sgRNA sequence of the CRISPRi system. Fortunately, the small RNA (sRNA) DicF, a natural RNAi sequence, was found to inhibit ftsZ mRNA in E. coli.
We combine CcaR/CcaS optical system with RNAi and CRISPRi to target genes and loci related to cell division, so as to achieve the purpose of controlling cell division. Through the optical system, we have realized the process of reversible regulation of cell division.And we have achieved that under the green light of 520nm, the division of MG1655 was inhibited, and the division of MG1655 returned to normal under the red light of 660nm. We hope that the strategy can facilitate some basic research and biological fermentation process.
Figure1. Schematic representation of light-controlled CRISPRi system.
Figure2. Schematic representation of light-controlled RNAi system.
References
[1]. Zheng, H. et al. Interrogating the Escherichia coli cell cycle by cell dimension perturbations. Proc. Natl. Acad. Sci. U. S. A. 113, 15000–15005 (2016).
[2]. Wallden, M., Fange, D., Lundius, E. G., Baltekin, Ö. & Elf, J. The Synchronization of Replication and Division Cycles in Individual E. coli Cells. Cell 166, 729–739 (2016).
[3]. Wu, F. et al. Cell Boundary Confinement Sets the Size and Position of the E. coli Chromosome. Curr. Biol. 29, 2131-2144.e4 (2019).
[4]. Magnan, D. & Bates, D. Regulation of DNA replication initiation by chromosome structure. J. Bacteriol. 197, 3370–3377 (2015).
[5]. Rocheleau, C. E. et al. Wnt Signaling and an APC-Related Gene Specify Endoderm in Early C. elegans Embryos as P 2-EMS signaling. Previous studies suggested that P 2-EMS signaling. Cell 90, 707–716 (1997).
[6]. Weiss, D. S. Bacterial cell division and the septal ring. Mol. Microbiol. 54, 588–597 (2004).
[7]. Québatte, M. & Dehio, C. Systems-level interference strategies to decipher host factors involved in bacterial pathogen interaction: from RNAi to CRISPRi. Curr. Opin. Microbiol. 39, 34–41 (2017).
[8]. Schmidl, S. R., Sheth, R. U., Wu, A. & Tabor, J. J. Refactoring and optimization of light-switchable Escherichia coli two-component systems. ACS Synth. Biol. 3, 820–831 (2014).
[9]. Murashko, O. N. & Lin-Chao, S. Escherichia coli responds to environmental changes using enolasic degradosomes and stabilized DicF sRNA to alter cellular morphology. Proc. Natl. Acad. Sci. U. S. A. 114, E8025–E8034 (2017).
[1]. Zheng, H. et al. Interrogating the Escherichia coli cell cycle by cell dimension perturbations. Proc. Natl. Acad. Sci. U. S. A. 113, 15000–15005 (2016).
[2]. Wallden, M., Fange, D., Lundius, E. G., Baltekin, Ö. & Elf, J. The Synchronization of Replication and Division Cycles in Individual E. coli Cells. Cell 166, 729–739 (2016).
[3]. Wu, F. et al. Cell Boundary Confinement Sets the Size and Position of the E. coli Chromosome. Curr. Biol. 29, 2131-2144.e4 (2019).
[4]. Magnan, D. & Bates, D. Regulation of DNA replication initiation by chromosome structure. J. Bacteriol. 197, 3370–3377 (2015).
[5]. Rocheleau, C. E. et al. Wnt Signaling and an APC-Related Gene Specify Endoderm in Early C. elegans Embryos as P 2-EMS signaling. Previous studies suggested that P 2-EMS signaling. Cell 90, 707–716 (1997).
[6]. Weiss, D. S. Bacterial cell division and the septal ring. Mol. Microbiol. 54, 588–597 (2004).
[7]. Québatte, M. & Dehio, C. Systems-level interference strategies to decipher host factors involved in bacterial pathogen interaction: from RNAi to CRISPRi. Curr. Opin. Microbiol. 39, 34–41 (2017).
[8]. Schmidl, S. R., Sheth, R. U., Wu, A. & Tabor, J. J. Refactoring and optimization of light-switchable Escherichia coli two-component systems. ACS Synth. Biol. 3, 820–831 (2014).
[9]. Murashko, O. N. & Lin-Chao, S. Escherichia coli responds to environmental changes using enolasic degradosomes and stabilized DicF sRNA to alter cellular morphology. Proc. Natl. Acad. Sci. U. S. A. 114, E8025–E8034 (2017).