Team:OUC-China
Overview
In order to build our “Disp” platform to rescue every fermentation kingdom,we “Dispers” created a treasure called “CHIP”: C stands for “control”,h stands for “hold”, I stands for “inhibition” and P stands for “transport”.Every part of this treasure are shown below.
Control-Number of copies
The introduced production genes are copied and lost in the continuous DNA replication as cells proliferate.As the clerks and R&D staffs of microbial fermentation factories told us, this is a very critical reason why engineered bacteria production decline when long-term cultured. So we decided to design a gene circuit that can maintain the number of copies of production genes. We try to knock out a gene necessary for the growth and introduced it into the plasma with a weak expression promoter added before it. If the copy number was too low after the production gene introduced into the engineered bacteria, the engineered bacteria would die. In this project, we knock out phosphofructokinase, which is the key enzyme for engineering bacteria to use glucose in the culture medium.
Hold-quorum sensing
We communicated with our PI and the R&D staffs of microbial fermentation factories about our preliminary project design which used a temperature control system to regulate production and growth.They all advised us to figure out another way because the temperature control system wasted too much energy and the temperature span is too small leading to an inaccurate adjustment.So we selected quorum sensing to regulate growth and production metabolism automatically.
When microorganisms are used for production, the yield is often reduced due to resource competition between growth and production metabolism.In order to avoid such adverse effects, we can achieve the purpose of increasing the yield by dynamically regulating the growth and production metabolism of engineered microorganisms. Quorum sensing- mediated dynamic control is a very useful strategy to balance the growth of cells and production of metabolites. Unlike most dynamic regulation strategies that require addition of an inducer or need pathway-specific parts such as special transcription factors which respond to products or key intermediate metabolites in metabolic pathways, quorum sensing-based dynamic regulation is pathway-independent and inducer-free. It is triggered when a certain cell density is reached, which is an important parameter in many metabolic engineering applications.
We use cytokinin-mediated quorum sensing circuits from Arabidopsis thaliana[1] makes our engineered microorganisms only reaches a certain population density will start fermentation process, used for decoupling competition between cell growth and fermentation. Cytokinin isopentenyladenine (IP), as quorum sensing molecule, is the product of ATP catalyzed by AtlPT4. When the signal molecule IP reaches a certain concentration, it activates the SSRE promoter and increases the expression of production gene controlled by SSRE promoter. In our experiment, to better characterize SSRE promoter and quorum sensing circuit, we put eGFP gene at the downstream of SSRE promoter.
Inhibition-Kill switch
We must inhibit engineering bacteria with no production from producing more offspring. To enable the engineered microorganism to initiate suicide at low yields, we designed kill switch whose aptamer faction binding to the product would activate ribozyme activity[2], causing the designed mRNA to self-cleavage and preventing YopE expression. The YopE cytotoxin of Yersinia pseudotuberculosis is an essential virulence determinant that is injected into the eukaryotic target cell via a plasmid-encoded type III secretion system[5]. Injection of YopE into eukaryotic cells induces depolymerization of actin stress fibres[6].
By using a RNA-based gene-regulatory platform, we design a ribozyme switch linked behind YopE mRNA[2], it contains a sensor domain, comprised of an aptamer sequence, and an actuator domain, comprised of hammerhead ribozyme sequence. 
Transport-transport RNA
The accumulation of products in engineering bacteria generally has a negative feedback regulation effect on the production of related enzymes, and the extraction process of crushing engineering bacteria to extract products is complicated. This part is committed to solve these problem.
The transport RNA can bind to membrane structure and specifically to the product by simply replacing the product binding aptamer sequence in RNA10[3], then transport the product along the concentration gradient. In detail, the transport RNA is composed of RNA9 and RNA10.Two RNA 9 molecules interact via right-hand loops, and the left-hand loop of the middle RNA 9 molecule interacts with the left-hand loop of RNA 10[4].
Improvement
Toxic protein
After communicating with other teams, we found that using YopE as the suicide protein in our design not only posed a security threat to the platform's R&D personnel, but also posed a greater security problem to the staff of the microbial factory. After communicating with the DUT-China team to test the sacB toxic protein in their project, we decided to use sacB as the final suicide protein of our platform because of its good killing effect and better biosafety. The sacB gene encodes a secretory sugar polysaccharide, which can catalyze the hydrolysis of sucrose into glucose and fructose, and polymerize fructose into high molecular weight fructose and the accumulation of high molecular weight fructans has potential toxic effects on cells, which can cause cell death. So we decided to test the toxic protein to see if it is capable to replace YopE.
Prevent escape
Considering that the engineering bacteria designed on the platform will be used in the fermentation tank of the factory, in order to prevent a large number of production engineering bacteria from escaping into the environment and causing pollution, we decided to design a switch to prevent escape after communicating with other iGEM teams. We design another kill riboswitch switch which can detect the concentration of an important element in culture medium If it spreads into environment accidentally, our kill switch can detect the low concentration of the important element and kill the engineered bacteria. This suicide switch is coupled with the copy number system. We designed to place it in the reintroduced PFK gene. When the engineering bacteria are stored in the fermentation tank, because there are enough detected substances, the PFK gene is normally expressed. When the engineering bacteria escape to the environment, the lack of detected substances causes the PFK gene expression to be closed and the engineering bacteria to die.
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References arrow_downward
- Yang, X., Liu, J., Zhang, J., Shen, Y., Qi, Q., Bao, X. and Hou, J. (2021). Quorum sensing-mediated protein degradation for dynamic metabolic pathway control in Saccharomyces cerevisiae. Metabolic Engineering, 64, pp.85–94. doi:10.1016/j.ymben.2021.01.010.
- Win, M.N. and Smolke, C.D. (2007). A modular and extensible RNA-based gene-regulatory platform for engineering cellular function. Proceedings of the National Academy of Sciences, 104(36), pp.14283–14288. doi:10.1073/pnas.0703961104.
- Wang, Y., Wang, J., Li, R., Shi, Q., Xue, Z., & Zhang, Y. (2017). ThreaDomEx: a unified platform for predicting continuous and discontinuous protein domains by multiple-threading and segment assembly. Nucleic acids research, 45(W1), W400-W407.
- JANAS, T. (2004). A membrane transporter for tryptophan composed of RNA. RNA, 10(10), pp.1541–1549. doi:10.1261/rna.7112704.
- Vlassov, A., Khvorova, A. and Yarus, M. (2001). Binding and disruption of phospholipid bilayers by supramolecular RNA complexes. Proceedings of the National Academy of Sciences, 98(14), pp.7706–7711. doi:10.1073/pnas.141041098.
- Von Pawel-Rammingen, U., Telepnev, M.V., Schmidt, G., Aktories, K., Wolf-Watz, H. and Rosqvist, R. (2002). GAP activity of the Yersinia YopE cytotoxin specifically targets the Rho pathway: a mechanism for disruption of actin microfilament structure. Molecular Microbiology, 36(3), pp.737–748. doi:10.1046/j.1365-2958.2000.01898.x.
- Leadsham, J.E., Kotiadis, V.N., Tarrant, D.J. and Gourlay, C.W. (2009). Apoptosis and the yeast actin cytoskeleton. Cell Death & Differentiation, 17(5), pp.754–762. doi:10.1038/cdd.2009.196.
