Team:NCTU Formosa/Design
- ☰
Biobrick Design Concept
Securing biosafety is one of the most critical problems that iGEMers will face while building up their projects. During brainstorming, we have learned different ways to provide biosafety from former iGEMers. One of them is to express the toxin gene. The method works easily, in which bacteria will express the toxin gene under specific promoter control. However, we noticed that these biosafety designs were "bio-not-so-safe" for the fact that the bacteria would mutate itself and proliferate after the selection.
Figure 1: The designed biobrick of E.Phoenix and toxin gene we use
How we choose our toxin genes?
In our project, five kinds of toxin genes are selected as candidates for bioassays, including Mazf, ccdB, ydfD, YafQ, and ChpBK. Except for ydfD, all toxic genes used are members of the toxin-antitoxin system (TA system), which is composed of the toxin gene and its cognate antitoxin gene. It exists in bacteria, archaea, and fungi, and these toxins will lead to cell growth arrest or even cell death, while antitoxin will bond to the corresponding toxin to form stable toxin-antitoxin complex and thus neutralize the toxicity of toxin. There are at least 33 TA systems in E. coli. Take ccdB and MazF, two of the toxin genes used in our project, for example, under normal conditions, ccdB and MazF will bind with antitoxin ccdA and MazE respectively so the growth of E. coli will not be affected. However, once E. coli faces severe conditions, ccdA and MazE will lose their function as they are degraded by protease. Therefore, the growth of E. coli will be suppressed. Researches showed that the TA system plays a vital role in the organism as it may help the maintenance of plasmid and also allow the organism to survive under stress.
Figure 2: Type I TA system.
Figure 3: Type II TA system.
Figure 4: Type III TA system.
Reference
1. Masuda, H., et al. (2016). "ydfD encodes a novel lytic protein in Escherichia coli." FEMS microbiology letters 363(6): fnw039.
2. Prysak, M. H., et al. (2009). "Bacterial toxin YafQ is an endoribonuclease that associates with the ribosome and blocks translation elongation through sequence‐specific and frame‐dependent mRNA cleavage." Molecular Microbiology 71(5): 1071-1087.
3. Tripathi, A. K. and H. D. Kumar (1986). "Mutagenesis by ethidium bromide, proflavine and mitomycin C in the cyanobacterium Nostoc sp." Mutation Research Letters 174(3): 175-178.
4. Vermeulen, N., et al. (2008). "The bactericidal effect of ultraviolet and visible light onEscherichia coli." Biotechnology and Bioengineering 99(3): 550-556.
5. Yamaguchi, Y., et al. (2011). "Toxin-Antitoxin Systems in Bacteria and Archaea." 45(1): 61-79
6. Zhang, Y., et al. (2005). "Characterization of ChpBK, an mRNA Interferase from Escherichia coli." Journal of Biological Chemistry 280(28): 26080-26088.
Go to >>>
