Part:BBa_K4990016
MazEF system
To mitigate leakage risks such as engineering bacteria in hypoxic environments like sewers after fecal excretion, we developed a temperature-responsive suicide switch. For our external suicide switch design, we drew on BNU-China 2019 project research, specifically their use of the TA system which piqued our interest. However, it should be noted that according to Fudan's 2020 proposal, the RelE/RelB module used by BNU-China in 2019 has further effects on the human gut. RelE possesses specific activity in eukaryotes and has been observed to cause cell apoptosis in cultured human cell lines. Additionally, RelE can accumulate unexpectedly in bacteria and affect the bacteria intricately involved in the shaping of human gut microbiota.
After conducting research, it has been determined that the Type II TA system is the most thoroughly examined system. Additionally, among these systems, the MazE/MazF module is considered to be relatively well-explored. Consequently, we have opted to implement the MazEF module in our suicide switch design. For regulation at the translation level, we have installed a temperature-sensitive RNA switch upstream of the antitoxin mazE. When the engineered bacteria are exposed to a high-temperature environment (37℃; in vivo), the RNA switch is activated, resulting in normal translation of both the antitoxin and toxin. The antitoxin typically hinders the toxin's effects, and the genetically modified bacteria can endure. At low-temperature environment(25℃; in vitro), the RNA switch forms a hairpin structure obstructing the antitoxin's translation and granting way to regular toxin translation and start suicide program.
I.Structure
Each MazF monomer forms a single globular domain with overall dimension of 40 × 40 × 20 Å, consisting of a seven-stranded, twisted antiparallel β sheet with three α helices. The two monomers interact with each other via intimate hydrophobic contacts, primarily involving segments of β strand S6 and α helix H3.
The MazE homodimer forms an intertwined β barrel of overall dimensions 20 × 30 × 25 Å, with two extended C-terminal segments that project laterally away from the crystallographic 2-fold symmetry axis and interact with the MazF homodimer. Within the N-terminal globular region, each monomer contributes two pairs of β strands connected by a short α helix to an intimate barrel-like structure with a single hydrophobic core. In addition, it is likely that the C-terminal regions are flexible in solution and readily susceptible to proteolytic digestion by the bacterial proteasome in vivo.
In solution，MazE/MazF complex form a heterologous hexamer of 2:4，consisting of three alternating homodimers MazF2-MazE2-MazF2.
II.Functions and mechanisms
MazF used to be believed that cleaves at ACA sites at or closely upstream of the AUG start codon of some specific mRNAs and thereby generates leaderless mRNAs. Moreover, we provide evidence that MazF also targets 16S rRNA within 30S ribosomal subunits at the decoding center, thereby removing 43 nucleotides from the 3′ terminus, which comprises the anti-Shine-Dalgarno (aSD) sequence that is required for translation initiation on canonical mRNAs. Resent research conducts global analysis of MazF targets through RNA seq and ribosomal analysis, indicating that that MazF leads to global RNA cleavage and efficiently blocks ribosome biogenesis, probably through the cleavage of rRNA precursors and ribosomal protein transcripts, which together will contribute significantly to a suppression of cell growth.
III.RNA thermometer
RNA thermometers have been extensively employed in iGEM projects as cis-acting elements to impact gene expression through post-transcriptional regulation.
Our initial design featured the historic FourU, but we later switched to the updated NoChill-19 based on the response temperature.
In a high-temperature environment (37 ℃ in vivo), the RNA thermometer can unfold, allowing for the typical mazE expression that engineers bacteria depend on for survival, with mazF activity serving a crucial role. Conversely, in a low-temperature environment (25 ℃ in vitro), the RNA thermometer transforms into a hairpin and closes over the RBS, impeding mazE translation and then mazF remains ensuring that the modified bacteria are unable to survive once separated from the body.
Iv. TA system library
In the development of the suicide switch in the CPU-CHINA 2023 project, we utilised various components from the TA system and discovered its versatile applications beyond this scope. Therefore, we have created a library for the TA system and provided a concise introduction to it, which briefly arranged and condensed its composition, types, targets, present application scenarios, and techniques to benefit future teams in utilizing these components.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 137
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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