To prevent our genetically modified probiotic bacteria from escaping the intestine to the environment, they were provided with the disabling mechanism, which could be activated after exposure to some environmental factors. This goal was obtained by implementation of the newly described type II toxin-antitoxin (TA) system VapXD from non-typeable Haemophilus influenzae .
Vilnius-Lithuania iGEM 2021 project AmeByelooks at amebiasis holistically and comprehensively, therefore target E. histolytica from several angles: prevention and diagnostics. Our team's preventive solution consists of probiotics engineered to produce naringenin - an antiprotozoal compound. Two strains of genetically modified microorganisms were chosen as the main chassis - world-renowned Lactobacillus casei BL23 (Lactobacillus paracasei) and Escherichia coli Nissle 1917. Furthermore, the team made specific gene deletions to enhance naringenin production and adapted a novel toxin-antitoxin system to prevent GMO spreads into the environment. The diagnostic part includes a rapid, point of care, user-friendly diagnostic test identifying extraintestinal amebiasis. The main components of this test are aptamers specific to the E. histolytica secreted proteins. These single-stranded DNA sequences fold into tertiary structures for particular fit with target proteins.
Usage and Biology
The cold-inducible promoter is a part of the VapXD kill-switch system and regulates toxin VapD expression. This promoter naturally occurs in Escherichia coli and regulates cold shock protein (CspA) expression as a response mechanism to instantaneous changes in temperature (from 37 °C to 20 °C, in particular) .
Sequence and Features
- 10COMPATIBLE WITH RFC
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- Bertelsen, M. B., Senissar, M., Nielsen, M. H., Bisiak, F., Cunha, M. V., Molinaro, A. L., Daines, D. A., & Brodersen, D. E. (2021). Structural Basis For Toxin Inhibition in the VapXD Toxin-Antitoxin System. Structure, 29(2). https://doi.org/10.1016/j.str.2020.10.002
- oldenberg D, Azar I, Oppenheim AB, Brandi A, Pon CL, Gualerzi CO. Role of Escherichia coli cspA promoter sequences and adaptation of translational apparatus in the cold shock response. Mol Gen Genet. 1997 Oct;256(3):282-90. doi: 10.1007/s004380050571. PMID: 9393453.