Part:BBa_K3904002

Bile-inducible promoter

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 [1].

Introduction

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

Bile-induced promoter is a part of the VapXD kill-switch system and regulates anti-toxin VapX expression. This promoter was characterized in Lactobacillus casei BL23 more thoroughly but it was showed to work in other Lactobacillus species as well. Induction can be performed in the presence of bile salt concentrations as low as 0.025 and 0.05%. What is more, the best induction results can be seen while using choline acid in particular [2].

For experimental results see BBa K3904005 and BBa K3904006.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 179
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

1. 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
2. Martínez-Fernández, J.A., Bravo, D., Peirotén, Á. et al. Bile-induced promoters for gene expression in Lactobacillus strains. Appl Microbiol Biotechnol 103, 3819–3827 (2019). https://doi.org/10.1007/s00253-019-09743-w