Part:BBa_K3904004

Bile-regulated VapXD kill-switch

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

This part was designed with the ability to change promoter before the toxin VapD. In our project context, promoters with a variety of strengths were tested for the most effective kill-switch system work. What is more, this part was used in later VapXD system measurements as a control for anti-toxin production (see BBa_K3904005).

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 199
  Illegal BamHI site found at 397
  Illegal XhoI site found at 442
  
• 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