Part:BBa_K1189032

EHEC TALE1 (binds to 19bp of stx2 gene)

Purpose

The Calgary iGEM 2013 team sought to detect pathogenic enterohemorrhagic E. coli (EHEC) DNA using pairs of TALEs designed to pathogenic E. coli markers. This TALE was designed based on a sequence alignment of the stx2 gene found in EHEC organisms (Perna et al., 2000). It binds to a 19 bp region of the toxin, will detect EHEC strains including E. coli O157:H7, as well as several other EHEC strains. This TALE, along with the complementary BBa_K1189033, will eventually be integrated into the final lateral flow strip FerriTALE prototype.

Design details

The sequence includes a KasI restriction enzyme cut site so that other TALEs may be substituted with the C-terminal linker and K coil. This is one element in which the 2013 Calgary team attempted to make their DNA detection system modular and useful for detection of other DNA sequences based on the selection of an appropriate TALE. The target site was specifically selected to increase binding strength of the TALE to DNA as per Meckler et al. (2013). Please see the iGEM Calgary 2013 Wiki for more details about how these TALEs were engineered for optimal binding.

Figure 1. Sequence alignment used to select a pair of TALEs to detect a large group of EHEC.

Progress

The Calgary team has tested their DNA detection system using proof of concept TALEs designed by the 2012 Slovenia iGEM team. They are working on assembling BBa_K1189032 and aim to express, test, and integrate this detector protein into the final system.

References

Meckler, J.F., Bhakta, M.S., Kim, M.S., Ovadia, R., Habrian, C.H., Zykovich, A., ... Baldwin, E.P. (2013). Quantitative analysis of TALE-DNA interactions suggests polarity effects. Nucleic Acids Research, 41(7), 4118–28. doi:10.1093/nar/gkt085

Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 2404
  Illegal BamHI site found at 2539
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]