Part:BBa_K1189033
EHEC TALE2 (binds to a 19bp region 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_K1189032, 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. Final size of the EHEC TALE 2 generated from IDT Gene Blocks using SOE PCR.
Progress
The Calgary team has tested their DNA detection system using proof of concept TALEs designed by the 2012 Slovenia iGEM team. They have assembled BBa_K1189033 as shown in Figure 2 and are working on expressing, testing, and integrating these protein components into the final system.
Figure 2. Final size of the EHEC TALE 2 generated from IDT Gene Blocks using SOE PCR.
References
Heckman, K. L., & Pease, L. R. (2007). Gene splicing and mutagenesis by PCR-driven overlap extension. Nature protocols, 2(4), 924-932.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2404
Illegal BamHI site found at 2539 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |