Part:BBa_K2203004
ZIKV_27B_lacZalpha
This composite part consists of a T7 promoter (BBa_J64997) in front of the toehold, lacZalpha (BBa_I732006) downstream and a T7 terminator (BBa_K731721). The sequence of toehold 27B is detecting a part of the Zika virus. This part can now be used in combination with cell-free extracts to detect the presence of Zika virus in a sample.
The reporter system in our toeholds(K2203004, K2203005 for zika detection and K2203003, K2203006 for hepatitis C detection) works as follows:
- M15 cells have a lacZ delta mutation which makes them encode a form of beta-galactosidase lacking residues 11-41.
- Beta-galactosidase produced without those residues is missing a small part and is thus not functional.
- Upon the annealing of a trigger zika sequence to the toehold switch, the toehold unfolds and the lacZalpha coding sequence is translated.
- The production of the lacZaplpha part and the indigenous presence of the lacZ omega part in M15 cells allows for alpha complementation and production of a functional beta-galactosidase that can be detected by the addition of a colorimetric substrate CPRG Chlorophenol red-β-D-galactopyranoside.
Characterization of the 27B_Zika Toehold in T7M15 lysate in the presence of a single stranded DNA trigger
Expression of LacZalpha was measured on a plate reader. Toehold and trigger were added to a usual lysate reaction as described here. Reactants were kept at 37°C for five hours.
The toehold is successfully triggered and LacZalpha translated.
Incubation reaction of 27B_Zika Toehold in T7M15 lysate in the presence of a single stranded DNA trigger
Right: Toehold LacZ alpha with no trigger added: the alpha part cannot be translated
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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