Difference between revisions of "Part:BBa K2203005"

Latest revision as of 17:38, 29 October 2017

ZIKV_32B_lacZalpha

This composite part consists of a T7 promoter (BBa_J64997) in front of the toehold, lacZalpha coding sequence (BBa_I732006) downstream and a T7 terminator (BBa_K731721) at the end. The sequence of toehold 32B 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 32B_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.