Difference between revisions of "Part:BBa K2203006"

Latest revision as of 17:39, 29 October 2017

HepHCV_ToeholdB

This composite part consists of a T7 promoter (BBa_J64997) in front of the toehold, a lacZalpha coding sequence (BBa_I732006) downstream and a T7 terminator (BBa_K731721) at the end. An RNA toehold switch with lacZalpha as a reporter added after the switch. When the trigger sequence in form of RNA or ssDNA is present in the sample, the toehold will unfold and lacZalpha will be translated. For more information about toeholds, go to 2017.igem.org/Team:EPFL

A toehold switch works according to this scheme: when the trigger sequence in form of RNA or ssDNA is present in the sample, the toehold will unfold and lacZalpha will be translated. For more information about toeholds, go to 2017.igem.org/Team:EPFL Sequence of toehold HepC_Toehold_A detecting a part of the Hepatitic C virus. This part can now be used in combination with cell-free extracts to detect the presence of Hepatitis C 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 HepC_Toehold_B 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.

Sequence and Features