Difference between revisions of "Part:BBa K3725010"

Revision as of 02:19, 21 October 2021

Phytophthora Toehold w/ GFP Reporter

Overview

The Phytophthora Toehold w/ GFP Reporter part is designed to be used in conjunction with the T7 Phytophthora trigger (BBa_K3725040) in order to express GFP as a part of the engineered toehold switch system. The described structure was designed using NUPACK (Nucleic Acid Package) software’s Design feature. This was done by finding a unique gene of the P. cryptogea species that encodes for pathogenicity: the X24 gene. After inputting the X24 gene sequence into the NUPACK software using an input code given by Takashi et. al., pairs of trigger sequences and switch sequences were outputted. The trigger sequences given were 36 base-pair long sequences from the X24 gene, and the switch sequences given were reverse complementary to the trigger sequences. The pairs were ordered by normalized ensemble defect, so the pair with the lowest normalized ensemble defect was used. The switch sequence was ordered as an insert in a pUCIDT Amp plasmid from Integrated DNA Technologies.

Description

Toehold biosensors, which are composed of a switch and trigger, are highly orthogonal riboregulators that activate translation in response to a specific RNA sequence. The switch is composed of a hairpin loop structure that represses translation through its complementary bases in between the ribosomal binding site and the start codon, which is followed by a linker sequence. Once the toehold is exposed to the trigger sequence, the complementary base pairs on the trigger will bind to the toehold, which exposes the ribosomal binding site. RNA polymerase can then bind to the RBS and initiate translation of the reporter protein.

Sequence and Features