Difference between revisions of "Part:BBa K3725040"

Revision as of 16:32, 21 October 2021

T7 Fusarium Trigger

Overview

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.

Design

The construction of a disease-specific biosensor required us to find a gene unique to the pathogen. When the switch turns on and GFP is expressed, we can confirm that the specific pathogen is present. For the detection of Phytophthora cryptogea, Lambert iGEM focused on the X24 gene. This gene was selected because it was required for pathogenicity and was unique to the species of interest. Biosafety note, the trigger sequence is not the full transcript sequence and therefore poses limited biosafety. We obtained the sequence via UniProt, an online database of protein sequences. Lambert iGEM used the code from Takahashi et. al provided by Megan McSweeney from the Styczynski Lab at the Georgia Institute of Technology to design the switch and trigger sequences on NUPACK. The team selected the pair from NUPACK with the lowest normalized ensemble defect (NED) to maximize the chances of successful compatibility. Once we obtained the sequences for the toehold pair, we constructed the toehold and trigger via SnapGene.

Sequence and Features

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T7 Fusarium Trigger

Overview

The T7 Phytophthora Trigger is designed to be used in conjunction with the Phytophthora Toehold w/ GFP Reporter (BBa_K3725010) to induce GFP expression for Lambert iGEM’s P. Cryptogea toehold biosensor. When the trigger RNA sequence is present, it binds to the complementary sequence in the toehold switch and unravels the hairpin loop allowing the reporter protein (GFP) to be expressed, producing green fluorescence. The sequence was designed via NUPACK using an input code provided by Takahashi et. al. We ordered the insert in a pUCIDT Kan plasmid from Integrated DNA Technologies.

Figure 1. The diagram above shows the construct of Part BBa_K3725220.

This plasmid was shared by Dr. Adam Silverman from Jewett Lab at Northwestern University.

Usage and Biology

Figure 2. The diagram above shows NarX (the native E. coli Nar membrane-bound sensor protein) and NarL (the corresponding DNA-binding response regulators), as well as how it works with our pNar Composite Part.

Lambert_GA iGEM 2021’s nitrate biosensor is modeled after E.coli’s natural nitrate sensor, the Nar Operon, which includes the membrane-bound sensor protein NarX and its corresponding DNA-binding response regulator NarL. The NarL/NarX system detects nitrate by using phosphorylated NarL as an inducer of promoter part BBa_K3725210 (Promoter pNar). In the presence of nitrate, NarX phosphorylates NarL, which activates pNar, and sfGFP is transcribed. Conversely, in the absence of nitrate, NarX does not phosphorylate NarL and sfGFP is not transcribed. To analyze the concentration of nitrate present in a solution, nitrate levels need to be correlated with a reporter protein, or a promoter responsive to phosphorylated NarL and report downstream GFP, a well-characterized fluorescent protein.

Sequence and Features