Part:BBa_K3725010
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.
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.
Experience
The T7 Phytophthora Trigger BBa_K3725040 and the Phytophthora Toehold with GFP Reporter BBa_K3725010 are intended to be compatible with each other and used in conjunction. To test the compatibility of the trigger sequence with the toehold sequence, a dual-plasmid transformation was performed, and fluorescence was measured in comparison with cells transformed with only the toehold part and pUC19 (positive control). All samples were divided by optical density to obtain a standardized unit. The fluorescence per optical density unit measurement of cells transformed with both the toehold and trigger sequences was greater and had a statistically significant difference in comparison to cells transformed with only the toehold part and pUC19 plasmid.
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