Part:BBa_K5096076

Overview The fifth inhA Toehold w/ GFP Reporter part, is designed to be used in conjunction with the Lambert iGEM inhA trigger (Part BBa_K5096045) 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 M. tuberculosis species that encodes for an essential component of the M. TB's cell wall: the inhA gene. After inputting the inhA 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 inhA gene, and the switch sequences given were reverse complementary to the trigger sequences. The pairs were ordered by normalized ensemble defect. We ordered the insert as a linear DNA construct designed with T7 promoters and terminators from TWIST Biosciences.

Description Toehold switches are ribo-regulators that consist of a specific RNA switch site and a ribosomal binding site (RBS). These switches have a complementary trigger that “activates” the quantification system when present. Toehold switches utilize a special RNA sequence that forms a hairpin loop, preventing translation of the downstream reporter gene, which, in our case, is Green Fluorescent Protein (GFP). However, when the trigger binds to the switch sequence, the toehold structure elongates. This exposes the RBS, allowing for the translation of the reporter gene (Green et al., 2014).

This detection system quantifies our CRISPRi system's ability to downregulate the inhA effector gene of M. tuberculosis. Simply stated, when the target gene is present – the switch is activated. However, when the gene is successfully downregulated, the switch remains inactive, resulting in negligible GFP output.