Difference between revisions of "Part:BBa K5096076"
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<figcaption>Figure 1. Construct map for part BBa_K5096040, inhA toehold.</figcaption> | <figcaption>Figure 1. Construct map for part BBa_K5096040, inhA toehold.</figcaption> | ||
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<figcaption>Figure 2. Characterization curve for inhA toehold 5 with 10 nM to 20 nM trigger concentration</figcaption> | <figcaption>Figure 2. Characterization curve for inhA toehold 5 with 10 nM to 20 nM trigger concentration</figcaption> | ||
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<figcaption>Figure 9. Deterministic ODE model simulating our inhA toehold reaction</figcaption> | <figcaption>Figure 9. Deterministic ODE model simulating our inhA toehold reaction</figcaption> | ||
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Revision as of 16:45, 1 October 2024
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.
Experience
To test the compatibility of the M. TB trigger sequence (Part BBa_K5096045) with the toehold sequence, the toehold and trigger constructs were added to the myTXTL Pro cell-free lysate kit with all necessary reagents. All 3 out of 6 inhA toeholds designed by Lambert iGEM were tested in cell-free this varying concentration of trigger DNA. Fluorescence was measured in comparison with a cell-free reaction with only the P70a-deGFP plasmid (positive control). Reactions were measured for fluorescence output in a plate reader across the 16 hour reactions. The relative fluorescence value of our fifth inhA toehold was significantly higher than reactions containing other inhA toeholds, confirming that this specific toehold-trigger pair is compatible.
