Toeholds
RNA-based Riboregulators Activated by Complimentary Triggers for Gene Quantification
Design
NUPACK
To design the necessary toehold switch and loop sequences for experimentation we utilized the in-silico web, Nucleic Acid Package (NUPACK). NUPACK is the leading software available for in-silico design and analysis of toeholds and their various components. The NUPACK software allows users to define specific domain restrictions to achieve a desired final structure. Due to this customizable interface, the length of domains such as the switch and conserved loop can be standardized, ensuring consistency across various developed outputs.
NUPACK requires the user to provide a source sequence to develop a toehold from. In the case of our team, we provided our target, the inhA gene sequence, as the source sequence to ensure that the developed switch sequence is complementary to our desired target RNA. An advantage of the NUPACK software is that it takes into account an efficiency score, which is calculated by taking the total number of nucleotides in the final secondary structure, or N, and striving to develop structures with a score below that of, N/100 (Zadeh et al., 2022). The NUPACK utilities tool allows for the visualization of the developed toehold’s structure and outlines the equilibrium probabilities for each base pair in the sequence (See Fig. 1 and Fig. 2). Based on recommendations from Dr. Megan McSweeney, a postdoctoral scholar in bioengineering at Stanford and previously from the Georgia Institute of Technology, we designed six separate toehold sequences using NUPACK for our experiments.
NUPACK Guide
Sequence Table
| Part Name | Part Type |
|---|---|
| BBa_K5096012 | Toehold Switch |
| BBa_K5096044 | Toehold Trigger |
| BBa_K5096040 | Toehold Switch Construct |
| BBa_K5096045 | Toehold Trigger Construct |
Toehold and Trigger Design
Lambert iGEM designed two constructs to test the functionality of the CRISPRi system. The first DNA construct contains a T7 promoter, our toehold sequence contains the switch, Green Fluorescent Protein (GFP), and a T7 terminator (See Fig 4.). This DNA construct is transcribed in our cell-free system to produce our RNA toehold.
Our second construct was a RNA trigger that includes a T7 promoter, a ribosomal binding site, the reverse complement of the toehold switch, and a T7 terminator (See Fig. 5). This construct generates viral RNA, which binds to and activates the toehold switch. (Soudier et al., 2022). We designed our complementary triggers to be between 18 and 22 nucleotides long to decrease the risk of secondary folding during the toehold reaction (McSweeney et al., 2023).
Justification
If the CRISPRi system fails, the dCas9-sgRNA complex does not bind to the target gene, allowing RNA polymerase to transcribe the gene. This permits the binding of the target RNA at the switch domain. This binding event will result in the elongation of the toehold structure, thereby revealing the GFP gene and allowing transcription to occur (Li et al., 2023) (See Fig. 6).
If the CRISPRi system functions correctly, target RNA will not be produced in the cell-free system. RNA polymerase will still initiate the formation of the toehold structure, but without target RNA, the structure remains intact, preventing GFP production (Li et al., 2023) (See Fig. 7).
By understanding the interaction between the dCas9-sgRNA complex from the CRISPRi system and the toehold biosensor, GFP allows us to track the functionality of the CRISPRi system. In the absence of fluorescence, we know our CRISPRi complex is working properly, while in the presence of fluorescence, we know it is not working properly.
Experimental Design
Cell-Free Expression
Lambert iGEM utilized commercial cell-free systems to test our toehold and trigger mechanisms due to their low cost and efficiency (Vezeau, 2020). Among the various commercially available cell-free systems, we selected the Arbor Biosciences Cell-free transcription-translation (TXTL) kits for our experiments. Specifically, we performed our experiments using the myTXTL Pro Expression Kit due to its T7 RNA polymerase promoter which is stronger compared to E. coli RNA polymerase (Tegel et al., 2010). Additionally, it supports the use of a commercial T7p14-deGFP plasmid from Arbor Biosciences to produce green fluorescence, which we utilized in the initial rounds of testing to establish a positive control.
Other Reagents
During our experimentation, we utilized various reagents from Arbor Biosciences’ myTXTL Kits. The Pro Kit Master Mix contains all necessary enzymes, cofactors, and other elements required for transcription and translation. Additionally, we used the T7p14-deGFP HP, a commercial plasmid that provides Green Fluorescent Protein as a positive control in our reactions. We also incorporated the Pro Helper Plasmid, which facilitates T7 promoter-based transcription of genes, along with Chi6 to prevent the degradation of linear DNA in the myTXTL reaction.