Team:Lambert GA/Design

PROJECT DESIGN

LABYRINTH is a continuation of Lambert iGEM’s 2018 project which used toehold switches to detect cholera. Due to Lambert iGEM’s familiarity with the toehold mechanism, our team decided to use a similar design to detect helminth infections in contaminated feces. In addition, toehold switches can be further implemented in cell-free systems. This would eliminate the need for refrigerated/frozen solutions that restrict in-field operations; thus, the system can be used for in-field testing of contaminated samples in the future.

How Toehold Switches Work

Toehold switches are riboregulators that activate translation in response to a distinct RNA sequence. It is comprised of a switch and a trigger. The switch is composed of a hairpin loop structure that represses translation through its complementary bases in between the ribosomal binding site and start codon, which is followed by a 21 nucleotide linker sequence. These sequences ensure that the toehold switch structure will be maintained while coding for low-molecular weight amino acids that would not interfere with the switch’s function. The toehold domains at the beginning of the hairpin are 12 to 18 nucleotides long and are designed to be complementary to the trigger in order to initiate linear RNA binding. The trigger contains complementary sequences to the toehold domain that will bind to the hairpin stem and unbind the loop once they are in the presence of the switch. This exposes the ribosomal binding site and start codon, allowing translation of the reporter protein to occur.

The 2019 Lambert iGEM team utilized Toehold Switches in two aspects: developing a C. elegans switch and characterizing a new switch construct with a weaker promoter.

A toehold switch works by keeping the sequence that is to be expressed, GFP, closed off from potentially being translated. Once a target sequence, the trigger, binds to the toehold, the toehold unwinds, allowing the GFP sequence to be accessed and expressed.

C. elegans Switch

To address the lack of a feasible in-field diagnostic tool for neglected tropical diseases, specifically parasitic worm infections, Lambert iGEM developed a toehold switch with the intention to detect helminths in feces samples. We decided to use C. elegans as a model organism for our proof of concept toehold switch. By using the lin-4 gene pre-mRNA primary transcript F59G1.6 sequence found on WormBank, we designed our toehold switch RNA sequence using NUPACK software and software code provided by Yan Zhang. In addition to gaining the toehold switch sequence, we also obtained a complementary RNA trigger sequence. By inputting the minimum free energy structures for each of the switch and trigger combinations, NUPACK showed the possible structure, allowing us to determine whether or not each switch and trigger would have a high probability of working. The minimum free energy shown below demonstrates the strength of repression for the switch RNA and the single-strandedness of the trigger RNA for the activated complex. A negative ∆GRBS-linker value is correlated to a lower switch dynamic range.

Lambert iGEM obtained BLAST results for C. elegans lin-4 gene pre-mRNA primary transcript F59G1.6 (non-toxic).


NUPACK provided the complementary trigger RNA sequence derived from C. elegans lin-4 gene pre-mRNA primary transcript F59G1.6 (non-toxic)


Additionally, NUPACK calculated optimal toehold switch structure design.


Using NUPACK, we designed DNA sequences for both the C. elegans toehold and trigger inserts. These inserts were then assembled into pSB3C5 and pSB1A3 respectively. After assembly of each part separately, we performed a dual plasmid transformation in order to have the trigger in the presence of the toehold. This should allow for the GFP reporter gene to be expressed.

This graphic displays the design of the C. elegans toehold switch. We inserted this toehold switch into pSB3C5, which is a high-copy plasmid.


Improvement with Promoter Change

In order to induce strong expression of the downstream reporter, the 2018 team used a strong T7 promoter (BBa_J23100) from the Anderson Promoter family. Promoter BBa_J23100 had a reported strength of 1, making it the strongest promoter of the family barring the consensus strain. However, the construct experienced overexpression and induced downstream transcription without the presence of the trigger sequence, leading to “leakiness”.

This table shows the Anderson Series of Promoters with BioBrick name, sequence, and measured strength.


In Lambert iGEM’s 2018 project, the toehold relied on the reporter gene LacZ, a lactose operon encoding the Beta-galactosidase protein (BBa_I732005). When this protein is expressed, it breaks down X-gal into galactose to produce blue pigmentation.

Promoter BBa_J23106 had a measured strength of 0.47, making it a medium strength-promoter, which was optimal for the constraints of the project. The promoter would potentially induce eGFP expression and allow the toehold mechanism to be successful in a dual plasmid transformation with its complementary trigger (BBa_K2550001), while preventing overexpression of the reporter.

Using SnapGene software, promoter BBa_J23106 was inserted in the T7 Promoter Toehold Ribosome Switch with LacZ expression (BBa_K2550000), replacing the BBa_J23100 promoter. In addition, the LacZ Reporter Gene (BBa_K2550201) was identified, deleted, and replaced with eGFP (BBa_E0040) obtained from the iGEM parts registry to create the new parts.

REFERENCES

[1]Alexander A. Green, Pamela A. Silver, James J. Collins, Peng Yin, Toehold Switches: De-Novo-Designed Regulators of Gene Expression, Cell, Volume 159, Issue 4, 2014, Pages 925-939, ISSN 0092-8674, https://doi.org/10.1016/j.cell.2014.10.002

[2]Ausländer, S., & Fussenegger, M. (2014). Toehold gene switches make big footprints. Nature,516(7531), 333-334. doi:10.1038/516333a

[3]Badelt, S., Flamm, C., & Hofacker, I. L. (2016). Computational Design of a Circular RNA with Prionlike Behavior. Artificial Life,22(2), 172-184. doi:10.1162/artl_a_00197

[4]Green, A., Silver, P., Collins, J., & Yin, P. (2014). Toehold Switches: De-Novo-Designed Regulators of Gene Expression. Cell,159(4), 925-939. doi:10.1016/j.cell.2014.10.002