RIBOTOX

Contribution

We contributed in two different aspects, informational and characterisation in silico. In total, we have added significant information for 49 individual registry parts, in the hopes that future teams will be able to benefit from better part documentation.

Informational contributions

We investigated 26 parts on the registry’s main pages which were poorly documented, before utilising resources such as UniProt and other existing scientific databases to add information to help potential future users. Illustrated schematics from these sources were also included where relevant. This is the full list of parts we contributed information to:

Parts (Contributors):

Part	Contributor
BBa_K3317053	Lucas (Ng)
BBa_K108020	Lucas
BBa_K1500994	Lucas
BBa_K2142004	Lucas
BBa_M1437	Lucas
BBa_K116601	Julian (Chen)
BBa_I712020	Julian
BBa_K112001	Julian
BBa_K108022	Julian
BBa_K108024	Haotong (Xiong)
BBa_K174000	Haotong
BBa_K137067	Lucas
BBa_K137000	Lucas
BBa_K1044003	Janusan (Jeyananthan)
BBa_K137009	Janusan
BBa_K137006	Janusan
BBa_I723025	Janusan
BBa_I723024	Janusan
BBa_I716152	Janusan
BBa_T9150	Lucas
BBa_K302010	Janusan
BBa_K108021	Lucas
BBa_K091001	Lucas
BBa_K1486000	Julian
BBa_K864204	Lucas
BBa_J07006	Lucas

Characterisation in silico

Our software package ToeholdTools provides the novel ability to rapidly test the specificity of toehold switches to their target RNA. To demonstrate its utility, we’ve tested 23 toehold switches from previous teams. The script we wrote for this can be found here and was run in the cloud via GitHub Actions.

To do this we designed the specification for TOML configuration files which work with the script we used to run these simulations. This enables future teams to replicate our results and also to easily test the parts of other toehold switch teams. More information on the specifics of that can be found in our GitHub repository.

The following parts were analysed:

CLSB 2017

BBa_K2206000
BBa_K2206012
BBa_K2206001
BBa_K2206010

SASTRA Thanjavur 2019

BBa_K3154000
BBa_K3154003

CSMU Taiwan 2020

BBa_K3431001
BBa_K3431005
BBa_K3431033
BBa_K3431006
BBa_K3431034
BBa_K3431002
BBa_K3431003
BBa_K3431004
BBa_K3431032
BBa_K3431011
BBa_K3431035
BBa_K3431012
BBa_K3431036
BBa_K3431013
BBa_K3431037
BBa_K3431014
BBa_K3431038

One pertinent example was the 2017 team CLSB’s first series toehold switch detecting hsa-miR-15b-5p. Concordantly with their experimental results, we found that when their toehold switch is in solution with this miRNA at the intended usage temperature of 37°C, the switch has a target miRNA specificity of 81 ± 30 % (95% confidence limits), and a relatively high activation level.

Interestingly, their other switches did not show this level of specificity to their respective target RNAs, which could have manifested itself in the difficulties they had with miRNA homologs. Their second series for hsa-miR-27b-3p was an improvement on its first series version, but it did not come close to beating their first series switch for hsa-miR-15b-5p.