Toolkit Items

2A is a short viral oligopeptide sequence that mediates a ribosome skipping effect during translation, causing co-translational cleavage of polyproteins. It is used in heterologous co-expression systems to separate proteins of biotechnological interest from a single coding sequence.

The blue-light-activated DNA template ON switch is a light-responsive DNA construct that enables transcription to be initiated by blue light. In the reported combined system, it was used with a separate light-controlled OFF switch to start transcription with one wavelength and later halt translation of the corresponding mRNA-to-protein output with a different wavelength.

CaRTRIDGE is a mammalian synthetic biology framework that repurposes CRISPR-associated proteins as translational modulators. In this system, Cas proteins repress or activate translation of mRNAs carrying a cognate Cas-binding RNA motif in the 5′ untranslated region, and the platform can be combined with other Cas-based regulatory layers.

Click-labelling in this context is a Bacillus subtilis genetic code expansion platform that incorporates noncanonical amino acids for click-chemistry-based protein labelling. In the cited 2021 study, it was implemented within broad and efficient stop-codon suppression systems and used alongside photo-crosslinking and translational titration applications.

cLIPS1 is a photoactivated translation inhibitor built by fusing a segment of 4EBP2 to a circularly permuted Avena sativa LOV2 domain. It binds human eIF4E in a light-dependent manner and inhibits translation in a yeast system engineered to harbor human eIF4E.

cLIPS2 is a light-responsive multi-component switch identified from small libraries of cLIPS1 variants in a higher-throughput yeast screen. It binds human eIF4E in a light-dependent manner in vitro and inhibits translation in vivo in yeast harboring human eIF4E, with improved optical control relative to screened cLIPS1 variants.

The combined EL222 and LOVdeg system is a light-responsive multi-component optogenetic system developed in Escherichia coli by pairing the existing EL222 module with the LOVdeg blue-light-inducible degradation tag. It is intended to enhance optogenetic performance by combining EL222-based control with post-translational light-inducible protein degradation.

Genetic code expansion in Bacillus subtilis is a translation-engineering method for site-specific incorporation of non-standard amino acids into proteins in vivo. The reported system achieved broad and efficient incorporation of 20 distinct non-standard amino acids in B. subtilis using three families of genetic code expansion systems and two codon choices.

GLIMPSe is a generalizable light-modulated protein stabilization system for optogenetic control of intracellular protein abundance independent of the target protein’s intrinsic function. It is presented as a method for light-mediated post-translational stabilization of a wide array of target proteins in live cells.

Human opsins are protein domains used as optogenetic tools in visual restoration strategies. The supplied evidence indicates that applying human opsins can improve light sensitivity and wavelength sensitivity in optogenetic systems, and places these tools within ongoing clinical translation for retinal therapy.

Inteins are internal protein domains that mediate conditional protein splicing as a post-translational control strategy. The supplied evidence describes switchable inteins as being developed to control splicing in ways compatible with applications in living cells.

The light-inducible split Cre recombinase is an optogenetic multi-component switch in which split Cre recombinase fragments are coupled to light-inducible dimerization modules to achieve inducible post-translational control of Cre activity. It was characterized by comprehensive screening of split sites across the Cre protein using a pooled, sequencing-based domain insertion profiling approach.

Long noncoding RNAs are RNA elements with broad regulatory functions in gene expression and cellular activity. The cited review describes lncRNAs as biomolecule-interacting regulators that affect mRNA stability, translational control, splicing, DNA triplex formation, and chromatin organization.

The LOV-intein fusion protein is a light-responsive chimera in which DnaE split inteins flank the Avena sativa LOV2 photosensory domain. It enables blue-light upregulation of intein splicing to control post-translational formation of a functional protein, and this behavior was reported in bacterial and human cells.

LOVdeg is an engineered protein tag that is appended to a protein of interest to enable blue-light-inducible degradation in Escherichia coli. It provides optically controlled post-translational regulation by coupling light exposure to loss of the tagged protein.

The main ORF (mORF) is the protein-coding open reading frame in a transcript whose translation can be repressed by upstream ORFs (uORFs). In Arabidopsis, transcripts initiated from downstream alternative transcription start sites can bypass uORFs and thereby support expression of the mORF, including in blue-light-responsive genes.

Photo-caged mRNA is an mRNA engineering strategy in which small-molecule caging groups are tethered to the 5′ untranslated region to suppress translation until illumination. Photocleavage of the cages activates translation and enables single-cell spatiotemporal control in mammalian cells.

Photo-crosslinking in this context is an application of genetic code expansion in Bacillus subtilis that enables light-triggered covalent capture of molecular interactions. The reported system was part of a broader noncanonical amino acid incorporation platform used for photo-crosslinking, click-labelling, and translational titration.

Phytochrome-based reporters and biosensors are construct designs derived from phytochrome systems for near-infrared sensing applications. They have been described for detecting protein-protein interactions, proteolytic activities, and posttranslational modifications, particularly in contexts relevant to mammalian cells and in vivo use.

The pooled library approach is an engineering method for rapid generation and parallel screening of nearly all possible split-protein constructs, with sequencing-based readout. In the cited application, it was used with optogenetic dimers to comprehensively map split-site behavior across Cre recombinase and support inducible post-translational control design.

Proximity labeling is described here as a methodological approach proposed to define state-specific proteomic and post-translational signatures in studies evaluating the addivosome pathological condensate model. The supplied evidence does not identify a specific proximity-labeling enzyme, chemistry, or construct design.

Switchable inteins are conditional protein-splicing systems in which intein activity is regulated post-translationally to control the state of pre-existing proteins. The cited review specifically discusses strategies for controlling intein activity, with emphasis on approaches intended for use in living cells.

Translational AND gates are artificial mammalian gene circuit elements created by interconnecting Cas-mediated translational switches. They implement combinatorial logic at the level of mRNA translation using Cas proteins that repress or activate transcripts bearing Cas-binding RNA motifs in the 5'-UTR, and a set of 60 such AND gates was reported.

Translational titration is an application of genetic code expansion in Bacillus subtilis that modulates protein production at the level of translation. In the cited study, it was implemented within a broad and efficient noncanonical amino acid incorporation platform that also supported click-labelling and photo-crosslinking.

Upstream open reading frames (uORFs) are endogenous 5′-leader RNA elements that can take precedence over translation of the main ORF and reduce protein output. In Arabidopsis, blue light can increase use of downstream transcription start sites that bypass uORFs, enabling higher expression of light-responsive genes.

Viral vector technology for gene transfer is described as an enabling delivery platform for optogenetic neuromodulation by introducing light sensitivity into target cells. In the cited review, recent advances are reported to substantially reduce vector-associated cytotoxicity and immune responses, supporting possible clinical translation.

This tool is a pair of wavelength-selective photo-cages conjugated to the 5′-UTR of mRNA to suppress translation until illumination. Selective photocleavage with different wavelengths enables sequential optical activation of two distinct mRNAs in the same mammalian cell with single-cell spatiotemporal resolution.