Riboregulators for Precise Control of Gene Expression
Riboregulators of various classes have been developed to allow for the tuning of gene expression. The two classes we have designed are: cis-repressor/trans-activator pairs, and tuned standalone cis-repressors. The constructs have been designed to be universal irrespective of gene sequence. Cis-repressing riboregulators can be placed in front of genes in the genome of a target organism in their native context to regulate expression of the gene from very low to near native levels. Cis-repressors that completely repress expression have also been developed to work with trans-activator RNA (taRNA) elements. Here, specific taRNA sequences target a cis-repressed gene and activate expression to designed levels. Multiple cognate pairs can transcribed from a vector and be used to control multiple genes in a pathway and tune expression. Libraries of taRNA with differing activation profiles can be selected for a particular phenotype. These RNA constructs have been demonstrated to provide very consistent regulation independent of gene sequence with greater than 12 discrete levels available. These constructs allow for the creation of gene pathways with expression tuned to improve growth and productivity of engineered strains. Cis-repressors must be inserted into the genome using standard techniques for genetic manipulations limiting the diversity of regulatory states. Targeting sequences of taRNA/cisRNA pairs must be validated as neutral and specific to ensure consistent activation, also activation is dependent on the concentration and therefore will vary with vector copy number and promoter. Sequences and help with design and implementation are available without restriction.
References and Additional Information:
Krishnamurthy M, Hennelly SP, Dale T, Starkenburg SR, Martí-Arbona R, Fox DT, Twary SN, Sanbonmatsu KY, Unkefer CJ. Tunable Riboregulator Switches for Post-transcriptional Control of Gene Expression. ACS Synth Biol. 2015 Dec 18;4(12):1326-34.
Los Alamos National Laboratory