Optogenetic Control of Gene Expression in Corynebacterium glutamicum

Take a culture of Corynebacterium glutamicum, transformed with a plasmid carrying a fluorescent reporter gene downstream of the ribonucleic acid anti-terminator or RAT sequence, in a multi-well plate.

During transcription, the RAT-RNA forms a stem-loop structure that blocks RNA polymerase, halting reporter gene expression.

The plasmid also constitutively expresses LicV, a fusion protein comprising the RAT-RNA-binding protein, LicT, and the blue-light-sensitive VVD domain.

In darkness, LicV remains monomeric and does not bind to the RAT-RNA stem-loop, which blocks the transcription of the reporter gene.

Illuminate the culture with blue light to induce a conformational change in the VVD domain, which triggers LicV dimerization.

The dimerized LicV binds to the RAT-RNA, unfolding the stem-loop structure and allowing RNA polymerase to transcribe the reporter gene, producing the fluorescent reporter protein.

Observe the culture under a fluorescence microscope.

Strong reporter fluorescence after blue light illumination and minimal fluorescence in darkness confirms successful optogenetic control.