Fig. 1
From: A synbio approach for selection of highly expressed gene variants in Gram-positive bacteria
Testing of a translational coupling device in Bacillus subtilis and Lactococcus lactis. a To asses the efficacy of a specific translational coupling device in B. subtilis and L. lactis an mRNA hairpin structure was sandwiched between the gene encoding green fluorescent protein (gfp, green) and the chloramphenicol resistance gene (CmR, orange). Left side: the predicted structure of the translational coupling device. Presumably, the stem of the mRNA hairpin structure consists of 11 nucleotide pairs comprising the stop codon of the upstream gene (red box) and the start codon of the downstream gene (green box). The ribosome binding site (black box) of the downstream gene is designed to be masked by the secondary mRNA structure. Upper right side: when the upstream gene (green, gfp) is not translated, the mRNA hairpin structure will not be resolved and the ribosome binding site of the downstream gene (orange, CmR) remains inaccessible for the ribosome. Therefore, there is no translation of the downstream gene. Lower right side: when a ribosome translates gfp, the ribosome’s helicase activity will melt the secondary mRNA structure which makes the ribosome binding site accessible and the chloramphenicol resistance gene can be translated. The correlation between protein production, determined as fluorescence normalized for cell density, and chloramphenicol resistance, determined as minimal inhibitory concentration (MIC), was determined for genome-based expression in B. subtilis (b) and for plasmid-based expression in L. lactis (c)