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Codon-optimized mCherry for Bacillus subtilis

Brief introduction in Fluorescent Proteins

Fluorescent proteins are small proteins with β-barrel-fold topology. They are useful for tracking global expression of target genes and localizations of these genes inside/outside cells. The unique chromophore in each fluorescent protein, originates from three intrinsic amino acids, at positions 65–67. The chromophore is tightly enclosed inside the protein and its formation does not require any cofactors or enzymes but only molecular oxygen. The rigidity of the β-barrel protects the chromophore from the environment and from radiationless decay. It also restricts chromophore flexibility as the correct folding of the protein is required for the chromophore formation. Proper orientation of the amino acids is necessary for chromophore maturation as it catalyzes chromophore synthesis.

Overview of mCherry

mCherry is a red fluorescent protein that has an excitation peak 585 nm and a peak emission at 615 nm. It originates from a protein isolated Discosoma sp., which a mushroom coral, and is very stable and resistant to photobleaching. It matures very quickly after transcription, making detection very quick.

mCherry expression in Bacillus subtilis

For sfGFP, we used the Streptococcus pneumoniae codon adapted version, which was previously described to work best for B. subtilis (Overkamp et al. 2013). sfGFP was used to monitor the secretion of several different signal peptides and screen for best ones. To measure the contributions of sfGFP and native fluorescence at 511 nm, wild type and sfGFP-cloned B. subtilis were grown at density and then excited at 481 nm to obtain the emission peak amplitudes.

Figure 2: Fluorescence measured at 511 nm, excited at 481 nm, of sfGFP-cloned Bacillus subtilis and wild-type (WT) w168 strain, using a plate reader. The sfGFP gene was cloned in front of a Pveg promoter (BBa_K823003), a strong constitutive promoter in B. subtilis .

Secretion of sfGFP Analysis

References:

Overkamp, W. et al. Benchmarking various green fluorescent protein variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for live cell imaging. Appl. Environ. Microbiol. 79, 6481–6490 (2013).



Sequence and Features