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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 at 585 nm and a peak emission at 615 nm. It originates from a protein isolated from Discosoma sp., a mushroom coral, and it is very stable and resistant to photobleaching. It matures very quickly after transcription, making its detection very quick.

mCherry expression in Bacillus subtilis

The Codon Adaptation Index (CAI) proposed by Sharp and Li (1987) was used to quantify the adaption of FP-encoding genes to the B. subtilis codon usage. For CAI-BSU, codon frequencies were compared to those obtained from the Kazusa codon usage database, which is based on the analysis of all B. subtilis genes regardless of their expression levels. The CAI was calculated using a customized version of the AutoAnnotator created by the iGEM Team TU-Munich (2013). The FP-encoding genes were synthesized by GeneArt® and marked by addition of “BSU” to the protein name (except of GFPmut1, for which the optimized variant is marked by the addition of “LT”, because the LifeTech® codon adaptation algorithm was used). mCherry was codon-optimized for B. subtilis , via that method.

mCherry was used to monitor the secretion of several different signal peptides and screen for best ones. To measure the contributions of mCherry and native fluorescence at 615 nm, wild type and mCherry-cloned B. subtilis were grown at density and then excited at 585 nm to obtain the emission peak amplitudes.

Figure 2: Fluorescence measured at 615 nm, excited at 585 nm, of mCherry-cloned Bacillus subtilis and wild-type (WT) w168 strain, using a plate reader. The mCherry gene was cloned in front of a Pveg promoter (BBa_K823003), a strong constitutive promoter in B. subtilis .

Secretion of mCherry Screen Analysis

Figure 3: Secreted mCherry expression increase . The mCherry gene was cloned in front of a Pveg promoter (BBa_K823003), a strong constitutive promoter in B. subtilis .

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