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Codon-adapted 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

In our project, mCherry was decided to be used as a fluorescence marker to monitor the secretion levels generated by several different signal peptides and screen them to find a small group of signal peptides that gave the best tailored secretion. We used the Codon Adaptation Index (CAI-BSU), proposed by Sharp and Li (1987), to codon optimize mCherry for B. subtilis. CAI-BSU 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). Before secreting mCherry, the contributions of mCherry and native fluorescence at 615 nm had to be measured to see if there was any quenching of the mCherry and that the fluorescence of mCherry was significantly greater than in the wild-type strain, with no mCherry expression. By experimentation, wild type (W168) and B. subtilis expressing mCherry were grown at density and then excited at 585 nm to obtain the emission peak amplitude at 615 nm (Figure 2).

Figure 2: Corrected fluorescence, measured at 615 nm and excited at 585 nm, of Bacillus subtilis-expressing mCherry and wild-type (WT) W168 strain, using a plate reader. The mCherry gene was cloned ahead of a Pveg promoter (BBa_K823003), a strong constitutive promoter in B. subtilis.

Screening of mCherry Secretion in B. subtilis

A new evaluation vector (BBa_K2273107) was formulated, to screen for protein specific secretion in B. subtilis of a large variety of signal peptides. The vector was designed for quick detection of successful cloning of a signal peptide into the vector, by exchanging a red fluorescent protein cassette for a signal peptide cassette, causing the loss of red fluorescence in a correct clone. The goal was to create signal peptide toolbox for B. subtilis, a novel contribution to iGEM that will allow users to tailor their secretion of a target protein in B. subtilis to their needs. Uses of this toolbox can be used, for example, with the SpyCatcher/SpyTag system, as the ratio of secretion of two proteins is important for optimal complex formation. For more information of the Signal Peptide Toolbox, click here.

A total of 74 peptides were screened, to characterize their secretion levels of sfGFP, mCherry and other proteins. Below are the peptide secretion screening results for mCherry.

Figure 3: Corrected fluorescence of the supernatant at 615 nm of B. subtilis clones secreting mCherry, from a signal peptide library. mCherry was cloned ahead of a Pxyl promoter (BBa_K823015), a xylose-inducible promoter, and a variety of different peptides. Signal peptides were screened to observe peptides of high, medium, and low secretion of mCherry.

Figure 4: Fluorescence fold increase of the supernatant at 615 nm, over the wild-type (W168 strain) supernatant fluorescene, of B. subtilis mCherry secretion strains of a spectrum of secretion levels. mCherry was cloned ahead of a Pxyl promoter (BBa_K823015), a xylose-inducible promoter, and a variety of different peptides. Signal peptides, LipA, SacC, YkwD and YhcR, had the highest secretion of mCherry over all of the screen peptides, while PelB had a medium level and AmyE had a low secretion level.

Library of Signal Peptides for mCherry Secretion 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