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Codon-optimized super folder GFP (sfGFP) 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 sfGFP

A mutant of the wild-type green fluorescent protein from Aequorea victoria, called super folder GFP (sfGFP), is a new and robust derivation, designed for in vivo high performance analysis of protein expression levels. It demonstrates increased stability at higher temperatures and is able to tolerate protein tagging to poorly folding proteins while still maintaining fluorescence. It contains the important S65T mutation and F64L for red shift and folding respectively while it also has six additional mutations for enhanced folding: S30R, Y39N, N105T, Y145F, I171V and A206V. The absorption peak is at 480 nm while its emission peak is around 510 nm (Stephanenko OV et al. 2012).

sfGFP expression in Bacillus subtilis

In our project, sfGFP 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 Streptococcus pneumoniae codon adapted version, which was previously described to work best for B. subtilis, according to the literature (Overkamp et al. 2013). Before secreting sfGFP, the contributions of sfGFP and native fluorescence at 511 nm had to be measured to see if there was any quenching of the sfGFP and that the fluorescence of sfGFP was significantly greater than in the wild-type strain, with no sfGFP expression. By experimentation, wild type (W168) and B. subtilis expressing sfGFP were grown at density and then excited at 481 nm to obtain the emission peak amplitude at 511 nm (Figure 1).

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

Secretion of sfGFP Analysis

A new evaluation vector (BBa_K2273107) was formulated, to help reduce time in cloning, 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 incorporating a RFP gene that when a signal peptide is inserted, which will cause loss of red fluorescence. 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 sfGFP.

Figure 3: Normalized fluorescence of the supernatant at 511 nm of a variety of signal peptide expressing B. subtilis sfGFP strains and the wild-type (W168 strain) supernatant. sfGFP was cloned in front of a Pxyl promoter (BBa_K823015), a xylose-inducable promoter, and a variety of different peptides. Signal peptides were screened to observe peptides of high, medium and low secretion of sfGFP.

Figure 4: Fluorescence fold increase of the supernatant at 511 nm of sequenced secreting B. subtilis SP-sfGFP strains, over the wild-type (W168 strain) supernatant. sfGFP was cloned in front of a Pxyl promoter (BBa_K823015), a xylose-inducable promoter, and a variety of different peptides. Signal peptides, PenP and AmyE, had the highest secretion of sfGFP over all of the screen peptides, while BglS had low secretion.

Table of Sequenced Signal Peptides for sfGFP

References:

Stepanenko OV et al.. Distinct Effects of Guanidine Thiocyanate on the Structure of Superfolder GFP. PLoS ONE. 7(11): e48809 (2012).

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