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. 2017 Nov 8;7(1):15058.
doi: 10.1038/s41598-017-15107-z.

The Bacillus BioBrick Box 2.0: Expanding the Genetic Toolbox for the Standardized Work With Bacillus Subtilis

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Free PMC article

The Bacillus BioBrick Box 2.0: Expanding the Genetic Toolbox for the Standardized Work With Bacillus Subtilis

Philipp F Popp et al. Sci Rep. .
Free PMC article

Abstract

Standardized and well-characterized genetic building blocks allow the convenient assembly of novel genetic modules and devices, ensuring reusability of parts and reproducibility of experiments. In the first Bacillus subtilis-specific toolbox using the BioBrick standard, we presented integrative vectors, promoters, reporter genes and epitope tags for this Gram-positive model bacterium. With the Bacillus BioBrick Box 2.0, we significantly expand the range of our toolbox by providing new integrative vectors, introducing novel tools for fine-tuning protein expression, and carefully evaluating codon-adapted fluorescence proteins in B. subtilis, which cover the whole spectrum of visible light. Moreover, we developed new reporter systems to allow evaluating the strength of promoters and ribosome binding sites. This well-evaluated extension of our BioBrick-based toolbox increases the accessibility of B. subtilis and will therefore promote the use of this model bacterium and biotechnological workhorse as a host for fundamental and applied Synthetic Biology projects.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
New empty and reporter vectors in RFC10 standard. Red parts indicate features for cloning in E. coli: the bla gene mediating resistance against ampicillin, the origin of replication (ori), and the multiple cloning site (MCS) which contains a gene encoding the red fluorescent protein (RFP) for red/white screening. In blue, B. subtilis-specific parts are depicted: the dark blue arrow represents the resistance cassette. (A,C) The boxes represent flanking homology regions used as integration sites for double homologous recombination into the B. subtilis chromosome. (B) The box represents the B. subtilis-specific origin of replication (ori) and the light blue arrows show the reporter operon luxABCDE encoding the Photorhabdus luminescence luciferase and accompanying enzymes for substrate generation and recycling. (A) The two empty vectors pBS1E and pBS1K integrate into the amyE locus and confer resistance either to macrolide, lincosamide and streptogramin B antibiotics (MLS) if induced by erythromycin (pBS1E, mediated by erm) or kanamycin (pBS1K, mediated by kan), respectively. (B) The first replicative vector in our toolbox, pBS0E, is equipped with ori1030 and confers resistance against MLS. (C) The reporter vectors encode luciferase (luxABCDE), intergrate into the sacA locus and confer resistance either against MLS (pBS3Elux) or kanamycin (pBS3Klux).
Figure 2
Figure 2
Layout and evaluation of the Bacillus BioBrick Box 2.0 expression vectors. (A) Vector maps of the original backbones used to generate the expression vectors, pBS2E integrating into the genomic B. subtilis lacA locus (hence, it is a single copy version) and pBS0E as a multi copy number version. Both vectors confer resistance against MLS (mediated by erm). For the remaining features see legend of Fig. 1. (B) Schematic outline of the MCS including one of the three inducible promoters (PliaI, PxylA, or xylR with PxylA) and their inducing compounds. (C) Observed Miller units of the β-galactosidase assay with strains harboring the original expression vectors and lacZ as reporter gene. Gray and black bars represent non-induced and fully induced samples, respectively. Strains TMB3132, TMB3128, TMB3246, TMB3133 and TMB3245 were grown in MCSE medium at 37°C until mid-exponential growth phase, induced with either 30 µg ml−1 bacitracin or 0.5% xylose, and harvested 30 min or 60 min after induction, respectively. (D) β-galactosidase activities derived from strains harboring the second version of expression vectors (TMB3535 to TMB3537 and TMB3542, TMB3543), using experimental conditions as described in C. (E) DNA sequence of the BioBrick prefix for the original expression vectors and the version 2. The liaI promoter is depicted as example of the upstream promoter. (C,D) Show mean values and standard deviations of at least three biological replicates.
Figure 3
Figure 3
Evaluation of cat-lux vectors for promoter screening. (A) Vector map of cat-lux vectors, which confer resistance either against MLS (mediated by erm, pBS3Ecatlux) or kanamycin (mediated by kan, pBS3Kcatlux). Both vectors harbor the cat-lux reporter system and integrate into the sacA locus. For the remaining vector features see description of Fig. 1. (B,C,D) Four constitutive promoters of decreasing strengths (Pveg, PlepA, PliaG, and PJ23101) were cloned into both cat-lux vectors as well as pBS3Clux, which lacks the cat gene upstream of the luxABCDE cassette. Strains carrying the integrated plasmids (TMB3196-TMB3199 for pBS3Ecatlux, TMB3192-TMB3195 and TMB2940, TMB3090, TMB3212 and TMB3213, respectively) were analyzed for luminescence output (B) MIC of chloramphenicol (C) and the correlation of both values (D). (B) Relative luminescence values normalized to cell density (OD600) in mid-exponential growth phase were measured in a microtiter plate reader for growth in MCSE medium at 37 °C. (C) Determination of chloramphenicol MIC with E-tests® (bioMérieux) on solid MH medium in strains harboring the cat-lux vectors with one of the four different constitutive promoters and the wild-type. Representative pictures and mean MIC values are shown. (D) Correlation between observed MIC values and relative luminescence as shown in B and C, both on logarithmic scale. (B,C,D) Show mean values and (B,D) standard deviations of at least three biological replicates.
Figure 4
Figure 4
Evaluation and layout of the Bacillus BioBrick Box 2.0 RBS evaluation vectors. (A) Vector maps of the two RBS evaluation vectors differing in the reporter (light blue) of either lacZ or the luxABCDE operon. For the remaining features, see legend of Fig. 1. (B) Detailed scheme of the MCS comprising the sites for the insertion of a promoter and the RBS sequence upstream of the respective reporter. The promoter of choice replaces the RFP cassette and the RBS replaces the lacZα fragment, so both cloning steps can be independently selected for by red/white or blue/white screening, respectively. (C) Schematic representation of the design used for evaluation. Two constitutive promoters were chosen (Pveg and PlepA) both followed by one out of five different RBS sequences. The spacer sequence accounts for both vector types followed by the specific reporter on each vector. (D) Observed β-galactosidase activities of strains harboring the pBS1CαlacZ RBS evaluation vector with one of the two constitutive promoters Pveg (dark gray bars) or PlepA (light gray) and the different RBS sequences (1–5) or no inserted RBS (lacZα) (Pveg: TMB3182-TMB3186 and TMB2924, PlepA: TMB3172-TMB3176 and TMB2913). Experimental conditions are described in Fig. 2C. (E) Relative luminescence values divided by OD600 of strains harboring the pBS3Cαlux RBS evaluation vector with one of the two constitutive promoters Pveg (dark gray bars) or PlepA (light gray) and the different RBS sequences (1–5) or no inserted RBS (lacZα) (Pveg: TMB3162-TMB3166 and TMB2762; PlepA: TMB3152-TMB3156 and TMB2761). Experimental conditions are described in Fig. 3B (D,E) show mean values and standard deviations of at least three biological replicates.
Figure 5
Figure 5
Endpoint measurements of the fluorescent proteins contained in the Bacillus BioBrick Box 2.0. Relative fluorescent units normalized by optical density (OD600) are shown for cells with fully induced FP expression (colored bars), compared to the autofluorescence of B. subtilis wild type cells (white bars). Depending on the FP, original as well as optimized versions were tested. The excitation and emission wavelengths are depicted for each FP. Bar graphs represent mean values and standard deviations of at least three biological replicates. The corresponding strains (TMB3909-TMB3921) carry the genes encoding the respective FP (RFC 10) under control of the bacitracin-inducible promoter PliaI. For evaluation of the spectra (Figure S6) and endpoint measurements, cells were grown to exponential phase and expression of the FPs was induced by addition of bacitracin (final concentration 30 µg ml−1) for 75 min to allow proper folding of the FPs. After induction, cells were harvested and washed with PBS and all measurements were performed in a final volume of 200 µl in 96 well plates using a Synergy™ NeoalphaB plate reader.

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