Difference between revisions of "Part:BBa K2114000"

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PCotYZ-RBS

PCotYZ promoter from Bacillus subtilis with ribosome binding site.
This part is an improvement of BBa_K823031.

Usage and Biology

The PCotYZ promoter from Bacillus subtilis plays an important role in the sporulation of B. subtilis. The Promoter is located in the cotVWXYZ gene cluster and drives the expression of the late-stage spore crust proteins CotY and CotZ [1]. We included the naturally occurring ribosome binding site for cotZ to the promoter PCotYZ (BBa_K823031) while maintaining the BioBrick compatibility. This enables the expression of genes that are not containing a ribosome binding site. The improved promoter can be used for the 3A assembly with a coding region and cloned into an integration vector. The resulting device enables the transformation of B. subtilis for integration and expression of desirable genes.

Cloning strategy of the promoter improvement. PCotYZ was amplified with primers containing extensions in order to introduce the ribosome binding site. The resulting fragment was digested with XbaI and PstI and ligated into the linearized pSB1C3 vector with the corresponding overhangs.

Characterization

This promoter was used for the expression of fusion constructs in the spores of B. subtilis. The respective construct was assembled into an integration vector [2] alongside with the PCotYZ-RBS promoter by 3A assembly.

Expression of genes driven by PCotYZ-RBS

The promoter PCotYZ-RBS was cloned alongside with BBa_K2114001 into the integration vector pBS1C by 3A assembly. After transformation the cells were selected by chloramphenicol resistance and screened for the disruption of the amyE gene on starch agar plates. Subsequently the positive clones were further cultivated and sporulation was induced by nutrient starvation. The resulting spores were purified from vegetative cells with lysozyme and analyzed by SDS-PAGE and Western blotting. The immunostaining with anti-HA antibodies resulted in the visualization of the expected band at approximately 33 kDa. Additional bands at higher molecular weight were hypothesized to be results from the high cross-linking of spore coat proteins responsible for the enormous rigidity and stability of the spores [3].

Figure 5: Expression analysis of genes regulated by PCotYZ-RBS. The expression of BBa_K2114001 driven by the PCotYZ-RBS promoter resulted in the expected band at 33 kDa. Not transformed spores of B. subtilis were used as controls. As loading control the lysate was analyzed after SDS PAGE and coomassie staining in order to verify the presence of extracted spore coat proteins.

Flow cytometry analysis

The gene expression driven by the PCotYZ-RBS promoter for the display of fusion proteins on the surface of B. subtilis spores was analyzed by flow cytometry. After transformation and induction of sporulation the resulting spores were purified and stained with an anti HA antibody conjugated to AlexaFluor® 647 (Cell Signaling Technology®). The antibody could only access the surface-localized epitopes of the expressed fusion genes and could confirm the successful display of heterologous proteins on the surface of B. subtilis spores.

Figure 6. Flow cytometry analysis of displayed proteins. The expression of the part BBa_K2114002 was regulated by the PCotYZ-RBS promoter resulting in the surface display on spores B. subtilis. The containing HA epitope tag was was stained with anti-HA antibodies conjugated to AlexaFluor 647. Flow cytometry analysis could confirm the display of the fusion proteins.

References

1. Imamura, D., Kuwana, R., Takamatsu, H. & Watabe, K. Proteins involved in formation of the outermost layer of Bacillus subtilis spores. J. Bacteriol. 193, 4075–4080 (2011). 2. Radeck, J. et al. The Bacillus BioBrick Box: generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis. J. Biol. Eng. 7, 29 (2013).
3. Hinc, K., Iwanicki, A. & Obuchowski, M. New stable anchor protein and peptide linker suitable for successful spore surface display in B. subtilis. Microb. Cell Fact. 12, 22 (2013).

Sequence and Features