Part:BBa_K2114000

PCotYZ-RBS

PCotYZ promoter from Bacillus subtilis with ribosome binding site.

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 while maintaining the BioBrick compatibility. 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 [REF: BioBrickBox] alongside with the PCotYZ-RBS promoter by 3A assembly.

Expression of genes driven by PCotYZ-RBS

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 as described in the methods sectio n.

[Western Blot Image of ]

Additional bands at higher molecular weights were observable and can be attributed to the cross-linking of spore crust proteins responsible for the enormous rigidity and stability of the spores .

II)Localization analysis of displayed proteins 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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]