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Improved Biobricks

pelB_anti-GFPnano body

The discovery of camelid heavy-chain antibodies and their subsequent modification to single-domain antibodies also called nanobodies^1,2 provide researchers with a wide range of tools including labeling methods for imaging, receptor modulation or therapeutic agents. They exhibit a small size of only 15 kDa and their easy structure enables a high-efficiency production in bacterial strains such as E. coli. The anti-GFP nanobody represents an established and well characterized variant of those proteins. In order to directly utilize their functionality, the purification of the over-expressed nanobody provides the advantage of acquiring a highly concentrated protein solution. The BioBrick in the iGEM registry BBa_K929104 contains the full sequence of the anti-GFP nanobody. However, the purification of the nanobody from a bacterial lysate requires its export into the periplasmatic space in order to avoid inclusion bodies and provide an oxidative environment which promotes the formation of the characteristic disulfide bond. The anti-GFP nanobody was provided to us by our supervisor Dr. Maximilian Ulbrich. To avoid the formation of inclusion bodies and increase the yield of purified nanobodies the pelB leader sequence was included at the N-terminal section of the nanobody. This sequence facilitates the export of the protein into the periplasmatic space. The nanobody can be expressed in standard expression vectors such as pGEX or pET in an appropriate bacterial strain.

Characterization

I) Expression analysis

The anti-GFP nanobody was be expressed in pET303 including a C-terminal 10xHis-tag in the E. coli strain BL21 after induction with IPTG for 3 hours at 37 °C. The lysate was purified using nickel columns and analysed by SDS-PAGE and coomassie staining to verifiy the expected size of 17 kDa (figure 2 A). In comparison, the bacterial over-expression of the anti-GFP nanobody without pelB leader sequence results in low yields since a considerable amount of expressed proteins remains in inclusion bodies in the bacterial pellet and therefore it cannot be purified from the bacterial lysate (figure 2 B).

II) Evaluation of binding kinetic

The evaluation of the binding properties of the anti-GFP nanobody was performed with the Octet RED96 system based on biolayer interferometry. The biosensors were loaded with His-tagged GFP at a concentration of 1.25 µg/mL. After baseline adjustment the association rate ka and the dissociation rate kd of the anti-GFP nanobody at a concentration of 4.8 µg/mL were measured. The resulting sensogram contained intereference patterns over time and was analyzed by the supplemented software. The dissociation constant KD was determined at 1.57 nM.

P_CotYZ-RBS

The P_CotYZ 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 CotZ³. 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 a suitable integration vector for B. subtilis. The resulting device enables the transformation of B. subtilis for integration and expression of desirable genes⁴.

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 alongside with the P_CotYZ-RBS promoter by 3A assembly – See assembly of fusion genes.

I) Expression analysis of fusion constructs driven by P_CotYZ-RBS

The promoter P_CotYZ-RBS was cloned alongside with BBa_K214001 into the integration vector pBS1C⁴ 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 as described in the methods sectio n. 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 proteins⁵.

II) Localization analysis of displayed proteins

The gene expression driven by the P_CotYZ-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.