Difference between revisions of "Part:BBa K4863003"

Revision as of 12:31, 11 October 2023

PpsbA2_SpyTag_Slp

A SpyTag-Slp protein complex driven by a light-inducible promoter. SpyTag (BBa_K1159200) binds to the N-terminus of surface layer protein (Slp:BBa_K4863002) of Synechocystis PCC 6803 to be displayed on the cell surface of the engineered organism.

Usage and Biology

Slp is a native surface exposed protein that constitutes of the S-layer on the outermost surface of Synechocystis sp.. Fusion of a recombinant protein to Slp will yield a cell completely covered with the fusion protein on the outermost surface. SpyTag is suitable for fusion with Slp since fusion of larger proteins with Slp will likely impede growth of the cell as we have learned. Via the displaying SpyTag, a protein complex of SpyCatcher fused with a large protein can be displayed on the cell surface of engineered Synechocystis via covalent bonding between SpyTag and SpyCatcher.

SpyTag is a 13 amino acid-long peptide chain originally isolated from Streptococcus pyogenes. Slp is encoded by the gene sll1951 in Synechocystis PCC6803. This protein forms a lattice that is about 30nm wide that covers the cell surface, forming the paracrystalline S-layer. It has an undefined signal peptide at C-terminus.

Characterization

We designed a plasmid, pKeystone005, for expression of a SpyTag-Slp complex with SpyTag fused to the N-terminus of Slp, controlled by the light inducible promoter PpsbA2.

We transformed the plasmid into E. coli DH5𝛼 for plasmid amplification in large quantities. The gel electrophoresis of the colony PCR product of the transformed strain indicates successful construction of the plasmid (figure 1.1).

The plasmid was then transformed into Synechocystis PCC 6803, leading to the integration of exogenous DNA at the neutral site sll0168 and production of the SpyTag-Slp complex. The engineered organism will have all of s-layer displaying the fusion peptide. Correct sequence length was obtained from colony PCR of transformed Synechocystis as seen in figure 1.2.

The protein complex SpyTag-Slp was extracted and purified from engineered <it>Synechocystis</it> PCC 6803. Protein electrophoresis result show correct protein weight at 180.4 kDA, verifying success of expression (figure 2).

A purified sfGFP-SpyCatcher complex was mixed with engineered Synechocystis PCC 6803 expressing the protein complex SpyTag-Slp. If SpyTag is successfully displayed, the sfGFP-SpyCatcher complex will fuse with the surface displayed SpyTag via covalent bonding and the organism will obtain green florescence from sfGFP. The engineered Synechocystis PCC 6803 was immersed in purified solution of sfGFP-SpyCatcher overnight on a shaker, and florescence intensity of the supernatant was measured. Florescence intensity of the supernatant of SpyTag-Slp expressing Synechocystis show significant difference compared to Control and WildType, verifying feasibility of surface displaying a larger protein.

To further verify the binding of sfGFP-SpyCatcher complex to the cell surface, the cells were observed under a florescent microscope. Observation through a 40X florescence microscope (Laica) indicate clear green florescence signals on the cell surface of Synechocystis expressing SpyTag-Slp, demonstrating successful surface display of SpyTag through fusion to Slp.

Sequence and Features