Part:BBa_K4863003
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.
Fig.1 Gel electrophoresis result of colony PCR of organisms transformed with pKeystone005. q.1: Gel electrophoresis result of colony PCR of transformed E. Coli of upstream (US) and downstream (DS) sequences of pKeystone005. 4C.2: Gel electrophoresis result of colony PCR of transformed Synechocystis PCC 6803 of upstream (US) sequences of pKeystone005.
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).
Fig.2 Protein electrophoresis result of SpyTag-Slp complex.
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.
Fig.3: Florescence intensity of supernatant (absorbance wavelength: 488mm; excitation wavelength: 512mm) of Control (no bacteria added), WildType, and SpyTag-Slp.
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.
Fig.4: Florescence observed with WildType (4.1 and 4.2) and SpyTag-Slp expressing Synechocystis PCC 6803 (4.3 and 4.4). Red florescence is chlorophyll in Synechocystis cells excited by green light and green florescence is sfGFP anchored to the cell surface.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 3552
Illegal EcoRI site found at 5532
Illegal XbaI site found at 758
Illegal PstI site found at 874
Illegal PstI site found at 4474 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 3552
Illegal EcoRI site found at 5532
Illegal NheI site found at 1181
Illegal NheI site found at 1278
Illegal PstI site found at 874
Illegal PstI site found at 4474 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 3552
Illegal EcoRI site found at 5532
Illegal BglII site found at 2693
Illegal BamHI site found at 3599
Illegal BamHI site found at 4913
Illegal XhoI site found at 816 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 3552
Illegal EcoRI site found at 5532
Illegal XbaI site found at 758
Illegal PstI site found at 874
Illegal PstI site found at 4474 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 3552
Illegal EcoRI site found at 5532
Illegal XbaI site found at 758
Illegal PstI site found at 874
Illegal PstI site found at 4474
Illegal AgeI site found at 2580
Illegal AgeI site found at 2763
Illegal AgeI site found at 3111
Illegal AgeI site found at 3171
Illegal AgeI site found at 4692 - 1000COMPATIBLE WITH RFC[1000]
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