Part:BBa_K4863002
S-layer protein (Slp)
S-layer protein (Slp) is a native protein on the cell surface of cyanobacteria. It 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 (Trautner and Vermaas, 2013).
Fusion of a protein to the N-terminus of Slp is shown to be an effective system for functional surface display. This year, we display the α-carbonic anhydrase hpCA on the cell surface of Synechocystis PCC6803 via fusion with Slp. We also display the 13 amino acid-long peptide chain SpyTag (BBa_K1159200) on the cell surface of Synechocystis PCC6803 via fusion with Slp, thus achieving surface display of a hpCA-SpyCatcher complex (BBa_K4863000 and BBa_K1159201) via covalent bonding between the displayed SpyTag and the SpyCatcher.
Usage and Biology
Following the potential of cyanobacteria as a cell factory for biofuel or biomaterials, functional surface display of proteins in cyanobacteria is beneficial in many ways: it can allow for better enzyme performance via improved binding with substrate, achieve immobilization of cells, and more. Cyanobacteria have a thick peptidoglycan layer and an additional S-layer, making effective surface display difficult. Slp makes up the extracellular paracrystalline S-layer outside of the cyanobacterial cell wall and is the only component of this outermost layer. Slp has an undefined signal peptide at C-terminus, and fusion of another protein at the N-terminus of Slp would yield a Synechocystis cell completely covered with the fusion protein on the outermost layer and is thus an effective method for surface display (Cengic et al., 2018).
The S-layer of cyanobacteria, which entirely constitutes of the Slp protein, is believed to be involved in carbon sequestration and other physiochemical processes. Slp self-assembles into lattice form via an entropy-driven process in extracellular space(Šmarda et al., 2002). In Synechocystis PCC 6803, the sll1951 gene, which is a 5226bp open reading frame, encodes for the hemolysin-like S-layer protein.
Source
Synechocystis PCC6803
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 Synechocystis 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 3112
Illegal EcoRI site found at 5092
Illegal XbaI site found at 318
Illegal PstI site found at 434
Illegal PstI site found at 4034 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 3112
Illegal EcoRI site found at 5092
Illegal NheI site found at 741
Illegal NheI site found at 838
Illegal PstI site found at 434
Illegal PstI site found at 4034 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 3112
Illegal EcoRI site found at 5092
Illegal BglII site found at 2253
Illegal BamHI site found at 3159
Illegal BamHI site found at 4473
Illegal XhoI site found at 376 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 3112
Illegal EcoRI site found at 5092
Illegal XbaI site found at 318
Illegal PstI site found at 434
Illegal PstI site found at 4034 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 3112
Illegal EcoRI site found at 5092
Illegal XbaI site found at 318
Illegal PstI site found at 434
Illegal PstI site found at 4034
Illegal AgeI site found at 2140
Illegal AgeI site found at 2323
Illegal AgeI site found at 2671
Illegal AgeI site found at 2731
Illegal AgeI site found at 4252 - 1000COMPATIBLE WITH RFC[1000]
Reference
Cengic I, Uhlén M, Hudson EP. (2018). Surface Display of Small Affinity Proteins on Synechocystis sp. Strain PCC 6803 Mediated by Fusion to the Major Type IV Pilin PilA1. Journal of Bacteriology, 200(16). https://doi.org/10.1128/JB.00270-18.
Šmarda, J., Šmajs, D., Komrska, J., & Krzyžánek, V. (2002). S-layers on cell walls of cyanobacteria. Micron, 33(3), 257-277. https://doi.org/10.1016/s0968-4328(01)00031-2.
Trautner, C., & Vermaas, W. F. J. (2013). The sll1951 gene encodes the surface layer protein of synechocystis sp. strain PCC 6803. Journal of Bacteriology, 195(23), 5370-5380. https://doi.org/10.1128/jb.00615-13.
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