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Revision as of 12:29, 11 October 2023


PilA1

PilA1 is a native protein in cyanobacteria. It is a major protein subunit in Type IV pili (T4P), which is a surface-exposed structure responsible for various essential functions in cyanobacteria, including and not limited to mobility, DNA uptake, and protein secretion. T4P is formed by polymerization of several pilin monomers from intracellular space, which are held together via hydrophobic interactions in the core of the pilin filament. PilA1 constitutes the major pilin, which is the major structural component of T4P necessary for its biogenesis. PilA1 has been shown to act as a carrier protein for the surface display of small affinity proteins in Synechocystis sp. strain PCC 6803 via C-terminal fusion and this surface display system is shown to be stable and effective. However, only small affinity proteins less than or equivalent to 6.5 kDA can be functionally displayed, since displaying larger proteins may lead to incorrect folding of the pilus (Cengic et al., 2018).

This year, we display the 13-amino acid peptide SpyTag (BBa_K1159200) on the cell surface of Synechocystis PCC 6803 via fusion with PilA1, and thus achieve surface display of a hpCA-SpyCatcher complex (BBa_K4863000 and BBa_K1159201) with PilA1. Other teams can use this part for the surface display of small affinity proteins.

Usage and Biology

Following the potential of cyanobacteria as a cell factory for biofuel or biomaterials, functional surface display of proteins on 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. PilA1, which is the major type IV pilus subunit, can offer effective surface display since it is a polymeric structure that is exposed on the cell surface. Cengic et al. show that fusion to C-terminus of PilA1 is a highly stable and effective surface display system for small affinity proteins. We utilize this system for surface display of SpyTag (BBa_K1159200).

Type IV pili is a surface appendage of cyanobacteria that is necessary for mobility, DNA uptake, and more. PilA1 is a native cyanobacterial protein that is encoded by the gene sll1694 and constitutes the major pilin subunit of Type IV pili. Polymers of PilA1 form the main structure and is essential for the biogenesis of Type IV pili. In the assembly of Type IV pili, the PilA1 protein is first produced as a precursor, and is then modified and assembled via a complex ATP-driven machinery (Schuergers and Wilde, 2015).

Source

Synechocystis PCC 6803

Characterization

We designed a plasmid, pKeystone007, for expression of a PilA1-SpyTag complex with SpyTag fused to the C-terminus of PilA1, 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 PilA1-SpyTag complex. The engineered organism will have all Type IV pili fused with SpyTag. 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 organism transformed with pKeystone007. 1.1: Gel electrophoresis result of colony PCR of transformed E. Coli sequences of pKeystone007. 1.2: Gel electrophoresis result of colony PCR of transformed Synechocystis PCC 6803 of sequences of pKeystone007.


Extracted and purified from engineered Synechocystis PCC 6803, the PilA1-SpyTag protein complex did not show on the protein electrophoresis result, possibly because the total amount of expression of the fusion protein is below the level for detection.

Fig. 2: Protein electrophoresis result.


A purified sfGFP-SpyCatcher complex was mixed with engineered Synechocystis PCC 6803 expressing the protein complex PilA1-SpyTag. 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 PilA1-SpyTag expressing Synechocystis show difference compared to Control and WildType, primarily 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 PilA1-SpyTag.

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) show few green florescence signals on the cell surface of Synechocystis expressing PilA1-SpyTag, demonstrating successful surface display of SpyTag through fusion to PilA1.

Fig.4: Florescence observed with WildType (4.1 and 4.2) and PilA1-SpyTag 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


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]

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.

Melville, S., & Craig, L. (2013). Type IV pili in gram-positive bacteria. Microbiology and Molecular Biology Reviews, 77(3), 323-341. https://doi.org/10.1128/mmbr.00063-12.

Schuergers, N., & Wilde, A. (2015). Appendages of the cyanobacterial cell. Life, 5(1), 700-715. https://doi.org/10.3390/life5010700.