Difference between revisions of "Part:BBa K4863004"

 
 
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<partinfo>BBa_K4863004 short</partinfo>
 
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A PilA1-SpyTag complex with SpyTag fused to the C-terminus of PilA1 is constructed to achieve surface display of SpyTag on the cell surface of <i>Synechocystis</i> PCC 6803.  
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A PilA1-SpyTag complex with SpyTag(<partinfo>K1159200</partinfo>) fused to the C-terminus of PilA1(<partinfo>K4863001</partinfo>), driven by a light inducible promoter, is constructed to achieve surface display of SpyTag on the cell surface of <i>Synechocystis</i> PCC 6803.  
  
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===Usage and Biology===
 
===Usage and Biology===
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Fusion of a recombinant protein to the C-terminus of PilA1 will yield a cell covered with the fusion protein on its Type IV pili. Only small affinity proteins (<6.5kDA) can be functionally displayed via this system, or else the fusion protein would interfere with the biological functions of the pili. We present a system where SpyTag is modified to display on the cell surface of <i>Synechocystis</i> via fusion with PilA1.
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PilA1 is a major protein subunit in Type IV pili (T4P), which is a surface-exposed appendage responsible for various essential functions in cyanobacteria,formed by polymerization of several pilin monomers. PilA1 constitutes the major pilin, which is the major structural component of T4P necessary for its biogenesis. SpyTag is a 13 amino acid-long peptide chain originally isolated from <i>Streptococcus pyogenes</i>.
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===Characterization===
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<p>We designed a plasmid for expression of a PilA1-SpyTag complex with SpyTag fused to the C-terminus of PilA1, controlled by the light inducible promoter PpsbA2.</p>
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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).
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</p>
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The plasmid was then transformed into <i>Synechocystis</i> 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 <i>Synechocystis</i> as seen in figure 1.2.
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<img src="https://static.igem.wiki/teams/4863/wiki/bba-k4863001-figure1.png" width="500"/>
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<p style="font-size: smaller; margin-top: 10px;"> Fig.1: Gel electrophoresis result of colony PCR of PilA1-SpyTag. 1.1: Gel electrophoresis result of colony PCR of transformed <i>E. Coli</i> of upstream (US) and downstream (DS) sequences of pKeystone007. 1.2: Gel electrophoresis result of colony PCR of transformed <i>Synechocystis</i> PCC 6803 of upstream (US) sequences of pKeystone007. </p>
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Extracted and purified from engineered <i>Synechocystis</i> 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.
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<img src="https://static.igem.wiki/teams/4863/wiki/bba-k4863001-figure2.png" width="500"/>
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<div align="center">
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<p style="font-size: smaller; margin-top: 10px;"> Fig. 2: Protein electrophoresis result. </p>
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A purified sfGFP-SpyCatcher complex was mixed with engineered <i>Synechocystis</i> 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 obtain green florescence from sfGFP. The engineered <i>Synechocystis</i> 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 <i>Synechocystis</i> show difference compared to Control and WildType, primarily verifying feasibility of surface displaying a larger protein.
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<img src="https://static.igem.wiki/teams/4863/wiki/bba-k4863001-figure3.png" width="500"/>
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<div align="center">
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<p style="font-size: smaller; margin-top: 10px;"> Fig.3: Florescence intensity of supernatant (absorbance wavelength: 488mm; excitation wavelength: 512mm) of Control (no bacteria added), WildType, and PilA1-SpyTag.</p>
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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 <i>Synechocystis</i> expressing PilA1-SpyTag, demonstrating successful surface display of SpyTag through fusion to PilA1.
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<img src="https://static.igem.wiki/teams/4863/wiki/bba-k4863001-figure4.png" width="500"/>
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<p style="font-size: smaller; margin-top: 10px;"> Fig.4: Florescence observed with WildType (4.1 and 4.2) and PilA1-SpyTag expressing <i>Synechocystis</i> PCC 6803 (4.3 and 4.4). Red florescence is chlorophyll in <i>Synechocystis</i> cells excited by green light and green florescence is sfGFP anchored to the cell surface. </p>
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Latest revision as of 10:06, 11 October 2023


PpsbA2_PilA1_SpyTag

A PilA1-SpyTag complex with SpyTag(BBa_K1159200) fused to the C-terminus of PilA1(BBa_K4863001), driven by a light inducible promoter, is constructed to achieve surface display of SpyTag on the cell surface of Synechocystis PCC 6803.

Usage and Biology

Fusion of a recombinant protein to the C-terminus of PilA1 will yield a cell covered with the fusion protein on its Type IV pili. Only small affinity proteins (<6.5kDA) can be functionally displayed via this system, or else the fusion protein would interfere with the biological functions of the pili. We present a system where SpyTag is modified to display on the cell surface of Synechocystis via fusion with PilA1.

PilA1 is a major protein subunit in Type IV pili (T4P), which is a surface-exposed appendage responsible for various essential functions in cyanobacteria,formed by polymerization of several pilin monomers. PilA1 constitutes the major pilin, which is the major structural component of T4P necessary for its biogenesis. SpyTag is a 13 amino acid-long peptide chain originally isolated from Streptococcus pyogenes.

Characterization

We designed a plasmid 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 PilA1-SpyTag. 1.1: Gel electrophoresis result of colony PCR of transformed E. Coli of upstream (US) and downstream (DS) sequences of pKeystone007. 1.2: Gel electrophoresis result of colony PCR of transformed Synechocystis PCC 6803 of upstream (US) 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 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]