Contribution

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1. The expression of heterologous carbonic anhydrase in cyanobacteria may lead to problems with normal cyanobacterial photosynthesis. Since the cyanobacterial carbonic anhydrase is regulated by the cell, extracellular constitutive expression of heterologous carbonic anhydrase may give rise to peripheral HCO3– imbalance, triggering intracellular carbonic anhydrase expression imbalance and affecting photosynthesis.
2. We found that after installing homology arms of slr0168 integration site, the sequence for carbonic anhydrase was not correctly inserted into the site but was instead inserted into sll1951 site. This indicates that if the length of a single homology arm is too long, after exceeding 5000 bp, the integration efficiency is higher than that of double homology arms of 700 bp. However, the shorter sequence for PilA1 can be correctly integrated into slr0168 site
3. Regardless of using SLP surface display system or PilA1 surface display system, not all cells may express the target protein; the expression may be due to the heterozygous nature of cyanobacteria.
4. While we are constructing the plasmid pKeystone001 (slr0168:PpsbA2_hpCA_SLP), Gibson assembly and Golden gate methods yielded very few transformants; Moreover, the sequencing results of the transformants showed mutations. We therefore speculate that the SLP of Synechocystis PCC6803 is toxic to E. coli. Since the promoter for SLP is the photosensitive promoter PpsbA2 in cyanobacteria, we additionally verified the expression efficiency of PpsbA2 in E. coli.
   

   Fig. 1: Culturing plate of positive transformants.

   
   We replaced the J23102 promoter in pKeystone000 with the photosensitive PpsbA2 promoter to drive the expression of eforRed in E. Coli. We selected 8 positive transformants and transferred them to a new plate for culture. Among them, we selected 4 positive colonies for sequencing, which are respectively named "Angela," "Emma," "Nancy," and "Toby".
   

   Fig. 2: Sequencing results of transformants.

   
   The sequencing results illustrate that there was no mutation occuring in "Angela"; there was no mutation in "Emma"'s promoter sequence, but frameshift mutation occured in the eforRed sequence; an A mutated to C at -145bp in "Nancy"; and a T mutated to G at -93bp in "Toby" along with base depletion at -14bp~-16bp. Mutation occured in the open reading frame of "Emma" so the transformant appears to be in white colour; transformants "Angela," "Nancy," and "Toby" all appears to be in red colour, indicating that PpsbA2 initiated the expression of eforRed. Interestingly, the transformant "Toby" with a 3bp depletion before the RBS has the darkest colour among the three, which may bring a potential idea for modifying PpsbA2 to achieve stronger expression.

Parts

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Documenting new parts

Basic Parts	Part Name	Links
hpCA	BBa_K4863000	http://parts.igem.org/Part:BBa_K4863000
PilA1	BBa_K4863001	http://parts.igem.org/Part:BBa_K4863001
SLP	BBa_K4863002	http://parts.igem.org/Part:BBa_K4863002

Composite Parts	Part Name	Links
PpsbA2_SpyTag_SLP	BBa_K4863003	http://parts.igem.org/Part:BBa_K4863003
PpsbA2_PilA1_SpyTag	BBa_K4863004	http://parts.igem.org/Part:BBa_K4863004
T7_hpCA_SLP	BBa_K4863005	http://parts.igem.org/Part:BBa_K4863005

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    We incorporate the SpyCatcher-SpyTag covalent bonding system originally isolated from Streptococcus pyogenes in our surface display system for displaying hpCA on the cell surface of Synechocystis PCC680. This system is essential for our project since it maximizes the binding of the carbonic anhydrase to substrate while ensuring its stability. SpyTag is a 13-amino acid peptide chain that rapidly forms covalent bond with its 138-residue protein partner, SpyCatcher. This irreversible, spontaneous isopeptide bond formation can be applied, among other applications, for the formation of multi-protein complexes.

    We present a system in which the 13 amino acids-long SpyTag is fused to the native surface-exposed proteins SLP and PilA1 on Synechocystis PCC680 for surface display, and a hpCA-SpyCatcher complex—produced by E. Coli BL21 (DE3) and purified—is mixed with the engineered Synechocystis to bind to SpyTag displayed on the cell surface. Functional surface display of hpCA is successfully achieved this way.

    To test for the functionality of this surface display system, we mix a purified sfGFP-SpyCatcher complex with Synechocystis PCC 6803 expressing the protein complexes SpyTag-SLP and 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 due to sfGFP. Florescence intensity of the supernatant of SpyTag-SLP expressing Synechocystis has significant difference compared to the WildType control and florescence intensity of the supernatant of PilA1-SpyTag expressing Synechocystis show some difference, demonstrating that some sfGFP have binded to the cell surface via bonding between SpyCatcher and SpyTag.

    For further verification, the cells were observed under a 40X florescent microscope. Clear green florescence signals show on the cell surface of Synechocystis expressing SpyTag-SLP and few green florescence signals show on the cell surface of Synechocystis expressing PilA1-SpyTag, verifying successful display of SpyTag on the cell surface and successful fusion of the protein complex to the displayed SpyTag.

    Therefore, we demonstrate successful surface display of SpyTag through fusion to both SLP and PilA1 and show that the SpyTag-SpyCatcher system can be applied for the surface display of a large protein on the cell surface of Synechocystis.

Brick

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    In our drylab, we extracted some carbonic anhydrase from E. coli and used it in the making of our BioStone, and this time we used sodium alginate as thebinding material of our BioStone. We succeeded with this protocol during the first trial, and resulted with a strong and compressed BioStone that has highresemblance to concrete. However, later on, during the next trials, we observed that the resultant BioStone after using the same protocol had an inflated bodyin the center but thin borders on the outside just after soaking in CaCl2·2H2O.It was also considerably soft when we squeezed it in the center.

    Finally, after several more trials, we solved this problem by first making more of high concentration 0.5 molar CaCl2·2H2O and ensuring CaCl2·2H2O will no longer bere-used for different trials. To further solve the problem, we even printed a 3D model to contain the alginate, BG11 medium, and sand matrix. It was a rectangular prism with multiple holes on both bottom and top, so when placed ina CaCl3 solution, the solution will enter the model through the holes andsolidify the alginate and BG11 medium. This model proved to be efficient as it was able to solidify the matrix and also make the shape of the BioStone a perfect rectangle. Then we came up with the final protocol of making BioStone.