Engineering Success

Introduction

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    The earthquake in Turkey this year has given us the idea of using synthetic biology to assist in post-disaster reconstruction. Surprisingly, after conducting literature and social research, we have found that this seemingly far-fetched idea is actually feasible and has practical value.

    In our initial concept, we propose using drought-resistant algae to express extracellular carbonic anhydrase, which can promote the precipitation of calcium carbonate(CA), similar to the formation of natural calcite. This approach aims to produce renewable bio-concrete that not only has negative carbon economic value but also possesses self-healing capabilities. When cracks form, the drought-resistant cyanobacteria hidden within the concrete will be activated by air and water, releasing carbonic anhydrase to absorb carbon dioxide and fill the cracks through a precipitation process, thus meeting the requirements of sustainable development (Heveran et al., 2020).

    During our interview with Dr. Chen, we learned that gene editing technology for drought-resistant algae is still immature. Based on this, we suggest using Synechocystis to display CA on its surface. This approach not only protects carbonic anhydrase from deactivation (Zhu et al., 2021). but also avoids the accumulation of calcium carbonate inside the cyanobacterial cells (Sundaram et al., 2017).

Carbonic Anhydrase Characterization

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    We used the pET vector to induce the expression of hpCA. All vector backbones used were provided by our instructor. hpCA is derived from the alpha-carbonic anhydrase of Helicobacter pylori 26695 and has been successfully expressed in Escherichia coli (Chirica et al., 2002). hpCA was synthesized by Suzhou Atantares Co., Ltd., and connected to the pET vector using Gibson assembly (Fig 2A).

    The pKeystone014 plasmid was constructed in DH5α, and after verifying the presence of the plasmid (Fig 2B), it was transformed into BL21. The transformed cells were then cultured at 37℃ until reaching an OD600 of 0.5. Expression was successfully induced by adding IPTG to a final concentration of 0.2mM and ZnSO4 to a final concentration of 0.5mM (Fig 2C).

    Subsequently, we used the lysed bacterial solution to measure the esterase activity of carbonic anhydrase. The alpha-carbonic anhydrase can catalyze the conversion of ethyl acetate to p-nitrophenol (Jean-Yves Winum & Colinas, 2015). By monitoring the rate of increase in absorbance at 405 nm, we can assess the carbonic anhydrase activity. We observed a significant esterase activity with hpCA (Fig. 4D). Compared to the control reaction mixture, the absorbance of carbonic anhydrase at 405nm increases 3.8-fold within 5 minutes of incubation, demonstrating the substantive catalytic activity of our carbonic anhydrase.

Display of hpCA on the S-layer of PCC6803

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    After the preliminary validation of hpCA's functionality, we plan to display hpCA on the surface of PCC6803 cells. Therefore, we have selected the S-layer protein (SLP) outside of PCC6803 as an anchor (Trautner & Vermaas, 2013). It is encoded by sll1951 and has been confirmed to form a honeycomb-like structure approximately 30 nm thick on the surface of PCC6803 (Smarda et al., 2002). It has a potential signal peptide at the C-terminus, allowing us to fuse hpCA to the N-terminus of SLP.

    To achieve this goal, we first used Gibson assembly to integrate the upstream and downstream 700bp fragments from the neutral site of slr0168 in PCC6803 into the J23102 vector, which contains a chloramphenicol resistance marker. We named this plamisd pKeystone000.

    Subsequently, we used OE PCR to connect PpsbA2 from PCC6803, codon-optimized hpCA, and a flexible linker (GSSGSSS). We then assembled this construct with sll1951 and pKeystone000 using Gibson assembly. The newly constructed plasmid was named pKeystone001 (Fig. 3A).

    However, the number of colonies on the Gibson transformation plate of pKeystone001 was very low. After identifying the colonies (Fig. 3B), we sent the positive colonies for sequencing and found that both PpsbA2 and SLP had mutations. Therefore, we suspected that constitutive expression of PpsbA2 in Escherichia coli caused toxic effects on E. coli, resulting in mutations in both PpsbA2 and SLP (see Contribution section for details). We selected a strain of E. coli with minimal impact and extracted the plasmid from it, which had a mutation at position -128bp of PpsbA2 (A→G) and a mutation at residue 1186 of SLP (Asp→Asn). We then naturally transformed this plasmid into PCC6803. Our original plan was to recombine hpca-SLP into the slr0168 site through colony PCR screening. However, although we obtained several positive algae strains through screening, we could only obtain an upstream fragment of 1850 bp through PCR (Fig. 3C), and we were unable to obtain the downstream fragment of 769 bp (results not shown). Therefore, it is possible that hpCA has integrated into the sll1951 site (see Contribution section for details).

    After several rounds of subculturing, the growth of the transformants on plates deteriorated, the bacteria appeared yellow-green in color (results not shown). When we inoculated them into liquid BG11 medium containing chloramphenicol, the overall color of the culture turned yellow compared to the wild type (Fig. 3D), and this macroscopic observation was consistent with microscopic observations (Fig. 3E). Subsequently, we separated the cells from the culture medium and lysed them separately. We then performed 6% SDS-PAGE gel electrophoresis after ultrafiltration. The results showed that the fusion proteins SLP-hpCA were present in the culture medium, which is consistent with previous studies(Fig.3F). We speculated that constitutive expression of hpCA affected the photosynthetic activity of the cells (see Contribution section for details). However, we did not detect carbonic anhydrase activity in the culture medium, which could be due to protein folding or low enzyme concentration leading to reduced hpCA enzymatic activity.

Cyanobacteria surface display based on SpyTag-SpyCatcher

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    Since the fusion expression of SLP and hpCA did not yield satisfactory results, we turned our attention to the pili of cyanobacteria. PilA1 is the major pili protein on the surface of cyanobacteria, with its signal peptide located at the N-terminus (Melville & Craig, 2013). We were able to fuse the target protein to the C-terminus of PilA1. However, PilA1 cannot display proteins larger than 6.5 kDa (Cengic et al., 2018). Therefore, we introduced the SpyTag-SpyCatcher system, where a small Spytag is displayed on PilA1, and hpCA is displayed by binding it with SpyCatcher fused to hpCA. Consequently, we designed two new vectors, pKeystone005 and pKeystone007 (Fig. 4A). In pKeystone005, hpCA of pKeystone001 was replaced with SpyTag as a control, while in pKeystone007, spytag_slp of pKeystone005 was replaced with pilA1_spytag.

    Plasmid pKeystone005 was constructed by first using OE PCR to connect PpsbA2, spytag, and a flexible linker. It was then assembled with pKeystone000 and SLP using Golden Gate cloning. On the other hand, Plasmid pKeystone007 was constructed by first using OE PCR to connect PpsbA2, PilA1, a flexible linker, and spytag. Subsequently, it was assembled with pKeystone000. Both constructs underwent colony PCR (Fig. 4B) and sequencing verification. The sequencing results confirmed that pKeystone007 did not have any mutations, while pKeystone005 exhibited the same mutation as observed in pKeystone001 (results not shown). Consequently, we selected a strain of cyanobacteria that had no mutations in PpsbA2 but had 5 AA mutations in SLP to extract plasmids. These plasmids were then used to transform the cyanobacteria with pKeystone005 and pKeystone007.

    Similar to pKeystone001, colony PCR analysis of PCC6803 transformed with pKeystone005 only detected the upstream region of 1214 bp (Fig. 4C), while the downstream region was not detected (results not shown). Sequencing results revealed that a reversion mutation had occurred in the plasmid-borne SLP, confirming our hypothesis that the insertion site was in SLP. On the other hand, the strain transformed with pKeystone007 showed detection of the full-length sequence, and sequencing results indicated no mutations.

    To validate the surface display effect, we used E. coli to express sfGFP with SpyCatcher . We constructed a plasmid named pKeystone012, which contained sfGFP_spycatcher driven by J23102 promoter, using Gibson assembly (Fig. 4A). After colony PCR (Fig. 4B) and sequencing verification, we cultured the transformed cells and purified sfGFP-SpyCatcher using a nickel column(Fig. 4D).

    Subsequently, after passaging them on agar plates, we inoculated the transformed cells into liquid BG11 medium containing chloramphenicol. The cultures were grown under light conditions until they reached mid-log phase. The supernatant and cells were then separated by centrifugation and subjected to ultrafiltration and sonication. Next, 10% SDS-PAGE gel electrophoresis was performed. The target bands of SpyTag-SLP appeared in the cell lysate, but PilA1-SpyTag was not detected in either the culture medium or the cell lysate(Fig. 4D).

    Next, we mixed the purified sfGFP with the cyanobacteria that express SpyTag-SLP (6803:spytag_slp) and PilA1-SpyTag (6803: pilA1_spytag), as well as the wild-type strain, in a 1:1 ratio. The mixture was incubated at 30 degrees Celsius for 30 minutes. After centrifugation, the culture medium was separated from the cells. The absorbance of the culture medium at 512 nm was measured, and the cells were washed twice with 20% Tris-HCl. Finally, the cells were observed under a fluorescence microscope.

    The measurement of A512 in the culture medium showed that the fluorescence protein content in the supernatant of 6803:spytag_slp and 6803:pilA1_spytag was lower than that of the wild-type strain (WT). Notably, there was a significant difference in the fluorescence protein content in the supernatant of 6803:spytag_slp, indicating that the cells with spytag absorbed some of the fluorescent proteins present in the culture medium, resulting in a decrease in fluorescence signal in the culture medium(Fig 4E).

    Using both green and blue light excitation, we observed the cyanobacteria. Green light excites the chlorophyll in cyanobacteria, resulting in a red color, while blue light excites sfGFP-SpyCatcher , leading to a green color. We were able to observe green fluorescence on the cells of 6803:spytag_slp and 6803:pilA1_spytag, but not on the wild-type strain (WT) (Fig. 4F). This indicates that we successfully achieved protein display outside the cyanobacterial cells using SpyTag-SpyCatcher system. However, not all cells were able to display sfGFP-SpyCatcher, which may be due to our transformation of a heterozygous strain where not all cells express spytag. In comparison to 6803:spytag_slp, 6803:pilA1_spytag showed lower display efficiency, possibly due to low expression levels, consistent with the absence of the target band in protein electrophoresis (Fig. 4D).

CA-SpyCatcher Characterization

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    After validating that SpyTag-SpyCatcher can be used as a tool for surface display in cyanobacterial cells, we proceeded to design the expression plasmid for hpCA-SpyCatcher , named pKeystone013 (Fig. 5A) (Kang & Baker, 2011). We constructed the plasmid in DH5α by using Gibson assembly to assemble hpCA-SpyCatcher with the pET vector backbone. After colony PCR and sequencing verification (Fig. 5B), we extracted the plasmid and transformed it into BL21 expression strain. Following the same method as with hpCA, we induced the expression of hpCA-SpyCatcher . The results of 10% SDS-PAGE gel electrophoresis showed successful induction of hpCA-SpyCatcher expression(Fig. 5C).

    Subsequently, we used the same method to measure the carbonic anhydrase (CA) activity of hpCA-SpyCatcher . The A405 absorbance in the supernatant of the pKeystone013 strain increased by 2.9-fold within 5 minutes compared to the control (Fig. 5D). This indicates that CA retains its activity even after the addition of SpyCatcher .

    Finally, we added the dialyzed hpCA-SpyCatcher (~3 mL) to a 100 mL water solution of 0.5 M CaCl2 and let it stand overnight. The next day, we observed the presence of cloudiness in the solution to which hpCA-SpyCatcher was added, while the control group showed no cloudiness(Fig. 5E). This indicates that hpCA-SpyCatcher may have facilitated the precipitation of calcium carbonate.

Algae Brick

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    Before using aggregated Synechocystis PCC6803 with surface-displayed hpCA, we first explored a method for producing biobricks using aggregated engineered PCC6803 , purified carbonic anhydrase (hpCA), and sand. We used the wild-type PCC6803 strain and hpCA purified from pKeystone014. We collected beach sand from Dameisha Beach in Shenzhen as raw material to create "algal bricks".

    At first, we suggested two candidates as the binding material of our BioStone, which are gelatin system and sodium alginate system, and both systems could pass the preliminary experiment and they could successfully form the gel. However, after we put the gelatin system into the model, it displayed unsatisfactory hardness, and it appears gelatinous and even breaks when external force is applied. The failure of the first experiment suggests that the methodology of adding the different ingredients still need to be modified. Also, since carbonic anhydrase is missing in current experiment, the efficiency of artificial calcium carbonate precipitation is not ideal and hence the amount of calcium carbonate produced might be insufficient to provide significant structural enhancement to the BioStone.

    Therefore, we extracted some aqueous carbonic anhydrase from E. coli and used it in the making of our BioStone, and this time we used sodium alginate as the binding material of our BioStone. We succeeded with this protocol during the first trial, and resulted with a strong and compressed BioStone that has high resemblance to concrete. However, later on, during the next trials, we observed that the resultant BioStone after using the same protocol had an inflated body in 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 be re-used for different trials (Fig. 6A).

    Trying to figure out how to fit the brick into the rectangular shape, we have decided to use 3D modelling n our first mold, but we then experienced a leaking problem. Because the mold is made of two parts, most of the fluid is already leaked out from the mold before it solidify into a fixed shape. In the second version, it was a rectangular prism with multiple holes on both bottom and top, so when placed in a CaCl2 solution, the solution will enter the model through the holes and solidify the alginate and BG11 medium. In this new mold, we fixed the issue by making it into one single compartment where leaking won’t happen. In such way, the BioStone have formed in the mold and have been taken out easily (Fig. 6B), and this model was used in our final protocol.

Summary

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    Our engineering process showcases our path towards creating the sustainable building material that reduces environmental impact during production, seals cracks in building structures, and recycles existing building residues. Up until now, we have:

• Verified the ability of carbonic anhydrase (hpCA-SpyCatcher ) to induce calcium carbonate precipitation
• Achieved protein surface display on Synechocystis PCC6803 via the SpyTag-SpyCatcher system
• Invented the method of making BioStone (our building material product) using the sodium alginate system.

    Due to time constraints, these findings and achievements summarise our current progress towards making our proposed sustainable building material. However, inspired and encouraged by the positive feedback regarding the market for such a product we received from our Human Practices activities, we are determined to continue our investigation after the Grand Jamboree and ultimately create our BioStone, the building material that constructs a better shared future for the world. Our journey does not stop here, and we look forward to the final work we present.

References

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Cengic, I., Uhlén, M., & Hudson, E. P. (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

Chirică, L. C., Petersson, C., Hurtig, M., Jonsson, B., Borén, T., & Lindskog, S. (2002). Expression and localization of α- and β-carbonic anhydrase in Helicobacter pylori. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 1601(2), 192–199. https://doi.org/10.1016/s1570-9639(02)00467-3

Heveran, C. M., Williams, S. L., Qiu, J., Artier, J., Hubler, M. H., Cook, S. M., Cameron, J. C., & Srubar, W. V. (2020). Biomineralization and Successive Regeneration of Engineered Living Building Materials. Matter, 2(2), 481

Jean‐Yves Winum, & Colinas, P. A. (2015). Carbonic Anhydrases as Esterases and Their Biotechnological Applications. Elsevier EBooks, 361–371. https://doi.org/10.1016/b978-0-444-63258-6.00021-4

Kang, H. J., & Baker, E. N. (2011). Intramolecular isopeptide bonds: protein crosslinks built for stress? Trends in Biochemical Sciences, 36(4), 229–237. https://doi.org/10.1016/j.tibs.2010.09.007

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

Šmarda, J., Šmajs, D., Jiří Komrska, & Vladislav Krzyzanek. (2002). S-layers on cell walls of cyanobacteria. Micron, 33(3), 257–277. https://doi.org/10.1016/s0968-4328(01)00031-2

Sundaram, S., & Thakur, I. S. (2018). Induction of calcite precipitation through heightened production of extracellular carbonic anhydrase by CO2 sequestering bacteria. Bioresource Technology, 253, 368–371. https://doi.org/10.1016/j.biortech.2018.01.081

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

Zhu, Y., Liu, Y., Ai, M., & Jia, X. (2022). Surface display of carbonic anhydrase on Escherichia coli for CO2 capture and mineralization. Synthetic and Systems Biotechnology, 7(1), 460–473. https://doi.org/10.1016/j.synbio.2021.11.008