iGEM 2023 — Team keystone

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{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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Engineering

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Introduction

Carbonic Anhydrase Characterization

Display of hpCA on the S-layer of PCC6803

Cyanobacteria surface display based on SpyTag-SpyCatcher

CA-SpyCatcher Characterization

Algae Brick

Summary

## Introduction

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![](https://static.igem.wiki/teams/4863/wiki/engfig1a.png)

Fig. 1: Overview of Our Engineering Approach.

    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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![](https://static.igem.wiki/teams/4863/wiki/engi-part2.png)

Fig. 2: Results for hpCA 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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![](https://static.igem.wiki/teams/4863/wiki/part3allnew1.png)

Fig. 3: Results for 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](https://2023.igem.wiki/keystone/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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![](https://static.igem.wiki/teams/4863/wiki/part4allnew.png)

Fig. 4: Results for surface display on Synechocystis PCC 6803 based on SpyTag-SpyCatcher system.

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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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![](https://static.igem.wiki/teams/4863/wiki/engi-part5.png)

Fig. 5: Results for 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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![](https://static.igem.wiki/teams/4863/wiki/engi-part6.png)

Fig. 6: Development of the BioStone.

    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

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Experiments

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Extracted DNA PCR

Bacterial & Cyanobacterial Colonies PCR

Thermocycler PCR

Agarose Electrophoresis

Agarose DNA Recycling

SDS-PAGE Gel Electrophoresis

Enzyme activity

Protein Concentration Measurement

BioBrick Protocol

Gibson Assembly

Golden Gate Assembly

Overlap PCR Protocol

### Extracted DNA PCR System

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Prepare the PCR reaction system added according to the proportion below. Refer to different tables depending on the enzyme type used.

For 2 X Taq Plus PCR Master Mix, 20 ul\* PCR reaction system

|  |  |
| --- | --- |
| Reagent | Amounts (ul) |
| 2 X Taq Plus PCR Master Mix | 10 |
| ddH2O | 6 |
| Sample DNA template | 2 |
| Forward Primer | 1 |
| Reverse Primer | 1 |

For Lamp Master Mix, 20 ul\* PCR reaction system

|  |  |
| --- | --- |
| Reagent | Amounts (ul) |
| Lamp Master Mix | 10 |
| ddH2O | 7.4 |
| Sample DNA template | 1 |
| Forward Primer | 0.8 |
| Reverse Primer | 0.8 |

For Phusion Pfu DNA Polymerase mix, 20 ul\* PCR reaction system

|  |  |
| --- | --- |
| Reagent | Amounts (ul) |
| ddH2O | 6 |
| Pfu Buffer | 8 |
| Sample DNA template | 2 |
| Forward Primer | 1.6 |
| Reverse Primer | 1.6 |
| Pfu Polymerase | 0.8 |

\*For other amounts (ex. A 50 ul reaction system) multiply the amounts by a common factor.

     1. Shave ice into a Styrofoam container
     2. Label the PCR tubes.
     3. Add all the reagents described in the tables above into the tube.
     4. Put the tubes in the thermocycler.

Once completed, jump to the thermocycler protocol to edit the program in the thermocycler.

### Bacterial & Cyanobacterial Colonies PCR System

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To prepare the DNA template, you need to first extract DNA from a single colony. To do this, follow these instructions:

     2. Add it to 10 ul of ddH20 in a clean tube.
     3. Mix it in with your pipette tip by repeatedly blowing it. Make sure no bubbles are formed.
     4. Take the tube out of the clean bench and heat it for 11 minutes
     5. Centrifuge at 4000 rpm for 10 minutes and use the supernatant for the DNA template for the PCR.

\*Note: When picking the single colony, avoid selecting colonies in densely distributed areas as to avoid accidentally touching other colonies with the tip of your micropipette.

Next, add in the reagents into an empty PCR tube. Depending on the enzyme type used, refer to the tables below:

For 2 X Taq Plus PCR Master Mix, 20 ul\* PCR reaction system

|  |  |
| --- | --- |
| Reagent | Amounts (ul) |
| 2 X Taq Plus PCR Master Mix | 10 |
| ddH2O | 6 |
| Sample DNA template | 2 |
| Forward Primer | 1 |
| Reverse Primer | 1 |

For Lamp Master Mix, 20 ul\* PCR reaction system

|  |  |
| --- | --- |
| Reagent | Amounts (ul) |
| Lamp Master Mix | 10 |
| ddH2O | 7.4 |
| Sample DNA template | 1 |
| Forward Primer | 0.8 |
| Reverse Primer | 0.8 |

For Phusion Pfu DNA Polymerase mix, 20 ul\* PCR reaction system

|  |  |
| --- | --- |
| Reagent | Amounts (ul) |
| ddH2O | 6 |
| Pfu Buffer | 8 |
| Sample DNA template | 2 |
| Forward Primer | 1.6 |
| Reverse Primer | 1.6 |
| Pfu Polymerase | 0.8 |

\*Again, for other reaction system sizes (50 ul etc.), just multiply everything by a common factor.

### Thermocycler PCR

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Place the PCR tube in the thermocycler and start the program below.

Note depending on the enzyme used there may be slight variation, the one below is only an example of what the protocol could be. Check the notebook for more information on the specific thermocycler protocols for each step of our experiment.

Thermocycler PCR Protocol

|  |  |  |
| --- | --- | --- |
| Step | Degree(C) | Time |
| 1 | 94 | 10 min |
| 2 | 94 | 30sec |
| 3 | Tm-5 | 30sec |
| 4 | 72 | Xs |
| 5 | Go back to step 2 and complete 30-35 cycles. |
| 6 | 72 | 5min |
| 7 | 10 | Ifinity |

\*Tm means melting temperature of the primers used

\*Due to the general rule of the amplification efficiency of the enzyme as 1kb/min amplicon, the x value is equal to the length (kb) of the DNA fragment amplified by PCR

After finishing the entire PCR process, store the PCR product in a 4 C Fridge

### Agarose Gel Electrophoresis

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1. According to the amount of TAE buffer needed, mix the 1% of agarose powder with 1X TAE buffer.- Heat up the solution until it turns clear.- When the solution is cooled to around 60 degrees Celsius, add the DNA dye into the solution.- Pour the solution slowly into the gel chamber and put in the comb after making sure that the casting tray will not leak.- After the gel solidifies, remove the combs.- Use the pipette, insert the DNA marker into the first well and slight amounts of the samples into each well starting from the second well.- Run the gel in the voltage and time according to the need of the sample inserted.- Place the gel on the gel imaging system and view the results.

### Agarose Gel DNA Recycling

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1. Cut the target DNA strip from the agarose gel into a centrifuge tube and weigh it.- Add three times the volume of sol solution, 50 degrees Celsius of water bath for 10 minutes until the gel completely dissolves to liquid.- Add the gel liquid to spin column CA1 or CA2 and the column to the collection tube, centrifuge at 13,000 rpm for 30 seconds, pour out the waste liquid.- Add 700 uL rinse liquid PW, centrifuge at 13,000 rpm for 30 seconds, pour out the waste liquid.- Add 500 uL rinse liquid PW, centrifuge at 13,000 rpm for 30 seconds, pour the waste liquid. Centrifuge at 13,000 rpm for 2 minutes, then put the spin columns in room temperature until it dries.- Put the spin column into a clean centrifuge tube, elution buffer EB to it, centrifuge at 13,000 rpm for a minute and collect the DNQA solution.- Step 6 could be repeated to increase the recycled amount of DNA.

### SDS-PAGE Gel Electrophoresis

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Preparation of Gel:

1. Mix all reagents to prepare the gel, TEMED added at last.- Pour the gel into pouring chamber.- Add butanol to remove unwanted air bubbles- Insert the comb into the space between the glass plates.

Sample Preparation

1. Add pure water to the beaker.- Add 2-mercaptoethanol to the sample buffer.- Place the buffer solution in a microcentrifuge tube and add the protein sample to it.- Place the MW marker in a separate tube.- Boil the sample for less than 5 minutes to completely denature the protein.

Electrophoresis

1. Remove the gel cassette from the casting table and place it in the electrode assembly.- Secure the electrode assembly to the fixture rack.- Pour the 1x Electrophoresis Buffer into the opening of the casting frame to fill the gel wells.- Aspirate 30ml of denaturing sample into the wells.- Cover the tank with a lid and connect the device to the power supply.- Run the sample at 30mA for about 1 hour and see the bands under UV light.

### CA Enzyme activity

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Ingredients

|  |  |  |
| --- | --- | --- |
| Reagent name | Specification | Preservation condition |
| Extracting solution | Liquid 60mL x 1 bottle | 2-8˚C |
| Reagent 1 | Liquid 50mL x 1 bottle | 2-8˚C |
| Reagent 2 | Powder x 2 bottles | 2-8˚C |
| Standard substance | Liquid x 1 | 2-8˚C |

Carbonic Anhydrase (CA, EC4.2.1.1) is a metallic enzyme with Zn2+ as the active center, which can be used to efficiently catalyze reversible hydration reaction of carbon dioxide: CO2+H2O⇋HCO3-+H+, with a catalytic rate up to 107 times that of natural conditions. It is one of the fastest catalytic enzymes known.

Carbonic anhydrase can catalyze the reaction of acetic acid to nitrophenyl ester to produce p-nitrophenol. The activity of carbonic anhydrase is reflected by detecting the increasing rate of absorbance value at 405nm.

Protocols

Sample processing:

1. Tissue: According to tissue quality (g): extraction liquid volume (mL) is 1:5~10 (it is recommended to weigh about 0.1g tissue and add 1mL extraction liquid), ice bath homogenization is carried out. Centrifuge 8000g at 4°C for 10min, take the supernatant and put it on the ice to be measured.
2. Bacteria or cultured cells: Collect bacteria or cells into the centrifuge tube first, and discard the supernatant after centrifugation; According to the number of bacteria or cells (104): the extraction liquid volume (mL) is 500~1000:1 ratio (it is recommended that 5 million bacteria or cells be added to 1mL of extraction liquid), ultrasonic crushing bacteria or cells (ice bath, power 200W, ultrasonic 3s, interval 10s, repeat 30 times), 8000g, Centrifuge at 4°C for 10min, take the supernatant and put it on the ice to be measured.
3. Liquid: direct measurement. (If the solution is cloudy, centrifuge the supernatant and then determine).

Measuring procedure

1. Preheat visible spectrophotometer more than 30min, adjust the wavelength to 405nm, distilled water zero.

2. Standard tube determination:

1. Standard tube determination: add 100μL standard liquid in the cuvette, 900μL reagent 1, fully mixed at 405nm after determination of light absorption value, recorded as A\_standard.
2. Determination of standard blank tube: 100μL distilled water and 900μL reagent were added to the colorimetric dish, and the absorption value was determined at 405nm after full mixing, and was recorded as A\_standard blank.
3. Calculate ∆A\_standard = A\_standard - A\_standard blank. (Standard tubes and standard blank tubes only need 1-2 times.)

Operation table

|  |  |  |
| --- | --- | --- |
| Reagent | Measuring tube (μL) | Blank tube (μL) |
| Sample | 100 | - |
| Extract | - | 100 |
| Reagent 1 | 700 | 700 |
| Reagent 2 working fluid | 200 | 200 |

The above reagents were added into 1mL glass cuvettes successively according to the sample adding table, immediately and thoroughly mixed to determine the light absorption value A1 at 10s at 405nm, quickly placed in 37°C water bath or constant temperature incubator for 5min, and quickly wiped to determine the light absorption value A2 at 5min10s. Calculate A\_measure = A2\_- A1\_measure, A\_blank = A2\_blank -A1\_blank, ∆A = A\_determination - A\_blank

### Protein Concentration Measurement

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In our experiments, protein concentration is measured using the Enhanced BCA Protein Assay Kit. A brief overview of the procedures in this kit is provided below.

1. Add 0, 1, 2, 4, 8, 12, 16, and 20 μL protein standard into the standard wells on a 96 well plate, respectively. Add protein standard diluent to make up to 20 μL in each well. In other words, the concentration of protein standard in each standard well will be 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg/mL, respectively.- Add an appropriate volume of sample to the sample wells of the 96 well plate. If the sample is less than 20 μL, add protein standard diluent to make up to 20 μL. Record the sample volume.- Add 200 μL BCA working solution into each well and place the 96 well plate at 37˚C for 20-30 minutes.
       1. Note: the plate can also be placed at room temperature for 2 hours or at 60˚C for 30 minutes. When measuring protein concentration using the BCA method, the color of the mixture in each well will gradually deepen over time, and the color reaction will accelerate along with increases in temperature. If the sample concentration is low, it is better to incubate at a higher temperature or extend the incubation time appropriately.- Measure the absorbance at A562 or wavelength within the range of 540-595 nm, using a microplate reader.- Calculate the protein concentration of the sample based on the standard curve and the sample volume.

#### BioBrick Protocol

1. Mix 10g of alginate into 100ml of BG11, mix with a magnetic stirrer at 90°C.- After gelatin fully dissolves in the medium, add 0.1M NaHCO, into the solution until the pH value becomes 7.6- When the alginate solution cools to below 36˚C, add 20ml of cyanobacteria-containing liquid medium and mix with the alginate solution.- Add 40g of pre-treated sea sand (small grains) and river sand (large grains) mixture and mix well with the alginate and cyanobacteria mixture.- Note: treatment for aggregates procedure that follows:
           1. Soak sand in 4% HCl solution for 24h- Take out the sand and rinse with tap water until the pH value becomes 7.0- Put the sand into the furnace and bake for 48h at 80˚C.- Pour the mixture into a square mould and flatten the top surface.- Pour a certain amount of CaCl2 2H2O solution (1M) onto the surface of the mixture until the surface is completely covered. By this point, the surface should be quickly solidified.- Gently lift the hydrogel and pour more CaCI2-2H2O solution (1M) onto the bottom of the mould. Wait for a few minutes, then lift the hydrogel and immerse it in 0.5M of CaCI2:2H2O solution overnight.- Then, dry the hydrogel in a dryer at 60°C for about 12 hours to obtain dried Biostone.

#### CA effect on Biostone Desiccation

1. Soak fine sea sand in 4% HCl solution for 24h- Take out the fine sea sand and rinse with tap water until the pH value becomes 7.0- Put the sea sand into the furnace and bake for 48h at 80˚C.- Repeate steps 1-4 for coarse river sand- 50mL 5% sodium alginate gel using BG11 medium as the solvent. Put the processed sand into two square molds and then pour in the sodium alginate gel solution. Fully homogenize them afterwards.- One square mold adds purified aqueous CA and the other one without CA. The aqueous CA was added after the homogenization of sodium alginate solution and aggregates.- 0.1M (~1.0%) CaCl2·2H2O solution was added to both two BioStones by spraying at the end.

#### Carbonic Anhydrase Extraction Protocol

1. Preparing tissue samples, thaw them on ice before starting the extraction.- Place samples in a pre-chilled homogenization buffer.- Centrifuge the homogenate at a low speed for 10-15 minutes at 4°C- Collect the supernatant carefully and transfer it to a microcentrifuge tube- Measure the protein concentration in the supernatant using a protein quantification method- Aliquot the supernatant into smaller portions and store them at -80°C

#### Cyanobacteria Transformation Protocol

1. Prepare a healthy culture of cyanobacteria in the exponential growth phase.- Isolate or obtain the plasmid DNA containing the target gene. Verify its purity and concentration.- Harvest cyanobacterial cells by centrifugation at a low speed and wash them with fresh cyanobacterial growth medium- Mix the harvested cyanobacterial cells with the purified plasmid DNA. Adjust the DNA concentration based on cyanobacterial strain and transformation efficiency.- Load the cell-DNA mixture into a cuvette or transformation chamber. Use an electroporator following set the voltage, resistance, and capacitance parameters- Transfer the electroporated cells into a fresh, pre-warmed cyanobacterial growth medium.- Incubate cells under suitable conditions- Add the appropriate antibiotic to the recovery medium to select for transformed cells- Plate the transformed cells onto agar plates- Incubate the plates under the same controlled conditions- Analyze resulting colonies for the presence of the target gene using techniques such as PCR and sequencing

#### E.Coli Transformation Protocol

1. Prepare a fresh, frozen E. coli culture onto an LB agar plate. Incubate the plate at 37°C overnight.- Isolate or obtain the plasmid DNA containing the target gene. Verify its purity and concentration.- Take a tube of competent E. coli cells from the -80°C freezer and thaw it on ice.- Add 1-5 μl of the plasmid DNA to the thawed competent cells.- Incubate the cell-DNA mixture on ice for 30 minutes.- Heat shock the mixture by placing it in a 42°C water bath for 45 seconds- Transfer the tube back to ice and let it cool for 2 minutes.- Add 950 μl of pre-warmed SOC medium to the cell-DNA mixture.- Incubate the tube at 37°C in a shaking incubator for 1-2 hours- Plate a portion of the transformed cell suspension onto an LB agar plate containing the appropriate antibiotic. Spread the cells evenly using a sterile spreader.- Incubate the plates inverted at 37°C overnight or until colonies are visible.- Select colonies on the LB agar plate with antibiotic.- Verify the presence of the target gene by performing plasmid isolation and techniques such as PCR or DNA sequencing.

### Gibson Assembly

---

#### Protocol for Gibson Assembly

1. Set reaction on ice- Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at –20°C for subsequent transformation.
     1. Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases- Transform NEB 5-alpha Competent E. coli cells (provided with the kit) with 2 μl of the assembly reaction, following the transformation protocol.

### Golden Gate Assembly

---

#### Protocol for Golden Gate Assembly

1. Set up the following reaction mix on ice:
   1. X µl DNA Insert PCR reactions (150 ng of DNA each or 2:1 molar ratio, insert:plasmid)- 1 µl (~75 ng) Vector/Plasmid/Backbone
        1. Or Golden Gate kit pGGA destination plasmid- 1 µl Golden Assembly Master Mix
          1. Use 2 µl for assemblies of > 10 inserts- If you're using enzymes instead of master mix:
            1. Insert + Plasmid (2:1 molar ratio)- 2.5 µl T4 DNA Ligase Buffer (extremely heat sensitive, will not survive multiple heat/thaw cycles)- 0.5 µl T4 DNA Ligase (extremely heat sensitive)- 1.5 µl BsaI-HFv2 (relatively heat sensitive)- Make up to 20 µl of dH2O- 20 - X - 2 dH2O- Mix gently by pipetting up and down. Tap on the bench or briefly centrifuge (1 sec) to ensure no bubbles in the reaction mixture.- Transfer the reaction tube(s) to a thermal cycler and run the following program:
       1. (5 min 37°C → 5 min 16°C) x 30 cycles- 5 min 60°C- If you're running overnight, add a Hold at 4°C. The next morning, briefly heat the reaction to 60°C before proceeding.
       Tactical Nap Opportunity- Add 2 µl assembly reaction to 50 µl thawed chemically competent cells and allow to sit on ice for 15-30 minutes.
         1. Make sure you include a positive and negative control (2 µl uncut plasmid, 10 µl dH2O)- Take your esky of ice over to the 42°C waterbath or 42°C heat block. Put tubes in a floatie (or) hold in the water bath (or) push tubes into the slots of the heat block. Allow 45 seconds for heat shock. (Plus or minus 10 seconds, this needs to be exact!). Then transfer the cells straight back onto ice (embed into ice, as above, don’t just rest on top).- Allow transformation mixtures to sit for 2 min on ice, then add 1 ml sterile LB broth to each tube. You can also use more fancy media (e.g. SOC or SOB), but there is not that much difference.- Incubate on 37°C shaker for 1 hour. Put the tubes horizontal so they get good shaking action. eg. put the tubes laying flat on the shaker platform and masking-tape into place. Make sure the lids are tight! You can incubate without shaking, and you can incubate for less time (30 min), but it won’t work as well in these cases.
               1. The role of this ‘recovery’ step is to allow the cells to create the proteins required for antibiotic resistance.- Recovery is not necessary for Ampicillin resistance plasmids. Proceed to plating.- Label the LB-antibiotic plates before starting the next bit; you need two plates for each ligation condition or plasmid type, since we will plate out two different cell concentrations of each to ensure we get countable/pickable numbers of colonies. Double check the plates to ensure you are using the correct type of antibiotic(s) for the type of plasmid(s) you are using.- Pipette 100 µl of the first cell suspension onto one LB-antibiotic plate (label ‘100 µl’ in addition to other info) and spread it over the plate using the glass rod (liquid > solid) Sterile Technique.- Spread 100 µl of the remaining samples, each onto a separate, appropriately-labelled plate.- Centrifuge all the tubes at ~15,000 rpm for 1 minute in a micro-centrifuge. Pour off most of the supernatant into culture waste (being careful not to touch the tubes on the edge of the culture waste bottle). Leave a little bit of liquid behind (about one or two drops).
                       1. The purpose of this spin and second plating is to ensure you avoid getting too few colonies or a confluent lawn when you check your plates tomorrow.- Vortex the cells in the remaining liquid for about 10 seconds, until they are not sticking to tube anymore, and you have a nice smooth, even, cell suspension.- Pipette the cells from the first cell suspension onto the appropriate pre-labelled LB-antibiotic plate (label with ‘pellet’ in addition to other info), spread plate as above. Repeat for the remaining samples and controls.- Incubate all plates at 37°C overnight. Note that for some plasmids and ligations, it may be beneficial to instead try room temp for 2-3 days – this lowers the copy number of pUC type plasmids, and is useful to allow retrieval of clones that might be toxic to the host.

### Overlap PCR Protocol

---

#### Overlap PCR Protocol

We used the lamp enzyme for this. There are two rounds of PCRs to achieve the overlap results.

For round 1:

For a 20 ul reaction system:

|  |  |
| --- | --- |
| Reagent | Amounts (ul) |
| Lamp | 10 |
| DNA template A | 1 |
| DNA template B | 1 |
| Primer A F/R | 0.8 |
| Primer B F/R | 0.8 |
| ddH20 | up to 20 ul\* |

\*It’s written as up to 20 ul as depending on the number of DNA templates, the amount of ddH20 used may vary

     1. Shave ice into a Styrofoam container
     2. Label the PCR tubes.
     3. Add all the reagents described in the tables above into the tube.
     4. Put the tubes in the thermocycler and set the program accordingly. Refer to the Keystone lab notebook for specific details on what to enter into the thermocycler for different parts of the experiment.

Use gel electrophoresis to check if the results from the first round of PCR are what you want, if it is indeed what you want, purify the DNA out of the gel to prepare for the second round of PCR.

For round 2:

50 ul Reaction System:

|  |  |
| --- | --- |
| Reagent | Amounts (ul) |
| Lamp | 25 |
| DNA template | 2.5 |
| Primer A F | 2 |
| Primer B R | 2 |
| ddH20 | up to 50 ul\* |

Prepare this through the same series of steps as the ones written for round 1.

Use gel electrophoresis again and double check if the results are what you wanted. If so, purify the DNA out of the gel again and store for future use.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

# {{ self.title() }}

## What should this page contain?

---

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

---

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

---

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

## Inspirations

---

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

{% endblock %}