Engineering Success

The aim of our project was to create a low-carbon biological cement from existing agricultural waste.

Aggregate: sand, gravel etc.
Cement: calcium oxides that form crystals to bind the aggregate together.

Engineered B. subtilis with high biomineralisation activity for biocementation
A structurally strong aggregate mix that supports the growth of B. subtilis

To achieve this first goal, we aimed to express the Sporosarcina pasteurii’s urease enzyme complex in the soil bacterium Bacillus subtilis (RM125) to increase its ability to biomineralize. B. subtilis contains the urease enzymes ureA, ureB, ureC already in its genome, but we did not know if they were functional or what their activity was. We therefore cloned in the genes originally from S. pasteurii: ureA, ureB, ureC as one transcriptional unit (ureABC), and ancillary genes ureE, ureF and ureG as a separate transcription unit (ureEFG) into a shuttle plasmid to overexpress them in B. subtilis. We also wanted to improve SZU 2017’s carbonic anhydrase part (BBa_K2232014) by adding an inducible promoter and co-expressing it with the urease enzyme to increase the rate of biomineralisation as a whole.

In order to complete the synthetic biology behind our project, we needed several components:

A plasmid backbone compatible with B. subtilis (we chose to take this a step further by making the plasmid Type IIS compatible)
A plasmid with the S. pasteurii urease pathway
A plasmid with the carbonic anhydrase gene
Protocols for the non-biological components in our material, to add our B. subtilis to.

Each of these components came with its own set of challenges and obstacles, both in the design process and during practical implementation.

At the start of our project, we searched the registry for a functional BioBrick-compatible plasmid shuttle vector for B. subtilis and E. coli. There was nothing already available, so our first task was to create one. After careful research, we found pCT5-bac2.0, a shuttle plasmid from the Claudia Schmidt-Dannert's Lab compatible with both E. coli and B. subtilis, containing GFP (Green Fluorescent Protein) inducible by cumate, a cheap and commercially available molecule. The plasmid was sourced via Addgene. pCT5-bac2.0 was incompatible with iGEM’s Type-IIS BioBrick assembly standards because it contained three BsaI sites. We decided to use site-directed mutagenesis to sequentially remove those sites, then clone in our transcriptional units as cassettes flanked by SapI Type-IIS prefix and suffix regions. Ultimately, we designed the final plasmids to be able to co-express urease and carbonic anhydrase in a BioBrick-compatible format.	We designed and ordered back-to-back primers for each site, changing a nucleotide in the BsaI site but keeping the amino acid sequence the same. These were designed to remove 3 BsaI sites from the plasmid backbone.
After receiving our primers, we tested each at various annealing temperatures to increase yield. Primer pairs 1, and 3.1 appeared to be successful, but primer pair 2 showed no bands when we ran agarose gel electrophoresis. We decided to concurrently begin site directed mutagenesis and retry primer pair 2 at lower temperatures with 0.5 µL DMSO in a 15 µL PCR. We successfully mutated the first site using a shortened transformation protocol for E. coli DH5a (available our protocols page) and confirmed the success with antibiotic selection plates and a diagnostic digest with BsaI. This iteration of plasmid was called pCT5 SDM1. We then moved onto the third site. We were successful again, as confirmed with antibiotic selection plates and a diagnostic digest with BsaI. This iteration was called pCT5 SDM1,3. However, our original primer pair 2 continued to fail, even at lower temperatures with DMSO added. We still attempted site-directed mutagenesis, using selection plates with lower concentrations of antibiotics and increased time on ice during the transformation protocol, but we remained unsuccessful.	We eventually decided to redesign primer pair 2 to include the mutation on the reverse primer instead. Primer pairs 2.1 and 2.2 were visible at the right sizes after PCR and being run on an agarose gel, and we chose to use primer pair 2.1. The site-directed mutagenesis was successful, and we finally had our pCT5c (pCT5-compatible) plasmid. This was confirmed by E. coli DH5a growth on antibiotic selection plates and a diagnostic digest with BsaI. We suspect the issue with the original primer pair for the second BsaI site 2 was that it was too GC-rich, and shifting the primers along reduced the GC content. The successful SDM with this redesigned primer was later confirmed by sequencing.

At the start of our project, we searched the registry for a functional BioBrick-compatible plasmid shuttle vector for B. subtilis and E. coli. There was nothing already available, so our first task was to create one. After careful research, we found pCT5-bac2.0, a shuttle plasmid from the Claudia Schmidt-Dannert's Lab compatible with both E. coli and B. subtilis, containing GFP (Green Fluorescent Protein) inducible by cumate, a cheap and commercially available molecule. The plasmid was sourced via Addgene.

pCT5-bac2.0 was incompatible with iGEM’s Type-IIS BioBrick assembly standards because it contained three BsaI sites. We decided to use site-directed mutagenesis to sequentially remove those sites, then clone in our transcriptional units as cassettes flanked by SapI Type-IIS prefix and suffix regions.

Ultimately, we designed the final plasmids to be able to co-express urease and carbonic anhydrase in a BioBrick-compatible format.

We designed and ordered back-to-back primers for each site, changing a nucleotide in the BsaI site but keeping the amino acid sequence the same. These were designed to remove 3 BsaI sites from the plasmid backbone.

After receiving our primers, we tested each at various annealing temperatures to increase yield. Primer pairs 1, and 3.1 appeared to be successful, but primer pair 2 showed no bands when we ran agarose gel electrophoresis. We decided to concurrently begin site directed mutagenesis and retry primer pair 2 at lower temperatures with 0.5 µL DMSO in a 15 µL PCR.

We successfully mutated the first site using a shortened transformation protocol for E. coli DH5a (available our protocols page) and confirmed the success with antibiotic selection plates and a diagnostic digest with BsaI. This iteration of plasmid was called pCT5 SDM1.

We then moved onto the third site. We were successful again, as confirmed with antibiotic selection plates and a diagnostic digest with BsaI. This iteration was called pCT5 SDM1,3.

However, our original primer pair 2 continued to fail, even at lower temperatures with DMSO added. We still attempted site-directed mutagenesis, using selection plates with lower concentrations of antibiotics and increased time on ice during the transformation protocol, but we remained unsuccessful.

We eventually decided to redesign primer pair 2 to include the mutation on the reverse primer instead. Primer pairs 2.1 and 2.2 were visible at the right sizes after PCR and being run on an agarose gel, and we chose to use primer pair 2.1. The site-directed mutagenesis was successful, and we finally had our pCT5c (pCT5-compatible) plasmid. This was confirmed by E. coli DH5a growth on antibiotic selection plates and a diagnostic digest with BsaI. We suspect the issue with the original primer pair for the second BsaI site 2 was that it was too GC-rich, and shifting the primers along reduced the GC content. The successful SDM with this redesigned primer was later confirmed by sequencing.

Even before we had ordered any of our parts, we knew that we would need a way to quantify urease activity to show that our new parts increased urease activity in our host cells. Most existing urease assays used some type of colorimetric mechanism that reacts in response to pH change. The urease pathway produces ammonia as a byproduct, which increases the pH of the surrounding solution and can therefore be detected in the presence of compounds like phenol red.	We based our initial protocols on a cell-free endpoint assay containing phenol and nitroprusside blue. We adapted it to function outside of a medical context (the assay was originally looking at bacteria in urinary infections) and created a protocol that we could easily do in our lab. We also chose to adapt it from an endpoint assay to a longer one, because we wanted to see the rate at which the pH changed between wild-type and transformed cells.
Following our adapted protocol, we tried our first runs using LB as a background medium, but the resulting cell growth led to a high absorption signal at 562 nm (as opposed to a wide peak measured at 600-650nm). We tried the next run with tris buffer (what concentration) and water as substrates, because cells can survive in each of them without being promoted to grow. This time, the absorption at 562nm was due to the presence of phenol red reacting to a higher pH. To normalize the data, we divided it by the OD at 700nm, which also removed the inconsistencies caused by the slower cell growth that persisted in both tris and water. Interestingly, the water results were less clear, so we decided to stick with the tris buffer. This was also successful, but the starting pH of the tris buffer was already slightly basic, so the color change as pH increased was less obvious than it could have been. To counter this, we pHed the tris down to pH 7 before filter sterilising it. This made the color change more dramatic and visible to the naked eye. The last glaring issue with the assay was that reactions and cell growth were happening too fast. We noticed this when the first results that the plate reader was picking up were already showing discrepancies between different samples, just minutes after loading for a 9-hour protocol. This was addressed by reducing the concentration of cells to 0.25 OD600 and increasing the urea concentration for a more stable pH. The concentrations run were 0M urea, 0.4M urea, and 0.8M urea. Based on the first three trials of this assay, the new parameters should have been successful. However, the data collected was abnormal, even showing increasing readings for wells blanked with water. We unfortunately did not have time to re-run the assay but can assume there was an issue with the plate reader malfunctioning and still be confident that with a little more troubleshooting, our assay protocol would give us the type of data we were looking for.

Even before we had ordered any of our parts, we knew that we would need a way to quantify urease activity to show that our new parts increased urease activity in our host cells. Most existing urease assays used some type of colorimetric mechanism that reacts in response to pH change. The urease pathway produces ammonia as a byproduct, which increases the pH of the surrounding solution and can therefore be detected in the presence of compounds like phenol red.

We based our initial protocols on a cell-free endpoint assay containing phenol and nitroprusside blue. We adapted it to function outside of a medical context (the assay was originally looking at bacteria in urinary infections) and created a protocol that we could easily do in our lab. We also chose to adapt it from an endpoint assay to a longer one, because we wanted to see the rate at which the pH changed between wild-type and transformed cells.

Following our adapted protocol, we tried our first runs using LB as a background medium, but the resulting cell growth led to a high absorption signal at 562 nm (as opposed to a wide peak measured at 600-650nm).

We tried the next run with tris buffer (what concentration) and water as substrates, because cells can survive in each of them without being promoted to grow. This time, the absorption at 562nm was due to the presence of phenol red reacting to a higher pH. To normalize the data, we divided it by the OD at 700nm, which also removed the inconsistencies caused by the slower cell growth that persisted in both tris and water. Interestingly, the water results were less clear, so we decided to stick with the tris buffer.

This was also successful, but the starting pH of the tris buffer was already slightly basic, so the color change as pH increased was less obvious than it could have been. To counter this, we pHed the tris down to pH 7 before filter sterilising it. This made the color change more dramatic and visible to the naked eye.

The last glaring issue with the assay was that reactions and cell growth were happening too fast. We noticed this when the first results that the plate reader was picking up were already showing discrepancies between different samples, just minutes after loading for a 9-hour protocol. This was addressed by reducing the concentration of cells to 0.25 OD600 and increasing the urea concentration for a more stable pH. The concentrations run were 0M urea, 0.4M urea, and 0.8M urea.

Based on the first three trials of this assay, the new parameters should have been successful. However, the data collected was abnormal, even showing increasing readings for wells blanked with water. We unfortunately did not have time to re-run the assay but can assume there was an issue with the plate reader malfunctioning and still be confident that with a little more troubleshooting, our assay protocol would give us the type of data we were looking for.

When looking through the existing parts registry, we wanted something that would be complementary to the purpose of our project: biomineralization. Because the carbonic anhydrase pathway also results in the extracellular formation of CaCl2, we decided it would be an appropriate candidate to express alongside our urease plasmids. The original part was from the 2017 SZU iGEM team and showed promising results when expressed in B. subtilis.	To improve the part, we wanted to make sure it would work well in B. subtilis and was easily cloneable into our plasmid. We took the characterized part from the registry BBa_K2232014 and codon optimized it for B. subtilis. We also added regions to the ends of the gene so it could undergo PCR with the same universal PCR primers as our urease parts and be cloned using the same enzymes: BamHI and SacI. Lastly, we removed three SapI sites in the coding sequence to keep it Type IIS compatible and allow it to be cloned with ureABC (TU1/TU1B).
After ordering, we were told by IDT that our part was unable to be synthesized due to the complexity of the cumate promoter. We needed to redesign the part if we wanted it to be synthesized.	We maintained the codon optimization of our improved part, but instead inserted NdeI and SacI sites so the gene could be cloned in under the control of the cumate promoter already existing in the pCT5 plasmid. This was successful and we received the part (internally called CA22) towards the end of our project. We successfully cloned the original part termed BBa_K2232014 (internally called CA22) into pCT5. However, given our time constraints, we were unable to successfully transform pCT5-CA22 into our strains, and decided to focus our limited resources on our novel part.

When looking through the existing parts registry, we wanted something that would be complementary to the purpose of our project: biomineralization. Because the carbonic anhydrase pathway also results in the extracellular formation of CaCl2, we decided it would be an appropriate candidate to express alongside our urease plasmids. The original part was from the 2017 SZU iGEM team and showed promising results when expressed in B. subtilis.

To improve the part, we wanted to make sure it would work well in B. subtilis and was easily cloneable into our plasmid. We took the characterized part from the registry BBa_K2232014 and codon optimized it for B. subtilis. We also added regions to the ends of the gene so it could undergo PCR with the same universal PCR primers as our urease parts and be cloned using the same enzymes: BamHI and SacI. Lastly, we removed three SapI sites in the coding sequence to keep it Type IIS compatible and allow it to be cloned with ureABC (TU1/TU1B).

After ordering, we were told by IDT that our part was unable to be synthesized due to the complexity of the cumate promoter. We needed to redesign the part if we wanted it to be synthesized.

We maintained the codon optimization of our improved part, but instead inserted NdeI and SacI sites so the gene could be cloned in under the control of the cumate promoter already existing in the pCT5 plasmid. This was successful and we received the part (internally called CA22) towards the end of our project.

We successfully cloned the original part termed BBa_K2232014 (internally called CA22) into pCT5. However, given our time constraints, we were unable to successfully transform pCT5-CA22 into our strains, and decided to focus our limited resources on our novel part.

There were many materials identified as important to include in our bricks for strength and sustainability purposes. Sand is a popular aggregate in concrete because the rough surface of each grain allows for higher binding affinity with the wet components of the material. Sodium alginate hydrogel is a valuable component of many biomaterials because it can support cell growth and mix evenly with aggregates, sticking components together. CaCl2 is an important source of calcium ions to bind to the carbon dioxide released by the urease and carbonic anhydrase pathways. Urea is the substrate for the urease pathway and can also be found in agricultural waste. Mycelium provides structure with its high content of fibrous polymers, and finally, engineered cells provide biocementation through overexpression of biomineralisation pathways. Because creating brick compositions involves so many sequential steps, we went through many small engineering cycles to arrive at our final prototype.	The first materials: sand and hydrogel, were mixed in various ratios and left to dry for several days in ice cube trays. As this was preliminary brick formulation, testing was quantitative. By manually applying gentle pressure, the stronger bricks could be identified. Most disintegrated relatively easily, but some were more durable. The best ratios were selected to be used in the next step of biomaterial-making. The next variables were the addition of CaCl2 and urea as substrates for the urease pathway and biomineralisation. Various concentrations of each solution were added to the sand/hydrogel mixture, packed into ice cube trays, and left to dry for several days. Once extracted, the bricks with CaCl2 were observed to be extremely weak and turned to dust upon removal from the molds. Compositions of sand, hydrogel, and urea were extremely strong. We identified the best composition that excluded Cacl2. Because calcium ions crosslink the sodium alginate polymer chains, the outside of the hydrogel solidifies before it can be properly combined with sand. We realized we needed to find different ways of including calcium in the system or change our selected hydrogel.
To solve the problem of calcium inclusion in the brick, we created several new approaches. The first was using a sodium alginate/methyl cellulose composite hydrogel to avoid the issue of crosslinking calcium ions. The second was to soak the dried brick in CaCl2 solution, having given all the hydrogel content time to dry. The third was to put solid CaCl2 into the aggregate before mixing it with the hydrogel/urea solution. The fourth approach was to add small quantities of CaCl2 while the mixture was in motion.	We tried all four of the CaCl2 strategies with bricks of the same sand/hydrogel/urea compositions. The sodium alginate/methyl cellulose composite hydrogel was successful, but the resulting product was a little looser than we would have liked. Soaking the dried brick in CaCl2 solution was also successful, but was a very time-intensive process. Adding solid CaCl2 into the sand was unsuccessful and we ended up with an uneven texture of material again. Including small amounts of CaCl2 during the mixing process was successful and we discovered that after 500 uL, the mixture is adequately primed and the rest of the CaCl2 can be added without issues. This was also the easiest method to use. We left the bricks in the ice cube molds for another round of the drying process and the selected the most structurally sound bricks. We wanted to give our cells the highest chance of survival in our bricks. We choose to replace the water in our hydrogel with cell growth medium: essentially putting sodium alginate in Terrific Broth, which is known for supporting high cell densities and rapid growth. By manually pressure testing the dried bricks, we found the bricks had hardened successfully, though they were slightly more brittle than water-based hydrogel. We decided to stick with the Terrific Broth approach and move onto the penultimate component: mycelium.
The first step in mycelium inclusion is to learn the best way to culture our strain: Ganoderma tsugae. We investigated a variety of different mediums and decided to try multiple, including potato dextrose agar/broth, solid oats, porridge, sawdust, and sawdust agar.	We set up a variety of cultures, both liquid and solid, on a variety of substrates and left small samples of our mycelium to grow at 25oC until they could establish themselves. This was a broad undertaking before we could narrow our mediums of choice down for inclusion in the brick. After the leaving the mycelium to grow for several days, it successfully established itself in most of the media, most successfully on the solid oats, sawdust, and potato dextrose. Now that we had an idea of how to grow and culture mycelium, we decided to stick with all three of our successful medias for the final stages of brick composition testing. We blended up our grown mycelium and combined it with our previously established ratios of sand and hydrogel. After leaving the bricks to set for several days, we manually tested them and discovered that the bricks “exploded” when pressure is applied. This is because mycelium moves to the material’s surface and hardens, while the center of the brick becomes pure sand which cannot withstand heavily applied pressure. We decided we needed to include another cellulose source for the mycelium to help it distribute evenly, and opted to include sawdust as a replacement for part of the sand aggregate component of our bricks. We finally had all the component proportions needed to build the final prototype: engineered bacteria, mycelium, sawdust, sand, urea and hydrogel with CaCl2 included in the mixing process. We mixed the prototypes, left them to dry, then sent them to be professionally pressure tested using a hydraulic press. Getting a sense of their material properties gave us a better understanding of where our product could fit within the construction industry.
A key component of a successful product is feasibility of production. As part of our Brick prototype development strategy, we went through multiple variations of mold types to find the ideal candidate. We started with small ice cube molds because they were accessible and small enough to use without worrying about excessive wastage.	For every mold, there were advantages and disadvantages. Plastic ice cube molds: these were cheap, accessible, and came in a variety of shapes, but it was difficult to remove prototypes Silicon ice cube molds were also cheap and accessible, and it is easier to remove prototypes than from plastic molds. However, they were too flexible to tightly pack material, leading to weak brick prototypes. Cylindrical aluminium drink cans were sustainable and the bricks within were easily releasable by scalpel. However, the overall process was time intensive and limited to access to a consistent size of recycled cans. Cut PVC pipe was the appropriate shape for compression testing (according to UCL Civil Engineering and Dr Tamuli) and the size was easy to customise, but the molds were difficult to hold together and required larger amounts of brick materials. Petri dishes accessible in large quantities, and the appropriate shape for compression and Brazilian testing. They also had a large surface area to volume ratio for faster hardening and were easily sealable with parafilm to keep mycelium growth sterile. Our final choice was petri dishes due to their ease of use and appropriate shape/size. Material testing was an important consideration in creating a construction material, and according to the UCL Civil Engineering Department, tensile strength measurements are difficult to do because preparation of samples is challenging. The Brazilian test is widely used instead, as the sample preparation and testing procedure are far more efficient and conveniently, it requires disk-shaped samples.

There were many materials identified as important to include in our bricks for strength and sustainability purposes.

Sand is a popular aggregate in concrete because the rough surface of each grain allows for higher binding affinity with the wet components of the material. Sodium alginate hydrogel is a valuable component of many biomaterials because it can support cell growth and mix evenly with aggregates, sticking components together. CaCl2 is an important source of calcium ions to bind to the carbon dioxide released by the urease and carbonic anhydrase pathways. Urea is the substrate for the urease pathway and can also be found in agricultural waste. Mycelium provides structure with its high content of fibrous polymers, and finally, engineered cells provide biocementation through overexpression of biomineralisation pathways.

Because creating brick compositions involves so many sequential steps, we went through many small engineering cycles to arrive at our final prototype.

The first materials: sand and hydrogel, were mixed in various ratios and left to dry for several days in ice cube trays. As this was preliminary brick formulation, testing was quantitative. By manually applying gentle pressure, the stronger bricks could be identified. Most disintegrated relatively easily, but some were more durable. The best ratios were selected to be used in the next step of biomaterial-making.

The next variables were the addition of CaCl2 and urea as substrates for the urease pathway and biomineralisation. Various concentrations of each solution were added to the sand/hydrogel mixture, packed into ice cube trays, and left to dry for several days.

Once extracted, the bricks with CaCl2 were observed to be extremely weak and turned to dust upon removal from the molds. Compositions of sand, hydrogel, and urea were extremely strong.

We identified the best composition that excluded Cacl2. Because calcium ions crosslink the sodium alginate polymer chains, the outside of the hydrogel solidifies before it can be properly combined with sand. We realized we needed to find different ways of including calcium in the system or change our selected hydrogel.

To solve the problem of calcium inclusion in the brick, we created several new approaches.

The first was using a sodium alginate/methyl cellulose composite hydrogel to avoid the issue of crosslinking calcium ions. The second was to soak the dried brick in CaCl2 solution, having given all the hydrogel content time to dry. The third was to put solid CaCl2 into the aggregate before mixing it with the hydrogel/urea solution. The fourth approach was to add small quantities of CaCl2 while the mixture was in motion.

We tried all four of the CaCl2 strategies with bricks of the same sand/hydrogel/urea compositions.

The sodium alginate/methyl cellulose composite hydrogel was successful, but the resulting product was a little looser than we would have liked. Soaking the dried brick in CaCl2 solution was also successful, but was a very time-intensive process. Adding solid CaCl2 into the sand was unsuccessful and we ended up with an uneven texture of material again. Including small amounts of CaCl2 during the mixing process was successful and we discovered that after 500 uL, the mixture is adequately primed and the rest of the CaCl2 can be added without issues. This was also the easiest method to use.

We left the bricks in the ice cube molds for another round of the drying process and the selected the most structurally sound bricks.

We wanted to give our cells the highest chance of survival in our bricks. We choose to replace the water in our hydrogel with cell growth medium: essentially putting sodium alginate in Terrific Broth, which is known for supporting high cell densities and rapid growth.

By manually pressure testing the dried bricks, we found the bricks had hardened successfully, though they were slightly more brittle than water-based hydrogel.

We decided to stick with the Terrific Broth approach and move onto the penultimate component: mycelium.

The first step in mycelium inclusion is to learn the best way to culture our strain: Ganoderma tsugae. We investigated a variety of different mediums and decided to try multiple, including potato dextrose agar/broth, solid oats, porridge, sawdust, and sawdust agar.

We set up a variety of cultures, both liquid and solid, on a variety of substrates and left small samples of our mycelium to grow at 25oC until they could establish themselves. This was a broad undertaking before we could narrow our mediums of choice down for inclusion in the brick.

After the leaving the mycelium to grow for several days, it successfully established itself in most of the media, most successfully on the solid oats, sawdust, and potato dextrose.

Now that we had an idea of how to grow and culture mycelium, we decided to stick with all three of our successful medias for the final stages of brick composition testing.

We blended up our grown mycelium and combined it with our previously established ratios of sand and hydrogel.

After leaving the bricks to set for several days, we manually tested them and discovered that the bricks “exploded” when pressure is applied. This is because mycelium moves to the material’s surface and hardens, while the center of the brick becomes pure sand which cannot withstand heavily applied pressure.

We decided we needed to include another cellulose source for the mycelium to help it distribute evenly, and opted to include sawdust as a replacement for part of the sand aggregate component of our bricks.

We finally had all the component proportions needed to build the final prototype: engineered bacteria, mycelium, sawdust, sand, urea and hydrogel with CaCl2 included in the mixing process.

We mixed the prototypes, left them to dry, then sent them to be professionally pressure tested using a hydraulic press. Getting a sense of their material properties gave us a better understanding of where our product could fit within the construction industry.

A key component of a successful product is feasibility of production. As part of our Brick prototype development strategy, we went through multiple variations of mold types to find the ideal candidate. We started with small ice cube molds because they were accessible and small enough to use without worrying about excessive wastage.

For every mold, there were advantages and disadvantages.

Plastic ice cube molds: these were cheap, accessible, and came in a variety of shapes, but it was difficult to remove prototypes

Silicon ice cube molds were also cheap and accessible, and it is easier to remove prototypes than from plastic molds. However, they were too flexible to tightly pack material, leading to weak brick prototypes.

Cylindrical aluminium drink cans were sustainable and the bricks within were easily releasable by scalpel. However, the overall process was time intensive and limited to access to a consistent size of recycled cans.

Cut PVC pipe was the appropriate shape for compression testing (according to UCL Civil Engineering and Dr Tamuli) and the size was easy to customise, but the molds were difficult to hold together and required larger amounts of brick materials.

Petri dishes accessible in large quantities, and the appropriate shape for compression and Brazilian testing. They also had a large surface area to volume ratio for faster hardening and were easily sealable with parafilm to keep mycelium growth sterile.

Our final choice was petri dishes due to their ease of use and appropriate shape/size. Material testing was an important consideration in creating a construction material, and according to the UCL Civil Engineering Department, tensile strength measurements are difficult to do because preparation of samples is challenging. The Brazilian test is widely used instead, as the sample preparation and testing procedure are far more efficient and conveniently, it requires disk-shaped samples.

Aim

Creating a Type IIS-compatible B. subtilis vector for use by the iGEM community

Urease Assay Design and Redesign

Carbonic Anhydrase Improvement and Re-Design

Brick Composition Development