iGEM 2022 — Team ucl

Labratory Notebook
![](https://static.igem.wiki/teams/4417/wiki/bacteria.png)

In this file, we detail all the significant daily events surrounding the growth of our genetically engineered organisms as well as activities preformed weekly in the lab.

Images ringed in green show correct results. Images ringed in yellow show unconfirmed or partially correct results. Images ringed in red show incorrect results.

# Aim

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

Concrete has two main constituents:

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

To create an alternative material, we needed:

* 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.

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

| 1) Design + Research 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. ![](https://static.igem.wiki/teams/4417/wiki/engineering-success/cycle1desres.png) | 2) Build 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. ![](https://static.igem.wiki/teams/4417/wiki/engineering-success/cycle1build.png) |
| --- | --- |
| 3) Test 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. ![](https://static.igem.wiki/teams/4417/wiki/engineering-success/cycle1test.png) | 4) Improve 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. ![](https://static.igem.wiki/teams/4417/wiki/engineering-success/cycle1improve.png) ![](https://static.igem.wiki/teams/4417/wiki/engineering-success/cycle1improve2.png) |

## Urease Assay Design and Redesign

| 1) Design + Research 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. | 2) Build 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. |
| --- | --- |
| 3) Test + Improve x 3 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. ![](https://static.igem.wiki/teams/4417/wiki/engineering-success/cycle2testimprove.png) |

## Carbonic Anhydrase Improvement and Re-Design

| 1) Design + Research 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. | 2) Build 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). |
| --- | --- |
| 3) Test 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. | 4) Improve 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. ![](https://static.igem.wiki/teams/4417/wiki/engineering-success/cycle3improve.png) |

## Brick Composition Development

| 1) Design + Research 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. ![](https://static.igem.wiki/teams/4417/wiki/engineering-success/cycle4desres1.png) | 2) Build + Test + Improve x 2 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. ![](https://static.igem.wiki/teams/4417/wiki/engineering-success/cycle4buildtestimp1.png) |
| --- | --- |
| 3) Design + Research 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. | 4) Build + Test + Improve x 2 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. |
| 5) Design + Research 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. ![](https://static.igem.wiki/teams/4417/wiki/engineering-success/cycle4desres3.png) | 6) Build + Test + Improve 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. ![](https://static.igem.wiki/teams/4417/wiki/engineering-success/cycle4buildtestimp3.png) |
| 7) Design + Research 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. | 8) Build + Test + Improve 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. ![](https://static.igem.wiki/teams/4417/wiki/engineering-success/cycle4buildtestimp4.png) |

![](https://static.igem.wiki/teams/4417/wiki/biocrafter-brick-3.png)

[Electrophoresis](#first)
[Phenol Red Assay](#second)
[Golden Gate](#third)
[Promoter Assay](#fourth)

[Colourimetric Assay](#fifth)
[E.coli Transformation](#sixth)
[pH Assay](#seventh)
[Longer Protocols](#eight)

## Agarose Gel Electrophoresis

![agarosegelelectrophoresis](https://static.igem.wiki/teams/4417/wiki/screenshot-2022-10-08-at-13-05-52.png)

## Materials

* Agarose powder
* 1X TBE buffer
* 1% Ethidium bromide
* NEB 1 kb QuickLoad ladder
* 4 µl NEB bromophenol loading dye
* 5-20 µl of your DNA samples

* Microwave
* 250 ml conical flask
* Gel electrophoresis tank
* Gel tray
* Well comb and supports
* Power pack

## Protocol

### Agarose Gel

1. Weigh 0.5g of agarose (to make a gel of 1%) and mix with 50 ml 1x TBE buffer in a conical flask
2. Microwave for 50 seconds or until the agarose is completely dissolved. It is best to heat 30 seconds, stop and swirl towards a boil. Do not over boil and be careful stirring as eruptive boiling can occur. Placing cling film or a lid over the top can help (do not secure the lid, just place it on top).
3. Allow the agarose solution cool down until you can comfortably hold the flask, if necessary use heat-proof gloves. If it begins to solidify, it can be re-melted. It should take about 5 minutes to cool.
4. Add 5 μl of Ethidium Bromide. N.B.: Caution the dye is a known mutagen. Wear a lab coat, eye protection and gloves when working with this chemical. Dispose of gloves in appropriate container after.
5. Pour the agarose into a gel tray with the well comb and supports in place. Pour slowly to avoid bubbles. Bubbles can be popped or pushed to the side using a pipette tip or the comb end.
6. Let sit at room temperature for 20-30 minutes until completely solidified. Once the gel is set, remove the comb by carefully pulling it up, then remove the supports.

### Visualize DNA

1. Add 1 μl 6x loading dye per 5μl of each of your DNA samples to a PCR tube and mix well by pietting up and down.
2. Place gel (still on the tray) inside the chamber. Black is negative (cathode) and red is positive (anode). The wells should be facing towards the cathode so DNA will migrate towards the anode. Fill the electrophoresis chamber with 1X TBE buffer so that the buffer covers up the gel and fills the wells.
3. Gently load 5 μl of 1kb molecular weight ladder (NEB QuickLoad Ladder) into the first lane. N.B.: Maintain positive pressure once sample is pushed out and only release once the pipette tip is out of the buffer to prevent bubbles dislodging the DNA.
4. Gently load 5-25 μl your samples into the well. It is highly advised to record what you are loading into each well, so that you can label the photo accordingly.
5. Run the gel at 130V for about 70 minutes or until the dye line is 1-2 cm from the bottom of the gel. N.B: Set yourself a timer and keep on checking on the gel to ensure that the bands will not over run.
6. Turn OFF the power, disconnect the electrodes and carefully remove the gel and place on a tray.
7. Using a UV light device (transilluminator) visualise the DNA fragments. Print the photograph and label wells exactly with the samples and the control names, write down the date of the gel.

## Safety considerations

*Use safety goggles, face shield and gloves while using Ethidium Bromide and gels/buffers containing it. Dispose of PPE and gels in EtBr-marked bins.*
*Use a lab coat and blue-light glasses or covers to shield your skin and eyes from exposed UV light.*

## Phenol Red Urease Assay

![ph2](https://static.igem.wiki/teams/4417/wiki/result-interpretation-of-urease-test.jpg)

## Materials

* PEM buffer(20 mM sodium phosphate buffer pH 7.5, 10 mM β-mercaptoethanol, 20 mM EDTA)
* UPEM substrate (20 mM sodium phosphate buffer pH 7.5, 10 mM β-mercaptoethanol, 20 mM EDTA, 300 mM urea)
* Purified jack bean urease (Sigma) for urease activity controls, 1 U/mL in PEM buffer
* Pooled sterilized human female urine
* 0.1% (w/v) phenol red in deionized water, sterilized using a 0.22 μm syringe filter
* 5 M urea in deionized water, filter sterilized
* 0.9% saline, sterilized by autoclaving
* 1.5 mL sterile microfuge tubes.

* 15ml and 50mL sterile conical tubes.
* 96-well sterile microtiter plate with lid.
* 25-mL sterile serological pipette.
* Multichannel pipette (5–50 μL).
* Water bath + Static and shaking incubators set at 37 °C.
* Centrifuge and rotor suitable for 50-mL conical tubes.
* Microcentrifuge
* Plate reader (visible range, capable of kinetic reads, temperature controlled)

## Introduction

 *Colorimetric urease activity assay utilising the pH altering effect of urease activity to prompt phenol red colour change.*

## Protocol

Protocol adapted from Richmond's and Yep's paper on the ["Quantification of urease activity"](https://link.springer.com/protocol/10.1007/978-1-4939-9601-8_9)

The procedure for this assay involves cell culture growth, microtitre plate preparation, induction of urease and, finally, running the urease assay. To get more details, click on this ["Phenol red urease assay link."](https://benchling.com/stefaniefr/f/lib_33kqdC8Z-lab-work/prt_GyUH6H9Z-phenol-red-urease-assay/edit)

## Golden Gate Assembly

![ph2](https://static.igem.wiki/teams/4417/wiki/ae1600-1.jpg)

## Materials

* T4 DNA ligase
* 10X T4 DNA ligase buffer
* Type IIS enzyme
* Receiver plasmid
* DNA fragments with Type IIS overhangs
* MilliQ water

## Protocol

* Set up assembly reactions as follows in a PCR tube:
  + 0.5μL of T4 ligase
  + 2μL of 10X T4 ligase buffer
  + 0.5μL of Type IIS restriction enzyme
  + 100ng of receiver plasmid
  + Equimolar amounts of inserts
  + MilliQ for a total volume of 20μL
* Mix gently
* Place the tube on a thermocycler
![](https://static.igem.wiki/teams/4417/wiki/protocols/screenshot-2022-10-13-at-21-57-50.png)

## Pressure promoter Assay

![ph2](https://static.igem.wiki/teams/4417/wiki/screenshot-2022-08-19-at-19-15-34.png)

## CA Colourimetric Assay

![ph2](https://static.igem.wiki/teams/4417/wiki/ca-ocpc-kit-box.jpg)

## Materials

* Clarified cell lysate
* 4-Nitrophenyl acetate (4-NPA)/li>* ASSAY SOLUTION (MOPS, Na2SO4, EDTA buffer)

* DMSO
* Welled Plate + Plate reader

## Protocol

### Assay

* Prepare 182 ul in each well using clarified cell lysate (concentrated to a small volume - this would normally be purified enzyme), 50 mM, pH 7.5 MOPS, 33 mM Na2SO4, 1.0 mM EDTA buffer
* Add 18 ul of 5 mM 4-NPA solution in 10% DMSO, 90% assay buffer to start the reaction
* PImmediately start plate reader protocol, measuring absorbance at 348 nm every 12-15s for 5 min

### Calculations

* One unit of activity represents the amount of enzyme catalyzing to produce 1 μmol p-nitrophenol per min under the assay conditions

## DH5-alpha Chemically Competent E. coli Cells Transformation

![ph2](https://static.igem.wiki/teams/4417/wiki/transformationtips-videothumb-282x210-0616.jpg)

## Materials

* DH5-alpha Chemically Competent E. coli cells (GoldBio Catalog # CC-101)
* pUC19 Control DNA, 10 pg/μl
* Recovery medium (GoldBio Catalog # CC-300)
* Ampicillin (GoldBio Catalog # A-301ES)* LB agar selection plates
  * Microcentrifuge tubes
  * Shaker incubator

## Protocol

1. Remove competent cells from the -80°C freezer and thaw completely on ice (10-15 minutes).
2. Aliquot 1-5 μl (1 pg-100 ng) of DNA to the chilled microcentrifuge tubes on ice.
3. When the cells are thawed, add 50 μl of cells to each DNA tube on ice and mix gently by tapping 4-5 times. For the pUC19 control, add 1 μl of (10 pg/μl) DNA to the 50 μl of cells on ice. Mix well by tapping. Do not pipette up and down or vortex to mix, this can harm cells and decrease transformation efficiency. \*0.8μl β-Mercaptoethanol (β-ME) can be added to 50 μl competent cells to increase transformation efficiency. Incubate on ice for 10 minutes before adding DNA.
4. Incubate the cells with DNA on ice for 30 minutes.
5. After the 30-minute ice incubation, heat shock the cells at 42°C for 45 seconds.
6. Transfer the tubes to ice for 2 minutes
7. Add 950 μl of Recovery Medium or any other medium of choice to each tube.
8. Incubate tubes at 37°C for 1 hour at 210 rpm in a shaker incubator.
9. Spread 50 μl to 200 μl from each transformation on prewarmed selection plates. We recommend plating two different volumes to ensure that at least one plate will have well-spaced colonies. For the puc19 control, plate 50 μl on an LB plate containing 100 μg/ml ampicillin. Use a sterilized spreader or autoclaved plating beads to spread evenly.
10. Incubate the plates overnight at 37°C.

## CA pH Assay

![ph2](https://static.igem.wiki/teams/4417/wiki/promix-greenhouse-growing-water-alkalinity-vs-ph.jpg)

## Materials

* Clarified Cell Lysate
* 0.02 M Tris⋅HCl buffer (pH 8.0. Store in an ice bath at 0-4°C before and during use)
* Carbon dioxide saturated water (Bubble CO2 gas through 200 ml ice cold water for 30 minutes prior to assay. During saturation process, store water at 0-4°C in an ice bath)

## Introduction

The Wilbur-Anderson assay measures the time taken (in seconds) for carbonic anhydrase (CA) activity to cause a pH drop from 8.3 to 6.3 in CO2 saturated water.

Protocol based on [this paper on [Carbonic anhydrase assay]](https://www.worthington-biochem.com/products/carbonic-anhydrase/assay) and [the original 1948 paper on [ELECTROMETRIC AND COLORIMETRIC DETERMINATION OF CARBONIC ANHYDRASE].](https://www.sciencedirect.com/science/article/pii/S0021925818510115)

## Protocol

## T0 Determination

1. Add 6.0 ml of chilled 0.02 M Tris⋅HCl buffer, pH 8.0 to a 15-20 ml beaker. Maintain temperature at 0-4°C and record pH.
2. Withdraw 4 ml of chilled CO2 saturated water and add to Tris buffer. Immediately start a stop watch and record the time required for the pH to drop from 8.3 to 6.3. Record this time as T0

## Enzyme preparation

1. We wont do this since we're using clarified cell lysate instead of purified enzyme, but we should still keep it cold and work out how it should be diluted. In the original 1948 paper, 'mucosal and brain extracts were made by grinding fresh tissue thoroughly and diluting with about 15 volumes of distilled water'.
2. If we were going to use purified protein then: Dissolve lyophilized powder at a concentration of 0.1 mg/ml in ice cold water. Store in ice bath prior to use. IMMEDIATELY prior to use dilute suspensions or lyophilized materials to a concentration of approximately 0.01 mg/ml in ice cold water.

## Enzyme Activity Assay

1. Add 6.0 ml of chilled 0.02 M Tris⋅HCl buffer, pH 8.0 to a 20 ml beaker. Maintain temperature at 0-4°C and record pH.
2. Add 0.1 ml of freshly diluted enzyme. Quickly add 4 ml of CO2 saturated water and record the time required for the pH to drop from 8.3 to 6.3. Record this time as T.

## Units Calculation

1. One unit is defined as a pH drop from 8.3 to 6.3 per minute at 0 °C within a 20 mmol·L−1 Tris-HCl buffer solution saturated with CO2
2. If we were using purified enzyme, then we could say that units = (2 x (T0 -T1))/(T x mg enzyme in reaction mixture).

## Longer protocols

In this section, we have included direct links to the remaining protocols that we have used in our experimentation. These tend to have more details/diagrams or be a combination of other protocols mentioned before.

| Type of protocol | Purpose |
| --- | --- |
| ["Berthelot's Urease Activity Assay"](https://static.igem.wiki/teams/4417/wiki/protocols/berthelot-s-urease-activity-assay-created-by-ucl-igem-2022.pdf) | Berthelot's reagent is a mixture of phenol and hypochlorite. Ammonia reacts with Berthelot's reagent to form a blue product which then gives samples an OD measurement for colorimetric analysis. |
| ["Christensen's Urease Assay"](https://static.igem.wiki/teams/4417/wiki/protocols/christensen-s-urease-assay-protocol-wip-created-by-ucl-igem-2022.pdf) | The urease test is used to determine the ability of an organism to split urea, through the production of the enzyme urease. |
| ["Spizizen medium"](https://static.igem.wiki/teams/4417/wiki/protocols/spizizen-s-minimal-medium-2022-07-29-etr-mt08pvoa.pdf) | Spizizen medium (SM) is a popular minimal medium for the cultivation of B. subtilis. |
| ["Bacillus subtilis Transformation Protocol"](https://static.igem.wiki/teams/4417/wiki/protocols/bacillus-subtilis-transformation-protocol-created-by-ucl-igem-2022.pdf) | Transformation in B. subtilis is achieved by the uptake of donor DNA into recipient cells and the integration of part of this donor DNA into the host chromosome. |
| ["Bacillus subtilis - Clarified cell lysate"](https://static.igem.wiki/teams/4417/wiki/protocols/bacillus-subtilis-clarified-cell-lysate-created-by-ucl-igem-2022.pdf) | Development of a Bacillus subtilis cell-free transcription-translation system for prototyping regulatory elements. |
| ["Q5 Site-Directed Mutagenesis"](https://static.igem.wiki/teams/4417/wiki/protocols/q5-site-directed-mutagenesis-protocol-created-by-ucl-igem-2022.pdf) | This kit utilizes the robust Q5 Hot Start High-Fidelity DNA Polymerase along with custom mutagenic primers to create insertions, deletions and substitutions in a wide variety of plasmids. |
| ["Crowley-Gulamani assay"](https://static.igem.wiki/teams/4417/wiki/protocols/crowley-gulamani-assay-created-by-ucl-igem-2022.pdf) | An colorimetric assay to determine urease activity. The hydrolysis of urea to form ammonia causes a pH change which then causes phenol red to change the colour of the solution. Protocol has been designed based on the protocols 'Phenol Red Urease Assay' and 'Urease Assay Protocol (WIP)', both of which are on benchling. The aim of writing this novel protocol is to simplify those which already exist, excluding elements which aren't relevant for our purpose. |
| ["General PCR"](https://static.igem.wiki/teams/4417/wiki/protocols/general-pcr-created-by-ucl-igem-2022.pdf) | Polymerase Chain Reaction (PCR) is a powerful and sensitive technique for DNA amplification. |
| ["DNA ligation"](https://static.igem.wiki/teams/4417/wiki/protocols/dna-ligation-created-by-ucl-igem-2022.pdf) | DNA ligation is to join breaks in the phosphodiester backbone of DNA that occur during replication and recombination. |
| ["Miniprep"](https://static.igem.wiki/teams/4417/wiki/protocols/miniprep-overnights-created-by-ucl-igem-2022.pdf) | Miniprep is used to isolate small plasmid DNA from bacteria while limiting contaminating proteins and genomic DNA. |
| ["Gel extraction"](https://static.igem.wiki/teams/4417/wiki/protocols/gel-extraction-created-by-ucl-igem-2022.pdf) | Gel extraction is commonly used to isolate DNA from an agarose gel. |
| ["DNA clean up"](https://static.igem.wiki/teams/4417/wiki/protocols/dna-clean-up-created-by-ucl-igem-2022.pdf) | DNA cleanup is required for efficient removal of primers, nucleotides, enzymes, salts and other impurities from DNA samples prior to use in other purpose. |
| ["Gibson assembly"](https://static.igem.wiki/teams/4417/wiki/protocols/gibson-assembly-created-by-ucl-igem-2022.pdf) | An efficient and effective method that multiple overlapping DNA fragments can be joined in a single isothermal reaction. |
| ["SDS-PAGE"](https://static.igem.wiki/teams/4417/wiki/sds-page-created-by-ucl-igem-2022.pdf) | SDS-PAGE is used to obtain high resolution separation of complex mixtures of proteins. |
| ["Bricks protocol"](https://uploads.igem.org/teams/4417/wiki/protocols) | Bricks protocol. |
| ["Glycerol stock"](https://static.igem.wiki/teams/4417/wiki/protocols/prepare-glycerol-stock-created-by-ucl-igem-2022.pdf) | This protocol is for making cell glycerol stock. |
| ["Improved E. coli ransformation"](https://static.igem.wiki/teams/4417/wiki/protocols/e-coli-transformation-protocol-improved.pdf) | This protocol is an improved protocol for E. coli transformation. |

[pH Growth Curves](#first)
[Cumate Induction](#second)
[Natural Urease](#third)
[SDM](#fourth)

[Urease Constructs](#fifth)
[SDS-PAGE](#sixth)
[Biocementation](#seventh)
[Patton-Reeder Assay](#eight)

[Aggregate Measurements](#nineth)
[Urease Activity](#tenth)
[CA Activity](#eleventh)
[References](#twelfth)

# Primary Accomplishments

## Wet Lab

1. We mutated pCT5-bac2.0 to be TIIS compatible.
2. We demonstrated overexpression of our urease protein in E. coli DH5a transformed with Construct TU1, Construct TU2 and our Multiple Transcriptional Unit (Construct MTU).
3. We successfully showed increased urease activity in E. coli DH5a transformed with Construct TU1.
4. We showed increased levels of biomineralisation and aggregate precitipation in DH5a transformed with Construct TU1.

### pH growth curves

Because the urease and carbonic anhydrase pathways create alkaline environments, we needed to know that our bacteria could survive in such conditions. We set up growth curve experiments in microwell plates with WT BL21 and transformed DH5a to see their growth over time in pHs 6-11.

![graph](https://static.igem.wiki/teams/4417/wiki/results/resultsfig1.png)
 **Observations:**

The microwells with lower-pH medias were noticeably more cloudy when the plate was removed from the reader, suggesting that growth rates were higher under those conditions.

 **Conclusion:**

E. coli appears to grow best in pH 8, which is suitable for the conditions we need it to survive in. pH 9 showed a low but consistent growth rate, and pH 10 and 11 showed no growth at all.

### Cumate induction of pCT5-bac2.0

Our pCT5-bac2.0 plasmid is cumate-inducible, so we needed to know exactly how much cumate was needed to most effectively induce the gene under its control. We set up growth curves of E. coli DH5a with the original pCT5-bac2.0 GFP plasmid in different concentrations of cumate and let it run for 13 hours measuring OD600 for cell growth and fluorescence.

![image](https://static.igem.wiki/teams/4417/wiki/results/resultsfig2.png)

 **Observations:**

Visible green color was visible in all wells other than control samples, demonstrating that the cumate induction was successful.

 **Conclusion:**

The highest GFP expression appeared at 50uM cumate, so we used that for the rest of our induction needs.

### B. subtilis natural urease activity

![image](https://static.igem.wiki/teams/4417/wiki/results/resultsfig345.png)

We knew that B. subtilis had a natural level of urease activity, and we wanted an idea of how expressed it was. At the very least, we wanted to establish a distinction between cells with and without urease genes. To test this, we made Christensen’s Urea Agar plates with phenol red, which changes color in an alkaline environment, such as one created by the ammonia byproduct of the urease pathway. We plated wild-type B. subtilis and E. coli DH5a on these plates and on buffered LB with phenol red.

 **Observations:**

We saw a clear difference in activity as the DH5a plates remained yellow, while the B subtilis plates with urea were pinker than those without urea.

 **Conclusion:**

This qualitatively suggests that that B subtilis urease activity is upregulated in the presence of urea.

### Site-directed mutagenesis for a TII-S compatible shuttle plasmid

![image](https://static.igem.wiki/teams/4417/wiki/results/resultsfig67.png)

We wanted to mutate out the three existing BsaI sites in our shuttle plasmid. We followed the Q5 Site-Directed Mutagenesis protocol and confirmed success through antibiotic selection plates to ensure plasmid uptake, diagnostic restriction digestion to confirm mutagenesis, and Sanger sequencing for certainty.

![image](https://static.igem.wiki/teams/4417/wiki/results/resultsfig8.png)

 **Observations:**

Mutation of Site 1 and Site 3 was successful on the first attempt. The primers for Site 2 failed multiple times and were evnetually redesgined.

**Conclusion:** Our pCT5 plasmid has no BsaI sites and is now TII-S compatible. We have named it pCT5c (pCT5-compatible) internally.

### Cloning of urease constructs

![image](https://static.igem.wiki/teams/4417/wiki/results/resultsfig910.png)

We spent several weeks working on cloning our constructs into our plasmid and tweaking protocols. Our original workflow was PCR g-blocks and miniprep plasmid, then gel extract or PCR clean-up g-blocks. Thsi was followed by a restriction digest with BamHI and SacI, then ligation and transformation into E. coli DH5a.

 **Observations:**

After many failed attempts, we finally found success by using a sequential digest of g-blocks and pCT5. We were also successful using Gibson assembly during a separate workflow. After Gibson assembly, we carried out Golden Gate assembly to express TU1 and TU2 in the same plasmid.

 **Conclusion:**

We ultimately have urease ureABC, accessory proteins ureEFG, and both together cloned into our plasmid.

### SDS page

![image](https://static.igem.wiki/teams/4417/wiki/results/resultsfig1112.png)

In order to observe whether the ureABC and ureEFG were successfully expressed, we analysed our cell pellet using SDS PAGE. The pellet was obtained from the 10 mL LB overnight cultures, then resuspended in Tris Buffer Saline at an OD600. Once resuspended, the cell sample was lysed using sonication. Following sonication, the samples were spun down to separate the soluble and insoluble fragments from the whole cell lysate. 60 μL from each sample, both of crude extract and soluble extract, were obtained and stained with Laemmli reagent.

 **Observations:**

There was a darker band at 61.5 kDa which is the size of ureC, and at 24.9 kDa, which is the size of ureF.

 **Conclusion:**

Several of our samples showed increased protein expression at expected band sizes, suggesting successful upregulation of the urease pathway.

### Biocementation agar plates

The ultimate purpose of our project was bio-cementation, not just enzyme expression. By plating transformed and wild-type bacteria on LB-agar with and without urea, CaCl2, and cumate for induction, we were looking for visible signs of bio cementation as well as crystal under the microscope.

![graph](https://static.igem.wiki/teams/4417/wiki/fig1314new.png)

 **Observations:**

There was a visibly pale ring around where the cell solution was dotted after several days. Bacteria transformed with TU1 and SZU’s carbonic anhydrase part also showed more crystaline structures under a microscope

 **Conclusion:**

These things together sugested a diffusion of CaCO3 from increased levels of biomineralisation.

### Patton-Reeder calorimetric assay

An effective and quantitative way to measure the amount of calcium carbonate precipitated is to look at the changes in calcium ion concentration in a solution over time. We carried out this assay with Construct 1 (TU1), pCT5 SMD1,3-CA17, wild-type B. subtilis, and a negative control. Final concentration of Ca+ ions was significantly higher in untransformed cultures, suggesting that calcium carbonate precipitation was higher with the expression of CA17 and TU1 and that our and SZU’s constructs were functioning as planned.

![image](https://static.igem.wiki/teams/4417/wiki/results/resultsfig15.png)

 **Observations:**

We saw a clear difference in activity as the DH5a plates remained yellow, while the B subtilis plates with urea were pinker than those without urea.

 **Conclusion:**

This suggested that that B subtilis urease activity is upregulated in the presence of urea.

### Cell culture aggregate measurements

Because crystal formation is what we are looking for in the expression of carbonic anhydrase and urease, we decided to measure the weight of aggregates formed from overnight cultures. We picked colonies of DH5a wild-type, master plasmid, and pCT5 SDM1,3-CA17 into overnight cultures of LB, CaCl2, and urea (or just CaCl2 for CA17). We then spun the cultures down, allowed the precipitates to dry, and measured the weight. We found higher weights of aggregate for pCT5 SDM1,3-CA17 and master plasmid than in wild-type DH5a, implying higher levels of biocementation.

![image](https://static.igem.wiki/teams/4417/wiki/results/resultsfig1617.png)

 **Observations:**

We saw a clear difference in activity as the DH5a plates remained yellow, while the B subtilis plates with urea were pinker than those without urea.

 **Conclusion:**

This suggested that that B subtilis urease activity is upregulated in the presence of urea.

### Urease activity assay

We decided to evaluate the activity of urease activity via a colorimetric pH assay. The urease pathway causes pH to increase because it releases ammonia, and an increased pH leads to reaction with phenol red in the solution, turning it from yellow to pink. Our assay measures precisely this using OD readings at 700nm and 562 nm, to account for both cell growth and color change. We suspended cell culture in a Tris medium to limit its growth and added cumate to induce gene expression and urea as a substrate.

![](https://static.igem.wiki/teams/4417/wiki/screenshot-2022-10-14-at-03-07-01.png)

**Observations:** After the protocol was run for several hours, the wells (not including the blanks or controls) appeared to be noticeably pinker than at the beginning of the experiment.

**Conclusion:** Optical density was shown to be higher in E. Coli transformed with DH5a than in wild-type DH5a, suggesting that our construct upregulated the urease pathway to produce ammonium and increase the pH of the environment.

### Carbonic anhydrase activity assay

We measured activity of our engineered E. coli to overexpress carbonic anhydrase (CA). CA catalyzes hydration of CO2, but it can also act as an ester hydration catalyst. CA activity was proved by combining clarified cell lysate (of cultures with OD600 = 1.74) with 4-NPA, a non-physiological commercially available ester, giving a yellow solution. The amount of yellowing is quantified by OD348 measurements corrected for OD600 of cell cultures before lysing. Different concentrations of clarified cell lysate were tested to ensure proper assay sensitivity.

![](https://static.igem.wiki/teams/4417/wiki/protocols/screenshot-2022-10-14-at-03-43-46.png)

**Observations:** CA catalyzes hydration of this ester which results in yellowing of the solution which could be observed

**Conclusion:**CA17 genetic construct in pCT5c plasmid could upregulate carbonic anhydrase expression.

## References

1. Concrete needs to lose its colossal carbon footprint.(2021). Nature, [online] 597(7878), pp.593–594. doi:10.1038/d41586-021-02612-5.
2. Leetham, D. (2015). The Cement Manufacturing Process. [online] Advancing Mining. Available at: https://www.thermofisher.com/blog/mining/the-cement-manufacturing-process/.
3. Team, F.N. (2018). Study: Water Use in Concrete Production Higher Than Expected. [online] www.fluencecorp.com. Available at: https://www.fluencecorp.com/concrete-industry-water-use/.