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.

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.

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.

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.

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

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

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.

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.

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

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.

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.

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.

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.

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

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.

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.

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

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.

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

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

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.

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.

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

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.

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.

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

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.

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.

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.

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.