Registered part: https://parts.igem.org/Part:BBa_K4758000.
Carbonic Anhydrase is a ubiquitous protein that catalyses the reaction of carbon dioxide and water to carbonic acid in a reversible manner. This variant of carbonic anhydrase (28 kda) comes from a pathogenic bacteria H. pylori which uses the protein in conjunction with urease to reduce the acidic environment of the gut therefore causing diseases such as ulcers and other gastrointestinal problems.
In transforming this protein we want to make use of this natural process and its ability to biomineralise the process by which particulates are formed as a by-production of biological reactions. Carbonic anhydrase and Urease and in the presence of calcium ions produce calcium carbonate. This mineral has widespread industrial applications, from bioremediation to bio-consolidation and cementation. This part page explains our findings on our findings on the transformation, expression, and assaying of this enzyme to demonstrate whether it can serve to improve the catalytic efficiency of the MICP process.
Golden Gate assembly was performed to ligate de novo synthesised carbonic anhydrase strand. A 2:1 ratio of insert to plasmid (pkel10) was used. The insert was cloned in PCR at cycles of: (5 min 37°C → 5 min 16°C) x 30 cycles 5 min 60°C 2 μL of assembly mix is added to 50 μL of thawed chemically competent cell (NEB® Turbo Competent E. coli) and heat-shocked (ice at 30 min to 42°C water bath for 45 seconds). After two more minutes on ice, 1 mL of LB media was added and the tube was placed in a 37°C shaking incubator for an hour.
A patch plate was performed in another LB-AMP plate, to get a number of high-quality samples as shown in Figure 2 and grown overnight.
12 PCR reactions were set up with Forward and reverse primers corresponding to the spanning region of Insert for spanning PCR. The PCR mix was then run on agar gel to demonstrate insert presence. This is shown in Figure 3.
Finally, Colonies 1 and 2 from Figure 3 (well 2 & 3) were chosen after PCR to be sent sequencing at the Ramaciotti Genomics Centre at UNSW. The results are shown below It is unclear why however the spanning PCR showed incomplete insert for forward but not reverse primers. However, it confirmed insertion and showed no issues with transforming cell line.
Next, the assembly prepared in Golden Gate was inserted and heat-shocked into chemically competent cells that are optimised for t7 promotor expression (T7 Express Competent E. coli). The cells were once again plated on LB-AMP agar plates and incubated overnight. A successful colony was chosen and two 5 mL Luria broth-AMP seed cultures were inoculated in 15 mL falcon tubes and allowed to incubate overnight. The next day two LB-AMP flasks were prepared one was inoculated with 100 IPTG and the other was a control. The seed culture was then inoculated into 100 mL of LB-AMP flask and incubated overnight at 18°C.
The following day. The flasks were transferred to falcon tubes for both control and IPTG-induced and centrifuged to pelletise cells. The cells were then solubilised in a modified binding buffer (tris-sulfate buffer at pH 8, 5 mM imidazole, 0.5 M NaCl and 0.1 mM Nickel sulfate) and transferred to a round-bottom tube where they were lysed via a homogeniser. The lysis was then pelletised via centrifugation at 10,000 g and a small volume of supernatant of lysis for both control and IPTG induced were stored for later SDS-page in 4°C. Next, we passed through the supernatant through a syringe to remove particulates from both lysis supernatants.
Only IPTG induced after syringe passage was added to Ni-NTA resin in gravity column. The first run-through was collected and stored at 4°C. The protein of interest should no be ligated to the Ni-NTA resin. Next wash buffers containing different concentration of imidazole 40 mM, 60 mM, 100 mM and Elutation buffer at 1M imidazole were added to Ni-NTA resin in succession, collecting about the first 6 mL in 2 mL fractions. The gravity column where possible was kept temperature.
Bradford assay was performed to determine volume to be added into each well, BSA was used to draw standard curve.
Next SDS-PAGE was performed with the Control and IPTG induced samples prepared in the Ni-NTA assay step. These samples are highlighted below and are in order of well:
As can be seen in figure 5 significant band is experienced between 20 kda and 66 kda bands demonstrating high expression of protein which is likely to be the protein of interest.
Following this E1 Conc. was fractioned into a number of tubes and stored in -20°C with 20% glycerol for activity analysis.
Ni-NTA assay was repeated, starting with periplasmic extraction.
A periplasmic extraction was performed here, Sucrose tris buffer at pH 9 and the sequence placed and put in room temperature for 30 min. The tube was then spin down, media changed to magnesium sulfate and put on ice for 30 min. The result solution was mixed binding buffer and stored. This was followed subsequently with Ni-NTA assay.
The samples are listed below in order in which they were loaded (laddar is in well 1):
Likely due to human errors no protein was detected in any of the Ni-NTA buffer solutions.
To assay for the activity of carbonic anhydrase, a para-nitrophenyl acetate (pNPA) kinetic assay was used. pNPA is another substrate which can be metabolised by the CA enzyme. The product, para-nitrophenol, contains a chromophore which can be detected using spectroscopic means.
The first attempt was done true to the original protocol where a standard curve was constructed then enzyme samples of various concentrations were measured for their kinetics. Below are data for this initial trial.
The results were positive, however, below expectations. This is due to the enzyme not suited in the condition of the assay buffer. Literatures have varied on the most suitable pH level for this enzyme but all results seemed to indicate the optimal point lies somewhere between 6.0 to 9.0.
From this result, a comprehensive pH range assay was conducted to find out at what point do the enzymes have the highest function. Results are shown below.
Evidently pH of 7.3 showed the highest activity for the enzyme. Armed with this knowledge the search for the optimal buffer continues. It was at this moment when one of the team members returned from a seminar with Professor Morten Meldal the laureate for the Nobel prize of chemistry in 2022 as the speaker. He mentioned trying various different solvents for his experiment then finding out milliQ water to be the best solvent. This causes water with a pH of 7.35 to be used as a potential buffer.
The result exceeded expectations and water is found to be the most optimal buffer. It was the most fortunate of timing as the positive control for CA also arrived soon after. A test with HPCA and CA from humans was compared side-by-side and the result showed that in water, HPCA is about twice as effective as human CA.
A MALDI analysis for fingerprinting our protein was ordered however, due to time constraints it will not arrive in time this analysis will instead be something our team will update on during our judging session.