Biomineralisation is the process by which living organisms cause the formation of minerals as a by-product. In recent years biomineralisation processes such as Microbially Induced Calcite Precipitation (MICP) have gained significant traction in biotechnology as a way to achieve bioremediation, biocementation and bioconsolidation.
MICP is a simple process involving two enzymes, carbonic anhydrase and Urease. The enzymatic pathway is shown below.
Figure 1. Ureolytic MICP enzymatic pathway.
This process allows the creation of calcium carbonate, the major binding agent in cement. Of course, MICP has many issues. One of its biggest issues is the requirement for a lot of time and money. To improve this we use Carbonic anhydrase and Urease variants from H. pylori.
As MICP proceeds, the concentration of ammonia a by-product in the MICP process increases. The biggest effect of this is a rise in pH, to between 8 and 10. As pH rises the catalytic efficiency of enzymes drops and if it rises enough it causes osmotic shock and thus cell death. To complete this pathway we designed and attempted to express genes from the ammonium oxidation pathway and denitrification pathway. This pathway is shown below.
Identify, build, and characterise carbonic anhydrase and urease variants with improved catalytic characteristics for MICP.
Identify, build, and characterise enzymatic pathways to remove ammonia by-products produced by MICP.
After identifying our aims we began by doing a literature search for variants of carbonic anhydrase and urease that may have better catalytic properties. In our search, we determined that H. pylori presents itself as a great option. The urease variant of H. pylori has a much higher substrate specificity (Mobley et al, 2001) and has an optimum pH activity between 7.5 and 8.5 (Bauerfeind et al, 1997) ideal for denitrification that will follow MICP (discussed under Denitrification). Carbonic anhydrase from H. pylori presented a similar benefit with a pH optimum of 8.9 (Chirica et al, 2001) and synergism with Urease (Bury-Moné et al, 2008).
We first build an electronic assembly of a plasmid featuring our recombinant enzyme, carbonic anhydrase This assembly is shown below.
Carbonic anhydrase is an enzyme that is expressed in the periplasm of H. pylori. To enable this transport in E. coli, a signal peptide was incorporated into the sequence of carbonic anhydrase that enables that translocation to the periplasm, the TorA leader sequence. The sequence of carbonic anhydrase was further codon optimised for expression in E. coli cell lines, This was achieved using the in-built system in Benchling a platform for designing DNA sequences. A polyhistidine tag was attached to the end of the enzyme to enable the purification of the enzyme.
The Transformation and assembly of H. pylori Carbonic anhydrase (HPCA) took about two weeks. The de novo sequence was obtained from IDT, a supplier of oligonucleotides. Once our sequence had arrived at our lab, a golden gate assembly was performed to anneal synthetic sequence to backbone.
The recombinant DNA was then transformed into E. coli (T7 express, NEB Turbo), where its successful transformation was validated via sequencing. This was followed by recombinant protein expression via IPTG induction at the t7 promotor and purification. Purified protein was stored in -20°C fridge until Test phase.
The first enzyme to test is carbonic anhydrase (CA). Protocols already exist for testing CA but most are for enzymes expressed by other organisms. Much modifications are required to successfully assay HPCA. A standard way of measuring the activity of this enzyme is by a kinetics assay which measures the rate a certain substrate is consumed or product is made. This could be done with photospectrometry if one of the chemicals has a chromophore or a functional group that interacts with light. One of these chemicals is para-nitrophenyl acetate (p-NPA). The molecule appears colourless normally but when hydrolysed, turns into para-nitrophenyl (p-NP) and displays a yellow colour. CA can also metabolise this molecule and give off the same products. This allowed for the assaying of CA with simple processes. However, problems soon began to arise as attempts of assaying were made. These will be addressed in the section below. Through multiple cycles of trial and error as well as consulting with academics and literature, the enzymes were finally able to be assayed and characterised. This protocol was able to be transcribed into software to be executed by robotic systems such as the Opentron2 (OT2).
Figure 2. Para-nitrophenyl acetate hydrolysed to form para-nitrophenol and acetic acid.
This procedure was well suited for assaying CA from other organisms but the growth condition for H. pylori is different to the pH range of the assay buffer. The buffer has a natural pH of 9 and can only be acidified to pH 6.5. The activity for HPCA drastically decreased above pH of 7.5 contrary to the paper by Chirica et al. This problem was attempted to be addressed with trying different buffer combinations. Like all enzymes, CA has unique ranges of conditions where its activities are the highest. It is clear what this range is.
Since it was the first time everybody in the wet lab had been in the lab. We learnt a lot in the process of creating the carbon anhydrase part. The biggest issues we faced was the organisation of our team, we did not know where someone stored their reagents and where the cell lines were kept. The result of this was that some cloning and transformation steps had to be repeated.
Nonetheless, The Designing and Building of the enzyme were quite successful. We were able to achieve our goal of successfully expressing the enzyme in the cell line desired and were able to show a significant difference in expression compared to control. However on the other testing took quite a while. At the start it took a while to accumulate the reagents required for the assay since the lab needed to order them. The biggest issue may have been the lack of appropriateness of our assay for carbonic anhydrase or a lack of optimisation to perform it.
Again we first build an electronic assembly of a plasmid featuring our recombinant enzyme, carbonic anhydrase This assembly is shown below.
Similarly, Urease is also expressed in the periplasm of H. pylori. In building this construct, we contacted an academic here in UNSW, Dr Natalia Castano Rodriguez and an academic from the Louisiana State University Health Shreveport, Dr David Mcgee. In doing so David provided some advice on the importance of the Nix transporter and offered to send us the construct so we could work with it as a backbone (this unfortunately did not arrive). We were however able to arrange the genomic DNA of H. pylori with the help of Dr. Natalia Castano Rodriguez. This meant that DNA optimisation, attachment of leader sequence, and histidine tag would prove difficult. Due to this limitation, we planned to do our testing in lysis for later steps to confirm enzyme function.
We faced a number of issues when attempting to build our Urease assembly. Our first attempt at using primers to amplify the gene of interest Urease from the genomic DNA that we obtained from Dr Natalia from UNSW, resulted in failure, however our subsequent attempts were successful. The next problem we faced was the lack of pure linearised plasmid backbone, this meant we had to do plasmid miniprep, followed by linearisation of the plasmid. This also meant that a former insert of backbone remained and likely hindered our attempts at a gibson assembly.
The other issue we faced was that the large size of the insert made it less likely to anneal to the backbone. Before presenting our project, we were able to plate out a gibson plasmid. However, due to time constraints, we were not able to report this in detail.
We successfully built an assay to test whether the enzyme was active. However, we did not reach the testing stage of this assay. The procedure would use Christensen agar, which mixes phenol red into a diluted sample of LB agar. This is to ensure the bacteria have a stable medium, and stay alive during the assaying process. However, we ultimately decided that if we were going to perform this assay it would be more beneficial to do the cell in lysate, and thus only require phenol red solution. This protocol is described here.
As part of our second aim, we aimed to build, characterise and excecute a denitrification pathway to get rid of the excess ammonia by-product. Though we were able to get a start on the designing aspect in the background, we were unable to get to the build and test stage of this. We were able to develop putative assemblies, however we were unable to spend enough time to flesh these out. A picture of these assemblies is provided below. One limitation of this was that we planned to do our testing in lysis for later steps to confirm enzyme function.
