This page outlines the experimental framework for validating surface-displayed carbonic anhydrase (CA) constructs across E. coli, Caulobacter crescentus, and Synechococcus elongatus UTEX. It connects molecular-level validation, protein localization and enzymatic activity, with biomineralization, establishing a full workflow from expression to microbially induced calcium carbonate precipitation (MICP).
The experiments outlined on this page represent planned future work that has not yet been executed. All assay designs, materials, and analysis pipelines have been developed and are ready for implementation. Our team plans to carry out these experiments in the upcoming phase of the project.
Introduction
Validating the function of our surface-displayed carbonic anhydrase (CA) constructs is critical to bridging our molecular-level enzyme design with functional biomineralization. To do this, we established a cross-chassis validation pipeline spanning E. coli (benchmark model), Caulobacter crescentus , and Synechococcus elongatus UTEX 2973. Together, these systems allow us to test CA expression, localization, and enzymatic activity- culminating in characterizing microbially induced calcium carbonate precipitation (MICP) for each of our engineered-strains.
Having designed surface-display constructs for all three organisms, our goal was to connect protein-level validation with downstream biocementation outputs. This required demonstrating three things: (1) successful surface localization of CA, (2) retention of and improved enzymatic activity when displayed, and (3) correlation between enzyme activity and calcium carbonate precipitation. The following experimental workflow outlines how each layer of validation was performed- moving from molecular assays (SDS-PAGE, Western blot) to functional tests (CO₂ hydration kinetics) and, finally, to mineralization assays that quantify MICP efficiency.
Experimental Workflow Overview
We divided validation into three main phases: Surface Display Verification, CA Activity Measurement, and Calcium Carbonate Precipitation Testing- each designed to progressively link genotype to functional phenotype.
Surface Display Validation
To confirm that CA was correctly localized on the cell surface and exposed extracellularly, we combined cell fractionation, trypsin accessibility, and immunodetection assays.
Surface Display Assay:
Outer membrane (OM) extraction in E. coli, S-layer extraction in Caulobacter, and S-layer stripping in Synechococcus is performed to isolate surface protein fractions. Benchmark strain (E. coli) expressing known surface proteins served as positive controls, while strains lacking recombinant protein were used as negatives. This assay identifies whether the CA fusion protein is present in the surface layer but does not directly confirm its exposure to the extracellular environment.
Trypsin Accessibility Assay:
Localization to the outermost cell layer, does not necessarily confirm extracellular exposure. The protein may be embedded within the S-layer or outer membrane, with its active site facing inward or occluded by structural components, making it inaccessible to the external environment. Therefore, the trypsin accessibility assay is required to distinguish truly extracellularly exposed proteins from those merely incorporated into surface-associated layers.
Enzymatic cleavage using trypsin was used to demonstrate surface display of the CA. Surface-accessible proteins will be digested, while intracellular proteins remained protected. After isolating the surface protein fraction, trypsin-treated samples would show the absence of the CA surface display fusion protein band, whereas untreated samples retained it - confirming that the enzyme was surface displayed and accessible to extracellular digestion.
Protein Expression and Localization:
Whole-cell lysates, surface fractions, and trypsin-cleaved samples will be analyzed by SDS-PAGE and Western blot using anti-Myc antibodies and Colourmetric staining (Horseradish peroxidase and 4-Chloro-1-naphthol method) . These assays verified the presence, size, and enrichment of CA fusion proteins.
Carbonic Anhydrase Activity Assay
To evaluate the enzymatic performance of our engineered carbonic anhydrases (CAs), we combined direct and indirect assays that capture both catalytic activity and downstream biomineralization efficiency. CA activity was quantified using the Abcam colorimetric esterase-based kit for standardized benchmarking and an in-house high-throughput Wilbur—Anderson assay to measure CO₂ hydration kinetics across temperature and pH gradients. In parallel, calcium carbonate precipitation efficiency was assessed using a calcium depletion assay, which tracks soluble calcium depletion under varying environmental conditions. Together, these assays established a functional link between enzyme activity and mineralization potential- key for optimizing CA performance in biocementation applications.
Calcium Depletion Assay
Using High-Throughput Calcium Carbonate Precipitation Assay, we measured the extent of microbially induced calcium carbonate precipitation (MICP) efficiency by quantifying the formation of calcium carbonate crystals under controlled conditions across engineered strains.
Candidate clones would be screened using the O-cresolphthalein complexone (O-CPC) method, which measures calcium carbonate formation indirectly by detecting the depletion of soluble calcium ions from the medium.
Figure 1: Initial O-CPC—based calcium depletion assay showing differential color intensities across wells. Wells with lighter coloration represent greater calcium carbonate precipitation, corresponding to lower residual calcium levels in solution.
To ensure accuracy, the results would be cross-validated through gravimetric analysis, in which the precipitated calcium carbonate would be air dried and weighed to confirm the extent of mineralization. Several parameters affecting crystal morphology and microbial activity would be tested, including temperature, pH, and calcium ion concentration.
Outcomes and Parameters Tested:
This assay identified which CA-expressing clones most effectively catalyzed mineral formation. Variables such as incubation temperature, and pH would be systematically altered to optimize precipitation yield. To validate our assay we are benchmarking it against the commercial Abcam (ab102505) Calcium Assay Kit (Colorimetric).
Carbonic Anhydrase Activity Assay
To validate carbonic anhydrase (CA) activity across constructs, two complementary approaches were used. The first employed the Abcam colorimetric CA Activity Assay Kit (ab284550), which measures CA’s esterase activity on a proprietary ester substrate that releases a chromophore (nitrophenol) upon hydrolysis. (relies on secondary esterase activity characteristic of mainly α-CAs). The amount of product released, quantified by absorbance at 405 nm, correlates directly with enzymatic activity. This commercial kit was used as a positive benchmarks, providing standardized and reproducible activity measurements.
The second approach utilized an in-house modified Wilbur-Anderson pH indicator-based assay. This high-throughput, 96-well plate adaptation quantifies CO₂ hydration activity using phenol red as a pH indicator, monitored at 557 nm. The assay measures the rate of pH drop as CO₂ is converted to bicarbonate and protons, reflecting CA catalytic efficiency.
CO2+H2O→HCO3−+H+
Compared to the Abcam esterase assay, this method directly captures CO₂ hydration kinetics, offering broader applicability to both α- and non-α-class CAs.
Controls and Conditions:
Bovine CA served as a positive control; heat-inactivated and CA-null strains as negatives. Assays were performed under varying buffer compositions, pH, and temperature conditions (25-90 °C) to assess catalytic robustness and thermal stability - parameters critical for industrial biocementation processes. Activity was quantified by the time required for a pH shift from 8.3 to 6.3, allowing direct comparison of enzymatic efficiency across hosts.
