Problem Statement and Definition
Heavy metals affect the lifestyle of plants and animals in an ecosystem. Heavy metal pollution occurs mainly in marine regions where industrial effluents are released. Industries such as Non- ferrous refineries, batteries, textile and fertilizer manufacturers release effluents that possess traces of Cadmium. Prolonged exposure to Cadmium may lead to liver and kidney damage, respiratory ailments and even mutations in some cases. Curlim is a biological initiative taken up by team REC-CHENNAI to eradicate the presence of heavy metals in water.
Ideation
Project Curlim aims to precipitate the heavy metal Cadmium by means of genetic engineering. E. coli is a lab strain listed in the iGEM white list of organisms which is safe and easy to handle. The E. coli strain is genetically engineered in such a way that it produces a phosphatase enzyme. This enzyme forms complexes with Cd and settles down. Project Curlim is a cost-efficient and eco-friendly solution to control pollution.
Design and Prototype
A MBBR was designed to provide the optimum environmental conditions to carry out the scavenging process. Bio Carriers were added to provide anchorage support for the genetically modified biofilm. The E. coli K12 MG1655 strain was modified in such a way that it displays an efficient biofilm growth. An aphA coding sequence (BBa_K4509369) was added for the expression of Acid Phosphatase enzyme. An outer membrane protein tag, ompA was added to the plasmid construct to present the phosphatase enzyme on the cell surface for better Cadmium interaction. Promoters were added for better protein expressions.
Testing and Analysis
The transformed E.coli was sub-cultured and allowed to develop transformed into colonies. The culture broth was transferred in a microtiter plate and incubated at 28°C. Various assays were conducted to prove the expression of the enzymes. A lab scale model of the Moving Bed Biofilm Reactor was constructed with charcoal and plastic bio carriers. Substrate and feed were imputed into the MBBR The transformed cells are coated over the bio carriers and introduced into the reactor. The set-up was aerated and agitated with sparger and kept undisturbed for 24 hours. The medium was then taken out and given for testing and analysis
Proof of Concept with Lab work results
To prove the expression of acid phosphatase by the biofilm, PNPP assay and Biuret's tests were performed.
To test the removal of Cadmium from the effluent sample (artificial lab sample), Atomic Adsorption Spectroscopy is performed. The concentration of cadmium in the control sample and sample after 24 hours were plotted in a graph against time.
