Cloning and functional analysis of an arsB gene responsible for arsenic sequestration in Lysinibacillus sphaericus MB701A VT15: Master Degree Project in Molecular Biotechnology

a ABSTRACT Arsenic contamination in environment is serious threat to all living beings. Lysinibacillus sphaericus (B1-CDA) is a Gram positive bacterium that can grow in arsenic contaminated environment. The genes responsible for arsenic resistance of this bacterium have been identified as arsB, arsC and arsR by previous researchers. In the present study, arsB was cloned from genomic DNA of B1-CDA by PCR. The gene was characterized by in silico and in vivo experiments. In silico analysis by Iterative Threading ASSEmbly Refinement (I-TASSER) resulted that ArsB is an integral membrane protein and their putative function included cellular metal homeostasis. Results obtained in silico were in coherence with growth studies. The arsB cDNA was transferred into Escherichia coli JW3469-1, in which arsB gene was knocked out. Statistical analysis of growth indicated that there was significant difference in growth after complementation of arsB in Escherichia coli JW3469-1 when grown with 50 mM arsenic (p=0.04). Further analysis by Inductive Coupled Plasma-Mass Spectroscopy (ICP-MS) confirmed sequestration of arsenic inside the cells and thus its removal from growth media. Preliminary data obtained from ICP-MS indicated that arsenic concentration in cell free broth was decreased by 80.78% (from 50 mM to 9.61 mM) when treated for 24 hrs with transgenic cells (after complementation) and by 78.22% (from 50 mM to 10.89 mM) when treated with mutant cells (before complementation) but the difference was not significant. The results are inconclusive as there was a significant difference in growth between the transgenic and mutant cells but not significant in ICP-MS studies. Heavy metal poisoning is a serious threat to human; one such naturally occurring and highly toxic chemical is arsenic. Arsenic is widely distributed in water and soil, in some part of the few regions of United states) exceeding the maximum allowed limit which is 50 micrograms per cubic meter. As a result there are various diseases caused by arsenic exposure in human from skin rashes to cancer and multiple organ failure. Therefore, there is an alarming need to find ways to remove arsenic from the contaminated environment. There are many methods used for the arsenic removal from environment including oxidation, coagulation, precipitation, adsorption and membrane filtration and recently using microorganisms. Microbial or bio-remediation might be a successful method for avoiding arsenic contamination as they are cost effective and eco-friendly. This method uses microorganism that has resistance against heavy metals which can grow and uptake arsenics from the contaminated …