Bioremediation of both mineral and organic mercury via the construction of recombinant vector pET28a(+)-merA-merB

Subject Areas : Microbial Biotechnology

Hamide Baghi Sefidan ¹ , Alireza Tarinejad ²

1 - کارشناس ارشد، گروه بیوتکنولوژی کشاورزی، دانشکده کشاورزی، دانشگاه شهید مدنی آذربایجان
2 - دانشیار، گروه بیوتکنولوژی کشاورزی، دانشکده کشاورزی، دانشگاه شهید مدنی آذربایجان

Received: 2018-03-27 Accepted : 2018-06-11 Published : 2018-11-22

Keywords: cloning, Bioremediation, merA gene, merB gene,

Abstract :

Background & Objectives: Mercury due to stability and the high cost of conventional refinement methods is a major environmental problem in the world. Biological methods such as the use of bacterial-based bio-reactors or their enzymes are one of the bioremediation methods. MerA and MerB bacterial enzymes are used to decompose organic and inorganic compounds of mercury. This study was designed to clone merA and merB genes into the pET28a (+) expression vector for the production of MerA and MerB active enzymes.Material & Methods: At first merA and merB genes were isolated from mercury- resistant bacterial genome and subsequently cloned into pET28a(+) expression vector. Confirmation of cloning the target gene was achieved by PCR and restriction enzymes. Then pET28a(+)-merA-merB recombinant vector was transformed into E.coli strain BL21. To assess resistance to inorganic and organic mercury by transformed bacteria and the functionality of the enzyme produced by a recombinant vector, the growth of E.coli strain BL21 containing the recombinant vector and without it were measured by adding mercury into the environment during 48 h.Results: Recombinant bacterial growth in medium containing different levels of inorganic and organic mercury was measured at different times. The result showed that the growth of E. coli containing no target gene in the vector was affected after introducing mercury into the medium till 12 hours so that bacteria would not be able to grow at 10 and 20ppm mercury concentrations. However, transformed bacteria with pET28a(+)-merA-merB vector showed suitable growth in a mercury-containing medium. The SDS-PAGE analysis of extracted proteins from transformed bacteria with pET28a(+)-merA-merB vector on 12.5% acrylamide gel showed the highest MerA (62kDa) and MerB enzymes (23kDa) expression following 16 hours induction with 1mM IPTG at 37ºC.Conclusion: Growth ability of transformed E.coli with recombinant vector indicates MerA and MerB proteins function in transformed bacteria. Furthermore, increasing resistance of recombinant bacteria to inorganic and organic mercury indicates that heavy metal pollution in the environment can be cleaned up with proper management through the construction of a recombinant vector.

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