Abstract
In this study E. coli recombinant clones that express the EC20 synthetic phytochelatin intracellularly were constructed. The increasement of Cd2+ biosorption capacity, and, also, the increasement of resistance to this toxic metal were analyzed. A gene that encodes the synthetic phytochelatin EC20 was synthesized in vitro. The EC20 synthetic gene was amplified by PCR, inserted into the DNA cloning vectors pBluescript®KS+ and pGEM®-TEasy, and also into the expression vectors pTE [pET-28(a)® derivative] and pGEX-T4-2®. The obtained recombinant plasmids were employed for genetic transformation of E. coli: pBsKS-EC20 and pGEM-EC20, they were introduced into DH10B and DH5α strains, similarly to pTE-EC20 and pGEX-EC20 that were introduced into BL21 strain. The EC20 expression was confirmed by SDS-PAGE analysis. The recombinant clones’ resistances to Cd2+ were determined by MIC analyses. The MIC for Cd2+ of DH10B/pBKS-EC20 and DH10B/pGEM-EC20 were 2.5 mM (EC20 induced), and 0.312 mM (EC20 repressed); respectively, 16 and 2 times higher than the control DH10B/pBsKS (0.156 mM). The MIC for Cd2+ of BL21/pTE-EC20 was 10.0 mM (EC20 induced) and 2.5 mM (EC20 repressed), compared with the control BL21/pTE which was only 1.25 mM. Analysis of ICP-AES showed that BL21/pGEX-EC20, after growth on the condition of EC20 expression, absorbed 37.5% of Cd2+, and even when cultured into the non-induction condition of EC20 expression, it absorbed 11.5%. These results allow the conclusion that recombinant E. coli clones expressing the synthetic phytochelatin EC20 show increased capacity for Cd2+ biosorption and enhanced resistance to this toxic ion.
Highlights
Industrial and other human activities have been generating environmental pollution in a never observed amounts, creating demands for the development of new remediation techniques
The EC20 synthetic gene was inserted into two cloning plasmids pGEMTEasy (Promega®) and pBluescriptKS(+) (Stratagene®) resulting in the recombinant plasmids pGEM-EC20 (Figure 1-b) and pBsKS-EC20 (Figure 1-c)
These recombinant plasmids were used in the genetic transformation of E. coli DH10B and DH5α strains
Summary
Industrial and other human activities have been generating environmental pollution in a never observed amounts, creating demands for the development of new remediation techniques. A promising alternative that grows on demand and development is the use of biomaterials for biosorption of toxic heavy metals. These biomaterials are named as biosorbents (VOLESKY; HOLAN, 1995; GAVRILESCU, 2004; GADD, 2009; WANG; CHEN, 2009; GUPTA; NAYAK; AGARWAL, 2015), they are beginning to be used in bioremediation of metal contaminated waters (GAVRILESCU, 2004; AKPOR; MUCHIE, 2010; AYANGBENRO; BABALOLA, 2017). Compared to traditional physicochemical techniques, bioremediation of toxic metals presents advantages such as lower costs, superior performance and safety, besides being environmentally friendly (GADD, 2009; WANG; CHEN, 2009; GUPTA; NAYAK; AGARWAL, 2015; AYANGBENRO; BABALOLA, 2017)
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