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Cloning and Protein Overexpression of Chlamydomonas reinhardtii's Novel Gene Cia7
Author(s) -
GonzalezCantu Hector,
Vazquez Erick,
Ynalvez Ruby
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.928.6
Subject(s) - chlamydomonas reinhardtii , chemistry , gene , biochemistry , mutant , biology , expression vector , microbiology and biotechnology , recombinant dna
Heavy metal contamination presents a threat to biological systems. These metals interact with the catalytic‐domain of enzymes, can bind irreversibly to enzymatic residues, and interfere with their normal rate of activity. Biological systems have adapted certain mechanisms to attenuate this; one of them being the expression and synthesis of low‐molecular weight, thiol‐rich proteins, termed as phytochelatins and metallothioneins. One such organism, which may present such metal‐defense mechanism, is Chlamydomonas reinhardtii . C. reinhardtii is an excellent model organism used in studies of heavy metal tolerance, and, with the discovery of a putative, metal‐interacting gene, Cia7 in C. reinhardtii , the objectives of this study are (1) overexpress CIA7 in E. coli , for downstream metal‐binding assays (2) the nuclear transformation of C. reinhardtii , in order to generate insertional mutants, subsequently screened for antibiotic resistance, and heavy metal tolerance, in order to assess CIA7's role in metal homeostasis. The gene Cia7 was cloned into a protein overexpression vector, pMal C5X, in fusion, with a maltose‐binding protein, to be further purified, for downstream metal‐binding assays. From pMal C5X‐Cia7, the gene was subcloned and ligated to psL72 vector, in order to transform it into C. reinhardtii via electroporation. This study will report the cloning and overexpression of the novel gene CIA7, as well as the screening of insertional‐cia7 mutants. This study is of significance within the context of bioremediation and heavy metal contamination, specifically from metal‐contaminated bodies of water. The characterization of CIA7 as a metal‐binding protein would allow for its potential use as a metal sequestering agent. This would correlate to metal‐remediated ecosystems, which would eliminate the potential for metal poisoning in humans, as well as a method to quantify heavy metal contamination, by having C. reinhardtii serve as a biological marker for heavy metal contamination. Support or Funding Information Norman Hackerman Advanced Research Program