CLONING AND CHARACTERIZATION SYNTHETIC PROMOTER OF ZEBRAFISH METALLOTHIONEIN-II GENE IN RECOMBINANT Escherichia coli

• Main Author: Deky Junihadi, Gusti
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/34369
• Institution: Institut Teknologi Bandung

Heavy metal pollutant is very dangerous, because it is toxicity, carcinogenic, and can undergo bioaccumulation and biomagnification. Metallothionein (MT) is a low molecular weight proteins (60-63 amino acids) can bind heavy metals and is involved in detoxification of heavy metals. MT is usually used as a biomarker to measure the level of heavy metal pollution in the waters. The expression of zebrafish Metallothionein-II gene (zMT-II) regulated by a promoter that can be induced by heavy metals. Activities zMT-II gene promoter can be analyzed by studying the transient expression of reporter gene regulated by a promoter in eukaryotic cells. In addition, gene promoter activity ZMT-II is thought to be studied in prokaryotes. Therefore, this study aimed to analyze the activity of MT-II gene promoter of zebrafish in Escherichia coli to control the expression of reporter gene Enhanced Green Fluorescent Protein (EGFP). Promoter Gene sequences zMT-II with accession number EU847278.1 was synthesized by the addition of restriction side for AseI (5 'end) and NheI (3' end). Synthetic promoter gene sequences zMT-II then cloned into the expression plasmid pEGFP-N1 section upstream of the reporter gene Enhanced Green Fluorescent Protein (EGFP). Promoter motif analysis was performed by using comparative study and Transfac bioinformatics program. Activities of the promoter was carried out by induction the promoter by using CdCl2.H2O then the transient expression of EGFP was measured by using Spectrofluorophotometer in Escherichia coli. Promoter synthetic of zMT-II was successfully cloned 1517 bp in length. Motif analysis showed the zMT-II promoter has elements such as TATA box, GC box, four regions of binding activator protein 1 (Ap1) and 10 Metal Response Elements (MRE) that are target binding of a transcription factor MRE-binding transcription activating factor (MTF-1 ). MTF-1 is a necessary transcription factor to activate transcription induced by heavy metal. zMT-II promoter activities in Escherichia coli with CaCl2.H2O exposure as much as 0, 25 and 50 ppm showed an increase in fluorescence intensity that allegedly because the expression of the EGFP fluorescence intensity is high at 25 ppm Cd induction. These results indicate that the zMT-II promoter can be induced by exposure to heavy metals and functional in prokaryotic cells.