Kloning dan Karakterisasi Gen Penyandi α-amilase (amyA) Aspergillus niger serta Konstruksi Ekspresi dengan Teknologi Gateway

Abstract

Cloning of the a-amylase gene of Aspergillus niger chromosomal DNA on pDEST 24 plasmid to increase the enzyme activity has been investigated. The transformation was conducted with several steps, including isolation of the messenger RNA, ligation by T4-DNA ligase, transformation of the recombinant DNA, and selection of the transformants using PCR colony. Gene encoding α- amylase isolated using RT-PCR (Reverse Transcriptase Polymerase Chain Reaction technique) and inserted into the pGEM-T Easy vector. Electrophoresis of the PCR products showed that the gene has a size of approximately 1650 bp. BLAST analysis of the DNA sekuen showed that this gene is homologous with the gene encoding α-amylase accessible from the gene bank. Expression of the gene encoding α-amylase of Aspergillus niger used gateway cloning technology. It showed that this gene has been successfully cloned in the cells of Escherichia coli through destination vector (pDEST 24). Electrophoresis of the PCR colony showed that the gene has a size of approximately 1650 bp.