Assessment of Chloroplast Expression Factors in Escherichia coli

Abstract

Background: Genetic manipulation of chloroplast in higher plants offers a number of unique prerogatives, including; undesirable of pleiotropic genome and gene silencing effects and also use as an important agronomic trait for producing essential biomaterials and industrial enzymes. In order to manipulate chloroplast genome, specific vectors are required. These vectors can be transformed and expressed in Escherichia coli due to the same evolutionary origin of bacteria and chloroplasts. Objectives: The aim of the present study was to construct chloroplast vector specified for spinach and assessing the chloroplast regulatory elements in a prokaryotic expression host, E. coli. Materials and Methods: Flanking sequences (INSL+INSR) were isolated by PCR from the spinach chloroplast genome and blunt-end ligated into the PvuII site of pUC19 vector to form an intermediate vector, pUCINS. Then the selectable marker cassette (including aadA gene, Prrn promoter and rbcL terminator) was isolated via PCR and blunt-end cloned into the unique PvuII site of pUCINS to make the final chloroplast vector, named pCSI. Results: The constructed vector transformed to E.coli strain DH5α and several procedures such as colony PCR, digestion and sequencing were assigned to confirm the consequence of the construct. Conclusions: The appearance of bacterial colonies on the plate containing different concentrations of streptomycin indicated the strength of resistance to streptomycin which showed the bacterial cells capability to express aadA gene under the controls of chloroplast regulatory elements.