Philippine E-Journals

Construction and Phenotypic and Molecular Assessment of a Bidirectional Plasmid Vector Incorporating the Oryza sativa L. spp. japonica BIP1 Bidirectional Promoter and Antibiotic Resistance Genes

Thomas Gabriel H. Desengano | Sofia Philine H. Abayos | Evangeline D. Pascual | Bernabeth Jo T. Tendero | Jorge Gil Angeles

 

Abstract:

Bidirectional promoters are promoters that allow dual-direction gene expression. Often, plasmid vectors with unidirectional promoters are available thus, it would be more advantageous if plasmid vectors can drive gene expression in both directions. This study aims to construct a bidirectional plasmid vector utilizing antibiotic resistance genes and a reported bidirectional Oryza sativa L. spp. japonica (OsBIP1) promoter. The OsBIP1 promoter obtained from the Nipponbare variety and the ampicillin (amp) and chloramphenicol acetyltransferase (cat) antibiotic resistance reporter genes were amplified using primers appended with different restriction sites to facilitate directional ligation and employed optimized PCR conditions for annealing temperature (Tm) and MgCl2 and dNTP concentrations. The amplified OsBIP1 bidirectional promoter and these antibiotic resistance genes were restriction digested and directionally-ligated (OsBIP1-sense and OsBIP1-antisense orientations) into the pBI121 plasmid backbone using various insert: vector ratios to assess for promoter activity and directionality. The ligation products were transformed into competent E.coli DH5α and grown in media supplemented with ampicillin and/or chloramphenicol to phenotypically assess the developed bidirectional plasmid. Cell growth was observed in the ampicillin-only and in the ampicillin-chloramphenicol plates. Maximal bacterial colony growth for OsBIP1-sense and OsBIP1-antisense on these double antibiotic plates was detected using the 1:1 insert: vector (v/v) ratio. PCR confirmation detected the expected amplicon for OsBIP1 promoter::amp using the extracted plasmid DNA from randomly picked bacterial clones. The growth of the transformed bacteria in the double antibiotic plates and the detection of the expected OsBIP1 promoter::amp amplicon verify the construction of the bidirectional vector. This bidirectional vector may be used as a baseline for future studies.