Chemical approach for optimization of translation of β-galactosidase

Abstract

The two synthetic DNA duplexes, the one as control having an optimized sequence and the other possessing partially random sequence, corresponding to the region of ribosome binding site were synthesized through the phosphite method on solid support. The synthetic RBS DNA duplexes having Eco RI and HindIII cohesive ends were cloned into the previously engineered gap in plasmid pMKT2 possessing 1pp promoter and the $\beta$-galactosidase gene. This construction gave a direct screening system for the expression level on the plate by the difference of color intensities of colonies formed. The colonies which showed the; high expression of $\beta$-galactosidase are characterized in terms of the activities and sequence. The effects of the sequence on RBS region to the translational efficiency are discussed.