tetA Optimization for Escherichia coli by Engineering Protein Structure and Promoter-RBS

Abstract

The tetA, which encodes a tetracycline resistant protein, is a very useful antibiotic resistance gene. It exhibits stable selectivity for tetracycline, and has been particularly used for genome engineering by using tetracycline-nickel dual selection. A dual selection system using a single gene for both positive and negative selection is considered as a more promising strategy in selecting desired recombinants because of its double functionality and elimination of inactive-marker genes caused by non-specific mutations. However, it has been reported that tetA negative selection is much less effective in Escherichia coli than in other bacteria such as Salmonella. There are a few reasons for the ineffective negative selection in E. coli, such as low activity of TetA, inadequate promoter and signal peptide, and so on. We propose three strategies to solve the problem; First, engineering of its promoter-RBS to optimize the expression of tetA in the genome of E. coli. Second, engineering of the signal peptide of the TetA protein to increase membrane integration efficiency. Finally, engineering TetA enzyme to increase Ni+ uptake activity. The effective tetracycline-nickel dual selection based on tetA gene would be very useful for genome editing in E. coli.