Leaky recombinant expression reveals design constraints of bicistronic synthetic operons in Escherichia coli

The simultaneous expression of multiple recombinant proteins in Escherichia coli is commonly achieved by synthetic operons containing one promoter, multiple ribosomal binding sites, and multiple recombinant genes. In these operons, the expression of the recombinant proteins is regulated, whereby operator/repressor combinations are often used for induction. Different approaches can be employed for co-expression of two or more recombinant proteins. Within this work, we created synthetic bicistronic systems with a single induction system. At the same time, the use of bicistronic systems can lead to increased complexity and potential regulatory issues in transcription and translation. In BL21(DE3), which is a common E. coli expression strain, leaky expression is associated with the basal expression of the T7 RNA polymerase (T7 RNAP). In addition, our research indicates that leaky expressions can also occur independently of the T7 RNAP and should be taken into consideration when designing synthetic bicistronic operons. By designing and testing multiple variants of one bicistronic system in two different E. coli strains, we identified potential promoter sequences causing leaky expression. We demonstrate that the design of a bicistronic expression system is critical for both the leaky and the induced expression of recombinant proteins.