A new method for the construction of translationally coupled operons in a bacterial chromosome

Abstract

A new method for the construction of translationally coupled operons in a bacterial chromosome was developed on the basis of the recombineering approach. The method includes the in vitro construction of an artificial operon with an efficiently translated proximal cistron, its insertion into the Escherichia coli chromosome, the modification of the operon via Red-driven insertion of a special “Junction” with an excisable selective marker into the intercistronic region of the initial operon, and the excision of the marker. The Junction structure was designed and tested. The Junction consists of three components. The first component is the E. coli rpl C- rpl D intercistronic region and serves for placing the TAA codon of the proximal gene in the SD sequence (TAAGGAG) of rpl D. The second component is the Cm R gene flanked by λ att L/R sites in such a fashion that the residual λ att B site after λInt/Xis-driven excision of the marker does not contain termination codons in frame with ATG of rpl D. The third component is the E. coli trp E- trp D intercistronic region which is added so that TGA of trp E acts a termination codon of the new open reading frame (ORF), while the overlapping (TGATG) ATG of trp D is in the position of the initiation codon of the distal gene of the original operon. The general design of the Junction provides the conversion of the original two-cistron operon into a three-cistron operon with translationally coupled genes, where the coupling of the artificial ORF ( rpl D’-λ att B-’ trp E) with the proximal gene is due to the rpl C- rpl D intercistronic region and its coupling with the distal gene is due to trp E- trp D. The strategy was experimentally implemented to construct an artificial operon P tac - aro G4- ser A5, where the expression the distal ser A5 gene was optimized owing to translational coupling in a three-cistron operon.