Control of cloned gene expression by promoter inversion in vivo: construction of the heat-pulseactivated att-nutL- p- att- N module

Abstract

We have constructed a prototype of gene-expression plasmids with three novel properties: (i) its “OFF phase” is absolute in all common hosts because the expression promoter is facing away from the studied gene and is blocked by a strong terminator; (ii) the “ON phase” is attained by the rapid and efficient inversion of the promoter; (iii) only a short heat pulse or exposure to other inducing agents is required to initiate this two-stage process. In the first stage, synthesis of the phage λ Int protein is induced by the transient derepression of the properly engineered λ xis −cIts857 prophage. In the immediately following second stage, Int causes inversion of a promoter cloned between the inverted P′OP phage att site and the normally oriented ΔPOΔP′ pseudo-bacterial att site. The inverted promoter can now control the expression of the studied gene and also of the λ N gene cloned in tandem. The N product, in conjunction with the nutL site placed downstream of the promoter, permits efficient antitermination of any terminators present in the att sites, in the plasmid or in the cloned DNA, making this system efficient and of practical value. Employing the promoter-inverting plasmid, it was possible to obtain rapid onset and a high level of galactokinase synthesis from the cloned galK gene. Only a transient, 10-min induction at 42°C was employed, permitting protein synthesis at 30° C, which might be of importance for thermosensitive products. Furthermore, the entire promoter-inversion module can be transferred to any plasmid as a 1.3-kb AvaI-Cla I fragment (see Fig. 1). The promoter-inverting plasmid provides also a simple method for measuring the Int function by assaying the galactokinase activity.