Methods and means relating to quiescent cells and uses thereof

#The present invention relates to cells in culture. In particular, it relates to bacterial cells in broth culture which may produce a heterologous (cloned) gene product, and synchronisation of cell division. Methods and means are provided for inducing and capitalising on quiescence of cells.

#The development of recombinant DNA technology over the last 20 years has made it possible to identify and isolate genes from any organism and express products in bacteria (the most common host organism is the enteric bacterium Escherichia coli). To achieve this, the gene is first inserted into a vector and then introduced into the bacterium by a method such as transformation or electroporation. Cloning and expression vectors are generally derivatives of plasmids: autonomously-replicating DNA circles which are found extensively in natural populations of bacteria. Typically, vectors carry some kind of marker to facilitate selection of vector-containing cells (for example an antibiotic resistance gene) and expression signals which direct the host bacterium to synthesise exogenous genes.

#A number of factors may reduce the efficiency with which the products of cloned genes are expressed in a bacterial host:

#The invention in various aspects will now be described further by way of exemplification. Figures referred to are as follows.

#FIG. 1 shows the structure of the ColE1 cer site. Conversion of plasmid dimers to monomers is accomplished by site-specific recombination at this site. The position of DNA strand exchange is shown by a cross. The Pcer promoter is active in plasmid dimers and directs the synthesis of the Rcd transcript, (approximately 80 bases).

#FIG. 2 shows the sequence and structure of the rcd transcript RNA (SEQ ID NO. 4).

#All references mentioned in the text are hereby incorporated by reference.

#Those skilled in the art are well aware of methods which can be employed to generate hns− or other mutant strains of E. coli or equivalents for other bacteria. One well-known technique which is particularly suitable is the use of “P1 transduction” (Miller 1972) to move transposon-inactivated hns. The technique involves introducing into host cells, via phage P1 infection, a defective hns gene containing an inserted transposon which includes a selectable marker (e.g. a gene which confers antibiotic resistance). Recombination within cells results in the replacement of the wild-type hns gene with the transposon-inactivated gene. Selection for the marker enables identification of successful recombination events. Only when the transposon is inserted into the host chromosome is the host-positive for the marker. Selected cells are tested for H-NS− phenotype and suitability for use in the present invention (Rcd sensitivity).

#We have primarily used the hns− derivative of a strain (DS941) which had been shown already to be Rcd-sensitive (by testing on agar plates). Some mutational event in some laboratory strains has been found to cause some resistance to the effects of rcd. However, this in no way detracts from the generality of the present invention, since those strains which are useful in the invention can easily be identified using no more than routine trial and error.