Abstract
In order to increase the cytotoxic activity of a Salmonella strain carrying a salicylate-inducible expression system that controls cytosine deaminase production, we have modified both, the vector and the producer bacterium. First, the translation rates of the expression module containing the Escherichia coli codA gene cloned under the control of the Pm promoter have been improved by using the T7 phage gene 10 ribosome binding site sequence and replacing the original GUG start codon by AUG. Second, to increase the time span in which cytosine deaminase may be produced by the bacteria in the presence of 5-fluorocytosine, a 5-fluorouracyl resistant Salmonella strain has been constructed by deleting its upp gene sequence. This new Salmonella strain shows increased cytosine deaminase activity and, after infecting tumour cell cultures, increased cytotoxic and bystander effects under standard induction conditions. In addition, we have generated a purD mutation in the producer strain to control its intracellular proliferation by the presence of adenine and avoid the intrinsic Salmonella cell death induction. This strategy allows the analysis and comparison of the cytotoxic effects of cytosine deaminase produced by different Salmonella strains in tumour cell cultures.
Highlights
Bacteria can be adapted to synthesize proteins with relevant biotechnological applications
In order to increase the amount of cytosine deaminase (CD) produced keeping standard induction conditions, we improved both the producing Salmonella strain and the CD expression plasmid
We transferred the new genome-integrated regulatory module previously developed in our laboratory (Medina et al, 2011) to the SL7207 Salmonella strain, generating the MPO375 strain
Summary
Bacteria can be adapted to synthesize proteins with relevant biotechnological applications. In order to assess the effects of CD produced by improved strains and plasmids in tumour cell cycle distribution and bystander activity in long-term cell cultures, a purD mutation has been generated in the producer strains to avoid cell death induced by intracellular Salmonella proliferation (Leung and Finlay, 1991; Mesa-Pereira et al, 2013).
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