Introduction

The Gifsy promoters with their respective repressors and anti-repressors are important regulatory elements in the switch and this is why we wanted to characterize them and submit them as BioBricks. We already know that this system is tight in its natural settings in ''Salmonella'', but to our knowledge no one before us has tried to characterize the system in ''Escherichia coli''. A good way to characterize it in E. coli is to test whether the repression of the pR promoters is tight, and if the antirepressors indeed do antirepress. Because of previous knowledge of the strength of the promoters in ''Salmonella'' (reference, spørg seb) all characterization had to be done in low copy number plasmids. We have characterized the following BioBricks:

• BBa_K374008
• BBa_K374009
• BBa_K374010

Construction of BioBricks

BioBricks BBa_K374008 , BBa_K374009

In wildtype Salmonella enterica Serovar Typhimurium (strain ATCC 14028) the Gifsy immunity region, consisting of the two divergent promoters pR and pRM and the repressor gogR/gtgR downstream of the pRM promoter, are present in the chromosome. We designed a primer pair to amplify the immunity region from Gifsy 1 and Gifsy 2, and by adding the standardized prefix and suffix as a tail, the amplicons were made Biobrick compatible. The amplicons were made by PCR. After the PCR the fragments were cleaned up using a PCR clean-up kit.

The amplicon as well as the linearized backbone plasmid pSB1C3 were PCR clean-up kitdigested with the restriction enzymes EcoRI and PstI leaving sticky ends. Both the digested PCR product and the digested plasmid were run on a gel in order to estimate DNA concentration.

Characterization

Strategy

Results