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 digested 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.

The ligation was made using T4 ligase with a 5:1 ratio of insert to backbone. After the ligation the plasmid was transformed into electrocompetent DH5α E. coli cells. After an hour of recovery in LB media at 37 °C the cells were plated on LB plates containing chloramphenicol (Cam).

The next day five colonies on the plates were selected and restreaked on LB+Cam plates in order to assure pure colonies. Overnight cultures were made of the transformants by taking one colony from each restreak and inoculating it in LB+Cam at 37 °C over night. Minipreps were made from the overnight cultures and a verification PCR was run on these in order to assure that the plasmid had the expected insert.

BioBrick BBa_K374010

The Gifsy1 anti-repressor AntO was amplified by PCR using a plasmid containing the anti-repressor as template (Sebastién Lemire). The primers had tails corresponding to the standardized prefix and suffix making the amplicon BioBrick compatible. Once we had the amplicon it was digested, ligated and transformed into DH5α E. coli cells following the same procedure as in the above mentioned BioBricks. The results from the verification PCR can be seen in figure 1.

Characterization

Strategy

Results