Results

Gel photo after PCR cloning of the individual gyrase binding sites

The three bands below the 400 bp mark on the gel photo indicate successful PCR of our gyrase binding sites

Chloroquine gel

Using chloroquine agarose gel to assay supercoiling, we demonstrated Mu GBS and GBS from pSC101 significantly increase supercoiling of the host plasmid by 42% and 25% respectively. The presence of the pBR322 GBS has significantly altered the level of migration.

As we intended at the design stage, introduction of a mu phage GBS (to make BBa_K676013) has significantly increased supercoiling of the 2009 Device, thus increasing its manufacturability as a commercial pDNA product. The presence of the GBS increased migration (supercoiling) by 10%.

The effect of the presence of a Mu GBS in a plasmid on the stress response

The graph indicates a clear distinction in the level of GFP expression between cell with Spy device and a Mu GBS/Spy device ligation. So this clearly proves that the presence of the Mu bacteriophage gyrase binding site creates a difference in the level of expression of GFP from the plasmid which is conclusive towards an increased stress response.

Effect of GBS on the level and quality of supercoiling in a plasmid molecule