Team:UCL London/Research/MagnetoSites/Results

From 2011.igem.org

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The three bands below the 400 bp mark on the gel photo indicate successful PCR of our gyrase binding sites
The three bands below the 400 bp mark on the gel photo indicate successful PCR of our gyrase binding sites
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'''Chloroquine gel displaying the difference in plasmid supercoiling between control and the three GBS'''
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'''Chloroquine gel'''
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[[File:Ucl-content-Magnetogel2.png|center]]
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[[File:Ucl-content-Magnetogel555.png|center]]
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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.
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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%.
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This gel clearly displays the superior property of the Mu GBS in introducing negative supercoils into a plasmid, as the bands for this plasmid travelled the farthest from the well. Although the pSC101 counterpart did manage to improve the supercoiling to a similar extent, the quality and consistency was still below that for Mu GBS. On the other hand the pBR322 GBS proved to be worse at introducing supercoils into the plasmid when compared to the control (pSB1C3 with RFP coding insert).
 
<h2>The effect of the presence of a Mu GBS in a plasmid on the stress response</h2>
<h2>The effect of the presence of a Mu GBS in a plasmid on the stress response</h2>

Revision as of 03:58, 22 September 2011

Contents

Results

Gel photo after PCR cloning of the individual gyrase binding sites

Ucl-content-Magnetogel1.png

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

Chloroquine gel

Ucl-content-Magnetogel555.png

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.

Ucl-content-Magnetograph.png

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

Ucl-content-Magnetogel3.png
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