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Team:UCL London/Research/Supercoilometer/Experiments
From 2011.igem.org
Building a Supercoilometer in the lab
The first step to construct the device was to clone the tyrT promoter from the E coli genome by performing PCR. The relevant primers (with necessary prefix and suffix for BioBrick standardisation) was designed and synthesised in order to clone out the promoter sequence of interest. Following this a 3A assembly was carried out using the cloned tyrT promoter, a CFP expression cassette (from iGEM 2011 Kit Plate 1 - 8M) and standard plasmid backbone (pSB1C3) to construct the final device.
For characterisation, the device was further ligated to a plasmid backbone containing the Mu gyrase binding site. Two shake flask cultures were carried out, one with the device on a normal plasmid and another with device ligated to the Mu GBS. Cell samples were collected from both flasks every 2 hours for a duration of 24 hours, and the CFP fluorescence level was measured to detect a difference. The remaining cell sample at the end of the culture would be mini-prepped and run on a chloroquine gel to look at the difference in the level of supercoiling density. Further work will be needed to carry
