IGEM:IMPERIAL/2009/M2/Assays/3.1

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Rationale

We want to monitor real time rate of production of trehalose per cell. To do this, we need two pieces of information. Firstly, we need to monitor cell growth to know the approximate cell count. This can be done by OD to obtain cell count. Secondly, we need to know the rate of production of trehalose. This can be obtained from performing the trehalose assay at regular time intervals of 30 minutes. From the second piece of information, we can plot a graph of trehalose concentration against time. The gradient of this graph would give us the total activity of cells. Dividing this by the number of cells will give us activity per cell per unit time. This should resemble a hill function.

We can also perform the above mentioned two steps for different concentration of HSL. This will also allow us to characterise our biobrick.

Method
1) After activation of the CRP sensitive promoter, take samples at regular intervals.

  • Take 0.5ml sample from the culture and perform OD(600) to get cell count. Make sure OD is within linear portion of cell growth.
  • Take another 1ml sample and lyse using a sonicator (pulsed sonication, in an ice bath to reduce heating.)
  • Measure trehalose content using the trehlose method shown below:

Trehalose assay

  • Take samples at 30 minute time intervals, until a constant level of trehalose is reached.
  • Plot the increase in trehalose concentration against time. This will allow characterisation of the trehalose output with POPs input.
  • Functionality of the trehalose can be determined by the trehalose functionality assay.
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