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Part:BBa_K353002:Experience

Designed by: Alejandro Virrueta   Group: iGEM10_Stanford   (2010-10-25)
Revision as of 21:30, 6 November 2010 by Axva1663 (Talk | contribs) (→‎Applications of BBa_K353002)

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Applications of BBa_K353002

Part BBa_K353002 is modification of earlier parts characterizing the pBAD (BBa_I0500) promoter. It consists of pBAD (BBa_I0500) promoter, an RNA tag/RSID (K353010), GFP (E0040), and a terminator (B1006). The parts were characterized on plasmid 1C3 in BW27783 cells. GFP fluorescence data was collected on a plate reader overnight (99 reads every thirteen minutes) after induction with varying does of arabinose. See dose-response curve for details. To gather flow cytometry data, fluorescence was read and plotted with number of cells as a function of intensity. The arabinose induction concentrations are provided as percentages. The top graph represents a fluorescence read for a single sample while the bottom graph represents a normalized fluorescence reading for the same data.

Below is a graph of our flow cytometry data for part K353002 plotting GFP output for BW27783 cells: Realdata.jpg

Both of these graphs plot the output of part K353002 in BW27783 cells induced with a series of concentrations of L-arabinose, measured in molarity. The first is a histogram showing the distribution of expression levels at each concentration of L-arabinose. The second is the same data displayed as a cumulative distribution so that population medians are more apparent.

740px-Flow data.jpg

We measured a dose-response-curve of median output vs L-arabinose concentration in triplicate. This curve constitutes the basic characterization of part K353002, including measurement of the concentration of L-arabinose which gives half-maximal output, the steepness of transition from off to on, and the parts dynamic range. Fitting a Hill curve provided the following values plus or minus 95% confidence intervals:

Half-max induction: 143 +- 36 uM

Hill coefficient: 1.4 +- .6

Fold Induction: 860


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The next step in our characterization of this part was to transform cells with both K353002 and K353009. This enable us to test a redundant component of our full sRNA system and to determine if the the device does measure a ratio. The experimental setup consisted of a 15 by 16 array of wells spanning several 96 plates. We repeated this array in triplicate. Each well contained 20 uL of cells at an OD of approximately .3, 100-200 uL of inducer, and M9 media filled up to 1 mL. The wells contained the following concentrations of AHL:

1e-12, 1e-11, 3.2e-11, 1e-10, 3.2e-10, 1e-9, 1.78e-9, 3.2e-9, 5.7e-9, 1e-8, 3.2e-8, 1e-7, 3.2e-7, e-6, and 1e-5

and L-arabinose:

1e-7, 1e-6, 3.2e-6, 1e-5, 3.2e-5, 1e-4, 1.78e-4, 3.2e-4, 5.7e-4, 1e-3, 3.2e-3, 1e-2, 3.2e-2, and 1.e-1. .

These plates were then incubated over night again, transferred 200 uL of cultured solution from each well to 96 clear-bottom well plates, and measured using a plate reader. The graph below displays the results:


This graph displays the same data, but with the x-axis displaying a ratios of Arabinose over AHL concentrations:

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