Part:BBa_K353002:Experience
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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:
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.
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:
The above graph illustrates three behaviors: 1) At very high concentrations of AHL, our device produces a lot of sRNA1. These sRNAs then inhibit the GFP mRNA transcripts, leading to suppression of GFP fluorescence.
This is indicated by the three lines at the bottom of the graph.
2) At very low concentrations of AHL, our device produces very few to none sRNA1 transcripts. Hence, most to all of the GFP mRNA transcripts are translated, leading to a
high level of fluorescence. This is indicated by the three lines at the top of the graph.
3) At intermediate levels of AHL, we see intermediate levels of GFP fluorescence. This can be explained with the same logic as above. This also illustrates the operational
range of our device as well.
The graph below displays the same data, but with the x-axis displaying the ratio of Arabinose to AHL concentration that lead to each GFP fluorescence signal:
As before, this graph illustrates the same three behaviors as above. The lines near the bottom and to the left of the graph represent very high concentrations of AHL, which leads to the suppression of GFP fluorescence. The lines near the top and towards the right of the graph represent very low concentrations of AHL, which leads to a high level of GFP fluorescence. The lines at the middle of the graph represent intermediate levels of GFP fluorescence as a function of intermediate AHL and arabinose induction. The main fact conveyed by this information is that our device is able to detect a ratio (of approximately 5e2 - 5e3) within a specific range of inducer inputs by switching from a non-fluorescent state to a fluorescent one.
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