Difference between revisions of "Part:BBa K353002:Experience"

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Characterization 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 for part K353002 in BW27783 cells was collected on a plate reader overnight (99 reads every thirteen minutes) after induction with a series of concentrations of L-arabinose, measured in percentage dilution. To gather flow cytometry data, fluorescence was read and plotted as a histogram showing the distribution of expression levels at each concentration of L-arabinose. The second figure 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

Characterization of BBa K353002 in the presence of BBa K353009

Basic Validation of K353002 and K353009: Microscopy and GFP Fluorescence

The next step in our characterization of this part was to transform cells with both K353002 and K353009. This enabled us to test a redundant component of our full sRNA system and to determine if the the device is capable of consistently measure a ratio at many absolute levels of induction of the L-arabinose and AHL inputs.

The above figure is a graph of the GFP fluorescence measured in two different cell types grown overnight in AHL and then back-diluted in L-arabinose M9 media. This data demonstrated that GFP inhibition depended on the sRNA construct (K353009) and was not an artifact of the GFP construct (K353002). The only condition that displayed notable GFP induction was GFP only-containing cells (K353002) with high arabinose.

We inoculated two cultures of combo cells over night at 37 C: one with just arabinose (2%) and another with arabinose (2%) and AHL (.1%) in M9 media. We added 100 uL of the arabinose cell solution to four microscopy pads, two containing just arabinose (2%) and two more with arabinose (2%) and AHL (.1%). This lead to the following experimental set up:

Movie G1: Cells grown with arabinose O/N, placed on a pad with arabinose (control).

Media:G1_Ara_Ara.mov

Movie G4: Cells grown with arabinose O/N, placed on a pad with arabinose and AHL.

Media:G4_Ara_AraAHL.mov

Movie G3: Cells grown with arabinose and AHL O/N, placed on a pad with arabinose and AHL (control).

Media:G3_AraAHL_AraAHL.mov

Movie G2: Cells grown with arabinose and AHL O/N, placed on a pad with arabinose.

Media:G2_AraAHL_Ara.mov

Movies G1 and G4 demosntrate that the addition of AHL leads to the inhibition of fluorescence signal. Hence, with more sRNA present than GFP mRNA transcripts, fluorence decreases. G1 is included as an AHL-free control.

Movies G2 and G3 demosntrate that the removal of AHL leads to increased production of fluorescence signal. Hence, with less sRNA present than GFP mRNA transcripts, fluorence increases. G3 is included as an AHL-control.

These experiments serve as a visual indicator of the dynamic behavior of our system.

Validation of Ratio Measurement

The last experimental setup consisted of measuring GFP fluorescence while induced with both input chemicals over specific ranges. We created a a 15 by 16 array of wells spanning several 96 plates. We repeated these measurement in triplicate. Each well contained 20 uL of cells at an OD600 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 Molar

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 Molar

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 above graph illustrates the following behaviors:

1. Even in the presence of K353002, the sRNA-expressing construct, K353002 is induced by L-arabinose to produce fluorescence in a dose-dependent manner.
2. At high concentrations of AHL, our device should produce a high level of sRNA1. This sRNA then inhibits translation of the GFP mRNA transcripts, leading to suppression of GFP fluorescence. Maximal inhibition is indicated by the three curves of high AHL at the bottom of the graph.
3. At low concentrations of AHL, our device should produce only a small number of sRNA1 transcripts. At these levels of AHL, we see a high level of fluorescence, which corresponds to most to all of the GFP mRNA transcripts being translated. This is indicated by the three curves of low AHL at the top of the graph.
4. At intermediate levels of AHL, we see intermediate levels of GFP fluorescence. This corresponds to the inducible range of both L-arabinose and AHL-responsive promoters, and it is the operational range of our device.

The graph below displays the same two-dimensional array of data, but with the x-axis displaying the ratio of Arabinose to AHL concentration that lead to each GFP fluorescence signal:

This graph illustrates the same 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.

Below, we show that our device is able to consistently detect a ratio of approximately 5e2 - 5e3 [L-arabinose]/[AHL] in the concentrations of the inputs. Our device detects this range of ratios, spanning one order of magnitude, over a range of AHL concentrations spanning at least two orders of magnitude: 1e-9 to 1e-7 M.

If our device did not perform a ratiometric calculation on the L-arabinose and AHL inputs, we would expect the range of detected ratios to equal the range of AHL input. However, the range of detected ratios is less than the range of AHL input, indicating our device's calculation of a stoichiometric ratio.

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