Difference between revisions of "Part:BBa K554010:Experience"

Revision as of 11:32, 19 October 2011

SoxR device

This part is composed by Constitutive Promoter linked to RBS+SoxR gene and Terminator. It was used to [http://2011.igem.org/Team:UNICAMP-EMSE_Brazil/Results#Device_2_testing:_SoxR.2FSoxS_system_regulating_GFP_production Device 2 testing: SoxR/SoxS system regulated GFP production]

Experimental results

In order to test the ability of the SoxR/SoxS sensor effector in sensing NO levels and activating genes linked to SoxS promoter, a Device testing was assembled, with GFP linked to SoxS promoter, as shown in the following schema (Figure 1):

The ability to recognize NO (nitric oxide), an inflammation signal molecule, was characterized for SoxR/SoxS sensor, and found to be FUNCTIONAL. NO is a product related to inflammatory response in vivo. To test the sensor in vitro, we used http://en.wikipedia.org/wiki/Paraquat Paraquat as inducer, a molecule which imposes superoxide stress within the cell in a similar manner as nitric oxide. In the section below we present the detailed results.

Methods

Competent E. coli DH5α strain cells were transformed with a pSB1A2 vector (Ampicillin resistant) carrying both the sensor (Strong_Constitutive_promoter + RBS + SoxR + Terminator) and the effector (SoxS_promoter + RBS + GFP + HlyA + Terminator) devices using a chemical shock protocol. Transformed bacteria were plated on solid LB medium with 50 μg/mL Ampicillin and grown at 37ºC overnight. Surviving colonies were grown on liquid LB medium containing 50 μg/mL Ampicillin. Oxidative stress was induced by adding increasing concentrations of Paraquat (Methyl viologen dichloride hydrate - Sigma), an oxidative stress inducer in bacteria. Final Paraquat concentrations were: 0 μM (control), 5 μM, 10 μM, 20 μM, 30 μM and 40 μM. Induction of the designed sensor/effector mechanism was accessed by fluorescence using a fluorometer (SLM – Aminco; 4 nm bandpass and 10 mm) with excitation in 500 nm and emission spectra from 508-550 nm. In order to access if increasing concentrations of Paraquat could inhibit culture growth due to it’s toxicity, optical density (OD) levels of the culture were measured during the incubation time with Paraquat. The GFP fluorescence was also detected in cells by fluorescence microscopy (Olympus).

Results

Achieved results indicated that the designed sensor/effector device system was capable of inducible production of GFP (or another generic protein controlled by the sensor). In addition, protein induction can be modulated through varying inducer concentration (Figure 2). Higher concentrations of Paraquat exhibited higher fluorescence levels, which indicates increased GFP concentrations. No plateau was achieved using the highest tested concentrations.

The experiment indicated that our sensor was capable of inducible production of GFP (or another protein controlled by the sensor). In addition, the protein induction can be modulated through varying the inducer concentration (as shown by Figure 2). Higher concentrations of Paraquat exhibited higher fluorescence levels, which indicates increased GFP concentrations.

The experimental concentrations of Paraquat did not show significant differences in cell growth as shown by OD levels in Figure 3. The [http://2009.igem.org/Team:Stanford/ProjectPage#Results_and_Analysis Stanford team (iGEM 2009)] showed that Paraquat can be toxic to E. coli cells, with growth inhibition at 60 – 80 µM.

Additionally, as complementary results for device 2 testing, we used Fluorescence Microscopy to detect the presence of fluorescence in the cultures containing the sensor and GFP production devices when induced with 40 uM of Paraquat against the non-induced cultures. The results also indicated that the bacteria was able to sense NO induced by Paraquat and respond through production of GFP, since microscopy data revealed GFP expression in the Paraquat induced cultures (Figure 5) but not in the non-induced one (Figure 6). This is an additional evidence that the NO sensor system and Sox driven effector (in this case, GFP) synthesis worked as expected.

Discussion

The NO is a product related to inflammatory response in vivo. To test the sensor in vitro, we used [Paraquat] as inducer, a molecule which imposes superoxide stress within the cell in a similar manner as nitric oxide. The experiment indicated that our sensor was capable of inducible production of GFP (or another protein controlled by the sensor). In addition, the protein induction can be modulated through varying the inducer concentration (as shown by Figure 2). Higher concentrations of Paraquat exhibited higher fluorescence levels, which indicates increased GFP concentrations. The experimental concentrations of Paraquat did not show significant differences in cell growth as shown by OD levels in Figure 3. The Stanford team (iGEM 2009) showed that Paraquat can be toxic to E. coli cells, with growth inhibition at 60 – 80 µM.

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