Difference between revisions of "Part:BBa K554000:Experience"
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===Experimental results===
 
===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):
+
In order to test the ability of Jedi Bacteria in sensing NO levels and activating genes linked to SoxS promoter, we built a Device testing, with GFP linked to SoxS promoter, as it is shown in the following schema (Figure 1):
  
−
[[Image:UNICAMP_EMSE_GFP_SOX_device.jpg|center|700px]]
+
[[Image:UNICAMP_EMSE_GFP_SOX_device.jpg|center|660px]]
 
<div align=center> '''Figure 1: Testing [http://2011.igem.org/Team:UNICAMP-EMSE_Brazil/Project#Device_2:_NO_sensor.2FIL-10_producer Device 2] through replacement of IL-10 to GFP. '''</div>
 
<div align=center> '''Figure 1: Testing [http://2011.igem.org/Team:UNICAMP-EMSE_Brazil/Project#Device_2:_NO_sensor.2FIL-10_producer Device 2] through replacement of IL-10 to GFP. '''</div>
  
−
The ability to recognize NO (nitric oxide), an inflammation signal molecule, was characterized for SoxR/SoxS sensor, and <font color=red>'''found to be FUNCTIONAL'''</font>. 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===
  
−
====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_pormoter + 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 [http://en.wikipedia.org/wiki/Paraquat 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.
−
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====
+
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 ability to recognize NO (nitric oxide), an inflammation signal molecule, was characterized for SoxR/SoxS sensor, and <font color=red>'''found to be FUNCTIONAL'''</font>. In the section below we present the detailed 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.
+
===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.  
 +
  
−
[[Image:UNICAMP_EMSE_GFP_SOX_device_result1.jpg|center|600px]]
 
−
<div align=center>'''Figure 2: SoxS/SoxR fluorescence data for concentrations 0, 5, 10, 20, 30 and 40 µM of inducer.'''</div>
 
  
−
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.
+
[[Image:UNICAMP_EMSE_GFP_SOX_device_result1.jpg|center|500px]]
 +
<div align=center>'''Figure 2: SoxS/SoxR fluorescence data for concentrations 0, 5, 10, 20, 30 and 40 µM of inducer (Paraquat).'''</div>
 +
<br>
 +
 
 +
Moreover, we tested if Paraquat could be toxic for the bacteria cells, and we found that the experimental concentrations of Paraquat did not show significant differences in cell growth as shown by OD levels in Figure 3.  
  
−
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.
 
  
 
[[Image:UNICAMP_EMSE_GFP_SOX_device_result2.jpg|center|600px]]
 
[[Image:UNICAMP_EMSE_GFP_SOX_device_result2.jpg|center|600px]]
−
<div align=center>'''Figure 3: SoxS/SoxR optical density data for concentrations 0, 5, 10, 20, 30 and 40 µM of inducer.'''</div>
+
<div align=center>'''Figure 3: SoxS/SoxR optical density data for concentrations 0, 5, 10, 20, 30 and 40 µM of inducer (Paraquat).'''</div>
 +
 
  
 
[[Image:UNICAMP_EMSE_GFP_SOX_device_result3.jpg|center|600px]]
 
[[Image:UNICAMP_EMSE_GFP_SOX_device_result3.jpg|center|600px]]
−
<div align=center>'''Figure 4: Determination of specific growth rate (μ) for cells in the control (0 μM) and 40 μM experiment. X is the cellular concentration per volume. Specific growth rate (μ) is equal to the slope of the plot lnX x time, described by the equation lnX= lnX0 + μt.'''</div>
+
<div align=center>'''Figure 4. Determination of specific growth rate (μ) for cells in the control (0 μM) and experimental (40 μM) conditions. X is the cellular concentration per volume. Specific growth rate (μ) is equal to the slope of the plot lnX x time, described by the equation lnX= lnX0 + μt.'''</div>
 +
 
 +
<br>
 +
 
 +
Fluorescence Microscopy experiments also corroborates GFP production through Paraquat induction of SoxR/SoxS sensor/effector system. Fluorescence was detected in the cultures containing the sensor and GFP production devices when induced with 40 uM of Paraquat (Figure 5), but not in non-induced cultures(Figure 6). This is an additional evidence that the NO sensor system and Sox driven effector (in this case, GFP) synthesis worked as expected.
  
−
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.
 
  
 
[[Image:picind.png|center|600px]]
 
[[Image:picind.png|center|600px]]
−
<div align=center>'''Figure 5: GFP fluorescence assessed by microscopy in Paraquat induced cells. A) Fluorescence microscopy 40X Exp.: 0.478 ms; B) Light microscopy 40X; C) Fluorescence microscopy 100X Exp.: 0.478 ms; D) Light microscopy 100X.'''</div>
+
<div align=center>'''Figure 5: GFP fluorescence assessed by microscopy in 40 μM Paraquat induced culture. A) Fluorescence microscopy 40X Exp.: 0.478 ms; B) Light microscopy 40X; C) Fluorescence microscopy 100X Exp.: 0.478 ms; D) Light microscopy 100X.'''</div>
 +
 
  
 
[[Image:picnotind.png|center|600px]]
 
[[Image:picnotind.png|center|600px]]
−
<div align=center>'''Figure 6: GFP fluorescence assessed by microscopy in non-induced cells. A) Fluorescence microscopy 40X Exp.: 0.478 ms; B) Light microscopy 40X; C) Fluorescence microscopy 100X Exp.: 0.478 ms; D) Light microscopy 100X.'''</div>
+
<div align=center>'''Figure 6: GFP fluorescence assessed by microscopy in non-induced culture. A) Fluorescence microscopy 40X Exp.: 0.478 ms; B) Light microscopy 40X; C) Fluorescence microscopy 100X Exp.: 0.478 ms; D) Light microscopy 100X.'''</div>
−
 
+
  
 
===Discussion===
 
===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 SoxR/SoxS system is one of the best characterized redox-sensing mechanisms in bacteria. Under oxidative stress conditions, activation of this system results in a cascade effect that ends in the activation of more than 16 genes that can counteract the harmful effect of superoxide and other oxidative radicals (Pomposiello and Demple 2001).
−
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.  
+
This device is a modification of Stanford team anti-inflammatory device (iGEM 2009 - http://2009.igem.org/Team:Stanford/ProjectPage), which comprises SoxR gene (BBa_K223047) under control of a Constitutive Promoter (BBa_J23119) and SoxS promoter (BBa_K223001 – deleted part). Although this system has already been designed and used in previously iGEM competitions (iGEM 2009 - http://2009.igem.org/Team:Stanford/ProjectPage), we took advantage of the availability of synthesized sequences to design new parts (SoxR and SoxS) that conforms the common biobrick standards in the iGEM registry. Our team has improved the parts as described in [http://2011.igem.org/Team:UNICAMP-EMSE_Brazil/Project#UNICAMP-EMSE_Brazil_Parts_Design:_why_are_they_different_to_Stanford_2009_team_ones.3F Innovation].
 +
 
 +
These newly designed parts were capable of sensing different concentrations of the inducer, resulting in an increased production of the protein under control of the SoxS promoter (in this case, GFP).
  
 +
Under the experimental conditions, no cell growth inhibition was observed and confirmed by the calculations of the specific growth rate (μ). No significant difference was found in control experiment (0 μM of Paraquat) and cell induced with 40 μM of Paraquat, both presented μ= 0,18 h-1. Although the Stanford 2009 team showed that Paraquat concentrations between 60 μM and 80 μM can inhibit E. coli cell growth due to enhanced toxicity, but these concentrations were not included in our tests.
  
 
===User Reviews===
 
===User Reviews===

Latest revision as of 22:32, 28 October 2011

SoxS promoter

This part 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 Jedi Bacteria in sensing NO levels and activating genes linked to SoxS promoter, we built a Device testing, with GFP linked to SoxS promoter, as it is shown in the following schema (Figure 1):

UNICAMP EMSE GFP SOX device.jpg
Figure 1: Testing [http://2011.igem.org/Team:UNICAMP-EMSE_Brazil/Project#Device_2:_NO_sensor.2FIL-10_producer Device 2] through replacement of IL-10 to GFP.


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_pormoter + 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 [http://en.wikipedia.org/wiki/Paraquat 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 ability to recognize NO (nitric oxide), an inflammation signal molecule, was characterized for SoxR/SoxS sensor, and found to be FUNCTIONAL. In the section below we present the detailed results.

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.


UNICAMP EMSE GFP SOX device result1.jpg
Figure 2: SoxS/SoxR fluorescence data for concentrations 0, 5, 10, 20, 30 and 40 µM of inducer (Paraquat).


Moreover, we tested if Paraquat could be toxic for the bacteria cells, and we found that the experimental concentrations of Paraquat did not show significant differences in cell growth as shown by OD levels in Figure 3.


UNICAMP EMSE GFP SOX device result2.jpg
Figure 3: SoxS/SoxR optical density data for concentrations 0, 5, 10, 20, 30 and 40 µM of inducer (Paraquat).


UNICAMP EMSE GFP SOX device result3.jpg
Figure 4. Determination of specific growth rate (μ) for cells in the control (0 μM) and experimental (40 μM) conditions. X is the cellular concentration per volume. Specific growth rate (μ) is equal to the slope of the plot lnX x time, described by the equation lnX= lnX0 + μt.


Fluorescence Microscopy experiments also corroborates GFP production through Paraquat induction of SoxR/SoxS sensor/effector system. Fluorescence was detected in the cultures containing the sensor and GFP production devices when induced with 40 uM of Paraquat (Figure 5), but not in non-induced cultures(Figure 6). This is an additional evidence that the NO sensor system and Sox driven effector (in this case, GFP) synthesis worked as expected.


Picind.png
Figure 5: GFP fluorescence assessed by microscopy in 40 μM Paraquat induced culture. A) Fluorescence microscopy 40X Exp.: 0.478 ms; B) Light microscopy 40X; C) Fluorescence microscopy 100X Exp.: 0.478 ms; D) Light microscopy 100X.


Picnotind.png
Figure 6: GFP fluorescence assessed by microscopy in non-induced culture. A) Fluorescence microscopy 40X Exp.: 0.478 ms; B) Light microscopy 40X; C) Fluorescence microscopy 100X Exp.: 0.478 ms; D) Light microscopy 100X.

Discussion

The SoxR/SoxS system is one of the best characterized redox-sensing mechanisms in bacteria. Under oxidative stress conditions, activation of this system results in a cascade effect that ends in the activation of more than 16 genes that can counteract the harmful effect of superoxide and other oxidative radicals (Pomposiello and Demple 2001).

This device is a modification of Stanford team anti-inflammatory device (iGEM 2009 - http://2009.igem.org/Team:Stanford/ProjectPage), which comprises SoxR gene (BBa_K223047) under control of a Constitutive Promoter (BBa_J23119) and SoxS promoter (BBa_K223001 – deleted part). Although this system has already been designed and used in previously iGEM competitions (iGEM 2009 - http://2009.igem.org/Team:Stanford/ProjectPage), we took advantage of the availability of synthesized sequences to design new parts (SoxR and SoxS) that conforms the common biobrick standards in the iGEM registry. Our team has improved the parts as described in [http://2011.igem.org/Team:UNICAMP-EMSE_Brazil/Project#UNICAMP-EMSE_Brazil_Parts_Design:_why_are_they_different_to_Stanford_2009_team_ones.3F Innovation].

These newly designed parts were capable of sensing different concentrations of the inducer, resulting in an increased production of the protein under control of the SoxS promoter (in this case, GFP).

Under the experimental conditions, no cell growth inhibition was observed and confirmed by the calculations of the specific growth rate (μ). No significant difference was found in control experiment (0 μM of Paraquat) and cell induced with 40 μM of Paraquat, both presented μ= 0,18 h-1. Although the Stanford 2009 team showed that Paraquat concentrations between 60 μM and 80 μM can inhibit E. coli cell growth due to enhanced toxicity, but these concentrations were not included in our tests.

User Reviews

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