Difference between revisions of "Part:BBa K554009:Experience"

Revision as of 10:43, 19 October 2011

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Please enter how you used this part and how it worked out.

TolC

This part was used to [http://2011.igem.org/Team:UNICAMP-EMSE_Brazil/Results#Device_3_testing.2C_Protein_Secretion_System Device 3 testing, Protein Secretion System]

Device 3 testing, Protein Secretion System

The assembled devices related to secretion system (Hemolysin secretion system under control of SoxS – SoxS-HlyB-HlyD-TolC) were tested under laboratory conditions using GFP as reporter, coupled to the secretion signal sequence HlyA.

UNICAMP EMSE secretion device paraquat schema.jpg

Figure 1: Testing [http://2011.igem.org/Team:UNICAMP-EMSE_Brazil/Project#Device_3:_Secretion_system Device 3] through replacement of IL-10 to GFP. This is a representation of device 3, the protein secretion system coupled to Sox regulated GPF production device (Part BBa_K554012). To export a protein, the bacteria must have the HlyD, HlyB and TolC proteins and the target protein must have a signal sequence (HlyA tail), as shown here in a light red oval. In this case, the target protein to be secreted is GFP.

Methods

To access GFP secretion, competent E. coli DH5α strain cells were transformed simultaneously with a pSB1C3 vector (Chloramphenicol resistant) carrying both the sensor (Strong_Constitutive_promoter + RBS + SoxR + Terminator) and the effector (SoxS_promoter + RBS + GFP + HlyA + Terminator), and pSB1AK3 (Ampicillin resistant) carrying the secretion system (SoxS_promoter + RBS + HlyB + HlyD + TolC + Terminator). As a non-secretion control, E. coli harboring only the pSB1C3 vector with both sensor and effector systems was used.

Oxidative stress was induced by adding increasing concentrations of Paraquat (Methyl viologen dichloride hydrate - Sigma), an oxidative stress inducer in bacteria. The secretion was tested in cultures harboring A) and B) (Figure ) using 0 μM and 40 μM of Paraquat as described [http://2011.igem.org/Team:UNICAMP-EMSE_Brazil/Results#Methods in this section]. After 3 hours samples were collected, centrifuged (4000 rpm / 10 min; to avoid cell lysis) and the supernatant was collected and centrifuged again (13000 rpm / 10 min; to remove remaining cells). The supernatant fluorescence was measured in fluorometer (SLM – Aminco; 4 nm bandpass and 10 mm) with excitation in 500 nm and emission spectra from 508-550 nm. The GFP fluorescence was also detected in cells by fluorescence microscopy (Olympus).

Results

GFP secretion was confirmed by fluorescence emission and estimated to be approximately 6% of total protein, according to fluorescence levels. Significant levels of GFP fluorescence were found only in the supernatant of Paraquat induced cultures containing both the sensor/effector and the secretion systems but not in the non-induced cultures and in the cultures containing only the sensor/effector system (Figure 2).

In the black line we have our control, bacteria with the complete secretion system and without paraquat added. You see low fluorescence levels. The red line represents the culture induced with 40uM paraquat, with the fluorescence peak at 511nm. In order to check it this fluor. Peak is due to na artifact such as cell lysis we tested the cultures without secr. System with paraquat added. We saw no fluor. Peak and we may conclude that the observed for red is really due to GFP secretion.

Figure 2: Supernatant fluorescence emission from 508 to 540 nm, excitation in 500 nm. Red line (40 μM Paraquat added): sensor/effector with secretion system, showing a clear peak of fluorescence measurement at 511 nm; Black line (0 μM): sensor/effector with secretion system, non-induced. Blue line (40 μM Paraquat added): sensor/effector without secretion system;

From Figure 2, it is possible to observe that the red line shows a clear and high supernatant fluorescence peak at 511 nm, and the values are higher than the bacteria harboring the three systems (NO sensor, GFP producer and protein secretion) but whithout induction of NO by Paraquat (Black line), which may indicate that GFP is being exported. To test if the observed effect is not a False Positive (e.g. the same effect could be observed when bacteria suffer a cell lysis or if there is a secretion system in bacteria that could promote GFP export to the extracelular media) we decidede to check the supernatant fluorescence of bacteria with 40 uM Paraquat added and harboring only two systems: the NO sensor and GFP production devices but lacking the secretion system device (Blue line). This experiment showed that the fluorescence levels found in the Blue line are significantly lower than the observed for the red lines, which may indicate that the observed pattern of red line is not due to an error.

UNICAMP-EMSE Brazil Parts Design: why are they different to Stanford 2009 parts?

In the section below we aim to explain in details how our parts differ from Stanford 2009's, and why we decided to create novel parts to achieve similar objectives.

Secretion System Changes

Analyzing the pre-existing secretion system from Stanford Team 2009 we have decided to make several changes in order to make it more efficient and to prove that it actually works.

Change the RBS part and its disposition in the transcriptional unit.
1. Attempting to create a new secretion system, we began by choosing a different but still highly efficient RBS part. And according to the experience made upon RBS deposited parts (BBa_B0034), it was suggested that the RBS chosen by our team (B0030) is as efficient as the one used by Stanford 2009 team (B0034).
2. To improve translation efficiency, gene sequence fidelity (avoid errors or mistranscription during PCR) and save time during the devices assembling UNICAMP-EMSE team synthesized each coding sequence already fusioned with RBS sequence. On the other hand Stanford 2009 Team did not use translational units.

2. Biobrick Standard Compatibility

The secretion system developed by Stanford Team 2009 was not compatible with the systems 10, 12, 21, 23 nor 25. UNICAMP-EMSE Team has removed internal illegal restriction sites presented in the parts Bba_K223055 and BBa_K223057 by codon usage of these sites prior to synthesis. Therefore our team suited the secretion system to be compatible with 10, 12 and 23 standards.

3. Exclusion of non-essential genes

In our system we have reduced the number of genes to use only the essential ones, considering that “the simpler, the better”. Evidence (Fath et al, 1993) shows that only TolC, HlyB and HlyD are essential for the system to work, hence we have excluded the gene HlyC, used by Stanford 2009 Team (Part Bba_K223056). Hemolysin C (HlyC), which encodes a 170 aminoacids protein, has no secretion function but facilitates the activation of Hemolysin A protein which can act as an cytotoxic agent for many host cells.

4. HlyB and HlyD

We have opted not to use HlyB and HlyD as a fusioned unit, like Stanford 2009 Team did (Part BBa_K223057), but as two independent translational units (Part BBa_K554008 and Part BBa_K554007)

5. Experience

There is no data shown to indicate that Stanford 2009 Team secretion system has worked. On the other hand, UNICAMP-EMSE Team could successfully prove that our system worked, under the control of SoxS promoter (http://2011.igem.org/Team:UNICAMP-EMSE_Brazil/Results).

UNIQabcedc390efbd737-partinfo-00000000-QINU UNIQabcedc390efbd737-partinfo-00000001-QINU

@@ Line 2: / Line 2: @@
 This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 how you used this part and how it worked out.
+==TolC==
+This part was used to [http://2011.igem.org/Team:UNICAMP-EMSE_Brazil/Results#Device_3_testing.2C_Protein_Secretion_System Device 3 testing, Protein Secretion System]
 ==Device 3 testing, Protein Secretion System==
-The assembled devices related to secretion system (Hemolysin secretion system under control of SoxS – SoxS-HlyB-HlyD-TolC) was tested under laboratory conditions using GFP as reporter.
+The assembled devices related to secretion system (Hemolysin secretion system under control of SoxS – SoxS-HlyB-HlyD-TolC) were tested under laboratory conditions using GFP as reporter, coupled to the secretion signal sequence HlyA.
-[[Image:UNICAMP_EMSE_secretion_device_schema2.jpg|center|730px]]
+[[Image:UNICAMP_EMSE_secretion_device_paraquat_schema.jpg|center|700px]]
-<div align=center> '''Figure 1: Testing [http://2011.igem.org/Team:UNICAMP-EMSE_Brazil/Project#Device_3:_Secretion_system Device 3] through replacement of IL-10 to GFP. '''</div>
+<div align=center> '''Figure 1: Testing [http://2011.igem.org/Team:UNICAMP-EMSE_Brazil/Project#Device_3:_Secretion_system Device 3] through replacement of IL-10 to GFP. '''
+'''This is a representation of device 3, the protein secretion system coupled to Sox regulated GPF production device ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K554012 Part BBa_K554012]). To export a protein, the bacteria must have the HlyD, HlyB and TolC proteins and the target protein must have a signal sequence (HlyA tail), as shown here in a light red oval. In this case, the target protein to be secreted is GFP.</div>
 ===Methods===
@@ Line 13: / Line 17: @@
 To access GFP secretion, competent E. coli DH5α strain cells were transformed simultaneously with a pSB1C3 vector (Chloramphenicol resistant) carrying both the sensor (Strong_Constitutive_promoter + RBS + SoxR + Terminator) and the effector (SoxS_promoter + RBS + GFP + HlyA + Terminator), and pSB1AK3 (Ampicillin resistant) carrying the secretion system (SoxS_promoter + RBS + HlyB + HlyD + TolC + Terminator). As a non-secretion control, E. coli harboring only the pSB1C3 vector with both sensor and effector systems was used.
-Oxidative stress was induced by adding increasing concentrations of Paraquat (Methyl viologen dichloride hydrate - Sigma), an oxidative stress inducer in bacteria. The secretion was tested in cultures harboring A) and B) (Figure ) using 0 μM and 40 μM of Paraquat as described [http://2011.igem.org/Team:UNICAMP-EMSE_Brazil/Results#Methods above]. After 3 hours samples were collected, centrifuged (4000 rpm / 10 min; to avoid cell lysis) and the supernatant was collected and centrifuged again (13000 rpm / 10 min; to remove remaining cells). The supernatant fluorescence was measured in fluorometer (SLM – Aminco; 4 nm bandpass and 10 mm) with excitation in 500 nm and emission spectra from 508-550 nm. The GFP fluorescence was also detected in cells by fluorescence microscopy (Olympus).
+Oxidative stress was induced by adding increasing concentrations of Paraquat (Methyl viologen dichloride hydrate - Sigma), an oxidative stress inducer in bacteria. The secretion was tested in cultures harboring A) and B) (Figure ) using 0 μM and 40 μM of Paraquat as described [http://2011.igem.org/Team:UNICAMP-EMSE_Brazil/Results#Methods in this section]. After 3 hours samples were collected, centrifuged (4000 rpm / 10 min; to avoid cell lysis) and the supernatant was collected and centrifuged again (13000 rpm / 10 min; to remove remaining cells). The supernatant fluorescence was measured in fluorometer (SLM – Aminco; 4 nm bandpass and 10 mm) with excitation in 500 nm and emission spectra from 508-550 nm. The GFP fluorescence was also detected in cells by fluorescence microscopy (Olympus).
 ===Results===
-Microscopy data revealed GFP expression in the Paraquat induced cultures (Figure 2) but not in the non-induced one (Figure 3).
+GFP secretion was confirmed by fluorescence emission and estimated to be approximately 6% of total protein, according to fluorescence levels. Significant levels of GFP fluorescence were found only in the supernatant of Paraquat induced cultures containing both the sensor/effector and the secretion systems but not in the non-induced cultures and in the cultures containing only the sensor/effector system (Figure 2).
-[[Image:picind.png|center|600px]]
+In the black line we have our control, bacteria with the complete secretion system and without paraquat added. You see low fluorescence levels. The red line represents the culture induced with 40uM paraquat, with the fluorescence peak at 511nm.  In order to check it this fluor. Peak is due to na artifact such as cell lysis we tested the cultures without secr. System with paraquat added. We saw no fluor. Peak and we may conclude that the observed for red is really due to GFP secretion.
-<div align=center>'''Figure 2: 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>
-[[Image:picnotind.png|center|600px]]
-<div align=center>'''Figure 3: 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>
+[[Image:Unicamp-emse-graph-secr.png|center|600px]]
+<div align=center>'''Figure 2:  Supernatant fluorescence emission from 508 to 540 nm, excitation in 500 nm. Red line (40 μM Paraquat added):  sensor/effector with secretion system, showing a clear peak of fluorescence measurement at 511 nm; Black line (0 μM): sensor/effector with secretion system, non-induced. Blue line (40 μM Paraquat added): sensor/effector without secretion system; '''</div>
-GFP secretion was confirmed by fluorescence emission and estimated to be approximately 6% of total protein, according to fluorescence levels. Significant levels of GFP fluorescence were found only in the supernatant of Paraquat induced cultures containing both the sensor/effector and the secretion systems but not in the non-induced cultures and in the cultures containing only the sensor/effector system (Figure 4).
+From Figure 2, it is possible to observe that the red line shows a clear and high supernatant fluorescence peak at 511 nm, and the values are higher than the bacteria harboring the three systems (NO sensor, GFP producer and protein secretion) but whithout induction of NO by Paraquat (Black line), which may indicate that GFP is being exported. To test if the observed effect is not a False Positive (e.g. the same effect could be observed when bacteria suffer a cell lysis or if there is a secretion system in bacteria that could promote GFP export to the extracelular media) we decidede to check the supernatant fluorescence of bacteria with 40 uM Paraquat added and harboring only two systems: the NO sensor and GFP production devices but lacking the secretion system device (Blue line). This experiment showed that the fluorescence levels found in the Blue line are significantly lower than the observed for the red lines, which may indicate that the observed pattern of red line is not due to an error.
-[[Image:Unicamp-emse-graph-secr.png|center|600px]]
+==<font color=red>'''UNICAMP-EMSE Brazil Parts Design: why are they different to Stanford 2009 parts?'''</font>==
-<div align=center>'''Figure 4:  Supernatant fluorescence emission from 508 to 540 nm, excitation in 500 nm. Black squares (40 μM A):  sensor/effector with secretion system; Green triangles (40 μM B): sensor/effector without secretion system; Red circles (0 μM): sensor/effector with secretion system, non-induced.'''</div>
+In the section below we aim to explain in details how our parts differ from Stanford 2009's, and why we decided to create novel parts to achieve similar objectives.
+===='''Secretion System Changes'''====
+Analyzing the pre-existing secretion system from Stanford Team 2009 we have decided to make several changes in order to make it more efficient and to prove that it actually works.
+#'''Change the RBS part and its disposition in the transcriptional unit.'''
+## Attempting to create a new secretion system, we began by choosing a different but still highly efficient RBS part. And according to the experience made upon RBS deposited parts ([https://parts.igem.org/Part:BBa_B0034:Experience BBa_B0034]), it was suggested that the RBS chosen by our team (B0030) is as efficient as the one used by Stanford 2009 team (B0034).
+##To improve translation efficiency, gene sequence fidelity (avoid errors or mistranscription during PCR) and save time during the devices assembling UNICAMP-EMSE team synthesized each coding sequence already fusioned with RBS sequence. On the other hand Stanford 2009 Team did not use translational units.
+:2. '''Biobrick Standard Compatibility'''
+:*The secretion system developed by Stanford Team 2009 was not compatible with the systems 10, 12, 21, 23 nor 25. UNICAMP-EMSE Team has removed internal illegal restriction sites presented in the parts [https://parts.igem.org/wiki/index.php?title=Part:BBa_K223055 Bba_K223055] and [https://parts.igem.org/wiki/index.php?title=Part:BBa_K223057 BBa_K223057] by codon usage of these sites prior to synthesis. Therefore our team suited the secretion system to be compatible with 10, 12 and 23 standards.
+:3. '''Exclusion of non-essential genes'''
+:*In our system we have reduced the number of genes to use only the essential ones, considering that “the simpler, the better”. Evidence (Fath et al, 1993) shows that only TolC, HlyB and HlyD are essential for the system to work, hence we have excluded the gene HlyC, used by Stanford 2009 Team ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K223056) Part Bba_K223056]). Hemolysin C (HlyC), which encodes a 170 aminoacids protein, has no secretion function but facilitates the activation of Hemolysin A protein which can act as an cytotoxic agent for many host cells.
+:4. '''HlyB and HlyD'''
+:*We have opted not to use HlyB and HlyD as a fusioned unit, like Stanford 2009 Team did ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K223057 Part BBa_K223057]), but as two independent translational units ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K554008 Part BBa_K554008] and [https://parts.igem.org/wiki/index.php?title=Part:BBa_K554007 Part BBa_K554007])
+:5. '''Experience'''
+:*There is no data shown to indicate that Stanford 2009 Team secretion system has worked. On the other hand, UNICAMP-EMSE Team could successfully prove that our system worked, under the control of SoxS promoter (http://2011.igem.org/Team:UNICAMP-EMSE_Brazil/Results).