Difference between revisions of "Part:BBa K554013:Experience"
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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) was tested under laboratory conditions using GFP as reporter.
  
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[[Image:UNICAMP_EMSE_secretion_device_schema2.jpg|center|730px]]
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[[Image:UNICAMP_EMSE_secretion_device_paraquat_schema.jpg|center|700px]]
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<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>
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<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. '''
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'''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===
 
===Methods===

Revision as of 23:32, 18 October 2011

SoxS HlyB HlyD TolC device

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.

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 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).

Results

Microscopy data revealed GFP expression in the Paraquat induced cultures (Figure 2) but not in the non-induced one (Figure 3).

Picind.png
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.


Picnotind.png
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.


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).


Unicamp-emse-graph-secr.png
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.

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