Difference between revisions of "Part:BBa K554002:Experience"

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(→‎Improvement of Reading Frame Problems within this Part when Assembled with Other Coding Parts (Fusion) by UI-Indonesia 2014 Team)
 
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==Device 3 testing, Protein Secretion System==
 
==Device 3 testing, Protein Secretion System==
  
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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.
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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.  
  
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[[Image:UNICAMP_EMSE_secretion_device_paraquat_schema.jpg|center|700px]]
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[[Image:UNICAMP EMSE secretion device paraquat schema.jpg|center|730px]]
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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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<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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'''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>
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<div align=left>
 
===Methods===
 
===Methods===
  
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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.
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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 [http://en.wikipedia.org/wiki/Paraquat Paraquat] (Methyl viologen dichloride hydrate - Sigma), an oxidative stress inducer in bacteria, according to the following conditions:
 +
 
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*Sensor/Effector + Secretion system: 0µM Paraquat
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*Sensor/Effector + Secretion system: 40µM Paraquat
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*Sensor/Effector - Secretion system: 40µM Paraquat (non-secretion control)
  
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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).
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After 3 hours of induction, 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.  
 +
<div align=left>
  
 
===Results===
 
===Results===
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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). 
 
  
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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.
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GFP secretion was confirmed by fluorescence emission and estimated to be approximately 10% of total protein, according to fluorescence levels found in bacterial cultures (See figure 2, from Device 2 testing). 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.  
  
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[[Image:Unicamp-emse-graph-secr.png|center|600px]]
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<div align=center>'''Figure 2: Supernatant fluorescence emission from 508 to 540 nm, excitation in 500 nm. Red line (40 μM Paraquat): sensor/effector with secretion system, showing a clear peak at 511 nm on fluorescence spectrum; Black line (0 μM): non-induced control, sensor/effector with secretion system, non-induced; Blue line (40 µM Paraquat): non-secretion control, sensor/effector without secretion system.
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<div align=left>
  
  
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[[Image:Unicamp-emse-graph-secr.png|center|600px]]
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According to the graphic (Figure 2), the supernatant of the culture harboring the complete secretion system and induced with 40 µM of Paraquat (Red line) exhibited a well-defined fluorescence peak between 508 and 520 nm, which indicates the presence of GFP in the supernatant probably due to secretion.
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<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>
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Validating this result, there is no clear fluorescence peak both in the supernatant of non-induced control spectrum (Black line - complete secretion system with 0 µM of Paraquat) and non-secretion control spectrum (Blue line – sensor/effector without secretion system). The aforementioned controls corroborates the result of GFP secretion though the secretion system, eliminating false positive results due to cell lysis for example.
  
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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.
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===Discussion===
  
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This device is a modification of Stanford team 2009 secretion system composite [https://parts.igem.org/Part:BBa_K223062 BBa_K223062]. The secretion system was improved according to biobrick assembly standard 23, and modified by deletion of Hemolysin C (HlyC) which encodes a 170 aminoacids protein that is related to pathogenesis and has no secretion function. HlyC protein facilitates the activation of Hemolysin A protein (do not confuse with HlyA secretion signal peptide, used in fusion to GFP, and IL’s) (Fath et al, 1993). When HlyA protein is activated by HlyC enzyme it has cytotoxic and cytolytic effects, then HlyC is related only to pathogenesis and not secretion.
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We could show that this new secretion device (composed by secretion signal peptide HlyA, HlyD, HlyB and TolC) was able to secrete GFP fusioned to HlyA secretion peptide signal in response to Paraquat induction of the sensor/effector/secretion system, as showed by supernatant fluorescence. This also reinforces that HlyC is not necessary for secretion.
  
 
==<font color=red>'''UNICAMP-EMSE Brazil Parts Design: why are they different to Stanford 2009 parts?'''</font>==
 
==<font color=red>'''UNICAMP-EMSE Brazil Parts Design: why are they different to Stanford 2009 parts?'''</font>==
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:5. '''Experience'''
 
: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).
 
:*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).
 +
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==<font color=red>'''Improvement of Reading Frame Problems within this Part when Assembled with Other Coding Parts (Fusion) by [http://2014.igem.org/Team:UI_Indonesia UI-Indonesia 2014 Team]'''</font>==
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Our team identified a problem that cause reading frame shift in this part when assembled with other coding part (protein coding). We found it after we did sequencing for our parts (that assembled to fusion with this tag protein: [https://parts.igem.org/Part:BBa_K1344002 BBa_K1344002], [https://parts.igem.org/Part:BBa_K1344004 BBa_1344004]) and review [https://parts.igem.org/cgi/sequencing/one_blast.cgi?id=19978 iGEM's sequencing results].
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The prefix of this part using the "non-coding" part design, rather than "coding" part design. The figure below will explain the frame reading shift and including our solution:
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https://static.igem.org/mediawiki/parts/thumb/9/92/BBa_K554002-FrameShift.jpg/800px-BBa_K554002-FrameShift.jpg
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<div align=center> '''Figure A,B,C. Frame Reading Shift BBa_K554002 After Assembled with Other Coding Parts'''</div>
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Figure A&B: 3A Assembly Result of Coding Parts + BBa_K554002 and its reading frame shift
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Figure B: Our solution by modify it before assembly
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#'''As described in Figure B, we propose a solution by adding 2 base pair (TG) in front of this part. Here is our forward primer design:'''
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<div align=center> '''5'-TGttagcctatggaagtcag-3' '''</div>
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:2. '''We are modify our coding parts that will be tagged by deleting our part's stop codons or terminators. It's important because if there's one, the HlyA tag protein would not be expressed. We intend to fusion the coding parts protein with this tag protein WITHOUT stop translation or transcription process. This is our example [https://parts.igem.org/Part:BBa_K1344002 BBa_K1344002], [https://parts.igem.org/Part:BBa_K1344004 BBa_1344004]'''
  
  

Latest revision as of 05:28, 21 October 2014

HlyA secretion signal peptide

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.

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.


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 [http://en.wikipedia.org/wiki/Paraquat Paraquat] (Methyl viologen dichloride hydrate - Sigma), an oxidative stress inducer in bacteria, according to the following conditions:

  • Sensor/Effector + Secretion system: 0µM Paraquat
  • Sensor/Effector + Secretion system: 40µM Paraquat
  • Sensor/Effector - Secretion system: 40µM Paraquat (non-secretion control)

After 3 hours of induction, 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.

Results

GFP secretion was confirmed by fluorescence emission and estimated to be approximately 10% of total protein, according to fluorescence levels found in bacterial cultures (See figure 2, from Device 2 testing). 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.

Unicamp-emse-graph-secr.png
Figure 2: Supernatant fluorescence emission from 508 to 540 nm, excitation in 500 nm. Red line (40 μM Paraquat): sensor/effector with secretion system, showing a clear peak at 511 nm on fluorescence spectrum; Black line (0 μM): non-induced control, sensor/effector with secretion system, non-induced; Blue line (40 µM Paraquat): non-secretion control, sensor/effector without secretion system.


According to the graphic (Figure 2), the supernatant of the culture harboring the complete secretion system and induced with 40 µM of Paraquat (Red line) exhibited a well-defined fluorescence peak between 508 and 520 nm, which indicates the presence of GFP in the supernatant probably due to secretion. Validating this result, there is no clear fluorescence peak both in the supernatant of non-induced control spectrum (Black line - complete secretion system with 0 µM of Paraquat) and non-secretion control spectrum (Blue line – sensor/effector without secretion system). The aforementioned controls corroborates the result of GFP secretion though the secretion system, eliminating false positive results due to cell lysis for example.

Discussion

This device is a modification of Stanford team 2009 secretion system composite BBa_K223062. The secretion system was improved according to biobrick assembly standard 23, and modified by deletion of Hemolysin C (HlyC) which encodes a 170 aminoacids protein that is related to pathogenesis and has no secretion function. HlyC protein facilitates the activation of Hemolysin A protein (do not confuse with HlyA secretion signal peptide, used in fusion to GFP, and IL’s) (Fath et al, 1993). When HlyA protein is activated by HlyC enzyme it has cytotoxic and cytolytic effects, then HlyC is related only to pathogenesis and not secretion.

We could show that this new secretion device (composed by secretion signal peptide HlyA, HlyD, HlyB and TolC) was able to secrete GFP fusioned to HlyA secretion peptide signal in response to Paraquat induction of the sensor/effector/secretion system, as showed by supernatant fluorescence. This also reinforces that HlyC is not necessary for secretion.

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.

  1. 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
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).


Improvement of Reading Frame Problems within this Part when Assembled with Other Coding Parts (Fusion) by [http://2014.igem.org/Team:UI_Indonesia UI-Indonesia 2014 Team]

Our team identified a problem that cause reading frame shift in this part when assembled with other coding part (protein coding). We found it after we did sequencing for our parts (that assembled to fusion with this tag protein: BBa_K1344002, BBa_1344004) and review iGEM's sequencing results.

The prefix of this part using the "non-coding" part design, rather than "coding" part design. The figure below will explain the frame reading shift and including our solution:

800px-BBa_K554002-FrameShift.jpg

Figure A,B,C. Frame Reading Shift BBa_K554002 After Assembled with Other Coding Parts

Figure A&B: 3A Assembly Result of Coding Parts + BBa_K554002 and its reading frame shift

Figure B: Our solution by modify it before assembly


  1. As described in Figure B, we propose a solution by adding 2 base pair (TG) in front of this part. Here is our forward primer design:
5'-TGttagcctatggaagtcag-3'


2. We are modify our coding parts that will be tagged by deleting our part's stop codons or terminators. It's important because if there's one, the HlyA tag protein would not be expressed. We intend to fusion the coding parts protein with this tag protein WITHOUT stop translation or transcription process. This is our example BBa_K1344002, BBa_1344004


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