Difference between revisions of "Part:BBa K808025"
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===== Experiment =====
 
===== Experiment =====
  
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Pieces of PET foil were added to a 2 µM solution of FsC and Est13. The solution was constantly agitated carefully. After 7 days the samples were washed with distilled water and dried in a cabinet dryer at 60 °C over night. Afterwards the samples are investigated using AFM. FsC induced surface modification of PET could be observed.
+
Pieces of PET foil were added to a 2 µmol/L solution of FsC. The solution was constantly agitated in 50 mL falcon tubes. After 7 days the samples were washed with distilled water and dried in a cabinet dryer at 60 °C over night. Afterwards the samples are investigated using AFM. FsC induced surface modification of PET could be observed.
 
The Pictures below show 3D models, height profiles and the cantilevers amplitude as well as the phase deviations. The 3D models give a first impression of the texture of the surface. Looking at the height profile and the amplitude deviation more detailed informations about the surface texture can be obtained. The reference samples show a very flat surface (±3 nm). Bigger deviations are due to manufactionary mistakes of the foil. FsC treated surfaces show a deviation ten times bigger than non treated ones (±30 nm). Comparing the phase deviation of FsC treated surfaces with the reference, there can be seen big diferences due to varying mechanical properties. These are caused by degradation by FsC. The Polymer chains are cut in to smaller pieces which then stand out of the surface, letting it swell. The resulting surface is not only rougher but persumably less dense and therefore less firm.
 
The Pictures below show 3D models, height profiles and the cantilevers amplitude as well as the phase deviations. The 3D models give a first impression of the texture of the surface. Looking at the height profile and the amplitude deviation more detailed informations about the surface texture can be obtained. The reference samples show a very flat surface (±3 nm). Bigger deviations are due to manufactionary mistakes of the foil. FsC treated surfaces show a deviation ten times bigger than non treated ones (±30 nm). Comparing the phase deviation of FsC treated surfaces with the reference, there can be seen big diferences due to varying mechanical properties. These are caused by degradation by FsC. The Polymer chains are cut in to smaller pieces which then stand out of the surface, letting it swell. The resulting surface is not only rougher but persumably less dense and therefore less firm.
  
 
'''AFM of PET foil after FsC exposure :'''
 
'''AFM of PET foil after FsC exposure :'''
  
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[[File:3D_FSC.png|220px]]
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<html>
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[[File:AFM_FSC_2.jpg|220px]]
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<img src=https://static.igem.org/mediawiki/2012/b/b8/3D_FSC.png width=220>
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[[File:AFM_FSC_2_Amplitude_auto.jpg|220px]]
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</html>
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[[File:AFM_FSC_2_Phase.jpg|220px]]
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<html>
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<img src=https://static.igem.org/mediawiki/2012/e/e5/AFM_FSC_2.jpg width=220>
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</html>
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<img src=https://static.igem.org/mediawiki/2012/2/2c/AFM_FSC_2_Amplitude_auto.jpg width=220>
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<img src=https://static.igem.org/mediawiki/2012/c/ca/AFM_FSC_2_Phase.jpg width=220>
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</html>
  
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[[File:3D_FSC_1_1.png|220px]]
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<html>
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[[File:AFM_FSC_1.jpg|220px]]
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<img src=https://static.igem.org/mediawiki/2012/6/61/3D_FSC_1_1.png width=220>
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[[File:AFM_FSC_1_Amplitude_auto.jpg|220px]]
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</html>
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[[File:AFM_FSC_1_Phase.jpg|220px]]
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<img src=https://static.igem.org/mediawiki/2012/7/7f/AFM_FSC_1.jpg width=220>
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<img src=https://static.igem.org/mediawiki/2012/0/08/AFM_FSC_1_Phase.jpg width=220>
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'''AFM of PET foil as reference :'''
 
'''AFM of PET foil as reference :'''
  
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[[File:3D_Referenz_2.png|220px]]
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<html>
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[[File:AFM_Referenz_1.jpg|220px]]
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<img src=https://static.igem.org/mediawiki/2012/1/1e/3D_Referenz_2.png width=220>
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[[File:AFM_Referenz_1_Amplitude_auto.jpg|220px]]
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</html>
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[[File:AFM_Referenz_1_Phase.jpg|220px]]
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<html>
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<img src=https://static.igem.org/mediawiki/2012/9/91/AFM_Referenz_1.jpg width=220>
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<img src=https://static.igem.org/mediawiki/2012/e/ed/AFM_Referenz_1_Amplitude_auto.jpg width=220>
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<img src=https://static.igem.org/mediawiki/2012/5/5c/AFM_Referenz_1_Phase.jpg width=220>
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</html>
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K808025 parameters</partinfo>
 
<partinfo>BBa_K808025 parameters</partinfo>
 
<!-- -->
 
<!-- -->

Revision as of 16:12, 26 September 2012

FsC: Cutinase PET cleaving enzyme

A cutinase is a cuticula degrading hydrolase from the fungus Fusarium solani pisi. It shows activity towards PET.


Usage and Biology

The characterization of FsC occured in 3 different ways.

Our [http://2012.igem.org/Team:TU_Darmstadt/Labjournal/Simulation Simulation Lab] performed

  • [http://2012.igem.org/Team:TU_Darmstadt/Modeling_Docking#Docking Docking Simulations]

Our [http://2012.igem.org/Team:TU_Darmstadt/Project/Material_Science Material Science Lab] performed

  • [http://2012.igem.org/Team:TU_Darmstadt/Labjournal/Material_Science#Surface_analysis_of_polyethylene_terephthalate_with_atomic_force_microscopy_.28AFM.29 Surface analysis of polyethylene terephthalat with atomic force microscopy]

Our [http://2012.igem.org/Team:TU_Darmstadt/Project/Degradation Degradation Lab]

  • [http://2012.igem.org/Team:TU_Darmstadt/Labjournal/Degradation#Enzyme_Activity_Assays Enzyme activity assay] using a [http://2012.igem.org/Team:TU_Darmstadt/Protocols/pNP_Assay#pNP-Assay pNP-Assay]

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 379
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 525
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 301

Docking Simulation

For the sake of convenience of this page, please visit our [http://2012.igem.org/Team:TU_Darmstadt/Modeling_Docking#Docking Simulation Lab > Docking Simulation] for further informtion.

Surface analysis of polyethylene terephthalat with atomic force microscopy

Atomic force microscopy is used to make very precise surface analysis up to nanometer scale. The goal was to indentify differences between different modifications of polyethylene terephthalate and to proof enzymatic degradation by changed surface properties.

Experiment

Pieces of PET foil were added to a 2 µmol/L solution of FsC. The solution was constantly agitated in 50 mL falcon tubes. After 7 days the samples were washed with distilled water and dried in a cabinet dryer at 60 °C over night. Afterwards the samples are investigated using AFM. FsC induced surface modification of PET could be observed. The Pictures below show 3D models, height profiles and the cantilevers amplitude as well as the phase deviations. The 3D models give a first impression of the texture of the surface. Looking at the height profile and the amplitude deviation more detailed informations about the surface texture can be obtained. The reference samples show a very flat surface (±3 nm). Bigger deviations are due to manufactionary mistakes of the foil. FsC treated surfaces show a deviation ten times bigger than non treated ones (±30 nm). Comparing the phase deviation of FsC treated surfaces with the reference, there can be seen big diferences due to varying mechanical properties. These are caused by degradation by FsC. The Polymer chains are cut in to smaller pieces which then stand out of the surface, letting it swell. The resulting surface is not only rougher but persumably less dense and therefore less firm.

AFM of PET foil after FsC exposure :

AFM of PET foil as reference :