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Team:Potsdam Bioware/Project/Details
From 2011.igem.org
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=== Phage Display === | === Phage Display === | ||
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<br> | <br> | ||
<br>'''Cloning'''<br> | <br>'''Cloning'''<br> | ||
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<br>'''ELISA'''<br> | <br>'''ELISA'''<br> | ||
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| - | The next step was the detection of the expression of the ''mdnA-myc-gene III''-fusion gene on the surface of the phage. So ''E. coli'' cells strains XL1-Blue and ER2738 were first transformed with the phagemid pPDV089 before they were infected with helper phages. ''E. coli'' cells containing both plasmids were selected. An ELISA test was performed to determine whether these cells are able to produce phage particles carrying the mdnA peptide on their surface. To perform this test anti-c-myc-antibodies were immobilized on ELISA plates and incubated with purified phages. For detection a second antibody coupled with horse radish peroxidase (HRP) was used which binds the gene VIII protein of the phages. The HRP substrate 3,3'-5,5'-Tetramethylbenzidine (TMB) was added and in case of binding a colour reaction was expected. The colour shift from achromatic to yellow in wells incubated with phages produced in XL1-blue cells showed the successful expression of | + | The next step was the detection of the expression of the ''mdnA-myc-gene III''-fusion gene on the surface of the phage. So ''E. coli'' cells strains XL1-Blue and ER2738 were first transformed with the phagemid pPDV089 before they were infected with helper phages. ''E. coli'' cells containing both plasmids were selected. An ELISA test was performed to determine whether these cells are able to produce phage particles carrying the mdnA peptide on their surface. To perform this test anti-c-myc-antibodies were immobilized on ELISA plates and incubated with purified phages. For detection a second antibody coupled with horse radish peroxidase (HRP) was used which binds the gene VIII protein of the phages. The HRP substrate 3,3'-5,5'-Tetramethylbenzidine (TMB) was added and in case of binding a colour reaction was expected. The colour shift from achromatic to yellow in wells incubated with phages produced in XL1-blue cells showed the successful expression of mdnA-c-myc-gene III-fusion protein on the phages.In wells incubated with infected ER2738 cells no colour change was obserevd. That might be due to the fact that a mistake during phage purification was noticed. <br> |
| - | For more precise results the absorption from 450 – 600 nm was measured | + | For more precise results the absorption from 450 – 600 nm was measured. The data were presented in a bar plot. As a negative control helper phages were added instead of produced phages. Furthermore two wells were prepared were the secondary antibody was not added. |
The graphic shows clearly the much higher absorption measured in wells, which were incubated with phage particles of interest produced in XL1-Blue cells. As has already pointed out this shows the succeeded expression of mdnA-c-myc-gene III-fusion protein on the surface of the phages. | The graphic shows clearly the much higher absorption measured in wells, which were incubated with phage particles of interest produced in XL1-Blue cells. As has already pointed out this shows the succeeded expression of mdnA-c-myc-gene III-fusion protein on the surface of the phages. | ||
| - | + | <div align="center"> | |
| - | [[File:UP Excel ELISA.png|400px]] | + | [[File:UP Excel ELISA.png|400px]]</div> |
[[File:UP absorptionELISA1.PNG|800px]] | [[File:UP absorptionELISA1.PNG|800px]] | ||
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So after that repeated and modified phage display a much higher ratio of cells grown on ampicillin before panning and cells grown on ampicillin after panning to cells grown kanamycin before panning and cells grown on ampicillin after panning could be noticed. This indicates that the unmodified mdnA expressed on the phages binds specifically to the immobilized trypsin and that phage display in general for screening a generated mdnA library is possible. | So after that repeated and modified phage display a much higher ratio of cells grown on ampicillin before panning and cells grown on ampicillin after panning to cells grown kanamycin before panning and cells grown on ampicillin after panning could be noticed. This indicates that the unmodified mdnA expressed on the phages binds specifically to the immobilized trypsin and that phage display in general for screening a generated mdnA library is possible. | ||
| - | [[File:UP | + | [[File:UP Phage display panning ratio.png|450px]] |
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=== In Vivo Selection === | === In Vivo Selection === | ||
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=== Modelling === | === Modelling === | ||
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<br>'''Motivation'''<br> | <br>'''Motivation'''<br> | ||
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[[Media:conzt3.m]]<br> | [[Media:conzt3.m]]<br> | ||
[[Media:plottfuntionendreiaufeinmal2.m]] | [[Media:plottfuntionendreiaufeinmal2.m]] | ||
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Latest revision as of 14:12, 21 September 2011
Details
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Microviridin
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Phage Display
In Vivo Selection
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