Difference between revisions of "Part:BBa K627006:Experience"

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===Applications of BBa_K627006===
 
===Applications of BBa_K627006===
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<br>'''ELISA'''<br>
+
<br>'''Control of expression of mdnA-myc-gene III in ''E. coli'''''<br>
 +
 
 +
The control of expression of the mdnA-myc-gene III fusion gene was done by western blotting analysis. ''E. coli'' cells transformed with the phagemid pPDV089 were harvested and lysated. The cell proteins were electrophoretically separated and transferred to a membrane. The mdnA-myc-gene III-fusion proteins were detected using specific anti-myc-antibodies and horseradish peroxidase (HRP)-linked antibodies as second antibodies. Enhanced chemiluminescence (ECL) was used to visualize the protein. ECL is based on the emission of light during the HRP -catalyzed oxidation of luminol, which was captured by a camera. The western blot analysis resulted in a band of a size just below the 30 kDa mark representing the mdnA-myc-gene III-fusion protein (24 kDa).
 
<br>
 
<br>
−
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>
+
<br>
−
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.
+
[[Image:UP_coomassie+western_blot.png‎|center|400px|thumb|'''Figure 1: Control of expression of mdnA-myc-gene III in ''E. coli'' by western blotting.''' For detection anti-myc-antibodies and secondary HRP-linked antibodies were used. The resulting band represents the mdnA-myc-gene III-fusion protein.]]
 +
<br>
 +
<br>
 +
<br>'''Detection of phages carrying mdnA on their surface by ELISA'''<br>
 +
<br>
 +
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 strain XL1-Blue 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 o-phenyldiamine (OPD) was added and in case of binding a color reaction was expected. The color 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.<br>
 +
For more precise results the absorption at 492 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.
 +
<br>
 +
<br>
  
  
−
[[Image:UP Elisa.png|400px]]
+
[[Image:UP_ELISA3_mit_fehlerindikator.png‎|center|400px|thumb|'''Figure 2: Detection of phages carrying mdnA on their surface by ELISA.''' The bar plot shows the absorption at 492 nm. Anti-myc-antibodies were immobilized. For detection a second antibody coupled with horse radish peroxidase (HRP) was used which binds the gene VIII coat protein of the phages. The left bar shows the absorption of the wells containing helper phages (negative control), the right bar shows the absorption of wells containing mdnA carrying phages.]]<br>
  
−
[[Image:UP tableELISA.PNG|800px]]
 
  
−
<br>'''Phage Display'''<br>
+
<br>'''Testing phage display with unmodified mdnA to examine its suitability as screening method '''<br>
 
<br>
 
<br>
−
To test the fundamental suitability of this screening method, phages representing unmodified mdnA on their surface and phages not representing mdnA in a ratio of one to one were incubated with immobilized trypsin which is known as a target of mdnA. The display was conducted in ELISA plates. The bound phages were eluted using a buffer with low pH value and neutralized afterwards. To check how many phages interacted with trypsin, ''E. coli'' cells were re-infected with eluted phages and plated on agar with different antibiotics. Cells infected with phages carrying mdnA are expected to grow on agar with ampicillin whereas cells containing phages without mdnA are expected to grow on agar with kanamycin. To control the success of the panning round additionally ''E. coli'' cells were infected with not panned phages and plated on agar. Subsequent the number of clones grew on ampicillin and kanamycin before and after panning was compared. During the running of this step it was noticed that much more cells infected with unpanned phages grew on kanamycin plates than on ampicillin plates. This is surprising and indicates that phages were not given in one to one ratio as calculated after nanodrop measuring but in a 1:400 ratio (phages carrying the protein of interest: helper phages). Thus the suggestion can be made that maybe the presented mdnA on the phages has some toxic effects on the cells. <br>
+
To test the fundamental suitability of this screening method, phages representing unmodified mdnA on their surface and phages not representing mdnA (helper phages) in a ratio of one to one were incubated with immobilized trypsin which is known as a target of mdnA. The display was conducted in ELISA plates. The bound phages were eluted using a buffer with low pH value and neutralized afterwards. To check how many phages interacted with trypsin, ''E. coli'' cells XL1-Blue were re-infected with eluted phages and plated on agar with different antibiotics. Cells infected with phages carrying mdnA are able to grow on agar with ampicillin whereas cells infected with helper phages are able to grow on agar with kanamycin. To control the success of the panning round additionally ''E. coli'' cells were infected with phage mix before panning and plated on agar. Subsequent the number of clones grew on ampicillin and kanamycin before and after panning was compared. During the running of this step it was noticed that much more cells were infected with helper phages than with phages carrying mdnA despite of the engaged 1:1 ratio. This was surprising and indicated that mdnA on the surface of the phages may inhibit their infectivity. After controlling the plates an  infection ratio of phages carrying mdnA to helper phages of 1:400 was calculated. This fact should be analyzed in further experiments.<br>
−
The results of the first phage display performed after one panning round are plotted in the figure below ("Grown clones after Phage Display 1"). It was expected that after panning the ratio of cells grown on ampicillin and cells grown on kanamycin increases. This is attributable to the fact that cells which grow on ampicillin should contain the phage particles carrying mdnA c-myc geneIII fusion protein on their surface and are expected to bind specifically to trypsin. Unfortunately this could not be observed in this experiment.<br>
+
The results of the first phage display are plotted in the figure below. After one panning round an enrichment of phages carrying mdnA was expected. This is attributable to the fact that phage particles carrying mdnA c-myc geneIII fusion protein on their surface are expected to bind specifically to the immobilized trypsin. Unfortunately this was not observed in this experiment. The ratio of cells growing on kanamycin agar before (4000) to cells growing on kanamycin agar (cells containing helper phages) after panning (750) was determined as 5:1. The ratio of cells growing on ampicillin agar before (12) to cells growing on ampicillin agar (cells containing mdnA carrying phages) after panning (2) was nearly equal. Thus no enrichment of mdnA carrying phages occurred in the first experiment. So it was decided to repeat this experiment under improved conditions. Therefor the number of washing steps during the described experimental procedure was increased. Here the ratio of cells growing on kanamycin agar before (3000) to cells growing on kanamycin agar (cells containing helper phages) after panning (29) was determined as 103:1. The ratio of cells growing on ampicillin agar before (26) to cells growing on ampicillin agar (cells containing mdnA carrying phages) after panning (2) was determined as 13:1. Thus an enrichment factor of eight was reached for the phages displaying mdnA on their surface. <br>
−
So it was decided to repeat this experiment under improved conditions. Therefore the number of washing steps during the described experimental procedure was increased.Results are shown in the figure below. 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.  
+
These results indicate that the unmodified mdnA expressed on the phages binds specifically to the immobilized trypsin. Therefore it can be deduced that mdnA is presented in a functional 3D structure. These findings suggest that phage display in general is an appropriate method for screening a recombinant mdnA library. Further experiments are required to optimize this system.
  
−
[[Image:UP Phage display panning ratio.png|450px]]
+
 
 +
[[Image:UP_panning_3.png‎|center|450px|thumb|'''Figure 3: Optimization of phage display.''' After optimized conditions (right) a clear concentration of phages carrying mdnA after one panning round was noted. ''E. coli'' cells were infected with phage mix (helper phages and phages carrying mdnA) before and after panning and plated on agar containing kanamycin or ampicillin. The ratio of cells growing on ampicillin or kanamycin agar before panning to cells growing on ampicillin or kanamycin agar after was calculated. Helper phages which acted as negative control have a kanamycine resistance whereby phages carrying mdnA have an ampicillin resistance.]]
 +
<br>'''Testing phage display with unmodified mdnA against further proteases'''<br>
 +
 
 +
Furthermore the interaction of unmodified mdnA with other proteases was determined. From the literature (Ziemert, 2010) the high inhibitory activity of microviridin L, besides trypsin, against chymotrypsin and elastase is also known. So a phage display with these enzymes was performed. Additionally papain, proteinase K, mycolysin and pepsin were tested for which no interaction was shown yet. For all enzymes an equal amount of ''E. coli'' cells and phages were used. In agreement with data from the literature interaction of microviridin with chymotrypsin and elastase was confirmed. Additionally an interaction with papain was noted. All other proteases were not bound by microviridin.
 +
<br>
 +
<br>
 +
[[Image:UP_test_of_different_enzymes.png‎|center|400px|thumb|'''Figure 4: Phage display against different proteases.''' After panning the number of clones was counted. In agreement with data from the literature interaction of microviridin with chymotrypsin and elastase was confirmed. Additionally an interaction with papain was noted.]]
  
 
===User Reviews===
 
===User Reviews===

Latest revision as of 20:54, 28 October 2011

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K627006


Control of expression of mdnA-myc-gene III in E. coli

The control of expression of the mdnA-myc-gene III fusion gene was done by western blotting analysis. E. coli cells transformed with the phagemid pPDV089 were harvested and lysated. The cell proteins were electrophoretically separated and transferred to a membrane. The mdnA-myc-gene III-fusion proteins were detected using specific anti-myc-antibodies and horseradish peroxidase (HRP)-linked antibodies as second antibodies. Enhanced chemiluminescence (ECL) was used to visualize the protein. ECL is based on the emission of light during the HRP -catalyzed oxidation of luminol, which was captured by a camera. The western blot analysis resulted in a band of a size just below the 30 kDa mark representing the mdnA-myc-gene III-fusion protein (24 kDa).

Figure 1: Control of expression of mdnA-myc-gene III in E. coli by western blotting. For detection anti-myc-antibodies and secondary HRP-linked antibodies were used. The resulting band represents the mdnA-myc-gene III-fusion protein.




Detection of phages carrying mdnA on their surface by ELISA

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 strain XL1-Blue 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 o-phenyldiamine (OPD) was added and in case of binding a color reaction was expected. The color 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.
For more precise results the absorption at 492 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.


Figure 2: Detection of phages carrying mdnA on their surface by ELISA. The bar plot shows the absorption at 492 nm. Anti-myc-antibodies were immobilized. For detection a second antibody coupled with horse radish peroxidase (HRP) was used which binds the gene VIII coat protein of the phages. The left bar shows the absorption of the wells containing helper phages (negative control), the right bar shows the absorption of wells containing mdnA carrying phages.



Testing phage display with unmodified mdnA to examine its suitability as screening method

To test the fundamental suitability of this screening method, phages representing unmodified mdnA on their surface and phages not representing mdnA (helper phages) in a ratio of one to one were incubated with immobilized trypsin which is known as a target of mdnA. The display was conducted in ELISA plates. The bound phages were eluted using a buffer with low pH value and neutralized afterwards. To check how many phages interacted with trypsin, E. coli cells XL1-Blue were re-infected with eluted phages and plated on agar with different antibiotics. Cells infected with phages carrying mdnA are able to grow on agar with ampicillin whereas cells infected with helper phages are able to grow on agar with kanamycin. To control the success of the panning round additionally E. coli cells were infected with phage mix before panning and plated on agar. Subsequent the number of clones grew on ampicillin and kanamycin before and after panning was compared. During the running of this step it was noticed that much more cells were infected with helper phages than with phages carrying mdnA despite of the engaged 1:1 ratio. This was surprising and indicated that mdnA on the surface of the phages may inhibit their infectivity. After controlling the plates an infection ratio of phages carrying mdnA to helper phages of 1:400 was calculated. This fact should be analyzed in further experiments.
The results of the first phage display are plotted in the figure below. After one panning round an enrichment of phages carrying mdnA was expected. This is attributable to the fact that phage particles carrying mdnA c-myc geneIII fusion protein on their surface are expected to bind specifically to the immobilized trypsin. Unfortunately this was not observed in this experiment. The ratio of cells growing on kanamycin agar before (4000) to cells growing on kanamycin agar (cells containing helper phages) after panning (750) was determined as 5:1. The ratio of cells growing on ampicillin agar before (12) to cells growing on ampicillin agar (cells containing mdnA carrying phages) after panning (2) was nearly equal. Thus no enrichment of mdnA carrying phages occurred in the first experiment. So it was decided to repeat this experiment under improved conditions. Therefor the number of washing steps during the described experimental procedure was increased. Here the ratio of cells growing on kanamycin agar before (3000) to cells growing on kanamycin agar (cells containing helper phages) after panning (29) was determined as 103:1. The ratio of cells growing on ampicillin agar before (26) to cells growing on ampicillin agar (cells containing mdnA carrying phages) after panning (2) was determined as 13:1. Thus an enrichment factor of eight was reached for the phages displaying mdnA on their surface.
These results indicate that the unmodified mdnA expressed on the phages binds specifically to the immobilized trypsin. Therefore it can be deduced that mdnA is presented in a functional 3D structure. These findings suggest that phage display in general is an appropriate method for screening a recombinant mdnA library. Further experiments are required to optimize this system.


Figure 3: Optimization of phage display. After optimized conditions (right) a clear concentration of phages carrying mdnA after one panning round was noted. E. coli cells were infected with phage mix (helper phages and phages carrying mdnA) before and after panning and plated on agar containing kanamycin or ampicillin. The ratio of cells growing on ampicillin or kanamycin agar before panning to cells growing on ampicillin or kanamycin agar after was calculated. Helper phages which acted as negative control have a kanamycine resistance whereby phages carrying mdnA have an ampicillin resistance.


Testing phage display with unmodified mdnA against further proteases

Furthermore the interaction of unmodified mdnA with other proteases was determined. From the literature (Ziemert, 2010) the high inhibitory activity of microviridin L, besides trypsin, against chymotrypsin and elastase is also known. So a phage display with these enzymes was performed. Additionally papain, proteinase K, mycolysin and pepsin were tested for which no interaction was shown yet. For all enzymes an equal amount of E. coli cells and phages were used. In agreement with data from the literature interaction of microviridin with chymotrypsin and elastase was confirmed. Additionally an interaction with papain was noted. All other proteases were not bound by microviridin.

Figure 4: Phage display against different proteases. After panning the number of clones was counted. In agreement with data from the literature interaction of microviridin with chymotrypsin and elastase was confirmed. Additionally an interaction with papain was noted.

User Reviews

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