Team:Potsdam Bioware/Project/Details

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=== Phage Display ===
=== Phage Display ===
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<br>
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<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-geneIII''-fusion gene on the surface of the phage. So ''E. coli'' cells 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-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 showed the successful expression of ''mdnA-gene III''-fusion protein on the phages. For more precise results the absorption  
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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 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>
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from 450 – 600 nm was measured and measured data was presented in a box plot. As a negative control helper phages were added instead of produced phages, carrying mdnA-myc-geneIII fusion protein on their surface. Furthermore two wells were prepared were the secondary antibody was not added.
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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.
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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-myc-gene-III-fusion protein on the surface of the phages.
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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.
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[[File:UP Excel ELISA.png|400px]]
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<div align="center">
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[[File:UP Excel ELISA.png|400px]]</div>
[[File:UP absorptionELISA1.PNG|800px]]
[[File:UP absorptionELISA1.PNG|800px]]
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<br>'''Phage Display'''<br>
<br>'''Phage Display'''<br>
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<br>
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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.
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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>
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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>
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So it was decided to repeat this experiment under improved conditions. Therefore the number of washing steps during the described experimental procedure was increased.
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Results are shown in figure <br>
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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.
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[[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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The equations above were solved using MATLAB. Using the Avogadro constant and the volume of a ''E.coli'' cell (10^-15L) and a periplam-volume of about 5%, following konstants were calculated and compared with similar literature values:<br>
The equations above were solved using MATLAB. Using the Avogadro constant and the volume of a ''E.coli'' cell (10^-15L) and a periplam-volume of about 5%, following konstants were calculated and compared with similar literature values:<br>
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k+1 =  1.e-6 (1/molecules*s)<br>
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k-1 = 2.e-4 (1/s)<br>
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kexpr.Prot =  1 (molecules/s)<br>
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kdegr1 =  3.e-3 (1/s)<br>
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k+2 =  5.e-7 (1/molecules*s)<br>
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k-2 = 4.e-4 (1/s)<br>
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kcat =  8 (1/s)<br>
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kexpr.Tor =  1.4 (1/s)<br>
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kdegr2 =  3.e-3 (1/s)<br>
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ktransTor =  3.e-3 (1/s)<br>
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ktransAmp = 3.e-3 (1/s)<br>
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Kcat2 = 0.3 (1/molecules*s)<br>
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MIC(0) = 8000 (molecules)<br>
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AMP(0) = 5000 (molecules)
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<br>
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[[File: UP_ode_solution_all_times.png|center|800px|thumb|'''Figure xxx:''' MATLAB numerical solution to our system of differential equations for substance concentrations (above) over all time segments.]]
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It can be seen that there is only a negligible amount of ampicillin in the periplam if the expression works as indicated. There is also a wide tolerance left for suboptimal conditions. The triggertime t1, t2 and t3 shal be about one hour after one another.
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In the first section the effect of the inhibition can clearly be seen: Even though the continuous expression of protease, only a very small number remains active in the cytoplasm. In the second section lactamase is expressed. Most of the lactamase in the cytoplasm is destroyed by the protease, however the number is by far large enough to steadily release molecules into the periplam where it cannot be affected by the protease any more. Because the volume of the periplasm is very small, the concentration there is even higher than the absolute amount of molecules in the graph suggests compared to the amount of molecules in the cytoplam. The third section shows that the concentrations remain very steady and the cells are ampicillin resistent.
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Cells passing this procedure are very ampicillin resistent and grow fast: They double their cell volume about every 20 minutes. If an error would appear and the ampicillin concentration inside the periplasma would increase over 2µg/ml, the growth rate would slow down drastically and the cells might die. This way defect cell cultures can easyly be seperated from the good ones.
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The ratio of ampicillin concentration in ''n µg/ml'' to the growth rate (cells double during this timespan) is approximatelly this: ''T(groth) = 20min+10*2^(n-1)''.
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<br>'''MATLAB code'''<br>
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[[Media:conzt1.m]]<br>
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[[Media:conzt2.m]]<br>
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[[Media:conzt3.m]]<br>
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[[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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Modelling

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