Difference between revisions of "Team:Freiburg/Results/Cellfree"

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For the optimal duration of cell-free expression in an iRIf to achieve the best protein expression in shortest time the reaction hadt to be observed in an time-resoluted manner.
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For the optimal duration of cell-free expression in an iRIf to achieve the best protein expression in shortest time the reaction had to be observed in an time-resoluted manner.
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For an easy and timly detection GFP as a reporter protein was choosen. Different kinds of vectors coding for GFP were analysed and compared for their ability to produce in the differnt cell-free expression test systems.
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For an easy and timely detection GFP as a reporter protein was chosen. Different kinds of vectors coding for GFP were analyzed and compared for their ability to produce in the different cell-free expression test systems.
 
First as an external standard an expression vector that was obtained from a group that had already worked for several years with this vector in a commercially available expression system and classified this vector as their best performing. This plasmid carried the gene for GFP with an N-terminal HA-tag and a C-terminal double 6xHis-tag. <img alt="FIXME" class="middle" src="/igem2015/lib/images/smileys/fixme.gif"/> (link)
 
First as an external standard an expression vector that was obtained from a group that had already worked for several years with this vector in a commercially available expression system and classified this vector as their best performing. This plasmid carried the gene for GFP with an N-terminal HA-tag and a C-terminal double 6xHis-tag. <img alt="FIXME" class="middle" src="/igem2015/lib/images/smileys/fixme.gif"/> (link)
 
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Secondly a from this years iGEM team especially for the application designed vector was used that carried a gene for GFP and an N-terminal 10X His and a Spy-tag on the other side. Non-coding areas surrounding the GFP gene were optimized for cell free expression. (link)
+
Secondly a from this years iGEM team especially for the application designed vector was used that carried a gene for GFP and an N-terminal 10xHis-tag and a Spy-tag on the C-term. Non-coding areas surrounding the GFP gene were optimized for cell free expression. (link)
 
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The vectors were tested with the from our iGEM team established cell-free expression system and additionally with an comercially available kit.
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The vectors were tested with the from our iGEM team established cell-free expression system and additionally with an commercially available kit.
 
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<em>FigureX: Cell-free expression of GFP-test vectors with our system or a commercial system over the time of 2 h in 50µl reactions at 37°C over 2 hors. Measurement was taken once a minute (Ex:480nm/Em:520nm. The experiment was performed in triplicates and the results normalized to the mean of air: A propagation of uncertainty was considered in the standard diviation.
+
<em>FigureX: Cell-free expression of GFP-test vectors with our system or a commercial system over the time of 2 h in 50 µl reactions at 37°C over 2 hours. Measurement was taken once a minute (Ex:480nm/Em:520nm. The experiment was performed in triplicates and the results normalized to the mean of air: A propagation of uncertainty was considered in the standard diviation.
 
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The expression of GFP was additionally verified with a western blot that shows the expected bands at a size of XXX which corresponded to the GFP with a Spy and 10xHis-Tag.
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The expression of GFP was additionally verified with a western blot that shows the expected bands at a size of XXX which corresponded to the GFP with a Spy and 10xHis-tag.
 
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<img alt="FIXME" class="middle" src="/igem2015/lib/images/smileys/fixme.gif"/>
 
<img alt="FIXME" class="middle" src="/igem2015/lib/images/smileys/fixme.gif"/>
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For an absolute measurement of GFP production the relative fluorescence levels were compared with and dilution series of GFP of known concentration. As figure x shows the achieved amount of product can be estimated as 100mM/µl.
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For an absolute measurement of GFP production the relative fluorescence levels were compared with dilution series of GFP of known concentration. As figure x shows the achieved amount of product can be estimated as 100 mM/µl.
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This could be also validated by a Western Blot that contained a diltution series of GFP.
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This could be also validated by a Western Blot that contained a dilution series of GFP.
 
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With our expression of GFP we were able to demonstrate that our system performs reliable and shows better results than a comercially available on. With the generated amount of protein an detection iRIf is possible due to the specific tagging system that enriches proteins at a specific surface.
+
With our expression of GFP we were able to demonstrate that our system performs reliable and shows better results than a commercially available one. With the generated amount of protein an detection iRIf is possible due to the specific tagging system that enriches proteins at a specific surface.
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Amidittly, our own designes Vector (Spy-GFP-10xHis) didn't performe as well as the external received Vector (HA-GFP-12xHis). This could be due to differences in the 5' upstream sequence which was demonstarted to have grat influence on the initiation of Transcription.
+
Amidittly, our own designed vector (Spy-GFP-10xHis) didn't perform as well as the external received vector (HA-GFP-12xHis). This could be due to differences in the 5' upstream sequence which was demonstrated to have influence on the initiation of transcription.
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Furthermore, the Tag in the beginning of the coding sequence also manipulates the expression level.
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Furthermore, the tag in the beginning of the coding sequence also manipulates the expression level.
 
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blots, platereader, iRIf
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blots, plate reader, iRIf
 
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luminescence curve, platereader
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luminescence curve, plate reader
 
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Revision as of 19:53, 13 September 2015

""

Results: Cell-free Expression

To establish a cell-free expression system, a bacterial lysate was produced and supplemented with several energy sources as well as co-factors and ions. GFP expression in our system was compared to a commercially available kit. In figure 2, the relative fluorescence of the samples is shown over time. After two hours the fluorescence indicates a x fold change of the amount of GFP in the sample compared to the beginning of the reaction. The commercially available kit reaches an x fold increase in relative fluorescence.
Additionally, it was shown that the expressed GFP is not only functional in terms of fluorescence but it also exhibits the same binding affinity to a commercial anti-GFP antibody as conventionally purified GFP (figure 3). Thus, our cell-free expression system can be used to mediate the copying process from a DNA template to a protein microarray.
The process of establishment and optimization of our cell-free expression system can be retraced here.

Figure 2: Comparison with self-made cell-free expression system and a commercially available kit. The relative fluorescence of cell-free expressed GFP is monitored over 2 hours in comparison with a commercial kit. For each system a no-DNA control was added enabling to calculate the fold change.
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Figure 3: Western Blot of conventionally and cell-free expressed GFP.

Proof of protein

Results

For the optimal duration of cell-free expression in an iRIf to achieve the best protein expression in shortest time the reaction had to be observed in an time-resoluted manner. For an easy and timely detection GFP as a reporter protein was chosen. Different kinds of vectors coding for GFP were analyzed and compared for their ability to produce in the different cell-free expression test systems. First as an external standard an expression vector that was obtained from a group that had already worked for several years with this vector in a commercially available expression system and classified this vector as their best performing. This plasmid carried the gene for GFP with an N-terminal HA-tag and a C-terminal double 6xHis-tag. FIXME (link)

Secondly a from this years iGEM team especially for the application designed vector was used that carried a gene for GFP and an N-terminal 10xHis-tag and a Spy-tag on the C-term. Non-coding areas surrounding the GFP gene were optimized for cell free expression. (link)

The vectors were tested with the from our iGEM team established cell-free expression system and additionally with an commercially available kit.

FigureX: Cell-free expression of GFP-test vectors with our system or a commercial system over the time of 2 h in 50 µl reactions at 37°C over 2 hours. Measurement was taken once a minute (Ex:480nm/Em:520nm. The experiment was performed in triplicates and the results normalized to the mean of air: A propagation of uncertainty was considered in the standard diviation.

Figure X clearly shows that the best performing expression mix/vector combination is the HA-GFP-(6xHis)² with our developed E. coli based DIAmix expression system.

The expression of GFP was additionally verified with a western blot that shows the expected bands at a size of XXX which corresponded to the GFP with a Spy and 10xHis-tag.

FIXME For an absolute measurement of GFP production the relative fluorescence levels were compared with dilution series of GFP of known concentration. As figure x shows the achieved amount of product can be estimated as 100 mM/µl. This could be also validated by a Western Blot that contained a dilution series of GFP.

FIXME

Discussion

With our expression of GFP we were able to demonstrate that our system performs reliable and shows better results than a commercially available one. With the generated amount of protein an detection iRIf is possible due to the specific tagging system that enriches proteins at a specific surface. Amidittly, our own designed vector (Spy-GFP-10xHis) didn't perform as well as the external received vector (HA-GFP-12xHis). This could be due to differences in the 5' upstream sequence which was demonstrated to have influence on the initiation of transcription. Furthermore, the tag in the beginning of the coding sequence also manipulates the expression level.

Step by step validation

Spotting of expressed GFP on slide

blots, plate reader, iRIf

On-slide expression of GFP

iRIf

In-chamber expression of GFP

iRIf

Cell-free expression of antigens

Blot, iRIf

Testing different conditions

luminescence curve, plate reader