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

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<p>
 
<p>
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.
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The purpose of cell-free expression in the DiaCHIP is to copy a DNA template into a protein microarray on demand. To enable antibody detection with this protein microarray, the single antigen spots have to be covered with a dense layer of antigens. Thus, the expression efficiency of the cell-free system has to be optimized to produce a sufficient amount of the target proteins within a timespan that is reasonable for DiaCHIP preparation in the suggested applications (LINK).
<br/>
+
 
+
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.
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</br>
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<a href="https://2015.igem.org/Team:Freiburg/Results/Cellfree">The process of establishment and optimization of our cell-free expression system can be retraced here</a>.
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</p>
 
</p>
  
<div class="flexbox">
 
<div class="thumb2 trien" style="width:410px"><div class="thumbinner"><a class="lightbox_trigger" href="https://static.igem.org/mediawiki/2015/5/50/Freiburg_2015_freiburg_cellfex_gfp_over_time.png"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/5/50/Freiburg_2015_freiburg_cellfex_gfp_over_time.png" width="400"/></a><div class="thumbcaption"><div class="magnify"><a class="internal" href="/igem2015/lib/exe/detail.php?id=results_overview&amp;media=2015_freiburg_cellfex_gfp_over_time.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div><strong>Figure 2: Comparison with self-made cell-free expression system and a commercially available kit.</strong> 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. </div></div></div><div class="thumb2 trien" style="width:310px"><div class="thumbinner">Invalid Link<div class="thumbcaption"><div class="magnify"><a class="internal" href="/igem2015/lib/exe/detail.php?id=results_overview&amp;media=figure_name.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div><strong>Figure 3: Western Blot of conventionally and cell-free expressed GFP.</strong> </div></div></div>
 
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<!-- EDIT1 SECTION "Results: Cell-free Expression" [1-46] -->
 
<h2 class="sectionedit2">Proof of protein</h2>
 
<div class="level2">
 
</div>
 
<!-- EDIT2 SECTION "Proof of protein" [47-76] -->
 
<h3 class="sectionedit3">Results</h3>
 
<div class="level3">
 
 
<p>
 
<p>
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.
+
One of the key factors influencing expression efficiency is the design of the expression vector. Therefore, we started with the design of a vector based on pSB1C3 that would allow successful cell-free expression (LINK). Additionally, we received an expression vector containing a GFP coding sequence form the BIOSS toolbox (LINK/MAP). The third vector we used for our experiments was pBEST<i>luc</i> (LINK/MAP) encoding a luciferase for performing the <a class=”wikilink1” href=”https://2015.igem.org/Team:Freiburg/Project/Cellfree_Expression”>Luciferase assay</a>.
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)
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</p>
 
</p>
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<p>
 
<p>
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)
+
However, the most important thing is of course the expression system itself. We obtained one commercially available expression kit based on an //E. coli// lysate, were provided with an established cell-free expression mix from the group of Bernhard (LINK!!) and additionally established a protocol for the production of such a system ourselves, based on [REF.]. We named it the DiaMIX and provide the <a href="https://2015.igem.org/Team:Freiburg/Protocols/Cellfex"> protocol </a> in order to give future iGEM Teams the possibility to produce their cell-free expression mix in a low-budget version themselves.
</p>
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<p>
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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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</p>
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<p>
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<a class="media" href="https://static.igem.org/mediawiki/2015/4/46/Freiburg_results-gfp_overtime.png" title="results:gfp_overtime.png"><img alt="" class="media" src="https://static.igem.org/mediawiki/2015/4/46/Freiburg_results-gfp_overtime.png" width="300"/></a>
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</p>
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<p>
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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 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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</em>
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</p>
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<p>
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Figure X clearly shows that the best performing expression mix/vector combination is the HA-GFP-(6xHis)² with our developed <em>E. coli</em> based DIAmix expression system.
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<a class="media" href="https://static.igem.org/mediawiki/2015/4/44/Freiburg_results-blot_gfp.png" title="results:blot_gfp.png"><img alt="" class="media" src="https://static.igem.org/mediawiki/2015/4/44/Freiburg_results-blot_gfp.png"/></a>
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</p>
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<p>
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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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</p>
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<p>
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<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 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 dilution series of GFP.
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</p>
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<p>
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<img alt="FIXME" class="middle" src="/igem2015/lib/images/smileys/fixme.gif"/>
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</p>
 
</p>
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<div class="image_box right">
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        <div class="thumb2 trien" style="width:400px"><div class="thumbinner">
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            <img src="https://static.igem.org/mediawiki/2015/0/0e/Freiburg_Cellfex_GFP_first_measurement.png"></img>
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        </div>
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            <strong>Figure 1: Cell-free expression of GFP.</strong> Fluorescing GFP could be detected via fluorescence microscopy after cell-free expression (A). Picture taken of the negative control (B). For detailed reaction performance see <a href=”https://2015.igem.org/Team:Freiburg/Labjournals/Cellfree/June”>our labjournal</a>.
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        </div>
 
</div>
 
</div>
<!-- EDIT3 SECTION "Results" [77-2379] -->
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<h3 class="sectionedit4">Discussion</h3>
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<div class="level3">
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<p>
 
<p>
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.
+
Before using our DiaMIX in the microfluidic chamber, we performed some initial experiments //in vitro// in well-plates. With our second cell-free expression experiment using all our self-prepared components we were already able to express GFP deriving from pQE60 (LINK). As negative control the reaction was performed in the same way, simply using water instead of DNA. After expression, a small amount of the reaction was directly pipetted onto a microscopy glass slide and analyzed under a fluorescence microscope. As it is clearly visible in figure 1, we could detect expressed GFP and therefore prove the functionality of our system for the first time!
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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</p>
 
</p>
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 +
<div class="image_box right">
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        <div class="thumb2 trien" style="width:400px"><div class="thumbinner">
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            <img src="https://static.igem.org/mediawiki/2015/7/71/Freiburg_pig15_104_5000ng_.png"></img>
 +
        </div>
 +
            <strong>Figure 2: Impact of Mg(OAc)2 addition during cell-free expression of tYFP.</strong> Mixtures of our own lysate with our premix (KK) and the lysate we obtained from Bernhard with our premix (BK) were tested. An impact of feeding with Mg(OAc)2 could be observed for the BK mixture. Validation of fluorescence at an excitation of 488nm.
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        </div>
 
</div>
 
</div>
<!-- EDIT4 SECTION "Discussion" [2380-3067] -->
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<h3 class="sectionedit5">Step by step validation</h3>
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<div class="level3">
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</div>
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<h4>Spotting of expressed GFP on slide</h4>
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<div class="level4">
+
 
<p>
 
<p>
blots, plate reader, iRIf
+
Nonetheless, there were of course many things that had to be optimized before performing the cell-free expression on a slide for a measurement with the iRIf (LINK was ist iRIF) device. At first, we investigated the influence of magnesium acetate (Mg(OAc)2) added to the reaction. In a work of ???? (LINK) we read about the enhancement of cell-free expression by regularly adding a particular amount of the chemical. We performed some expression experiments where some reactions were fed with small amounts of Mg(OAc)2 and some were not. Indeed, we could observe an effect of the addition of the supplement (see figure 2). However, the actual impact of the feeding varied a lot in the experiments (LINK zu labjournal), even among reactions with the same mix components. <br/>
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 +
<br/>
 +
<br/>
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</p>
 
</p>
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<div class="image_box left">
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        <div class="thumb2 trien" style="width:400px"><div class="thumbinner">
 +
            <img src="https://static.igem.org/mediawiki/2015/2/20/Freiburg_2015-08-10_105_lk.png"></img>
 +
        </div>
 +
            <strong>Figure 3: Variation of Mg(OAc)2 concentrations in cell-free expression.</strong> Cell-free expression of pBESTluc and subsequent validation via Luciferase assay.
 +
        </div>
 
</div>
 
</div>
<h4>On-slide expression of GFP</h4>
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<div class="level4">
+
 
<p>
 
<p>
iRIf
+
As supplying a reaction lasting for two hours with some additional chemicals every 20 minutes is quite time consuming, we thought about simply varying the initial concentration of Mg(OAc)2 in the reaction mix. For the expression mix we received from Bernhard, the optimal start concentration was stated in the range of 15-18mM (REF!!). Therefore, we expressed the pBESTluc (LINK) with our whole DiaMIX at concentrations between 14-18mM to compare the expression via <a href=”http://Team:Freiburg/Project/Cellfree_Expression”>Luciferase assay</a>. The result is shown in figure 3 and reveals the optimal amount of Mg(OAc)2 at a starting concentration of 14mM.
 
</p>
 
</p>
 +
 +
 +
<div class="image_box left">
 +
        <div class="thumb2 trien" style="width:400px"><div class="thumbinner">
 +
            <img src="https://static.igem.org/mediawiki/2015/1/11/Freiburg_Cellfex_DNA_variation.png"></img>
 +
        </div>
 +
            <strong>Figure 4: Variation of DNA concentration for cell-free expression.</strong> Results for concentrations of 0.02µg/µl (A), 0.04µg/µl (B), 0.1µg/µl (C) and the negative control (D). Validation was performed via Luciferase assay and analyzed in a microplate reader.
 +
        </div>
 
</div>
 
</div>
<h4>In-chamber expression of GFP</h4>
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<div class="level4">
+
 
 +
 
 
<p>
 
<p>
iRIf
+
After establishing the best concentration of Mg(OAc)2 in our reactions we further investigated the optimal concentration of DNA added for expression. Again, pBESTluc (LINK) was expressed to allow the analysis via Luciferase assay in a microplate reader. Triplicates for reactions of 50µl volumes were set up with amounts of DNA ranging from 1µg to 5µg, as well as a negative control without DNA. Exemplarily, the results for three different concentrations as well as the negative control are shown in figure 4, the other results can be found <a href=”https://2015.igem.org/Team:Freiburg/Labjournals/Cellfree/August”>in our labjournal</a>. The optimal amount of DNA established for further experiments was 2µg.
 
</p>
 
</p>
</div>
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<!-- EDIT5 SECTION "Step by step validation" [3068-3257] -->
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<h3 class="sectionedit6">Cell-free expression of antigens</h3>
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<div class="level3">
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<p>
 
<p>
Blot, iRIf
+
Having optimized our self-prepared low-budget cell-free expression mix, we were keen on comparing it to a commercially available kit and thereby making a statement about the functionality of our DiaMIX. As already shown on the <a class=”wikilink1” href=”https://2015.igem.org/Team:Freiburg/Results”> main results page </a> we set up an experiment using DNA of the pQE60 vector in order to express functional GFP and analyze it via fluorescence. The cell-free expression itself as well as the negative control for each mix were prepared in triplicates. <br/>
 +
Our self-produced DiaMIX performed about as good as the commercial kit as it is shown in figure X (Fig. X: Cell-free GFP expression over time. Comparison ot the commercial kit with the DiaMIX was performed using the pQE60 HA-GFP-His vector. Relative fluorescence was measured every minute over 2 hours. As negative control the fluorescence of the respective mix without adding DNA was recorded.). Compared to the background, an expression time of two hours resulted in a 2-fold increase in relative fluorescence in both systems. The background fluorescence was estimated by the negative control.
 
</p>
 
</p>
</div>
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<!-- EDIT6 SECTION "Cell-free expression of antigens" [3258-3313] -->
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<h3 class="sectionedit7">Testing different conditions</h3>
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<div class="level3">
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<p>
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luminescence curve, plate reader
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</p>
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<div class="tags"><span>
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<a class="wikilink1" href="/igem2015/doku.php?id=tag:info&amp;do=showtag&amp;tag=info" rel="tag" title="tag:info">info</a>
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</span></div>
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</div>
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<!-- EDIT7 SECTION "Testing different conditions" [3314-] -->
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</div>
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</html>
 
</html>
 
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{{Freiburg/wiki_content_end}}

Revision as of 12:42, 14 September 2015

""

Results: Cell-free Expression

The purpose of cell-free expression in the DiaCHIP is to copy a DNA template into a protein microarray on demand. To enable antibody detection with this protein microarray, the single antigen spots have to be covered with a dense layer of antigens. Thus, the expression efficiency of the cell-free system has to be optimized to produce a sufficient amount of the target proteins within a timespan that is reasonable for DiaCHIP preparation in the suggested applications (LINK).

One of the key factors influencing expression efficiency is the design of the expression vector. Therefore, we started with the design of a vector based on pSB1C3 that would allow successful cell-free expression (LINK). Additionally, we received an expression vector containing a GFP coding sequence form the BIOSS toolbox (LINK/MAP). The third vector we used for our experiments was pBESTluc (LINK/MAP) encoding a luciferase for performing the Luciferase assay.

However, the most important thing is of course the expression system itself. We obtained one commercially available expression kit based on an //E. coli// lysate, were provided with an established cell-free expression mix from the group of Bernhard (LINK!!) and additionally established a protocol for the production of such a system ourselves, based on [REF.]. We named it the DiaMIX and provide the protocol in order to give future iGEM Teams the possibility to produce their cell-free expression mix in a low-budget version themselves.

Figure 1: Cell-free expression of GFP. Fluorescing GFP could be detected via fluorescence microscopy after cell-free expression (A). Picture taken of the negative control (B). For detailed reaction performance see our labjournal.

Before using our DiaMIX in the microfluidic chamber, we performed some initial experiments //in vitro// in well-plates. With our second cell-free expression experiment using all our self-prepared components we were already able to express GFP deriving from pQE60 (LINK). As negative control the reaction was performed in the same way, simply using water instead of DNA. After expression, a small amount of the reaction was directly pipetted onto a microscopy glass slide and analyzed under a fluorescence microscope. As it is clearly visible in figure 1, we could detect expressed GFP and therefore prove the functionality of our system for the first time!

Figure 2: Impact of Mg(OAc)2 addition during cell-free expression of tYFP. Mixtures of our own lysate with our premix (KK) and the lysate we obtained from Bernhard with our premix (BK) were tested. An impact of feeding with Mg(OAc)2 could be observed for the BK mixture. Validation of fluorescence at an excitation of 488nm.

Nonetheless, there were of course many things that had to be optimized before performing the cell-free expression on a slide for a measurement with the iRIf (LINK was ist iRIF) device. At first, we investigated the influence of magnesium acetate (Mg(OAc)2) added to the reaction. In a work of ???? (LINK) we read about the enhancement of cell-free expression by regularly adding a particular amount of the chemical. We performed some expression experiments where some reactions were fed with small amounts of Mg(OAc)2 and some were not. Indeed, we could observe an effect of the addition of the supplement (see figure 2). However, the actual impact of the feeding varied a lot in the experiments (LINK zu labjournal), even among reactions with the same mix components.



Figure 3: Variation of Mg(OAc)2 concentrations in cell-free expression. Cell-free expression of pBESTluc and subsequent validation via Luciferase assay.

As supplying a reaction lasting for two hours with some additional chemicals every 20 minutes is quite time consuming, we thought about simply varying the initial concentration of Mg(OAc)2 in the reaction mix. For the expression mix we received from Bernhard, the optimal start concentration was stated in the range of 15-18mM (REF!!). Therefore, we expressed the pBESTluc (LINK) with our whole DiaMIX at concentrations between 14-18mM to compare the expression via Luciferase assay. The result is shown in figure 3 and reveals the optimal amount of Mg(OAc)2 at a starting concentration of 14mM.

Figure 4: Variation of DNA concentration for cell-free expression. Results for concentrations of 0.02µg/µl (A), 0.04µg/µl (B), 0.1µg/µl (C) and the negative control (D). Validation was performed via Luciferase assay and analyzed in a microplate reader.

After establishing the best concentration of Mg(OAc)2 in our reactions we further investigated the optimal concentration of DNA added for expression. Again, pBESTluc (LINK) was expressed to allow the analysis via Luciferase assay in a microplate reader. Triplicates for reactions of 50µl volumes were set up with amounts of DNA ranging from 1µg to 5µg, as well as a negative control without DNA. Exemplarily, the results for three different concentrations as well as the negative control are shown in figure 4, the other results can be found in our labjournal. The optimal amount of DNA established for further experiments was 2µg.

Having optimized our self-prepared low-budget cell-free expression mix, we were keen on comparing it to a commercially available kit and thereby making a statement about the functionality of our DiaMIX. As already shown on the main results page we set up an experiment using DNA of the pQE60 vector in order to express functional GFP and analyze it via fluorescence. The cell-free expression itself as well as the negative control for each mix were prepared in triplicates.
Our self-produced DiaMIX performed about as good as the commercial kit as it is shown in figure X (Fig. X: Cell-free GFP expression over time. Comparison ot the commercial kit with the DiaMIX was performed using the pQE60 HA-GFP-His vector. Relative fluorescence was measured every minute over 2 hours. As negative control the fluorescence of the respective mix without adding DNA was recorded.). Compared to the background, an expression time of two hours resulted in a 2-fold increase in relative fluorescence in both systems. The background fluorescence was estimated by the negative control.