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| − | <h1 class="sectionedit1">Methodology Cell-free Expression</h1>
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| − | <!-- EDIT1 SECTION "Methodology Cell-free Expression" [1-48] -->
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| − | <h3 class="sectionedit2">Why do we use cell-free expression?</h3>
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| − | <div class="kommentar">
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| − | Ramona
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| − | <p>
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| − | Cell-based protein expression is a well established method to obtain a large amount of a target protein. It enables to accumulate and purify the protein in quantities sufficient for various <i>in vitro</i> applications.
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| − | Nonetheless, it a tedious task to generate all the genetically modified organisms if a variety of proteins needs to be expressed. Additionally, protein purification is not a generalized procedure, but has to be optimized for every protein itself. </br>
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| − | Cell-free expression represents a possibility to overcome many challenges of conventional protein expression in general and offers many advantages concerning our project in particular.
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| − | Generally speaking, it saves a lot of time and money to avoid the generation of genetically modified organisms for every protein. Suitable DNA sequences are constructed once and can be stored until needed. In terms of purification, cell-free expression avoids the need for cell lysis preserving the protein from this harsh procedure. In cell-based expression, too strong induction often results in aggregating and therefore non-functional protein. This probability is minimized using cell-free expression because the expressed protein is dispersed in a far larger volume than the intracellular space. </br>
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| − | For copying a DNA template into a protein microarray in a microfluidic set-up, cell-free expression is the method of choice, because such a system is capable of expressing many different sequences at once. Additionally, the microfluidic system provides the opportunity to supplement the expression constantly with consumed (?) components, like dNTPs or amino acids, enabling high yields of protein expression.
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| − | <!-- EDIT2 SECTION "Why do we use cell-free expression?" [49-220] -->
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| − | <h3 class="sectionedit3">Our system</h3>
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| − | <p>
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| − | We developed our own way of expressing proteins in a prokaryotic cell-free expression system based on an <em>Escherichia coli</em> lysate.
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| − | There are mainly four cell types that are typically used to establish cell-free expression systems: Rabbit reticulocytes, wheat germ cells, insect cells and <em>E. coli</em> cells. Due to the prokaryotic origin of most of the proteins expressed during our project, we decided to prepare our lysate based on an <em>E. coli</em> culture.</br>
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| − | We used the <em>E. coli</em> strain BL21 for our lysate, which carries the gene coding for the T7 RNA polymerase which facilitates an improved expression of proteins during the cellfree expression. The cells were induced with IPTG prior to further treatment to initialize expression of the T 7 polymerase. </br>
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| − | Since the coding sequences we used during our project were all synthetically manufactured, the <em>E. coli</em> BL21 cells were additionally transformed with a plasmid (pRARE2) which contains the coding sequence of tRNAs for the translation of rare codons (AGA, AGG; AUA, CUA, GGA, CCC and CGG).</br>
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| − | Following an optimized protocol (link) adapted from EMBL Heidelberg the lysate was prepared from this E. coli BL21 cells, frozen in liquid nitrogen and stored at -80°C. This lysate contains the whole transcription and translation apparatus of the cell, which allows expression of proteins from DNA templates.</br>
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| − | However, the <em>E. coli</em> lysate is just one of the main components of the resulting cell-free expression mix. To be able to express proteins the system needs additional buffers, amino acids, nucleotides and energy sources. We obtained amino acid from ? and prepared an accurate mix ourselves. The energy sources (???) and buffers (see protocol), nucleotides and amino acids were prepared and added to the lysate directly before expression.</br>
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| − | The now complete cellfree expression mix was then used to express proteins. The DNA coding for the respective proteins was added to the mix, thus starting the reaction.</br></br>
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| − | We compared our self-made expression mix to commercially available kits to verify the functionality of our system. We could show (link to results) that the mix prepared with our own lysate produces high proteins yields comparable to those obtained from commercial kits.
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| − | <!-- EDIT3 SECTION "Our system" [221-540] -->
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| − | <h3 class="sectionedit4">Proof of protein</h3>
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| − | To validate expression of correctly folded proteins we used different methods like fluorescence measuring, luciferase assays and western blots. One of our first approaches was the expression of GFP, which can be detected via its fluorescence. For expression of GFP we used a plasmid (?) template that was added to the self-made expression mix. Expression was then visualized via a microplate reader measurement that monitored the increase in fluorescence with progression of the expression. To verify the obtained data from this measurement a western blot was done additionally. We used GFP antibodies(?) to show the expression of GFP in our mix (link zu results).</br>
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| − | As a second test for our system we perfomed a luciferase assay. In contrast to to the expression of GFP which can be followed by a gradually rising of GFP over time the luciferase assay is based on the emittance of light when luciferin is conversed into oxyluciferin. This reaction is catalysed by the enzyme luciferase and doesn’t allow a tracking of expression over time. We used the coding sequence of the firefly luciferase (pBEST<em>luc</em>) to express the protein with our cellfree expression system. The substrate luciferin was added to the mix after expression was finished and the luminescence was measured at once using a microplate reader (Link zu den results). This analysis helps to evaluate the concentration of expressed protein in the cellfree expression system.
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| − | <div class="thumb2 trien"><div class="thumbinner"><a class="media" href="https://static.igem.org/mediawiki/2015/1/10/Freiburg_luciferase_reaction.png" title="luciferase_reaction.png"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/1/10/Freiburg_luciferase_reaction.png" width=100%/></a><div class="thumbcaption"><div class="magnify"><a class="internal" href="https://static.igem.org/mediawiki/2015/1/10/Freiburg_luciferase_reaction.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div> explanation of luciferase</div></div></div>
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| − | <!-- EDIT4 SECTION "Proof of protein" [541-784] -->
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| − | <h3 class="sectionedit5">Step by step validation</h3>
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| − | <p>
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| − | Since we aim to use cell-free expression in the DiaCHIP we decided to validate the process in single steps.
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| − | Starting with expression in a tube we spotted already expressed proteins on iRIF slides to check wether or not we expressed detectable antigens that interacted with our antibodies at hand.
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| − | As a next step we spotted our expression system on the slide without starting the reaction beforehand. By letting it incubate on a specific surface we could check for further possibilities.
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| − | Lastly, we performed a cell-free expression from immobilized DNA in the finished setup of glass and PDMS slide.
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| − | <h4>Spotting of expressed GFP on slide</h4>
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| − | <p>
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| − | To establish, whether our cell-free expression system reaches protein concentrations high enough to detect in the iRIf, we spotted readily cell-free expressed GFP on iRIF slides. This not only gave us information about the amount of protein, but also on its folding. By seeing an interaction with a corresponding antibody in the iRIf measurement, we could draw conclusions on the epitope.
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| − | <h4>On-slide expression of GFP</h4>
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| − | Firstly we could detect wether the interaction between tagged proteins and the surface was strong enough to bind the proteins as soon as they were formed. This would show when measuring the slide in the iRIf afterwards.
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| − | Secondly we wanted to assess if the surface was specific enough to not bind components of the mix itself.
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| − | <h4>In-chamber expression of GFP</h4>
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| − | This step would show us if we would already get our setup to work as a whole. Moreover, we could estimate, whether the diffusion patterns in a flow chamber differed too much from that in a suitable reaction tube.
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| − | <!-- EDIT5 SECTION "Step by step validation" [785-2474] -->
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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>
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| − | Our biggest goal was to express antigens with the required tags in a concentration and folding state that suffices for an iRIf measurement. Here we focussed on a <em>C. tetani</em> epitope flagged by a double His6 and a Spy-Tag (Spy-tag überhaupt erwähnen?) For cloning of this construct we used our own cloning site (link) and a vector that we optimized for cellfree expression.</br>
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| − | The antigen was expressed in a tube reaction and afterwards spotted on a Ni-NTA surface. Sadly we could not show binding of the respective antibodies/serum(?) with our iRif measurement device. To validate this result we did a western blot, using our cellfree expressed <em>C. tetani</em> antigen and specific antibodies(anti-his?). With this western blot we could indeed show that the <em>C. tetani</em> antigen was expressed in our cell-free expression mix. (Bild oder link zu results) This result is directly contrary to the iRif measurement which could be explained by folding mistakes of the <em>C. tetani</em> antigen during cell free expression. Since we used denatured protein for the western blot the folding state of the protein has no effect on the binding of the antibody.</br>
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| − | Because of time constraints we were not able to optimize our system in a way that would solve this folding problem. Still we were able to express complex proteins such as antigens with our self-made expression mix.
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| − | <!-- EDIT6 SECTION "Cell-free expression of antigens" [2475-2829] -->
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| − | <h3 class="sectionedit7">Testing different conditions</h3>
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| − | To ameliorate the quantity of the cell-free expressed proteins, we varied MgOAc and DNA template concentrations. Here we tested for optimal amounts by using luciferase DNA for direct detection.
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| − | <a class="wikilink1" href="/igem2015/doku.php?id=tag:info&do=showtag&tag=info" rel="tag" title="tag:info">info</a>
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| − | </span></div>
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| − | <!-- EDIT7 SECTION "Testing different conditions" [2830-] -->
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| − | {{Freiburg/wiki_content_end}}
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