Revision as of 19:00, 12 September 2015

iGEM Bielefeld 2015

CFPS

Cell-Free Protein Synthesis

"Cell free protein synthesis" (CFPS) is the production of proteins from nucleic acid templates without living cells, but with the use of the transcription and translational "hardware" that originally is built up by cells. This is possible because this "hardware" does not necessarily need a whole cell, but only some well characterized components to function properly. Among these components, the most obvious are RNA-Polymerase and Nucleoside-Triphosphates (NTPs) for transcription, as well as ribosomes and amino acids for the translation. However, the production of proteins relies on many other molecules and the environment where the production takes place. In the following we present a short overview on all parts (and aspects) that are necessary and essential for in vitro transcription and translation. When using crude cell extracts of E. coli, most compounds are brought to the reaction via the extract itself.

Globally affecting factors

strain used

The early experiments on CFPS relayed on RNase- and protease-deficient strains (Zubay, 1973; Pratt, 1984). Since then, many different strains were tested and genetically improved to support the system.
Especially, strains that have less RNase- and protease activity, produce rare tRNAs, and carry mutations that counteract the depletion of amino acids during reaction build the best basis for an efficient in vitro protein synthesis. You can find detailed overviews in following publications:
- Jewett and Swartz, 2004; Calhoun and Swartz, 2006; Spirin and Swartz, 2008; Airen, 2011; Hong et al., 2015

pH value

For efficient protein synthesis, a pH value above 6.5 is indispensable (Kim et al., 2008; Jewett and Swartz, 2004). This is the main reason for the use of pH-adjusted washing buffers and phosphate buffer in the media, when E. coli extracts are prepared in-house. The optimal pH for the reaction itself varies depending on the protein to be produced. To prevent precipitation of the protein, the pH should generally be between 6.5 and 8.5 (Tokmakov et al., 2014).
Different approaches for stabilizing the pH value have been proposed, ranging from the use of buffers (Airen, 2011) to the use of engineered enzymes (Kim et al., 2015).

Energy

Life depends upon ATP as it is the main energy source in cellular organisms. To provide ATP throughout the CFPS-reaction, ATP has to be permanently regenerated. The translation machinery also needs two GTP for every amino acid that is incorporated into the growing peptide chain (Mavelli et al., 2015). Therefore, molecules with high energy content are needed that fuel phosphorylation of ADP (for example via creatin phosphate, abbreviated CP) or that fuel glycolysis (for example phosphoenolpyruvate, abbreviated PEP). In general, the use of the high energetic CP in combination with creatin kinase gives the best results (Shimizu and Ueda, 2010; Kazuta et al., 2014). However, CP and creatin kinase are very expensive compared to other reagents. In recent years many different energy sources have been established which give good results at less costs. You can find detailed information in reviews and particulary in these publications:
Kim and Swartz, 2001; Jewett and Swartz, 2004; Ma et al., 2010; Caschera and Noireaux, 2015a; Cai et al., 2015

Medium

2xYTP and 2xYTPG medium are most frequently employed for production of homemade E. coli cell extract (Caschera and Noireaux, 2015a; Kwon and Jewett, 2015). The phosphate buffer helps to maintain a stable pH during cultivation and cell harvest.

Transcription

template

The use of DNA as template has some advantages over the use of an mRNA template. For example, one needs only small amounts of DNA compared to mRNA to obtain the same output signal as long as transcription is not limiting (Rosenblum and Cooperman, 2014). Compared to mRNA, DNA, especially plasmid DNA, is more stable during the reaction (Spirin and Swartz, 2008). The use of linear DNA as a template can support protein production if the PURE system is used (Chizzolini et al., 2014). Higher amounts of DNA template result in higher amounts of mRNA produced, but this is only to a certain level advantageous for protein production (Chizzolini et al., 2014).
Apart from the later translated region, the 5'-untranslated region (5'-UTR) is of major importance for an efficient reaction (Spirin and Swartz, 2008; Karig et al., 2012; Lentini et al., 2013; Takahashi et al., 2013). You can find an overview about that topic here
Quality and purity of the DNA template is an important aspect. When using plasmid purification kits one has to keep in mind that RNase is part of the resuspension buffer. In the literature, opinions vary regarding which is the best method for purification (Kigawa et al., 2004; Calhoun and Swartz, 2005; Sun et al., 2013).

RNA-Polymerase

The most widely used RNA-Polymerase for cell free protein production is the T7-Polymerase from bacteriophage T7 which is highly efficient and provides fast transcription (Shin and Noireaux, 2010). The T7-Polymerase is highly specific to its T7-Promoter, and is commonly used to express proteins in vivo (Sousa and Mukherjee, 2003). Simple in-house production of T7-Polymerase has been described in many publications, so even E. coli strains without endogenous T7-Polymerase can be utilized (Yang et al., 2012a; Roos et al., 2014).
To overcome the limits of T7-Polymerase, Shin and Noireaux established CFPS based on endogenous polymerases and sigma factors from E. coli (Shin and Noireaux, 2010; Shin and Noireaux, 2012). With this work the possibilities of in vitro transcription-translation were extended; it became possible to characterize other than bacteriophage promotors in vitro (Chappell and Freemont, 2013). This lead to the analysis of genetic circuits outside of living cells (Chappell et al., 2013; Sun et al., 2013; Takahashi et al., 2015).

NTPs

The four nucleoside triphosphates (NTPs) ATP, CTP, GTP and UTP are the building blocks of RNA and therefore essential for transcription. They are one of the main cost factors, and it was shown that it is possible to use nucleoside monophosphates (NMPs) instead, although CFPS is less effective in this case (Calhoun and Swartz, 2005; Kim et al., 2008).

Translation

Amino acids

Nearly all proteins are built up by L-amino acids. Neglecting selenomethionine and pyrrolysine, there are 20 proteinogenic amino acids that all have to be brought to the cell free reaction. This is a difficult task for the experimenter, because these molecules have different solubilities and pKa-values. Recently, an easy procedure for the production of an amino acid mastermix has been presented (Caschera and Noireaux, 2015b).
Some amino acids – especially cysteine, tryptophan, arginine and serine – are depleted much faster in the cell free reaction than others, for example due to enzymatic reactions (Calhoun and Swartz, 2006). This is one of the biggest problems that occur during batch CFPS and leads to fast stop of protein production, unless appropriate knockout strains are used or amino acid concentrations are increased (Jewett and Swartz, 2004; Calhoun and Swartz, 2006; Pedersen et al., 2011).

Other components involved in translation

Amino acids are fused to corresponding tRNAs and thus activated for translation through the action of aminoacyl-tRNA-synthetases, a process that is called aminoacylation. Additional E. coli tRNA supplied to the CFPS-reaction is advantageous (Calhoun and Swartz, 2005), though it was observed that under certain conditions an addition has no effect (Shin and Noireaux, 2010; Cai et al., 2015)
Elongation and releasing factors coordinate translation. For example, the GTP-binding protein EF-Tu is the major elongation factor in E. coli (Airen, 2011). It has been shown that the supplementation of EF-Tu improves translation efficiency (Underwood et al., 2005), and that EF-Tu could mediate the incorpotation of non-natural aminoacids into proteins, for example selenocysteine (Miller et al., 2015)

The protein of interest

Proteins are a very wide group of molecules, different in amino acid composition, 3D-structure and physicochemical properties. Because of the individuality of proteins, it is necessary to optimize the cell free synthesis reaction in every case (Spirin and Swartz, 2008) A recent approach based on bioinformatics was made to better estimate how one can improve CFPS for a certain protein (Tokmakov et al., 2014). Generally, the production is more successful when the transcript is short and the protein folds fast and is soluble (Lentini et al., 2013; Chizzolini et al., 2014). Aspects like the formation of disulfide bonds or a chromophore have also to be considered.
CFPS can be used to produce proteins whose production is limited in vivo due to toxicity or membrane association, and also to incorporate unnatural or labeled amino acids (Kigawa et al., 2004; Xu et al., 2005; Bundy and Swartz, 2010; Quast et al., 2015b). It has to be considered that production of complex proteins that are further processed after translation, for example membrane-bound proteins, is in general more feasible in eukaryotic cell extracts (Quast et al., 2015a).

Additives

There are lots of possible additives that are known to contribute to a more efficient in vitro protein production, as well small substances like DTT as enzymes (a good overview in Spirin and Swartz, 2008 and Airen, 2011). Although some cofactors, for example coenzyme A, NAD and folinic acid, seem more important than others, the effects often differ depending on conditions like cell extract, scale, protein of interest and so on.

One advantage of the clearly defined environment the cell free reaction takes place in, is that "[...] it should be possible to build a complete mathematical model describing the cellular mimic that could aid in designing new features."(Lentini et al., 2013). Recent approaches have been made to mathematically model and characterize the PURE system (Karzbrun et al., 2011; Stögbauer et al., 2012; Mavelli et al., 2015).

Cost considerations

in vitro transcription and translation has been an expensive issue. Commercial systems composed of purified single components, like the PURE system (Shimizu and Ueda, 2010) or E. coli (T7) S30 Extract, work efficiently, but are often more than ten times more expensive than in-house generated reactions. A milestone for lowering the costs of selfmade extracts was the finding of Kim and coworkers that after lysis, highly active cell extracts can be produced with a single centrifugation step and a subsequent run-off reaction at 37 °C (Kim et al., 2006). The run-off reaction facilitates degeneration of endogenous DNA, whereby the background expression is reduced (Pratt, 1984). A final centrifugation step after the run-off reaction can be helpful (Kwon and Jewett, 2015).

Indeed, many researchers have proven that it is possible to further minimize the costs per reaction down to a fraction of cents (Calhoun and Swartz, 2005; Kim et al., 2011; Sun et al., 2013; Caschera and Noireaux, 2015). Protocols for preparation of cell extracts, especially for efficient lysis of harvested cells, have been described in recent years (Shrestha et al., 2012; Kwon and Jewett, 2015). With the Multiplex Automated Genome Engineering (MAGE) method, it became possible to tag and purify all necessary proteins for translation (Wang et al., 2012). This approach has the potential to simplify and reduce the costs for cell free in vitro reactions based on purified components (Kelwick et al., 2014) .

These papers inspired us to experiment with selfmade E. coli cell extracts, aiming to develop protocols for the production of cheap cell extracts and to open cell free protein synthesis to the iGEM community.

Advantages and possible uses

In general, the developments in CFPS proceed into two major directions: These are on the one hand the production of large quantities of proteins (Scale-up direction) without needing recombinant hosts. On the other hand the aim is to investigate distinct proteins or protein synthesis procedures at a small scale for a minimum of costs, and to enrich a protein of interest in a minimal volume (miniaturization direction) (Alexandrov and Johnston).

With in vitro Transcription-Translation, it long was difficult to produce identical or higher amounts of active proteins when compared to in vivo. Recent publications proof that this is now possible and that the scale-up of CFPS becomes more and more efficient (Caschera and Noireaux, 2014; Kazuta et al., 2014).

CFPS offers great advantages over classical overexpression in living cells: The reaction is not restricted to the experimenter by a cell wall, so one can add exactly the components to the reaction that are needed, for example additional co-factors, inducers or catalysts. One recent example is the use of an engineered enzyme to maintain a stable pH without the need of a chemical buffer (Kim et al., 2015). Depending on the system, it is possible to use linear DNA or RNA as a template, skipping ligation or reverse transcription (Rosenblum and Cooperman, 2014)

Regarding Synthetic biology, CFPS emerged as a valuable tool, with the long-term potential to not only be integrated into, but create whole new fields of research. With CFPS, the synthesis of proteins that would kill the cells used for production became possible, just to mention for example antibiotic-like proteins (Xu et al., 2005; Yang et al., 2012b). The incorporation of non-natural amino acids into proteins via in vitro translation with appropriate Aminoacyl-tRNA-synthetases enables construction of proteins with completely new functionalities (for a review, see Quast et al., 2015b).

The advantages of bacterial extracts are fast translation rate and flexibility. For example it is possible to synthesize complex molecules like proteins that contain disulfide-bonds and even membrane proteins with the help of E. coli extract (Billerbeck et al., 2013; Kimura-Soyema et al., 2014; Roos et al., 2014). CFPS with eukaryotic cells is another option. Recently, synthesis of 80 µg/mL eYFP was reported with the use of BY-2 cell lysates (Buntru et al., 2015). Compared to other eukaryotic lysates, Buntru et el. showed that BY-2-lysates are cost effective (3$ for a 15 µl reaction). It is interesting that it is possible to combine extracts from E. coli with eukaryotic extracts, and that this combination can help to produce active, properly folded protein (Zárate et al., 2010).

The level of miniaturization is becoming more and more “minimal”, as CFPS was reported to work in 300 nl-lipid droplets (Taylor and Sarles, 2015). This points out that CFPS-systems are a valuable part for the construction of artificial cells (Lentini et al., 2014). Other exciting new developments, for example DNA hydrogels based on X-DNA (Park et al., 2009; Zheng et al., 2012), reveal that CFPS is going to revolutionize research and applications around the world.

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Zubay, G. (1973): In vitro synthesis of protein in microbial systems. In Annual review of genetics 7, pp. 267–287. DOI: 10.1146/annurev.ge.07.120173.001411

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+   <div class="container">
+      <div class="jumbotron">
+         <h1>CFPS</h1>
+         <p>Cell-Free Protein Synthesis</p>
+      </div>
+    <nav id="pagenav" class="navbar navbar-fixed-bottom"><ul class="nav nav-tabs" role="tablist" style="margin-left: 0px">
+<li><a href="#globally">Globally affecting factors</a></li>
+<li><a href="#transcription">Transcription</a></li>
+<li><a href="#translation">Translation</a></li>
+    <li><a href="#additives">Additives</a></li>
+    <li><a href="#costs">Cost Considerations</a></li>
+    <li><a href="#advantages">Advantages and Possible Uses</a></li>
+</ul>
+</nav>
+<p> "Cell free protein synthesis" (CFPS) is the production of proteins from nucleic acid templates without living cells, but with the use of the transcription and translational "hardware" that originally is built up by cells. This is possible because this "hardware" does not necessarily need a whole cell, but only some well characterized components to function properly. Among these components, the most obvious are RNA-Polymerase and Nucleoside-Triphosphates (NTPs) for transcription, as well as ribosomes and amino acids for the translation. However, the production of proteins  relies on many other molecules and the environment where the production takes place.  In the following we present a short overview on all parts (and aspects) that are necessary and essential for in vitro transcription and translation. When using crude cell extracts of <i>E. coli</i>, most compounds are brought to the reaction via the extract itself.
+</p>
-<div class="container">
-<div class="row">
-<div class="col-md-12">
-<div class="jumbotron" style="background-image: url(URL des Bildes)">
-<div class="jumbotron-text">
-<h1 style="margin-bottom: 0px">Heavy Metals</h1>
-<p>We detect several heavy metals with a single test strip.</p>
-</div>
-</div>
-     <a href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/HeavyMetals#lead"><img src="https://static.igem.org/mediawiki/2015/7/71/Bielefeld-CeBiTec_heavymetals_teststrip_circle.png"></a>
+  <div id="globally">
+<h2> Globally affecting factors </h2>
+<p> <b>strain used</b></p>
+<ul>
+     <li> The early experiments on CFPS relayed on RNase- and protease-deficient strains <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Zubay1973">(Zubay, 1973</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Pratt1984">Pratt, 1984</a>). Since then, many different strains were tested and genetically improved to support the system.   </li>
+<li> Especially, strains that have less RNase- and protease activity, produce rare tRNAs, and carry mutations that counteract the depletion of amino acids during reaction build the best basis for an efficient in vitro protein synthesis. You can find detailed overviews in following publications:
+<ul style="list-style-type:circle">
+    <li> <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#JewettSwartz2004">Jewett and Swartz, 2004</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#CalhounSwartz2006">Calhoun and Swartz, 2006</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#SpirinSwartz2008">Spirin and Swartz, 2008</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Airen2011">Airen, 2011</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Hong2015">Hong et al., 2015 </a></li>
+</ul>
+ </li>
+      </ul>
+    <p><b>pH value</b></p>
+        <ul>
+            <li>For efficient protein synthesis, a pH value above 6.5 is indispensable (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Kim2008">Kim et al., 2008</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#JewettSwartz2004">Jewett and Swartz, 2004</a>). This is the main reason for the use of pH-adjusted washing buffers and phosphate buffer in the media, when <i>E. coli</i> extracts are prepared in-house. The optimal pH  for the reaction itself varies depending on the protein to be produced. To prevent precipitation of the protein, the pH should generally be between 6.5 and 8.5 (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Tokmakov2014">Tokmakov et al., 2014</a>).
+            </li>
+            <li>Different approaches for stabilizing the pH value have been proposed, ranging from the use of buffers  (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Airen2011">Airen, 2011</a>) to the use of engineered enzymes (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Kim2015">Kim et al., 2015</a>). </li>
+        </ul>
-     <img src="https://static.igem.org/mediawiki/2015/0/08/Bielefeld-CeBiTec_heavymetals_teststrip.png" width="600" alt="teststrip"
-usemap="#teststrip">
+     <p><b>Energy</b></p>
+     <ul>
+        <li> Life depends upon ATP as it is the main energy source in cellular organisms. To provide ATP throughout the CFPS-reaction, ATP has to be permanently regenerated. The translation machinery also needs two GTP for every amino acid that is incorporated into the growing peptide chain (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Mavelli2015">Mavelli et al., 2015</a>). Therefore, molecules with high energy content are needed that fuel phosphorylation of ADP (for example via creatin phosphate, abbreviated CP) or that fuel glycolysis (for example phosphoenolpyruvate, abbreviated PEP). In general, the use of the high energetic CP in combination with creatin kinase gives the best results (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Shimizu2010">Shimizu and Ueda, 2010</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Kazuta2014">Kazuta et al., 2014</a>). However, CP and creatin kinase are very expensive compared to other reagents. In recent years many different energy sources have been established which give good results at less costs. You can find detailed information in reviews and particulary in these publications:  </li>
+        <li><a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#KimSwartz2001">Kim and Swartz, 2001</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#JewettSwartz2004">Jewett and Swartz, 2004</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Ma2010">Ma et al., 2010</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#CascheraNoireaux2015a">Caschera and Noireaux, 2015a</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Cai2015">Cai et al., 2015</a></li>
+    </ul>
+<p> <b>Medium</b></p>
+<ul>
+    <li> 2xYTP and 2xYTPG medium are most frequently  employed for production of homemade <i>E. coli</i> cell extract (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#CascheraNoireaux2015a">Caschera and Noireaux, 2015a</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#KwonJewett2015">Kwon and Jewett, 2015</a>). The phosphate buffer helps to maintain a stable pH during cultivation and cell harvest.
+    </li>
+        </ul>
+      </div> <!-- end of tab 1 -->
+   <div id="transcription">
+<h2> Transcription </h2>
+    <p> <b>template</b></p>
+    <ul>
+        <li> The use of DNA as template has some advantages over the use of an mRNA template. For example, one needs only small amounts of DNA compared to mRNA to obtain the same output signal as long as transcription is not limiting (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Rosenblum2014">Rosenblum and Cooperman, 2014</a>). Compared to mRNA, DNA, especially plasmid DNA, is more stable during the reaction (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#SpirinSwartz2008">Spirin and Swartz, 2008</a>). The use of linear DNA as a template can support protein production if the PURE system is used (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Chizzolini2014">Chizzolini et al., 2014</a>). Higher amounts of DNA template result in higher amounts of mRNA produced, but this is only to a certain level advantageous for protein production (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Chizzolini2014">Chizzolini et al., 2014</a>).
+    </li>
+        <li>Apart from the later translated region, the 5'-untranslated region (5'-UTR) is of major importance for an efficient reaction (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#SpirinSwartz2008">Spirin and Swartz, 2008</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Karig2012">Karig et al., 2012</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Lentini2013">Lentini et al., 2013</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Takahashi2013">Takahashi et al., 2013</a>). You can find an overview about that topic <a >here</a></li>
+        <li>Quality and purity of the DNA template is an important aspect. When using plasmid purification kits one has to keep in mind that RNase is part of the resuspension buffer. In the literature, opinions vary regarding which is the best method for purification (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Kigawa2004">Kigawa et al., 2004</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#CalhounSwartz2005">Calhoun and Swartz, 2005</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Sun2013">Sun et al., 2013</a>).  </li>
+    </ul>
-<div class="text-center">
-<img src="https://static.igem.org/mediawiki/2015/0/08/Bielefeld-CeBiTec_heavymetals_teststrip.png" width="600" alt="teststrip"
-usemap="#teststrip">
-<map name="teststrip">
-<area shape="rect" coords="0,0,900,200" href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/HeavyMetals#lead" alt="TL resources">
-</map>
-</div>
-<ul class="nav nav-tabs">
+    <p> <b>RNA-Polymerase</b></p>
-  <li class="active"><a data-toggle="tab" href="#arsenic">Arsenic</a></li>
+        <ul>
-  <li><a data-toggle="tab" href="#chromium">Chromium</a></li>
+            <li> The most widely used RNA-Polymerase for cell free protein production is the T7-Polymerase from bacteriophage T7 which is highly efficient and provides fast transcription (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#ShinNoireaux2010">Shin and Noireaux, 2010</a>). The T7-Polymerase is highly specific to its T7-Promoter, and is commonly used to express proteins <i>in vivo</i> (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Sousa2003">Sousa and Mukherjee, 2003</a>). Simple in-house production of T7-Polymerase has been described in many publications, so even <i>E. coli</i> strains without endogenous T7-Polymerase can be utilized (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Yang2012a">Yang et al., 2012a</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Roos2014">Roos et al., 2014</a>). </li>
-  <li><a data-toggle="tab" href="#lead">Lead</a></li>
+            <li> To overcome the limits of T7-Polymerase, Shin and Noireaux established CFPS based on endogenous polymerases and sigma factors from <i>E. coli</i> (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#ShinNoireaux2010">Shin and Noireaux, 2010</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#ShinNoireaux2012">Shin and Noireaux, 2012</a>). With this work the possibilities of <i>in vitro</i> transcription-translation were  extended; it became possible to characterize other than bacteriophage promotors <i>in vitro</i> (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#ChappellFreemont2013">Chappell and Freemont, 2013</a>). This lead to the analysis of genetic circuits outside of living cells  (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Chappell2013">Chappell et al., 2013</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Sun2013">Sun et al., 2013</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Takahashi2015">Takahashi et al., 2015</a>).</li>
-  <li><a data-toggle="tab" href="#mercury">Mercury</a></li>
+        </ul>
-  <li><a data-toggle="tab" href="#nickel">Nickel</a></li>
-  <li><a data-toggle="tab" href="#copper">Copper</a></li>
-</ul>
-<div class="tab-content">
+    <p> <b>NTPs</b></p>
-  <div id="arsenic" class="tab-pane fade in active">
+    <ul>
-    <h3>Occurrence</h3>
+        <li>The four nucleoside triphosphates (NTPs) ATP, CTP, GTP and UTP are the building blocks of RNA and therefore essential for transcription. They are one of the main cost factors, and it was shown that it is possible to use nucleoside monophosphates (NMPs) instead, although CFPS is less effective in this case (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#CalhounSwartz2005">Calhoun and Swartz, 2005</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Kim2008">Kim et al., 2008</a>).  </li>
-<p>Arsenic is found in nature in both organic and inorganic forms, typically as arsenite (As<sup>III</sup>) or arsenate (As<sup>V</sup>). The average arsenic concentration in sea water is about 1-2 µg/L (<a href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/HeavyMetals#Kaur2015">Kaur et al. 2015</a>). Inorganic arsenic is naturally present at high levels in the groundwater of a number of countries, including Argentina, Bangladesh, China, India, Mexico, and the USA (<a href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/HeavyMetals#WHO2012">World Health Organization 2012</a>).  Arsenic contamination is most dramatic in Bangladesh, where over one million people suffer from arsenic poisoning. Strong local and seasonal fluctuations in arsenic concentrations make it necessary to test each well regularly (<a href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/HeavyMetals#vanderMeer2003">van der Meer 2003</a>).</p>
+    </ul>
-<h3>Health effects</h3>
-<p>Inorganic arsenic compounds are highly toxic. Acute effects of arsenic intake can range from gastrointestinal distress to death. Chronic exposure can result in skin lesions, vascular diseases and cancer. These chronic effects are referred to as arsenicosis, and there is no effective therapy for them. Due to its toxicity and frequency, arsenic ranks first on the Priority List of Hazardous Substances prepared by the US Environmental Protection Agency (EPA) and the Agency for Toxic Substances and Disease Registry (ATSDR). The World Health Organization recommends a limit of 10 µg/L in drinking water, but some countries have adopted a national standard of 50 µg/L (<a href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/HeavyMetals#WHO2012">World Health Organization 2012</a>; <a href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/HeavyMetals#Chen2014">Chen, Rosen 2014</a>).</p>
-<h3>Detection</h3>
-<p>Arsenic can be accurately detected by means of techniques such as atomic absorption spectroscopy (AAS), atomic fluorescence spectrometry or high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS). However, they are expensive and not suitable for field testing (<a href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/HeavyMetals#Chen2014">Chen, Rosen 2014</a>).
+      </div> <!-- end of tab 2 -->
-Chemical test kits are available, which mostly rely on the Gutzeit method. This method is based on the generation of arsine gas from a sample solution. Arsine then reacts with a mercuric bromide impregnated test strip, which results in a color change (<a href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/HeavyMetals#Kearns2010">Kearns 2010</a>). The accuracy and reliability of this method has been called into question (<a href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/HeavyMetals#Rahman2002">Rahman et al. 2002</a>).
-The need for an inexpensive and realible detecion method has led to the development of various arsenic biosensors. Among them are both whole-cell-based and cell-free biosensors. For a recent review, refer to <a href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/HeavyMetals#Kaur2015">Kaur et al. 2015</a>.</p>
+  <div id="translation">
-<h3>Our arsenic biosensor</h3>
+<h2> Translation </h2>
-<p>We choose to work with the chromosomal arsenic operon of <i>E. coli</i>, which was used by the team from Edinburgh in 2006. This operon encodes an efflux pump which confers resistance against arsenic. The expression is controlled by the repressor ArsR, which negatively autoregulates its own expression. As<sup>III</sup> can bind to three cysteine residues in ArsR. The resulting conformational change deactivates the repressor (<a href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/HeavyMetals#Chen2014">Chen, Rosen 2014</a>).</p>
+    <p> <b>Amino acids</b></p>
+    <ul>
+        <li> Nearly all proteins are built up by L-amino acids. Neglecting selenomethionine and pyrrolysine, there are 20 proteinogenic amino acids that all have to be brought to the cell free reaction. This is a difficult task for the experimenter, because these molecules have different solubilities and pKa-values. Recently, an easy procedure for the production of an amino acid mastermix has been presented (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#CascheraNoireaux2015b">Caschera and Noireaux, 2015b</a>).</li>
+        <li> Some amino acids – especially cysteine, tryptophan, arginine and serine – are depleted much faster in the cell free reaction than others, for example due to enzymatic reactions (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#CalhounSwartz2006">Calhoun and Swartz, 2006</a>). This is one of the biggest problems that occur during batch CFPS and leads to fast stop of protein production, unless appropriate knockout strains are used or amino acid concentrations are increased (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#JewettSwartz2004">Jewett and Swartz, 2004</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#CalhounSwartz2006">Calhoun and Swartz, 2006</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Pedersen2011">Pedersen et al., 2011</a>).</li>
+    </ul>
-<h3>References</h3>
-<div class="references">
-<p id="Chen2014">Chen, Jian; Rosen, Barry P. (2014): Biosensors for inorganic and organic arsenicals. In Biosensors 4 (4), pp. 494–512. DOI: 10.3390/bios4040494.</p>
-<p id="Kaur2015">Kaur, Hardeep; Kumar, Rabindra; Babu, J. Nagendra; Mittal, Sunil (2015): Advances in arsenic biosensor development--a comprehensive review. In Biosensors & bioelectronics 63, pp. 533–545. DOI: 10.1016/j.bios.2014.08.003.</p>
-<p id="Kearns2010">Kearns, James Kalman (2010): Field Portable Methods for the Determination of Arsenic in Environmental Samples. Dissertation.</p>
-<p id="Rahman2002">Rahman, Mohammad Mahmudur; Mukherjee, Debapriyo; Sengupta, Mrinal Kumar; Chowdhury, Uttam Kumar; Lodh, Dilip; Chanda, Chitta Ranjan et al. (2002): Effectiveness and Reliability of Arsenic Field Testing Kits: Are the Million Dollar Screening Projects Effective or Not? In Environ. Sci. Technol. 36 (24), pp. 5385–5394. DOI: 10.1021/es020591o.</p>
-<p id="vanderMeer2003">van der Meer, Jan Roelof (2003): EAWAG news 56e: Bacterial Biosensors to Measure Arsenic in Potable Water.</p>
-<p id="WHO2012">World Health Organization (2012): Arsenic fact sheet. Available online at http://www.who.int/mediacentre/factsheets/fs372/en/, checked on 8/12/2015.</p>
-</div>
-  </div>
-  <div id="chromium" class="tab-pane fade">
+    <p> <b>Other components involved in translation</b></p>
-    <h3>Occurrence</h3>
+        <ul>
-    <p>Chromium is an essential part of the earth´s crust.It is the sixth most abundant one and used inmetallurgical, chemical and refractory form. The three most important oxidative forms of chromium are the elemental metal (Cr), the trivalent (Cr<sup>III</sup>) and the hexavalent(Cr<sup>VI</sup>) (Mitchell D. Cohen et al.).</p>
+            <li> Amino acids are fused to corresponding tRNAs and thus activated for translation through the action of aminoacyl-tRNA-synthetases, a process that is called aminoacylation. Additional <i>E. coli</i> tRNA supplied to the CFPS-reaction is advantageous (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#CalhounSwartz2005">Calhoun and Swartz, 2005</a>), though it was observed that under certain conditions an addition has no effect (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#ShinNoireaux2010">Shin and Noireaux, 2010</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Cai2015">Cai et al., 2015</a>)  </li>
-  </br>
+            <li> Elongation and releasing factors coordinate translation. For example, the GTP-binding protein EF-Tu is the major elongation factor in <i>E. coli</i> (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Airen2011">Airen, 2011</a>). It has been shown that the supplementation of EF-Tu improves translation efficiency (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#UNderwood2005">Underwood et al., 2005</a>), and that EF-Tu could mediate the incorpotation of non-natural aminoacids into proteins, for example selenocysteine (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#MIller2015">Miller et al., 2015</a>)</li>
+        </ul>
- <h3>Health effects</h3>
- <p>While the trivalent form is an essential dietary mineral and themost common natural form, we are interested in the hexavalent form because of its potential toxicity and carcinogenetic effects. Most of it is produced trough industrial uses(Paustenbach et al. 2003).
+    <p> <b>The protein of interest</b></p>
-Chromium intoxication can result in damage to the nervous system, fatigue and mental instability (Singh et al. 2011). It´s potential cancerogenity results out of chromium VI being able to enter the cells while it is not possible for chromium III compounds. Chromium VI in the cells is reduced to chromium III and can´t leave the cells anymore (Mitchell D. Cohen et al.). Because of its toxicity the World Health Organization (WHO) recommends a limit of 50 µg/l chromium in drinking water. In contrast to this guideline concentrations as high as 120µg/l chromium were detected in drinking water (Guidelines for drinking-water quality 2011)
+    <ul>
-.</p></br>
+        <li> Proteins are a very wide group of molecules, different in amino acid composition, 3D-structure and physicochemical properties. Because of the individuality of proteins, it is necessary to optimize the cell free synthesis reaction in every case (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#SpirinSwartz2008">Spirin and Swartz, 2008</a>) A recent approach based on bioinformatics was made to better estimate how one can improve CFPS for a certain protein (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Tokmakov">Tokmakov et al., 2014</a>). Generally, the production is more successful when the transcript is short and the protein folds fast and is soluble (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Lentini2013">Lentini et al., 2013</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Chizzolini2014">Chizzolini et al., 2014</a>). Aspects like the formation of disulfide bonds or a chromophore have also to be considered. </li>
-<h3>Detection</h3>
+        <li> CFPS can be used to produce proteins whose production is limited <i>in vivo</i> due to toxicity or membrane association, and also to incorporate unnatural or labeled amino acids (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Kigawa2004">Kigawa et al., 2004</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Xu2005">Xu et al., 2005</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#BundySwartz2010">Bundy and Swartz, 2010</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Quast2015b">Quast et al., 2015b</a>). It has to be considered that production of complex proteins that are further processed after translation, for example membrane-bound proteins, is in general more feasible in eukaryotic cell extracts (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Quast2015a">Quast et al., 2015a</a>). </li>
-<p>Chromium in drinking water is detected trough atomic absorption spectroscopy (AAS) or Ion chromatography with post column derivatization and UV visible spectroscopic detection (U.S. EPA, OW, OGWDW, SRMD, Technical Support Center). Moreover chromium detection at home can be detected by a basic titrimetric method using a iodide reaction for measurement (GIORGIA).</p>
+    </ul>
-</br>
-<h3>Our chromium biosensor</h3>
-<p>We work with the chromate inducible operon of Ochrobactrumtritici5bvl1which enables a resistance for chromium VI and superoxide which was introduced to iGEM by BIT 2013 team. The parts which are of interest to us are the Chromium dependent Repressor ChrB and its associated Promoter (Branco et al. 2008) The output of our sensor system works through fluorescence.</p>
+      </div> <!-- this closes tab 3 -->
-</br>
-<h3>References</h3>
+<div id="additives">
-<p>Blaha, Didier; Arous, Safia; Blériot, Camille; Dorel, Corinne; Mandrand-Berthelot, Marie-Andrée; Rodrigue, Agnès (2011): The Escherichia coli metallo-regulator RcnR represses rcnA and rcnR transcription through binding on a shared operator site: Insights into regulatory specificity towards nickel and cobalt. In Biochimie 93 (3), pp. 434–439. DOI: 10.1016/j.biochi.2010.10.016.
-cavillona (2005): Nickel in Drinking-water, checked on 9/9/2015.
+<h2> Additives</h2>
-EPA, U. S.; OAR; Office of Air Quality Planning and Standards (2013): Nickle Compounds | Technology Transfer Network Air Toxics Web site | US EPA. Available online at http://www.epa.gov/airtoxics/hlthef/nickel.html, updated on 10/18/2013, checked on 9/10/2015.
+    <p> There are lots of possible additives that are known to contribute to a more efficient <i>in vitro</i> protein production, as well small substances like DTT as enzymes (a good overview in <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#SpirinSwartz2008">Spirin and Swartz, 2008</a> and <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Airen2011">Airen, 2011</a>). Although some cofactors, for example coenzyme A, NAD and folinic acid, seem more important than others, the effects often differ depending on conditions like cell extract, scale, protein of interest and so on.</p>
-Guidelines for Drinking-water Quality, Fourth Edition, checked on 9/9/2015.
+    <p> One advantage of the clearly defined environment the cell free reaction takes place in, is that "[...] it should be possible to build a complete mathematical model describing the cellular mimic that could aid in designing new features."(<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Lentini2013">Lentini et al., 2013</a>). Recent approaches have been made to mathematically model and characterize the PURE system (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Karzbrun2011">Karzbrun et al., 2011</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Stögbauer2012">Stögbauer et al., 2012</a>;<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Mavelli2015"> Mavelli et al., 2015</a>). </p>
-Iwig, Jeffrey S.; Rowe, Jessica L.; Chivers, Peter T. (2006): Nickel homeostasis in Escherichia coli - the rcnR-rcnA efflux pathway and its linkage to NikR function. In Molecular microbiology 62 (1), pp. 252–262. DOI: 10.1111/j.1365-2958.2006.05369.x.
-kreusche: Trinkwasser-Installation 27.6.07 Endfassung.qxd, checked on 9/10/2015.
+      </div>
-US: Technical Factsheet on: Nickel, checked on 9/9/2015.
+<div id="costs">
+<h2> Cost considerations</h2>
+    <p> <i>in vitro</i> transcription and translation has been an expensive issue. Commercial systems composed of purified single components, like the PURE system (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Shimizu2010">Shimizu and Ueda, 2010</a>) or <i>E. coli</i> (T7) S30 Extract, work efficiently, but are often more than ten times more expensive than in-house generated reactions. A milestone for lowering the costs of selfmade extracts was the finding of Kim and coworkers that after lysis, highly active cell extracts can be produced with a single centrifugation step and a subsequent run-off reaction at 37 °C (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Kim2006">Kim et al., 2006</a>). The run-off reaction facilitates degeneration of endogenous DNA, whereby the background expression is reduced (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Pratt1984">Pratt, 1984</a>). A final centrifugation step after the run-off reaction can be helpful (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#KwonJewett2015">Kwon and Jewett, 2015</a>).  </p>
+    <p>Indeed, many researchers have proven that it is possible to further minimize the costs per reaction down to a fraction of cents (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#CalhounSwartz2005">Calhoun and Swartz, 2005</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Kim2011">Kim et al., 2011</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Sun2013">Sun et al., 2013</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#CascheraNoireaux2015">Caschera and Noireaux, 2015</a>). Protocols for preparation of cell extracts, especially for efficient lysis of harvested cells, have been described in recent years (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Shrestha2012">Shrestha et al., 2012</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#KwonJewett2015">Kwon and Jewett, 2015</a>). With the Multiplex Automated Genome Engineering (MAGE) method, it became possible to  tag and purify all necessary proteins for translation (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Wang2012">Wang et al., 2012</a>). This approach has the potential to simplify and reduce the costs for cell free <i>in vitro</i> reactions based on purified components (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Kelwick2014">Kelwick et al., 2014</a>) .</p>
+<p>These papers inspired us to experiment with selfmade <i>E. coli</i> cell extracts, aiming to develop protocols for the production of cheap cell extracts and to open cell free protein synthesis to the iGEM community. </p>
+      </div>
+       <div id="advantages">
+<h2> Advantages and possible uses </h2>
+    <p>In general, the developments in CFPS proceed into two major directions: These are on the one hand the production of large quantities of proteins (Scale-up direction) without needing recombinant hosts. On the other hand the aim is to investigate distinct proteins or protein synthesis procedures at a small scale for a minimum of costs, and to enrich a protein of interest in a minimal volume (miniaturization direction) (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Alexandrov">Alexandrov and Johnston</a>). </p>
+    <p>With <i>in vitro</i> Transcription-Translation, it long was difficult to produce identical or higher amounts of active proteins when compared to <i>in vivo</i>. Recent publications proof that this is now possible and that the scale-up of CFPS becomes more and more efficient (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#CascheraNoireaux2014">Caschera and Noireaux, 2014</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Kazuta2014">Kazuta et al., 2014</a>). </p>
+    <p>CFPS offers great advantages over classical overexpression in living cells: The reaction is not restricted to the experimenter by a cell wall, so one can add exactly the components to the reaction that are needed, for example additional co-factors, inducers or catalysts. One recent example is the use of an engineered enzyme to maintain a stable pH without the need of a chemical buffer (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Kim2015">Kim et al., 2015</a>). Depending on the system, it is possible to use linear DNA or RNA as a template, skipping ligation or reverse transcription (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Rosenblum2014">Rosenblum and Cooperman, 2014</a>)</p>
+    <p>Regarding Synthetic biology, CFPS emerged as a valuable tool, with the long-term potential to not only be integrated into, but create whole new fields of research. With CFPS, the synthesis of proteins that would kill the cells used for production became possible, just to mention for example antibiotic-like proteins (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Xu2005">Xu et al., 2005</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Yang2012b">Yang et al., 2012b</a>). The incorporation of non-natural amino acids into proteins via in vitro translation with appropriate Aminoacyl-tRNA-synthetases enables construction of proteins with completely new functionalities (for a review, see <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Quast2015b">Quast et al., 2015b</a>). </p>
+    <p>The advantages of bacterial extracts are fast translation rate and flexibility. For example it is possible to synthesize complex molecules like proteins that contain disulfide-bonds and even membrane proteins with the help of <i>E. coli</i> extract (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Billerbeck2013">Billerbeck et al., 2013</a>;<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Kimura-Soyema2014"> Kimura-Soyema et al., 2014</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Roos2014">Roos et al., 2014</a>). CFPS with eukaryotic cells is another option. Recently, synthesis of 80 µg/mL eYFP was reported with the use of BY-2 cell lysates  (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Buntru">Buntru et al., 2015</a>). Compared to other eukaryotic lysates, Buntru et el. showed that BY-2-lysates are cost effective (3$ for a 15 µl reaction). It is interesting that it is possible to combine extracts from E. coli with eukaryotic extracts, and that this combination can help to produce active, properly folded protein (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Zarate2010">Zárate et al., 2010</a>). </p>
+    <p></p>
+    <p>The level of miniaturization is becoming more and more “minimal”, as CFPS was reported to work in 300 nl-lipid droplets (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Taylor2015">Taylor and Sarles, 2015</a>). This points out that CFPS-systems are a valuable part  for the construction of artificial cells (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Lentini2014">Lentini et al., 2014</a>). Other exciting new developments, for example DNA hydrogels based on X-DNA (<a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Park2009">Park et al., 2009</a>; <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Zheng2012">Zheng et al., 2012</a>), reveal that CFPS is going to revolutionize research and applications around the world.</p>
+      </div>
+<p></p>
+<h2 id="CFPSreferences">References</h2>
+    <div class="references">
+<p id="Airen2011">
+    Airen, I. O. (2011): Genome-wide Functional Genomic Analysis for Physiological Investigation and Improvement of Cell-Free Protein Synthesis. PhD thesis.
 </p>
-</div>
+<p id="Alexandrov">
+    Alexandrov, Kirill; Johnston, Wayne A.: Cell-free protein synthesis. Methods and protocols (Methods in Molecular Biology, Methods and Protocols, 1118).
+</p>
+<p id="Billerbeck2013">
+    Billerbeck, Sonja; Härle, Johannes; Panke, Sven (2013): The good of two worlds: increasing complexity in cell-free systems. In Current opinion in
+    biotechnology 24 (6), pp. 1037–1043. DOI: 10.1016/j.copbio.2013.03.007
+</p>
+<p id="BundySwartz2010">
+    Bundy, Bradley C.; Swartz, James R. (2010): Site-Specific Incorporation of p -Propargyloxyphenylalanine in a Cell-Free Environment for Direct
+    Protein−Protein Click Conjugation. In Bioconjugate Chem. 21 (2), pp. 255–263. DOI: 10.1021/bc9002844
+</p>
-<div id="lead" class="tab-pane fade">
+<p id="Buntru 2015">
-     <h3>Occurrence</h3>
+     Buntru, Matthias; Vogel, Simon; Stoff, Katrin; Spiegel, Holger; Schillberg, Stefan (2015): A versatile coupled cell-free transcription-translation system
-     <p>Lead is a heavy metal. Its widespread occurrence, relatively simple extraction and combination of desirable properties have made it useful to humans.
+     based on tobacco BY-2 cell lysates. In Biotechnology and bioengineering 112 (5), pp. 867–878. DOI: 10.1002/bit.25502
-But the frequency of occurrence is not as high as that of other metals. Nonetheless lead plays a major role in the industryand is one of the most used metals. For example, lead is important for the manufacture of batteries, which are used in vehicles.
-Lead is found in different parts of the environment, so it is in the air, the soil and in the water. Lead and lead compounds are used in a high variety of products, which are found in pipes and plumbing materials, solders, gasoline, batteries, ammunition and cosmetics, just as product that is found in and around our homes.
+</p>
-Due to the fact that lead is also occurring in water, if there are obstruct pipes that consist of lead or that has a part of lead, respectively.When lead is used in household plumbing, this allows water to be easily contaminated with the metal.Long time absorption could results in adverse health effects.
+<p id="Cai2015">
-</p></br>
+     Cai, Qi; Hanson, Jeffrey A.; Steiner, Alexander R.; Tran, Cuong; Masikat, Mary Rose; Chen, Rishard et al. (2015): A simplified and robust protocol for
-<h3>Health Effects</h3>
+     immunoglobulin expression in Escherichia coli cell-free protein synthesis systems. In Biotechnology progress. DOI: 10.1002/btpr.2082
-     <p>Lead has no biological role in the body, but it is a highly poisonous metal. The ingestion of lead could affect almost every organ and system in the body (Heavy Metals Testing ByUsp. Caspharma.com.). The main target for lead toxicity is the nervous system.It could have acute or chronic health effects. The acute health effects are occurring immediately after contact with lead. So it can irritate the eyes or can lead to headache, irritability, disturbed sleep, and mood and personality changes. Exposure to higher lead content over a long-term could cause serious damage to the brain and to the kidneys. The damage can finally cause death (Golub, M. S., 2005).
-The poisoning is mostly resulting of ingestion of water or food, which is contaminated with lead or lead compounds (Ferner D. J., 2001).It is taken up fast in the bloodstream and spread in the body(Bergeson, 2008).
-The World Health Organization recommends a limit of 10 µg/L in drinking water, concentrations in drinking water are generally below 5 μg/l. But there are much higher concentrations that have been measured if lead fittings are existing (WHO: Guidelines for Drinking-water Quality,fourth edition).
-</p></br>
-<h3>Detection</h3>
-    <p>Due to the effects on health the detection of lead in drinking water is of importance in all parts of the world. So, a simple system for fast review of the water quality is a worthwhile aim.
-The method for detection is currently on the principle of flame atomic absorption spectrometry (FAAS) (Abdallah, A. T. and Moustafa, M. A.,2002).
-This detection method is impossible in developing countries, for example, thus there are hardly quality standards.
-</p></br>
-<h3>Our lead biosensor</h3>
-     <p>For our biosensor, we use the chromosomal lead operon of Cupriavidusmetallidurans(Ralstoniametallidurans). The promoter that we use is PbrA. This part of the operon is regulated by the repressor pbrR. The PbrR protein is mediated Pb2+-inducible transcription. PbrR belongs to the MerR family, which are metal-sensing regulatoryproteins (Borremanset al., 2001).
-Our sensor system comprised PbrR, which is under the control of aconstitutive Promoter and PbrA and a 5’ untranslated region, which controls the transcription of a sfGFP. The sfGFP protein is the measuring output signal.
-</p></br>
-<h3>References</h3>
-     <p>Some content in menu 2.</p></br>
-  </div>
+</p>
+<p id="CalhounSwartz2005">
+    Calhoun, Kara A.; Swartz, James R. (2005): An economical method for cell-free protein synthesis using glucose and nucleoside monophosphates. In
+    Biotechnology progress 21 (4), pp. 1146–1153. DOI: 10.1021/bp050052y
-  <div id="mercury" class="tab-pane fade">
+</p>
-     <h3>Menu 1</h3>
+<p id="CalhounSwartz2006">
-     <p>Some content in menu 1.</p>
+     Calhoun, Kara A.; Swartz, James R. (2006): Total amino acid stabilization during cell-free protein synthesis reactions. In Journal of biotechnology 123
-  </div>
+     (2), pp. 193–203. DOI: 10.1016/j.jbiotec.2005.11.011
-  <div id="nickel" class="tab-pane fade">
-    <h3>Occurrence</h3>
-    <p>The amount of natural occurring nickel is quite low even if it is anelement of the earth’s crust. Therefore small amounts of it are found in food water soil and air.
-Nickel-concentration in drinking water is normally less than 0.02 mg/l, although troughreleases from taps and fittings the nickel may contribute to concentrations up to 1 mg/l. There may be higher concentrations in drinking-water in special cases of release from natural or industrial nickel deposits in theground and therefore a higher guideline value of 0.07 mg/l (70 μg/l)(Guidelines for Drinking-water Quality, Fourth Edition)
-</p></br>
- <h3>Health effects</h3>
-    <p>Evan if nickel is essential for mammals and part of human nutrition it may cause dermatitis as well as itching of fingers, hands and forearms in some people who had long term skin contact. The main source of nickel exposure is food or water but most people have contact to nickel trough everyday products as jewelry or stainless steel dishware or trough smoking tobacco(US; EPA et al. 2013).In Germany most drinking water pollutions by nickel happen in the last meters of the plumbing system. Wrong tapware is the main source of nickel contamination in drinking water (kreusche)</p>
-  </br>
- <h3>Detection</h3>
-    <p>The two most commonly used analytical methods for nickel in water are atomicabsorption spectrometry and inductively coupled plasma atomic emissionspectrometry. Inductively coupled plasma atomic emissionspectroscopy is used for the determination of nickel etection limit ofabout 10 μg/litre (ISO, 1996). Flame atomic absorption spectrometry is suitable in the range of 0.5–100 μg/litre (ISO, 1986). A limit of detection of 0.1 μg/ can be achieved using inductively coupled plasma mass spectrometry. Alternatively, electrothermal atomic absorption spectrometry can beused. (cavillona 2005)</p>
-  </br>
- <h3>Our nickel biosensor</h3>
-    <p>For our nickel sensor system we used the rcn-operon from E. coli which codes for a nickel- and cobalt-efflux system, which is highly sensitive to nickel . If Ni(II)-ions bind to the repressor RcnR, it cannot attach to DNA and RcnA the nickel responsive promoter is activated. In the absence of nickel or cobalt, RcnR is bound to RcnR operator and blocks RcnA transcription. (EPA et al. 2013; Blaha et al. 2011; Iwig et al. 2006) Our output signal works trough fluorescence.</p>
-  </br>
- <h3>References</h3>
-    <p>
-Blaha, Didier; Arous, Safia; Blériot, Camille; Dorel, Corinne; Mandrand-Berthelot, Marie-Andrée; Rodrigue, Agnès (2011): The Escherichia coli metallo-regulator RcnR represses rcnA and rcnR transcription through binding on a shared operator site: Insights into regulatory specificity towards nickel and cobalt. In Biochimie 93 (3), pp. 434–439. DOI: 10.1016/j.biochi.2010.10.016.
-cavillona (2005): Nickel in Drinking-water, checked on 9/9/2015.
-EPA, U. S.; OAR; Office of Air Quality Planning and Standards (2013): Nickle Compounds | Technology Transfer Network Air Toxics Web site | US EPA. Available online at http://www.epa.gov/airtoxics/hlthef/nickel.html, updated on 10/18/2013, checked on 9/10/2015.
-Guidelines for Drinking-water Quality, Fourth Edition, checked on 9/9/2015.
-Iwig, Jeffrey S.; Rowe, Jessica L.; Chivers, Peter T. (2006): Nickel homeostasis in Escherichia coli - the rcnR-rcnA efflux pathway and its linkage to NikR function. In Molecular microbiology 62 (1), pp. 252–262. DOI: 10.1111/j.1365-2958.2006.05369.x.
-kreusche: Trinkwasser-Installation 27.6.07 Endfassung.qxd, checked on 9/10/2015.
-US: Technical Factsheet on: Nickel, checked on 9/9/2015.
 </p>
-  </div>
+<p id="CascheraNoireaux2014">
+    Caschera, Filippo; Noireaux, Vincent (2014): Synthesis of 2.3 mg/ml of protein with an all Escherichia coli cell-free transcription-translation system. In
+    Biochimie 99, pp. 162–168. DOI: 10.1016/j.biochi.2013.11.025
- <div id="copper" class="tab-pane fade">
-    <h3>Occurrence</h3>
-    <p>Copper is an essential trace element for humans’ animals and plants. In the human body you have concentrations of 1.4 to 2.1 mg/kg body mass. Copper is ingested through the gut and transferred to most tissues trough the liver. It is used present in coatings and alloys and used to make pipes, valves and fittings. Moreover coppersulfate pentahydrate is used in algae control by adding it to surface water. Therefore copper concentrations in drinking water vary widely (Guidelines for Drinking-water Quality, Fourth Edition)</p>
-  </br>
-<h3>Health effects</h3>
-    <p>Copper is an essential for human health, but in to high doses it can cause anemia, kidney and liver damage as well as stomach and intestinal irritation and immunotoxicity(ATSDR). Some people are at greater risks off negative health effects caused by copper overexposure. A with copper associated disease is Wilsons disease which results in a miss function, so that copper can´t be excreted by the liver into bile.If it´s not treated it can result in brain and liver damage.(US EPA ORD NCEA Integrated Risk Information System (IRIS) 2014)Some studies associate high copper levels with aging diseases as Atherosclerosis andAlzheimer’s Disease(Copper excess, zinc deficiency, and cognition loss in Alzheimer's disease - Brewer - 2012 - BioFactors - Wiley Online Library)</p>
-  </br>
-<h3>Detection</h3>
-    <p>The most important analytical methods for the detection of copper in water areinductively coupled plasma mass spectrometry (ICP-MS) which has the lowest detection limit (0.02 μg/litre) and Atomicabsorption spectrometry (AAS) with flame detection, which has the highest (20 μg/litre) as well as graphite furnace atomicabsorption spectroscopy, inductively coupled plasma atomic emission spectroscopy,and stabilized temperatureplatform graphite furnace atomic absorption(cavillona 2004).</p>
-  </br>
-<h3>Our copper Biosensor</h3>
-    <p>The operon we used for our biosensor is native to E.coli K12. The parts we used are CopA promoter (CopAP) and it´s regulator CueR. CueR is a MerR like regulator, which stimulates the transcription of CopA, a P-ype ATPase pump(Outten et al. 2000). CopA is the central component in obtaining copper homeostasis, it exports free copper from cytoplasm to periplasm. This is possible trough Copper induced activation of the operons transcription via CueR.CueR-Cu+ is the DNA-binding transcriptional dual regulator which activates transcription(Yamamoto, Ishihama 2005) To sum it up CueR regulon plays an important role in aerobic copper tolerance in E.coli(Grass, Rensing 2001).We combined CueR under the control of a constitutive promoter with CopAP a 5´ untranslated region for higher translation levels and sfGFP for measuring output signals</p>
-  </br>
-<h3>References</h3>
-    <p>
-ATSDR: TOXICOLOGICAL PROFILE FOR COPPER, checked on 8/27/2015.
-cavillona (2004): Copper in Drinking-water. Background document for development of WHO Guidelines for Drinking-water Quality, checked on 9/9/2015.
-Copper excess, zinc deficiency, and cognition loss in Alzheimer's disease - Brewer - 2012 - BioFactors - Wiley Online Library. Available online at http://onlinelibrary.wiley.com/doi/10.1002/biof.1005/abstract, checked on 8/28/2015.
-Grass, Gregor; Rensing, Christopher (2001): Genes Involved in Copper Homeostasis in Escherichia coli, checked on 8/26/2015.
-Guidelines for Drinking-water Quality, Fourth Edition, checked on 9/9/2015.
-Outten, F. W.; Outten, C. E.; Hale, J.; O'Halloran, T. V. (2000): Transcriptional activation of an Escherichia coli copper efflux regulon by the chromosomal MerR homologue, cueR. In The Journal of biological chemistry 275 (40), pp. 31024–31029. DOI: 10.1074/jbc.M006508200.
-US EPA ORD NCEA Integrated Risk Information System (IRIS) (2014): Copper (CASRN 7440-50-8) | IRIS | US EPA. Available online at http://www.epa.gov/iris/subst/0368.htm, updated on 10/31/2014, checked on 9/2/2015.
-Yamamoto, Kaneyoshi; Ishihama, Akira (2005): Transcriptional response of Escherichia coli to external copper. In Molecular microbiology 56 (1), pp. 215–227. DOI: 10.1111/j.1365-2958.2005.04532.x.
 </p>
-  </div>
+<p id="CascheraNoireaux2015a">
-</div>
+    Caschera, Filippo; Noireaux, Vincent (2015a): A cost-effective polyphosphate-based metabolism fuels an all E. coli cell-free expression system. In
+    Metabolic engineering 27, pp. 29–37. DOI: 10.1016/j.ymben.2014.10.007
+</p>
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