Team:Heidelberg/Project/Delftibactin

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                        <h2>Highlights</h2>
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                      <b> <h2>Highlights</h2>
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<li> Optimization of the Gibson assembly method for the creation of large plasmids (> 30 kbp) with high GC content.
<li> Optimization of the Gibson assembly method for the creation of large plasmids (> 30 kbp) with high GC content.
<li> Amplification and cloning of all components required for recombinant delftibactin production.  
<li> Amplification and cloning of all components required for recombinant delftibactin production.  
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<li> Transfer of the entire pathway from <i>D. acidovorans</i> for the synthesis of delftibactin to <i>E. coli</i>.
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<li> Transfer of the entire pathway from <i>Delftia acidovorans</i> for the synthesis of delftibactin to <i>Escherichia coli</i>.
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                         <h2>Abstract</h2>
                         <h2>Abstract</h2>
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                            Efficient recycling of gold from electronic waste using recombinant delftibactin<br><br>
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Undoubtedly, <b>gold</b> is one of the most precious materials on earth. Besides its common use in art and jewelry, gold is also an essential component of our modern computers and cell-phones. Due to the fast turn-over of today’s high-tech equipment, millions of tons of <b>electronic waste</b> accumulate each year containing tons of this valuable metal. The main approach nowadays to recycle gold from electronic waste is by electrolysis. Unfortunately, this is a highly inefficient and expensive procedure, preventing most of the gold from being recovered.<br/><br/>
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Undoubtedly, gold is one of the most precious materials on earth. Besides its common use in art and jewelry, gold is also an essential component of our modern computers and cell-phones. Due to the fast turn-over of today’s high-tech equipment, millions of tons of electronic waste accumulate each year containing tons of this valuable metal. The main approach nowadays to recycle gold from electronic waste is by electrolysis. Unfortunately, this is a highly inefficient and expensive procedure, preventing most of the gold from being recovered.<br><br>
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In this subproject we want to demonstrate that the gold-precipitating natural secondary metabolite <b>delftibactin</b>, a non-ribosomal peptide produced by the bacterium <i>Delftia acidovorans</i>, can be used for the <b>efficient recovery of gold</b> from electronic waste.<br/><br/>
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Earlier this year, a publication in Nature Chemical Biology reported the existence of a non-ribosomal peptide – delftibactin - which has the astonishing property to specifically precipitate elemental gold from gold-ion containing solutions. Naturally, delftibactin is produced by <i>Delftia acidovorans</i>, an extremophile bacterium, which secretes delftibactin to complexate and dispose of toxic gold ions present in its environment. Although the exact delftibactin production pathway is not known, bioinformatic predictions claim a non-ribosomal peptide synthesis pathway encoded on a large, 59 kb gene cluster (the del-cluster) to be responsible for delftibactin production. <br><br>
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Moreover we want to show that very large constructs such as the genes needed for the production of delftibactin which are encoded on a 59 kbp long gene cluster  can be succesfully inserted into <i>Escherichia coli</i>. Furthermore the aim is to <b>recombinantly express</b> the responsible <b>NRPSs</b> with the promising perspective that delftibactin could readily be produced and used as an efficient way of gold recycling from electronic waste.<br/><br/>
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In this subproject we want to demonstrate that the natural secondary metabolite delftibactin can be efficiently produced in <i>E. coli</i> and used for the recycling of gold from electronic waste. To this end, we developed a cloning strategy based on an optimized Gibson Assembly protocol, enabling the cloning of large, GC-rich genomic regions onto regular low-copy plasmids. We thereby engineered three different plasmids (about 70 kb in total size) enabling the expression of the predicted del-cluster from regular <i>E. coli</i> promoters along with the methylmalonyl-CoA pathway providing the basic delftibactin building blocks and a NRPS activating PPTase, Sfp from Bacillus subtilis. <br><br>
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We managed to introduce and express the genes needed for delftibactin production. Furthermore the <b>recycling of gold from electronic waste</b> with delftibactin was <b>successful</b>. Consequently the industrial usage of recombinantly expressed delftibactin as an efficient method to recover gold becomes conceivable. <br/><br/>
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We want to show that these large constructs can be potentially inserted and expressed by <i>E. coli</i> with the promising perspective that delftibactin could readily be used as an efficient way of gold recycling from electronic waste.
 
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                     <h2 id="introduction">Introduction</h2>
                     <h2 id="introduction">Introduction</h2>
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<p>The quest for a magical substance to generate gold from inferior metals stirred the imagination of generations. However, this substance, the Philosopher’s Stone, stands for more than just the desire to produce gold. In the old days, the fabled Philosopher’s Stone also represented wisdom, rejuvenation and health. Nowadays, gold is still of great importance for us as it is needed for most of our electronic devices.</p>
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<p>The quest for a magical substance to generate gold from inferior metals stirred the imagination of generations. However, this substance, the Philosopher’s Stone, stands for more than just the desire to produce gold. There was a time when the fabled Philosopher’s Stone also represented wisdom, rejuvenation and health. Nowadays, gold is still of great importance for us as it is needed for most of our electronic devices.</p>
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<p>In 2007, more than two tons of gold, worth $ 92 million, were discarded hidden in electronic waste in Germany. Most of the precious element ends up on waste disposal sites as only a minor fraction of 28 % of the gold is recycled also due to the small amounts per device. Since our planet’s gold supplies are limited, the metal is more and more depleted and the value of gold continously reaches all-time highs. In order to satisfy our society’s need for gold, we have to develop heavy mining techniques involving strong acids, causing devastating impact on human and environment <span class="citation">[1]</span> <span class="citation">[2]</span> <span class="citation">[3]</span>.</p>
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<p>Besides economical usage of the resource gold, one way to reduce global demands for gold is elevation of gold recovery <span class="citation">[4]</span>. Intriguingly, nature itself offers a structure that has been reported to efficiently extract pure gold from solutions containing gold ions. This fascinating molecule is called Delftibactin and is in fact a small peptide secreted by a metal-tolerant bacterium called <em>Delftia acidovorans</em>.</p>
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<p>This extremophile has the incredible ability to withstand toxic amounts of gold ions in contaminated soil. If one could culture these bacteria and produce Delftibactin in large scales, could one potentially recover gold from electronic waste in a cost- and energy-efficient way? But what is the special feature of Delfibactin to precipitate gold that efficiently?</p>  
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<br/>
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<p>In 2007, more than two tons of gold, worth $92&nbsp;million, were discarded hidden in electronic waste in Germany [1]. Most of the precious element ends up on waste disposal sites as only a minor fraction of 10-15% [2] of the gold is recycled also due to the small amounts per device. Since our planet’s gold supplies are limited, the metal is more and more depleted and the value of gold continuously reaches all-time highs. In order to satisfy our society’s need for gold, we have to develop heavy mining techniques involving strong acids, causing devastating impact on humans and environment [3] [4] [5].</p>
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<center>
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<a class="fancybox fancyGraphical" href="https://static.igem.org/mediawiki/2013/4/47/Heidelberg_goldrecycling.png" title="Industrial application of delftibactin for gold recovery from electronic waste.">
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    <img style="width:60%; margin-bottom:10px; padding:1%;border-style:solid;border-width:1px;border-radius: 5px;" src="https://static.igem.org/mediawiki/2013/4/47/Heidelberg_goldrecycling.png" ></img>
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<p>Besides economical usage of the resource <i>gold</i>, one way to reduce global demands for gold is elevation of gold recovery [6]. Intriguingly, nature itself offers a structure that has been reported to efficiently extract pure gold from solutions containing gold ions. This fascinating molecule is called delftibactin and is in fact a small peptide secreted by a gold-ion-tolerant bacterium called <i>Delftia acidovorans</i> [7].</p>
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    <figcaption style="width:60%;"><b>Fig. 1</b> Industrial application of delftibactin for gold recovery from electronic waste. The electronic waste would be dissolved and the supernatant of a bacteria culture producing delftibactin would be added. This would allow the easy recovery of the precipitated pure gold.</figcaption>
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<p>This extremophile has the incredible ability to withstand toxic amounts of gold ions in contaminated soil. If one could culture these bacteria and produce delftibactin in large scales, could one potentially recover gold from electronic waste in a cost- and energy-efficient way? But what is the special feature of delfibactin to precipitate gold that efficiently?</p>
-
<p>Delftibactin is no ordinary peptide but a non-ribosomal peptide (NRP) <span class="citation">[5]</span> <span class="citation">[6]</span>. The efficient and non-pollutant large-scale production of this NRP in <em>E. coli</em> could revolutionize the recovery of gold from electronic waste and additionally highlight the plethora of versatile applications for non-ribosomal peptide synthetases (NRPSs). The most sriking feature of these non-ribosomal synthetases is their ablity to incorporate far more than the 21 common amino acids into peptides. They make use of numerous modified and even non-proteinogenic amino acids <span class="citation">[7]</span> to assembly peptides of diverse functions.</p>
 
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<br>
 
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<center>
 
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<a class="fancybox fancyGraphical" href="https://static.igem.org/mediawiki/2013/9/96/Heidelberg_Del_Cluster.png" title="Cluster of genes responsible for Delftibactin production.">
 
-
    <img style="width:60%; margin-bottom:10px; padding:1%;border-style:solid;border-width:1px;border-radius: 5px;" src="https://static.igem.org/mediawiki/2013/9/96/Heidelberg_Del_Cluster.png" ></img>
 
-
    <figcaption style="width:60%;"><b>Fig. 2</b> Cluster of genes responsible for Delftibactin production. The cluster consists of the genes Daci_4754 to Daci_4765. The proteins DelE, DelF, DelG and DelH are directly responsible for the production of delftibactin. Domain architecture of the NRPS-PKS hybrid assembly-line is shown which consists of adenylation (A), thiolation (T), condensation, (C), ketosynthase (KS), acyltransferase (AT), ketoreductase (KR) and thioesterase domains. The predicted structure of delftibactin is shown below. <span class="citation">[5]</span></figcaption>
 
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</a>
 
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</center>
 
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<br>
 
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<p>Delftibactin is a NRP produced by a hybrid NRPS/ polyketide synthase (PKS) system. In their recent publication, Johnston <em>et al.</em> <span class="citation">[5]</span> predicted that the enzymes responsible for producing delftibactin are encoded on a single gene cluster, hereafter referred to as Del cluster. It comprises 59 kbp encoding for 21 genes. DelE, DelF, DelG and DelH constitute the hybrid NRPS/ PKS system producing delftibactin, with DelE, DelG and DelH being NRPS and DelF the PKS. The remaining enzymes involved in the Delftibactin synthesis pathway are required for NPRS/ PKS maturation or post-synthesis modification of Delftibactin. The predicted activities of the assumed proteins are:</p>
+
<p>Delftibactin is a non-ribosomal peptide (NRP) [7] [8]. The efficient and non-pollutant large-scale production of this NRP in <i>Escherichia&nbsp;coli</i> could revolutionize the recovery of gold from electronic waste and additionally highlight the plethora of versatile applications for non-ribosomal peptide synthetases (NRPSs). The most striking feature of these non-ribosomal synthetases is their ability to incorporate far more than the 21&nbsp;common amino acids into peptides. They make use of numerous modified and even non-proteinogenic amino acids to assemble peptides of diverse functions [9].</p>
 +
 
 +
<p>Delftibactin is a NRP produced by a hybrid NRPS/polyketide synthase (PKS) system. In their recent publication, Johnston and colleagues [7] predicted that the enzymes responsible for producing delftibactin are encoded on a single gene cluster, hereafter referred to as del cluster
 +
 
 +
(<a class="fancybox fancyFigure" title="Cluster of genes responsible for Delftibactin production. The cluster consists of the genes Daci_4754 to Daci_4765. The proteins DelE, DelF, DelG and DelH are directly responsible for the production of delftibactin. Domain architecture of the NRPS-PKS hybrid assembly-line is shown which consists of adenylation (A), thiolation (T), condensation, (C), ketosynthase (KS), acyltransferase (AT), ketoreductase (KR) and thioesterase domains. The predicted structure of delftibactin is shown below." href="https://static.igem.org/mediawiki/2013/9/96/Heidelberg_Del_Cluster.png" rel="gallery1">Fig.&nbsp;1</a>).  
 +
 
 +
It comprises 59&nbsp;kbp encoding for 21&nbsp;genes. DelE, DelF, DelG and DelH constitute the hybrid NRPS/ PKS system producing delftibactin, with DelE, DelG and DelH comprising the NRPS and DelF the PKS. The remaining enzymes involved in the delftibactin synthesis pathway are required for maturation or post-synthesis modification of delftibactin. The predicted activities [7] of the proteins are:</p>
<p>
<p>
<ol style="list-style-type: decimal">
<ol style="list-style-type: decimal">
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<li>DelA: MbtH-like protein, most likely required for efficient delftibactin synthesis <span class="citation">[8]</span></li>
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<li>DelA: MbtH-like protein, most likely required for efficient delftibactin synthesis <span class="citation">[10]</span></li>
<li>DelB: thioesterase</li>
<li>DelB: thioesterase</li>
<li>DelC: 4’-phosphopanteinyl transferase: required for maturation of ACP/PCP subunits</li>
<li>DelC: 4’-phosphopanteinyl transferase: required for maturation of ACP/PCP subunits</li>
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<p>We aimed to introduce the large Del cluster into the commonly used, easy-to-culture model organism <em>E. coli</em> to produce Delftibactin. This target bacterium already possesses many components needed for the functionality of non-ribosomal-peptide synthetases. Although this del-cluster contains a PPTase native for <em>D. Acidovorans</em> we introduce the sfp phosphopanteinyl transferase from <em>Bacillus subtilis</em>. Furthermore DelF (the PKS of the system) requires methylmalonyl-CoA as a substrate. This metabolite, from now on abbreviated as MMCoA is not produced by <em>E. coli</em>. Therefore we also transferred the MMCoA synthesis pathway from <em>B. subtilus</em> which is able to activate a wide variety of PKSs including those from <em>Saccharomyces cerevisiae</em> <span class="citation">[9]</span> which as been proven to work in <em>E. coli</em> by the indigoidine project</p>
 
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<p>The resulting engineered <em>E. coli</em> could be used as host for the delftibactin synthesis pathway, possibly also eliminating the need to introduce DelC. As promoters of the Del cluster were only predicted <span class="citation">[10]</span> for Daci_4750 (DelK) and Daci_4760 (DelA) and the cluster is transcribed starting with Daci_4760, we assumed that the entire sequence stretch of approximately 40 kbp is transcribed as single polycistronic mRNA.</p>
 
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<p>Facing these challenges, we decided to approach the project straight forward by cultivation of <em>D. acidovorans</em> and the isolation of native Delftibactin to reproduce the findings of Johnston <em>et al.</em> <span class="citation">[5]</span></p>
 
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<h2 id="experiments">Experiments</h2>
 
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<p>Our aim is to express delftibactin in <em>E. coli</em>. This will be achieved by introducing three different plasmids which contain parts of the delftibactin-cluster [File:Del cluster.gb] ,a Methylmalonyl-CoA pathway, a Pptase which replaces the DelC-function and a permeability device for the export of the desired NRP.</p>
 
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<a class="fancybox fancyGraphical" href="https://static.igem.org/mediawiki/2013/0/03/Heidelberg_Delftibactin_Intro.png" title="Cloning strategy for production of Delftibactin in <i>E. coli</i>.">
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<a class="fancybox fancyGraphical" href="https://static.igem.org/mediawiki/2013/9/96/Heidelberg_Del_Cluster.png" title="Cluster of genes responsible for Delftibactin production. The cluster consists of the genes Daci_4754 to Daci_4765. The proteins DelE, DelF, DelG and DelH are directly responsible for the production of delftibactin. Domain architecture of the NRPS-PKS hybrid assembly-line is shown which consists of adenylation (A), thiolation (T), condensation, (C), ketosynthase (KS), acyltransferase (AT), ketoreductase (KR) and thioesterase domains. The predicted structure of delftibactin is shown below.">
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     <img style="width:50%; margin-bottom:10px; padding:1%;border-style:solid;border-width:1px;border-radius: 5px;" src="https://static.igem.org/mediawiki/2013/0/03/Heidelberg_Delftibactin_Intro.png" ></img>
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     <img style="width:60%; margin-bottom:10px; padding:1%;border-style:solid;border-width:1px;border-radius: 5px;" src="https://static.igem.org/mediawiki/2013/9/96/Heidelberg_Del_Cluster.png">
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     <figcaption style="width:60%;"><b>Fig. 3</b> Cloning strategy for production of Delftibactin in <i>E. coli</i>. The genes DelA to DelH, DelL, DelO and DelP from the cluster for delftibactin production are introduced in two plasmids (shown in yellow). Additional genes needed for the production and export of delftibactin are located on a third plasmid (shown in blue). </figcaption>
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     <figcaption style="width:60%;"><b>Figure 1: Cluster of genes responsible for Delftibactin production.</b> The cluster consists of the genes Daci_4754 to Daci_4765. The proteins DelE, DelF, DelG and DelH are directly responsible for the production of delftibactin. Domain architecture of the NRPS-PKS hybrid assembly-line is shown which consists of adenylation (A), thiolation (T), condensation, (C), ketosynthase (KS), acyltransferase (AT), ketoreductase (KR) and thioesterase domains. The predicted structure of delftibactin is shown below. Figure adopted from <span class="citation">[7]</span>.</figcaption>
</a>
</a>
</center>
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<br/>
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<ol style="list-style-type: decimal">
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<p>We introduced the large del cluster into the commonly used, easy-to-culture model organism <i>E.&nbsp;coli</i> with the aim of recombinant delftibactin expression. Although the del cluster contains the native PPTase of <i>D. acidovorans</i> we additionally introduced the sfp phosphopanteinyl transferase from <i>Bacillus subtilis</i> as this PPTase is able to activate a wide variety of PKSs including those from <i>Saccharomyces cerevisiae</i>. Importantly, it has been proven to work in <i>E.&nbsp;coli</i> by the <a href='https://2013.igem.org/Team:Heidelberg/Project/Tag-Optimization'>indigoidine project</a> of our own team [11]. Additionally, DelF, the polyketide synthetase of the del cluster requires methylmalonyl-CoA as substrate. This metabolite, from now on abbreviated as mmCoA is not produced by <i>E.&nbsp;coli</i>. Therefore, we also transfer the mmCoA synthesis pathway from <i>B.&nbsp;subtilis</i> into <i>E.&nbsp;coli</i>. This should allow for efficient production of recombinant delftibactin.</p>
-
<li>Methylmalonyl-CoA, ppTase &amp; permeability device</li>
+
 
-
<li>DelH</li>
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<p>As the del cluster starts with Daci_4760 (DelA; Daci IDs are NCBI Gene gene symbols, Del* gene names as referred to in [12]) and promoters within the Del-cluster were bioinformatically predicted upstream of Daci_4750 (DelK), Daci_4760 (DelA) and Daci_4746 (DelO) we assumed that the entire sequence from Daci_4760 (DelA) to Daci_4753 (DelH) is transcribed as a single polycistronic mRNA of approximately 40 kbp in size [12].</p>
-
<li><p>DelA-P - The rest of the genes of the Del-cluster</p></li>
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<li>Our first aim was to achieve a genomic integration of the genes that encode for components of the Methylmalonyl-CoA pathway into <em>E. coli</em>. The presence of this pathway is required for the production of NRPs. Because the genomic integration turned out to be more challenging than expected a new strategy was developed. Therefore, two plasmids were created (pIK2) containing MethylmalonylCoA amplified from Streptomyces coeliolor and a ppTase amplified from <em>Bacillus subtilis</em> in the Biobrick Backbone pSB3C5 and the permeability device (BBa_I746200) for the outer membrane of <em>E. coli</em> was inserted in another plasmid (pIK1). Team Cambridge revealed in 2007 that Bba_I746200 is toxic. It was itherefore inserted into pIK2 between the two terminators driven by a weak promoter (BBa_J23114) and a weak RBS (Bba_B0030), yielding pIK8 with a total size of 9,467 bp, which was inserted in DH10ß and BL21DE3 via electroporation.</li>
+
<p>Facing these challenges, we decided to approach the project by cultivation of <i>D.&nbsp;acidovorans</i> and the isolation of native delftibactin to reproduce the findings of Johnston and colleagues [7].</span></p>.
-
<li>As the gene encoding DelH alone has a size of 18 kb we decided to clone and introduce this huge gene on a separate plasmid. The first restriction enzyme strategy was problematic because of DelH amplification and the low yield in the ligation. A new GibsonAssembly-strategy was performed and DelH amplified in smaller pieces. It seemed to appear the same problem of as in the pIK1 that <em>E. coli</em> is selecting out the mutated DelH-constructs or is activly mutating it for toxic reasons. A plasmid was designed with the same low copy promotor as in the pIK8 and a low copy RBS [BBa_B0032]. Another shot was a plasmid without promotor so that <em>E. coli</em> has no need to express and mutate DelH. Finally DelH is going to be inserted in <em>E. coli</em> DH10ß and BL21 via electroporation.</li>
+
 
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<li><p>DelA-P (the remaining genes of the Del-cluster) [File:Del cluster.gb] was amplified with different primer combinations out of <em>D.acidovorans</em>, and a plasmid was created containing these genes on the pSB4K5 Backbone with lacI promotor and without mRFP. The was transformed into DH10ß and BL21(DE3) via electroporation.</p></li>
+
<p>In order to achieve recombinant expression of delftibactin, we decided to introduce constructs coding for the delftibactin-cluster the methylmalonyl-CoA pathway and the PPTase sfp. In addition, we transformed the permeability device <a href='http://parts.igem.org/Part:BBa_I746200'>BBa_I746200</a> from the parts registry for the export of recombinant delftibactin out of the target organism <i>E.&nbsp;coli</i>. The desired genes from the del cluster were subdivided onto two different plasmids in order to decrease plasmid size and thereby avoid the intricacies expected for cloning of a single 59&nbsp;kbp plasmid as well as to allow for faster trouble shooting in case issues with the cloning of particular genes occur:</p>
 +
 
 +
<ol>
 +
<li>Plasmid: methylmalonyl-CoA pathway, PPTase sfp & permeability device <a href='http://parts.igem.org/Part:BBa_I746200'>BBa_I746200</a>, transcription regulated by inducible lac promoter, chloramphenicol resistance;</li>
 +
<li>Plasmid: DelH, transcription regulated by inducible lac promoter, ampicillin resistance;</li>
 +
<li>Plasmid: DelA-P, genes of the del cluster required for production of delftibactin, transcription regulated by inducible lac promoter, kanamycin resistance.</li>
</ol>
</ol>
-
<p>All three plasmid were then electroporated into <em>E. coli</em> BL21 DE3.</p>
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 +
<p>Here we show successful amplification, cloning and transformation of plasmids above 30&nbsp;kbp in size as well as expression of the desired genes of the del cluster from its natural host <i>D.&nbsp;acidovorans</i>. Furthermore, we demonstrate efficient recovery of purified gold from electronic waste using the non-ribosomal peptide delftibactin. Additionally, we proved toxicity of DelH for our target organism <i>E.&nbsp;coli</i> when expressed as the only gene of the del cluster: cloning of DelH into a construct without promoter lead to depletion of DelH from our target system which previously had been selecting for mutated versions of DelH.</p>
 +
 
                     <h2>Results</h2>
                     <h2>Results</h2>
-
                           <h3>Efficient Recycling of Gold from Electronic Waste using Delftibactin</h3>
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                           <h3>Efficient Recycling of Gold from Electronic Waste Using Endogenously-Derived Delftibactin</h3>
 +
 
 +
<p>As a first step into the direction of an environmentally friendly procedure for recycling gold from gold-containing waste, we wanted to show that the non-ribosomal peptide delftibactin can be used to precipitate gold from gold ion-containing solutions.</p>
-
<p>As first, important step in direction of an environmental friendly procedure for recycling gold from gold-containing waste, we wanted to show that the non-ribosomal peptide delftibactin can be used to precipitate gold from gold ion-containing solutions.</p>
+
<p>We obtained <i>D.&nbsp;acidovorans</i> DSM-39 from the DSMZ and successfully reproduced the paper by Johnston and colleagues [7]. What they had been able to show was that delftibactin selectively precipitates gold from gold solution. In our experiments, precipitation on agar plates worked even better than described by Johnston <em>et al.</em>  
-
<p>We obtained <i>D. acidovorans</i> DSM-39 from the DSMZ and successfully reproduced the paper by Johnsson <i>et al.</i> [5]. In our experiments, precipitation on agar plates worked even better than described in the paper as shown in Figure 1. <i>D. acidovorans</i> is capable to precipitate solid gold from gold chloride solution as purple-black nanoparticles. Already at low concentrations of gold chloride, gold nonaparticles are precipitated increasing with concentration of gold chloride in solution (Fig. 2).</p>
+
(<a class="fancybox fancyFigure" title="ACM agar plate with D.&nbsp;acidovorans DSM-39 overlaid with 0.2% HAuCl4 in 0.5% agarose. <i>D.&nbsp;acidovorans</i> had only been growing on the upper part of the shown plate. One can clearly see that gold nanoparticles are exclusively formed on the part of the plate harboring bacteria." href="https://static.igem.org/mediawiki/2013/6/66/Heidelberg_IMAG0449.png" rel="gallery1">Fig.&nbsp;2</a>).
 +
<i>D.&nbsp;acidovorans</i> is capable to precipitate solid gold from gold chloride solution as purple-black nanoparticles.</p>
<br/>
<br/>
<center>
<center>
-
<a class="fancybox fancyGraphical" href="https://static.igem.org/mediawiki/2013/6/66/Heidelberg_IMAG0449.png" title="ACM agar plate with D. acidovorans (left) overlaid with 0.2% HAuCl4 in 0.5% agarose.">
+
<a class="fancybox fancyGraphical" href="https://static.igem.org/mediawiki/2013/6/66/Heidelberg_IMAG0449.png" title="Figure 2: <i>D.&nbsp;acidovorans</i> precipitates elementary gold from gold solution.</b> ACM agar plate with <i>D.&nbsp;acidovorans</i> DSM-39 overlaid with 0.2% HAuCl<sub>4</sub> in 0.5% agarose. <i>D.&nbsp;acidovorans</i> had only been growing on the upper part of the shown plate. One can clearly see that gold nanoparticles are exclusively formed on the part of the plate harboring bacteria.">
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     <img style="width:30%; margin-bottom:10px; padding:1%;border-style:solid;border-width:1px;border-radius: 5px;" src="https://static.igem.org/mediawiki/2013/6/66/Heidelberg_IMAG0449.png"></img>
+
     <img style="width:30%; margin-bottom:10px; padding:1%;border-style:solid;border-width:1px;border-radius: 5px;" src="https://static.igem.org/mediawiki/2013/6/66/Heidelberg_IMAG0449.png">
-
     <figcaption style="width:60%;"><b>Fig. 5</b> ACM agar plate with <i>D. acidovorans</i> DSM-39 overlaid with 0.2% HAuCl<sub>4</sub> in 0.5% agarose. <i>Delftia acidovorans</i> had only been growing on the upper part of the shown plate. One can clearly see that gold nanoparticles only formed on the half with the bacterium. </figcaption>
+
     <figcaption style="width:60%;"><b>Figure 2: <i>D.&nbsp;acidovorans</i> precipitates elementary gold from gold solution.</b> ACM agar plate with <i>D.&nbsp;acidovorans</i> DSM-39 overlaid with 0.2% HAuCl<sub>4</sub> in 0.5% agarose. <i>D.&nbsp;acidovorans</i> had only been growing on the upper part of the shown plate.One can clearly see that gold nanoparticles are exclusively formed on the part of the plate harboring bacteria. </figcaption>
</a>
</a>
</center>
</center>
<br>
<br>
-
<p style="clear:both">Using supernatants from the new <i>Delftia acidovorans</i> strain SPH-1, we showed precipitation of gold chloride solution to gold nanoparticles. Furthermore, we melted the purple-black nanoparticles to shiny solid gold as shown in figures 6 to 8.</p>
+
<p style="clear:both">We also showed that another strain, <i>D.&nbsp;acidovorans</i> SPH-1, is also able to precipitate gold ions to gold nanoparticles. When using the supernatant of a culture gold nanoparticles were precipitated at an amount which caused a color change to black already at low concentrations of 0.35 µg/ml of gold chloride. With increasing concentration of gold chloride more nanoparticles formed, even though the process became slower above a certain concentration (Video 1 and
 +
 
 +
<a class="fancybox fancyFigure" title="Supernatant of <i>D.&nbsp;acidovorans</i> culture is sufficient for gold precipitation. a) Sequences of movie over time showing gold precipitation in D.&nbsp;acidovorans supernatant using different concentrations of HAuCl<sub>4</sub>. From left to right: 0 µg/ml, 0.15 µg/ml, 0.35 µg/ml, 0.55 µg/ml, 0.75 µg/ml, 0.95 µg/ml, 1.15 µg/ml, 1.35 µg/ml, 1.55 µg/ml, 1.75 µg/ml, 1.95 µg/ml, 2.15 µg/ml, 2.35 µg/ml and 2.55 µg/ml. b) Sparkling gold appearing in the melting pot after the precipitation of gold ions using the supernatant of <i>D.&nbsp;acidovorans</i> SPH-1. c) Final recovered solid gold with Delftibactin collected in tube. d) Solid gold covering the walls of a 2 ml tube after the application of delftibactin to gold solution." href="https://static.igem.org/mediawiki/2013/8/8e/Zusammenstellung1.png" rel="gallery1">Fig.&nbsp;3a</a>). An optimum is visible at a concentration of about 1.15 µl/ml of gold solution. Johnston <i>et al.</i> [7] described that the optimal ratio of gold ions to delftibactin is 1:1. Therefore it can be concluded that 1.96 nmol of delftibactin was present in 1 ml of <i>D. acidovorans</i> supernatant.
 +
Furthermore, we melted the purple-black nanoparticles to shiny, solid gold as shown in  
 +
<a class="fancybox fancyFigure" title="Testing the precipitation gold with <i>D.&nbsp;acidovorans</i> supernatant. a) Sequences of movie over time showing gold precipitation in D.&nbsp;acidovorans supernatant using different concentrations of AuCl4. From left to right: 0 µg/ml, 0.15 µg/ml, 0.35 µg/ml, 0.55 µg/ml, 0.75 µg/ml, 0.95 µg/ml, 1.15 µg/ml, 1.35 µg/ml, 1.55 µg/ml, 1.75 µg/ml, 1.95 µg/ml, 2.15 µg/ml, 2.35 µg/ml and 2.55 µg/ml. b) Sparkling gold appearing in the melting pot after the precipitation of gold ions using the supernatant of <i>D.&nbsp;acidovorans</i> SPH-1. c) Final recovered solid gold with Delftibactin collected in tube. d) Solid gold covering the walls of a 2 ml tube after the application of delftibactin to gold solution." href="https://static.igem.org/mediawiki/2013/8/8e/Zusammenstellung1.png" rel="gallery1">Fig.&nbsp;3b,c,d</a>.</p>
<br/>
<br/>
<center>
<center>
-
<a class="fancybox fancyGraphical" href="https://static.igem.org/mediawiki/2013/8/8e/Zusammenstellung1.png" title="ACM agar plate with D. acidovorans (left) overlaid with 0.2% HAuCl4 in 0.5% agarose.">
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<div>
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     <img style="width:50%; margin-bottom:10px; padding:1%;border-style:solid;border-width:1px;border-radius: 5px;" src="https://static.igem.org/mediawiki/2013/8/8e/Zusammenstellung1.png"></img>
+
<iframe id="video" width="480" height="360" src="//www.youtube.com/embed/xsB9_7Acuyk" frameborder="0" allowfullscreen></iframe>
-
     <figcaption style="width:60%;"><b>Fig. 4</b> Sequences of movie showing gold precipitation in <i>D. acidovorans</i> supernatant using gold concentrations ranging from 0 to 2.55 µg/ml. <b>Fig. 6</b> Sparkling gold appearing in the melting pot. <b>Fig. 7</b> Final recovered solid gold collected in tube.<b>Fig. 8</b> Solid gold recovered from nanoparticles in 2 ml tube.</figcaption>
+
<p style="margin-left:20%; margin-right:20%; margin-top:5px"><b>Video 1: Precipitation of elementary gold by delftibactin is dependent on the ratio of peptide to gold ions</b>. Different concentrations of gold solution applied to the supernatant of a <i>D.&nbsp;acidovorans</i> SPH-1 culture. From left to right: 0 µg/ml, 0.15 µg/ml, 0.35 µg/ml, 0.55 µg/ml, 0.75 µg/ml, 0.95 µg/ml, 1.15 µg/ml, 1.35 µg/ml, 1.55 µg/ml, 1.75 µg/ml, 1.95 µg/ml, 2.15 µg/ml, 2.35 µg/ml and 2.55 µg/ml HAuCl<sub>4</sub>. Black gold nanoparticles form due to the precipitation of solid gold from solution by the NRP delftibactin. According to Johnston and colleages, gold precipitation is most sufficient at peptide to gold ions ration of 1:1 [7], suggesting an amount of peptide of 1.96 nmol. The process is shown in time-lapse and had an actual duration of 8min 23s.</p>
 +
</div>
 +
 
 +
<a class="fancybox fancyGraphical" href="https://static.igem.org/mediawiki/2013/8/8e/Zusammenstellung1.png" title="Supernatant of <i>D.&nbsp;acidovorans</i> culture is sufficient for gold precipitation. a) Sequences of movie over time showing gold precipitation in <i>D.&nbsp;acidovorans</i> supernatant using different concentrations of AuCl4. From left to right: 0 µg/ml, 0.15 µg/ml, 0.35 µg/ml, 0.55 µg/ml, 0.75 µg/ml, 0.95 µg/ml, 1.15 µg/ml, 1.35 µg/ml, 1.55 µg/ml, 1.75 µg/ml, 1.95 µg/ml, 2.15 µg/ml, 2.35 µg/ml and 2.55 µg/ml. b) Sparkling gold appearing in the melting pot after the precipitation of gold ions using the supernatant of <i>D.&nbsp;acidovorans</i> SPH-1. c) Final recovered solid gold with Delftibactin collected in tube. d) Solid gold covering the walls of a 2 ml tube after the application of delftibactin to gold solution.">
 +
     <img style="width:50%; margin-bottom:10px;margin-top:5%; padding:1%;border-style:solid;border-width:1px;border-radius: 5px;" src="https://static.igem.org/mediawiki/2013/8/8e/Zusammenstellung1.png">
 +
     <figcaption style="width:60%;"><b>Figure 3: Supernatant of <i>D.&nbsp;acidovorans</i> culture is sufficient for gold precipitation. </b> a) Sequences of movie over time showing gold precipitation in <i>D.&nbsp;acidovorans</i> supernatant using different concentrations of HAuCl<sub>4</sub>. From left to right: 0 µg/ml, 0.15 µg/ml, 0.35 µg/ml, 0.55 µg/ml, 0.75 µg/ml, 0.95 µg/ml, 1.15 µg/ml, 1.35 µg/ml, 1.55 µg/ml, 1.75 µg/ml, 1.95 µg/ml, 2.15 µg/ml, 2.35 µg/ml and 2.55 µg/ml. b) Sparkling gold appearing in the melting pot after the precipitation of gold ions using the supernatant of <i>D.&nbsp;acidovorans</i> SPH-1. c) Final recovered solid gold with Delftibactin collected in tube. d) Solid gold covering the walls of a 2 ml tube after the application of delftibactin to gold solution.</figcaption>
</a>
</a>
</center>
</center>
<br>
<br>
-
<p style="clear:both">Next, we established purification of Delftibactin using HP20 resins. Additionally, we proved precipitation of gold by the purified Delftibactin (figure 6) and detected it by Micro-TOF. Moreover, we showed precipitation of dissolved gold recovered from electronic waste by <i>D. acidovorans</i>. Adding this solution to <i>D. acidovorans</i> agar plates resulted in the formation of solid gold nanoparticles. Taken together, we have successfully established a method enabling the recycling of pure gold from electronic waste using delftibactin produced by <i>D. acidovorans</i>.  
+
<p style="clear:both">Next, we established a purification protocol for delftibactin using HP20 resins. Additionally, we proved precipitation of gold by the purified delftibactin
 +
(<a class="fancybox fancyFigure" title="Purified Delftibactin precipitates gold from gold solution. From left to right: ddH2O, 1:10 ACM media in water, 1:10 supernatant of D.&nbsp;acidovorans SPH-1 in water, 1:10 filtered supernatant of D.&nbsp;acidovorans SPH-1 in water, 1:10 purified delftibactin in water; gold solution: 0.6 µg/ml HAuCl<sub>4</sub>." href="https://static.igem.org/mediawiki/2013/1/12/Heidelberg_IMG_4368.JPG" rel="gallery1">Fig.&nbsp;4</a>)
 +
and detected it by Micro-TOF  
 +
(<a class="fancybox fancyFigure" title="Micro-TOF results for ACM media (top) compared to purified supernatant of a <i>D.&nbsp;acidovorans</i> SPH-1 liquid culture (middle). Delftibactin is present in <i>D.&nbsp;acidovorans</i>SPH-1 supernatant but not in the pure ACM-media as can be seen from the peak at about 1055.5, 1033.5, 517.2 and 539.2 shown in the enlargements (bottom)." href="https://static.igem.org/mediawiki/2013/1/11/Heidelberg_Microtof-Delftia.png" rel="gallery1">Fig.&nbsp;5</a>).</p>
 +
 
 +
 
-
<br/>
 
<center>
<center>
-
<a class="fancybox fancyGraphical" href="https://static.igem.org/mediawiki/2013/1/11/Heidelberg_Microtof-Delftia.png" title="Micro-TOF result for D. acidovaorans">
+
<a class="fancybox fancyGraphical" href="https://static.igem.org/mediawiki/2013/1/12/Heidelberg_IMG_4368.JPG" title="Purified Delftibactin precipitates gold from gold solution. From left to right: ddH2O, 1:10 ACM media in water, 1:10 supernatant of D.&nbsp;acidovorans SPH-1 in water, 1:10 filtered supernatant of D.&nbsp;acidovorans SPH-1 in water, 1:10 purified delftibactin in water; gold solution: 0.6 µg/ml HAuCl<sub>4</sub>.">
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     <img style="width:60%; margin-bottom:10px; padding:1%;border-style:solid;border-width:1px;border-radius: 5px;" src="https://static.igem.org/mediawiki/2013/1/11/Heidelberg_Microtof-Delftia.png" ></img>
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     <img style="width:40%; margin-bottom:10px; padding:1%;border-style:solid;border-width:1px;border-radius: 5px;" src="https://static.igem.org/mediawiki/2013/1/12/Heidelberg_IMG_4368.JPG">
-
     <figcaption style="width:60%;"><b>Fig. 9</b> Micro-TOF result for <i>D. acidovorans</i></figcaption>
+
     <figcaption style="width:60%;"><b>Figure 4: Purified Delftibactin precipitates gold from gold solution.</b> From left to right: ddH<sub>2</sub>O, 1:10 ACM media in water, 1:10 supernatant of <i>D.&nbsp;acidovorans</i> SPH-1 in water, 1:10 filtered supernatant of <i>D.&nbsp;acidovorans</i> SPH-1 in water, 1:10 purified delftibactin in water; gold solution: 0.6 µg/ml HAuCl<sub>4</sub>.</figcaption>
</a>
</a>
</center>
</center>
-
<br>
+
<br/>
 +
<br/>
<center>
<center>
-
<a class="fancybox fancyGraphical" href="https://static.igem.org/mediawiki/2013/1/12/Heidelberg_IMG_4368.JPG" title="Test if purified Delftibactin (diluted 1:10 in H2O) is able to precipitate gold. From left to right: water, 1:10 ACM media, 1:10 supernatant D. acidovorans, 1:10 filtered supernatant D. acidovorans, 1:10 purified Delftibactin">
+
<a class="fancybox fancyGraphical" href="https://static.igem.org/mediawiki/2013/1/11/Heidelberg_Microtof-Delftia.png" title="<i>D.&nbsp;acidovorans</i> SPH-1 secretes delftibactin. Micro-TOF results for ACM media (top) compared to purified supernatant of a <i>D.&nbsp;acidovorans</i> SPH-1 liquid culture (middle). Delftibactin is present in <i>D.&nbsp;acidovorans</i> SPH-1 supernatant but not in the pure ACM-media as can be seen as peak of the Micro-TOF spectra at about 1055.5, 1033.5, 517.2 and 539.2 shown in the enlargements (bottom).">
-
     <img style="width:40%;" margin-bottom:10px; padding:1%;border-style:solid;border-width:1px;border-radius: 5px;" src="https://static.igem.org/mediawiki/2013/1/12/Heidelberg_IMG_4368.JPG" ></img>
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     <img style="width:60%; margin-bottom:10px; padding:1%;border-style:solid;border-width:1px;border-radius: 5px;" src="https://static.igem.org/mediawiki/2013/1/11/Heidelberg_Microtof-Delftia.png">
-
     <figcaption style="width:60%;"><b>Fig. 10</b> Test if purified Delftibactin (diluted 1:10 in H<sub>2</sub>O) is able to precipitate gold. From left to right: water, 1:10 ACM media, 1:10 supernatant <i>D. acidovorans</i>, 1:10 filtered supernatant <i>D. acidovorans</i>, 1:10 purified Delftibactin.</figcaption>
+
     <figcaption style="width:60%;"><b>Figure 5: <i>D.&nbsp;acidovorans</i> SPH-1 secretes delftibactin.</b> Micro-TOF results for ACM media (top) compared to purified supernatant of a <i>D.&nbsp;acidovorans</i> SPH-1 liquid culture (middle). Delftibactin is present in <i>D.&nbsp;acidovorans</i> SPH-1 supernatant but not in the pure ACM-media as can be seen as peak of the Micro-TOF spectra at about 1055.5, 1033.5, 517.2 and 539.2 shown in the enlargements (bottom).</figcaption>
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<p style="both:empty">An important prerequisite for efficient gold recycling from electronic waste is, undoubtedly, that gold precipitation should occur even if the concentration of the gold ions in solution would be rather low, as this would likely be the case for most gold solutions derived from electronic waste.</p>
+
<p style="clear:both">Moreover we were interested in the potential of delftibactin for the recycling of gold from electronic waste.  An important prerequisite for efficient gold recycling from electronic waste is the feasibility for gold precipitation from gold solutions of low concentration. Undoubtedly, this is likely the case for most gold solutions derived from electronic waste.Therefore, we used an old, broken CPU and established a protocol for dissolving gold from gold-containing metal waste. We incubated gold covered CPU pins in <i>aqua regia</i> resulting in a gold-ion containing solution
 +
(<a class="fancybox fancyFigure" title="<i>D.&nbsp;acidovorans</i> can be used for gold recovery from electronic waste. a) Pins removed from an old CPU. b) Green solution of dissolved pins in aqua regia. c) Gold chloride solution obtained after the dilution of an old CPU. d) "Dissolved electronic waste" and D.&nbsp;acidovorans. e) "Dissolved electronic waste" on D.&nbsp;acidovorans. f) Precipitated "dissolved electronic waste" and D.&nbsp;acidovorans." href="https://static.igem.org/mediawiki/2013/b/b1/Zusammenstellung2.png" rel="gallery1">Fig.&nbsp;6a-c</a>).
 +
We showed precipitation of dissolved gold recovered from electronic waste by <i>D.&nbsp;acidovorans</i>. Adding this solution to <i>D.&nbsp;acidovorans</i> SPH-1 agar plates resulted in the formation of solid gold nanoparticles
 +
(<a class="fancybox fancyFigure" title="<i>D.&nbsp;acidovorans</i> can be used for gold recovery from electronic waste. a) Pins removed from an old CPU. b) Green solution of dissolved pins in aqua regia. c) Gold chloride solution obtained after the dilution of an old CPU. d) "Dissolved electronic waste" and D.&nbsp;acidovorans. e) "Dissolved electronic waste" on D.&nbsp;acidovorans. f) Precipitated "dissolved electronic waste" and D.&nbsp;acidovorans." href="https://static.igem.org/mediawiki/2013/b/b1/Zusammenstellung2.png" rel="gallery1">Fig.&nbsp;6d-f</a>). </p>
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<p>Therefore, we used an old, broken CPU and established a protocol for dissolving gold from gold-containing metal waste. We incubated golden CPU pins in aqua regia resulting in a gold-ion containing solution (figures 8 to 10).</p>
 
<br/>
<br/>
<center>
<center>
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<a class="fancybox fancyGraphical" href="https://static.igem.org/mediawiki/2013/b/b1/Zusammenstellung2.png" title="Pins removed from an old CPU.">
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<a class="fancybox fancyGraphical" href="https://static.igem.org/mediawiki/2013/b/b1/Zusammenstellung2.png" title="<i>D.&nbsp;acidovorans</i> can be used for gold recovery from electronic waste. a) Pins removed from an old CPU. b) Green solution of dissolved pins in aqua regia. c) Gold chloride solution obtained after the dilution of an old CPU. d) "Dissolved electronic waste" and D.&nbsp;acidovorans. e) "Dissolved electronic waste" on D.&nbsp;acidovorans. f) Precipitated "dissolved electronic waste" and D.&nbsp;acidovorans.">
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     <img style="width:70%; margin-bottom:10px; padding:1%;border-style:solid;border-width:1px;border-radius: 5px;" src="https://static.igem.org/mediawiki/2013/b/b1/Zusammenstellung2.png" ></img>
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     <img style="width:70%; margin-bottom:10px; padding:1%;border-style:solid;border-width:1px;border-radius: 5px;" src="https://static.igem.org/mediawiki/2013/b/b1/Zusammenstellung2.png">
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     <figcaption style="width:60%;"><b>Fig. 11</b> Pins removed from an old CPU.<b>Fig. 12</b> Green solution of dissolved pins.<b>Fig. 13</b> Gold chloride solution obtained from an old CPU.<b>Fig. 14</b> "Dissolved electronic waste" and <i>D. acidovorans</i>.<b>Fig. 15</b> "Dissolved electronic waste" on <i>D. acidovorans</i>.<b>Fig. 16</b> Precipitated "dissolved electronic waste" and <i>D. acidovorans</i>.</figcaption>
+
     <figcaption style="width:70%;"><b>Figure 6: <i>D.&nbsp;acidovorans</i> can be used for gold recovery from electronic waste.</b> a) Pins removed from an old CPU. b) Green solution of dissolved pins in aqua regia. c) Gold chloride solution obtained after the dilution of an old CPU. d) "Dissolved electronic waste" and <i>D.&nbsp;acidovorans</i>. e) "Dissolved electronic waste" on <i>D.&nbsp;acidovorans</i>. f) Precipitated "dissolved electronic waste" and <i>D.&nbsp;acidovorans</i>.</figcaption>
</a>
</a>
</center>
</center>
<br>
<br>
 +
<p style="clear:both">Taken together, we have successfully established a method enabling the recycling of pure gold from electronic waste using delftibactin produced by <i>D.&nbsp;acidovorans</i>. </p>
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<p style="clear:both">Although the recycling was been working efficiently in our hands, the approach of using the natural <i>D. acidovorans</i> bacterial strain for delftibactin production on a larger scale has several disadvantages:
+
<p>Although the recycling was working efficiently in our hands, the approach of using the natural <i>D.&nbsp;acidovorans</i> bacterial strain for delftibactin production on a larger scale has the following disadvantages:  
-
<li> <i>D. acidovorans</i> are relatively slow in growth (colony formation on plates occurs after 2-3 days)</li>
+
<ol>
-
<li> Efficient production of delftibactin requires the strain to be grown in ACM media, which is rather expensive compared to typical <i>E. coli</i> growth media, making the procedure less economic.</li></p>
+
<li> <i>D.&nbsp;acidovorans</i> are relatively slow in growth (colony formation on plates occurs after 2-3 days) </li>
 +
<li> Efficient production of delftibactin requires the strain to be grown in ACM media, which is moreexpensive, compared to typical <i>E.&nbsp;coli</i> growth media, making the procedure less economic.</li></ol></p>
-
<p style="clear:both;">Therefore, we wanted to engineer an <i>E. coli</i> strain producing delftibactin in high yields, thereby circumventing the abovementioned limitations. To this end, we first had to develop a thorough cloning strategy which would allow us to clone all necessary genes encoding the delftibactin-producing non-ribosomal peptide synthetases and polyketide synthetases from the del cluster (about 50 kb in total) and express them in ''E. coli'' alongside with the MethylMalonyl-CoA pathway providing one of the basic substrates in this pathway not naturally present in <i>E. coli</i>.</p>
+
<p style="clear:both;">Therefore, we wanted to engineer an <i>E.&nbsp;coli</i> strain producing delftibactin in high yields, thereby circumventing the above mentioned limitations.</p>
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                     <h3>Amplification of the Del Cluster Genes</h3>
+
                     <h3>Expression of Del Cluster Genes in <i>E.&nbsp;coli</i></h3>
-
<p>The first step towards introducing the Delftibactin expression pathway into <i>E. coli</i> was the amplification of the Del-cluster encoded on the genome of ''Delftia acidovorans''. To this end, we designed Gibson primers and amplified the genes of the non-ribosomal peptide synthetase and the polyketide synthase (PKS) pathway as well as of additional proteins, which were predicted to be necessary for the production of Delftibactin [5]. In the first weeks, PCRs were successfully established and optimized. At the same time, a separate plasmid was created encoding the PPTase from <i>Bacillus subtilis</i>, the MethylMalonyl-CoA pathway and a permeability device <a href='http://parts.igem.org/wiki/index.php?title=Part:BBa_I746200'>BBa_I746200</a> for the export of the synthesized delftibactin NRP. Additionally, low-copy plasmids from the <a href='http://parts.igem.org/Main_Page'> partsregistry</a> were successfully transformed and amplified using corresponding Gibson primers in order to generate the destination backbone fragments for the assembly.</p>
+
<p>We developed a cloning strategy which allowed us to clone all necessary genes encoding the delftibactin-producing non-ribosomal peptide synthetases and polyketide synthetases from the del cluster (about 59 kbp in total) and express them in <i>E.&nbsp;coli</i>. In addition, introducing the methylmalonyl-CoA pathway into <i>E.&nbsp;coli</i> provided one of the basic substrates for the del pathway which is endogenously not present in <i>E.&nbsp;coli</i>.</p>
 +
 
 +
<p>Therefore, our initial aim was the genomic integration of the genes encoding for the methylmalonyl-CoA pathway into <i>E.&nbsp;coli</i> using the lambda red system established by [13]. This pathway is required for sufficient delftibactin production, as it supplies the substrate methylmalonyl-CoA for DelF, the PKS of the delftibactin cluster. As several genomc integration attempts did not yield positive results, as verified by colony-PCR with the forward primer binding to the genomic region and the reverse primer to the insert (a representative gel image can be seen in the <a href="/Team:Heidelberg/Delftibactin/MMCoA#2013-07-02">lab journal</a>), a new strategy was developed. Two plasmids were created: pIK2 containing the mm-CoA pathway amplified from <i>Streptomyces coelicolor</i> as well as the PPTase sfp, amplified from <i>Bacillus subtilis</i> in the BioBrick backbone pSB3C5. The permeability device (<a href='http://parts.igem.org/wiki/index.php?title=Part:BBa_I746200'>BBa_I746200</a>) for the outer membrane of <i>E.&nbsp;coli</i> was cloned into another plasmid (pIK1). Since the iGEM Team Cambridge 2007 showed that <a href='http://parts.igem.org/wiki/index.php?title=Part:BBa_I746200'>BBa_I746200</a> is toxic if produced in higher quantities we inserted it into pIK2 between the two terminators driven by a weak promoter (<a href='http://parts.igem.org/Part:BBa_J23114'>BBa_J23114</a>) and a weak RBS (<a href='http://parts.igem.org/Part:BBa_B0030'>BBa_B0030</a>), resulting in the final plasmids pIK8 with a total size of 9,467 bp, which was transformed into the <i>E.coli</i> strains DH10ß and BL21 DE3 via electroporation.</p>
 +
 
 +
 
 +
<p>Details of our cloning strategy are shown in  
 +
<a class="fancybox fancyFigure" title="Cloning strategy for the production of delftibactin in <i>E.&nbsp;coli</i>. The genes DelA to DelH, DelL, DelO and DelP from the cluster for delftibactin production are introduced in two plasmids (shown in yellow). Additional genes needed for the production and export of delftibactin are located on a third plasmid (shown in blue)." href="https://static.igem.org/mediawiki/2013/0/03/Heidelberg_Delftibactin_Intro.png" rel="gallery1">Fig.&nbsp;7</a>.
 +
Notably, the three plasmids we wanted to assemble are of large size (23, 32 and 10 kbp in size) and cloning was further complicated due to the high GC content and presence of repetitive elements within the del genes.</p>
<br/>
<br/>
<center>
<center>
-
<a class="fancybox fancyGraphical" href="https://static.igem.org/mediawiki/2013/0/03/Heidelberg_Delftibactin_Intro.png" title="Overview on the cloning strategy for the introduction of the delftibactin production pathway in E. coli.">
+
<a class="fancybox fancyGraphical" href="https://static.igem.org/mediawiki/2013/0/03/Heidelberg_Delftibactin_Intro.png" title="Cloning strategy for the production of delftibactin in <i>E.&nbsp;coli</i>. The genes DelA to DelH, DelL, DelO and DelP from the cluster for delftibactin production are introduced in two plasmids (shown in yellow). Additional genes needed for the production and export of delftibactin are located on a third plasmid (shown in blue).">
-
     <img style="width:50%; margin-bottom:10px; padding:1%;border-style:solid;border-width:1px;border-radius: 5px;" src="https://static.igem.org/mediawiki/2013/0/03/Heidelberg_Delftibactin_Intro.png" ></img>
+
     <img style="width:50%; margin-bottom:10px; padding:1%;border-style:solid;border-width:1px;border-radius: 5px;" src="https://static.igem.org/mediawiki/2013/0/03/Heidelberg_Delftibactin_Intro.png">
-
     <figcaption style="width:60%;"><b>Fig. 17</b> Overview on the cloning strategy for the introduction of the delftibactin production pathway in <i>E. coli</i>.</figcaption>
+
     <figcaption style="width:60%;"><b>Figure 7: Cloning strategy for the production of delftibactin in <i>E.&nbsp;coli</i>.</b> The genes DelA to DelH, DelL, DelO and DelP from the cluster for delftibactin production are introduced in two plasmids (shown in yellow). Additional genes needed for the production and export of delftibactin are located on a third plasmid (shown in blue).</figcaption>
</a>
</a>
</center>
</center>
-
<br>
+
<br/>
-
 
+
-
<p style="clear:both">Details of our cloning strategy are shown in figure 14. Notably, the three plasmids we wanted to assemble are huge (23, 32 and 10 kbp in size) and was further complicated due to the high GC content and presence of repetitive elements in Del genes.</p>
+
                   <h3>Gibson Assembly & Transformation</h3>
                   <h3>Gibson Assembly & Transformation</h3>
-
<p>Assembly of plasmids above 20 kb in size and composed of multiple different fragments is challenging when using conventional restriction-enzyme based cloning. Thus, we have used the  <a href='https://2010.igem.org/Team:Cambridge/Gibson/Protocol'>Gibson Assembly method</a>, which was introduced to the iGEM community by Cambridge in iGEM 2010 as powerful alternative to such common cloning procedures. The assembled constructs of up to 32 kbp in size were transformed into E. coli via electroporation. Correct assemblies of the fragments was tested by analytical restriction digests. The exemplary restriction digest shown above (Fig. 16) confirmed the correct assembly of the three desired constructs as it shows the expected band pattern expected from in silico digestion. Clones (6 and 9) contained the plasmid that encodes for the Methylmalonyl-CoA pathway (Fig. 16a). The obtained DNA sequences were sent in for sequencing over the Gibson-assembled regions for confirmation. <a href='http://www.mwg-biotech.com/'>http://www.mwg-biotech.com/</a>, <a href='http://www.gatc-biotech.com/de/index/.html'>http://www.gatc-biotech.com/de/index/.html</a>. The sequencing confirmed the accuracy of the sequence.</p>
+
<p>Assembly of plasmids above 30 kbp in size composing of multiple fragments is challenging when using conventional restriction enzyme based cloning. Thus, we decided to use <a href='https://2010.igem.org/Team:Cambridge/Gibson/Protocol'>Gibson Assembly</a>, a method which was introduced to the iGEM community by Cambridge in iGEM 2010 as a powerful alternative to common cloning procedures. The assembled constructs of up to 32 kbp in size were transformed into <i>E.&nbsp;coli</i> via electroporation. Correct assembly of the fragments was tested by analytical restriction digests. The exemplary restriction digests shown above  
 +
(<a class="fancybox fancyFigure" title="Restriction digest of plasmids coding for MMCoA, DelH and DelRest which are needed in the NRPS/PKS pathway to generated Delftibactin. a) Digested minipreps of the pIK8-plasmid of four different clones. Clones 6 and 9 show the expected pattern. b) shows the minipreps of one clone of <i>E.&nbsp;coli</i> transformed with the 32 kbp DelRest plasmid which was digested with three different enzymes. All digests show the expected patterns. c) shows the restriction digest of the DelH plasmid with PvuI. The digest of clone 5 shows the anticipated bands and is probably positive." href="https://static.igem.org/mediawiki/2013/f/fa/Heidelberg_DelCluster_Digest.png" rel="gallery1">Fig.&nbsp;8</a>) confirmed the correct assembly of the three desired constructs as it displays the expected band pattern. For cloning of the mm-CoA pathway, clones (6 and 9) show the expected restriction pattern
 +
(<a class="fancybox fancyFigure" title="Restriction digest of plasmids coding for MMCoA, DelH and DelRest which are needed in the NRPS/PKS pathway to generated Delftibactin. a) Digested minipreps of the pIK8-plasmid of four different clones. Clones 6 and 9 show the expected pattern. b) shows the minipreps of one clone of <i>E.&nbsp;coli</i> transformed with the 32 kbp DelRest plasmid which was digested with three different enzymes. All digests show the expected patterns. c) shows the restriction digest of the DelH plasmid with PvuI. The digest of clone 5 shows the anticipated bands and is probably positive." href="https://static.igem.org/mediawiki/2013/f/fa/Heidelberg_DelCluster_Digest.png" rel="gallery1">Fig.&nbsp;8a</a>). Sequencing of the assembly sites of these constructs confirmed the restriction digest results.</p>
 +
 
 +
<p>Cloning of the DelRest plasmid was validated by different suitable analytic restriction digests
 +
(<a class="fancybox fancyFigure" title="Restriction digest of plasmids coding for MMCoA, DelH and DelRest which are needed in the NRPS/PKS pathway to generated Delftibactin. a) Digested minipreps of the pIK8-plasmid of four different clones. Clones 6 and 9 show the expected pattern. b) shows the minipreps of one clone of <i>E.&nbsp;coli</i> transformed with the 32 kbp DelRest plasmid which was digested with three different enzymes. All digests show the expected patterns. c) shows the restriction digest of the DelH plasmid with PvuI. The digest of clone 5 shows the anticipated bands and is probably positive." href="https://static.igem.org/mediawiki/2013/f/fa/Heidelberg_DelCluster_Digest.png" rel="gallery1">Fig.&nbsp;8b</a>)
 +
and also confirmed by sequencing. The sequence was compared with the available <i>D.&nbsp;acidovorans</i> SPH-1 reference sequence obtained from NCBI (for further information please visit our <a href='https://2013.igem.org/Team:Heidelberg/Delftibactin'>labjournal</a>). The high quality of the alignment shows that Gibson assembly is a suitable cloning approach for rapid assembly of large NRPS and PKS expression constructs.</p>
 +
 
 +
<p>Analytical restriction digest of DelH
 +
(<a class="fancybox fancyFigure" title="Restriction digest of plasmids coding for MMCoA, DelH and DelRest which are needed in the NRPS/PKS pathway to generated Delftibactin. a) Digested minipreps of the pIK8-plasmid of four different clones. Clones 6 and 9 show the expected pattern. b) shows the minipreps of one clone of <i>E.&nbsp;coli</i> transformed with the 32 kbp DelRest plasmid which was digested with three different enzymes. All digests show the expected patterns. c) shows the restriction digest of the DelH plasmid with PvuI. The digest of clone 5 shows the anticipated bands and is probably positive." href="https://static.igem.org/mediawiki/2013/f/fa/Heidelberg_DelCluster_Digest.png" rel="gallery1">Fig.&nbsp;8c</a>)
 +
also gave rise to a number of positive clones. However, the sequencing results derived from all DelH showed various mutations, which were exclusively located within the region of the first DelH forward primer. Most of these mutations are deletions present in the first 30 bp of the DelH coding region thereby resulting in frameshifts and formation of a stop codon disposing <i>E. Coli</i> of expression of DelH. As there was no clone without mutations, we proceeded with DelH clone C5, as this clone did not have any bp deletion but only harbored a substitution mutation at bp position 28 of the ORF, leading to the conversion of Alanine at position 10 to Threonine. Two representative sequences compared to <i>D.&nbsp;acidovorans</i> SPH-1 are listed below (Tab. 1) and display two of the observed mutations in different DelH clones. </p>
<br/>
<br/>
<center>
<center>
-
<a class="fancybox fancyGraphical" href="https://static.igem.org/mediawiki/2013/f/fa/Heidelberg_DelCluster_Digest.png" title="Restriction digest of three different plasmids needed for the NRPS/PKS pathway which to generated Delftibactin. a) Four digested colonies clones of the pIK8-plasmid, where clones 6 and 9 show the expected pattern. b) shows one clone of E. coli clone with the 32 kpb DelRest plasmid and was digested with three different enzymes and every lane shows the specific pattern for the according enzyme. c) shows the restriction digest of the DelH plasmid with PvuI. Clone 5 shows the expected pattern and is probably positive.">
+
<a class="fancybox fancyGraphical" href="https://static.igem.org/mediawiki/2013/f/fa/Heidelberg_DelCluster_Digest.png" title="Restriction digest of plasmids coding for MMCoA, DelH and DelRest which are needed in the NRPS/PKS pathway to generated Delftibactin. a) Digested minipreps of the pIK8-plasmid of four different clones. Clones 6 and 9 show the expected pattern. b) shows the minipreps of one clone of <i>E.&nbsp;coli</i> transformed with the 32 kbp DelRest plasmid which was digested with three different enzymes. All digests show the expected patterns. c) shows the restriction digest of the DelH plasmid with PvuI. The digest of clone 5 shows the anticipated bands and is probably positive.">
-
     <img style="width:60%; margin-bottom:10px; padding:1%;border-style:solid;border-width:1px;border-radius: 5px;" src="https://static.igem.org/mediawiki/2013/f/fa/Heidelberg_DelCluster_Digest.png" ></img>
+
     <img style="width:60%; margin-bottom:10px; padding:1%;border-style:solid;border-width:1px;border-radius: 5px;" src="https://static.igem.org/mediawiki/2013/f/fa/Heidelberg_DelCluster_Digest.png">
-
     <figcaption style="width:60%;"><b>Fig. 18</b> Restriction digest of three different plasmids needed for the NRPS/PKS pathway which to generated Delftibactin. a) Four digested colonies clones of the pIK8-plasmid, where clones 6 and 9 show the expected pattern. b) shows one clone of <i>E. coli</i> clone with the 32 kpb DelRest plasmid and was digested with three different enzymes and every lane shows the specific pattern for the according enzyme. c) shows the restriction digest of the DelH plasmid with PvuI. Clone 5 shows the expected pattern and is probably positive.</figcaption>
+
     <figcaption style="width:60%;"><b>Figure 8: Restriction digest of plasmids coding for MMCoA, DelH and DelRest which are needed in the NRPS/PKS pathway to generated Delftibactin.</b> a) Digested minipreps of the pIK8-plasmid of four different clones. Clones 6 and 9 show the expected pattern. b) shows the minipreps of one clone of <i>E.&nbsp;coli</i> transformed with the 32 kbp DelRest plasmid which was digested with three different enzymes. All digests show the expected patterns. c) shows the restriction digest of the DelH plasmid with PvuI. The digest of clone 5 shows the anticipated bands and is probably positive.</figcaption>
</a>
</a>
</center>
</center>
<br>
<br>
-
<p>The successful generation of DelRest plasmid was proven by different enzymatic restriction digests (Fig. 16b) and also attested by the sequencing. The sequence was compared with the available <i>D. acidovorans</i> SPH1 reference sequence of the Del cluster obtained from <a href='http://tools.neb.com/'>NCBI</a> (For further information please visit our labjournal). This shows, that Gibson assembly is a suitable cloning approach for rapid assembly of large NRPS and PKS expression constructs.</p>
 
-
<p>Analytical restriction digest of DelH (Fig.16c) also gave rise to a number of positive clones. However, in contrary to the two successfully assembled and sequenced plasmids discussed above, the sequencing results derived from all DelH clones showed various mutations, which were mostly located within the region of the first DelH forward primer. Most of these mutations were deletions present at the very beginning of the DelH coding region. Interestingly, one specific deletion in the DNA sequence was found consistently in several independent clones. As we did not have any clone without mutations, we proceeded with the a DelH clone (termed C5), as this clone did not have any bp deletion but only harbored a minor base-pair substitution (leading to conversion of the DelH Alanine at position 10 to Threonine). Some exemplary sequences are listed below (Tab. 1) of <i>Delftia acidovorans</i> and two observed mutations in different DelH clones. </p>
 
-
</html>''Tab.1 DelH 5’ sequence, in which most mutations were observed. The ATG start codon is depicted in bold. The table shows the sequence comparison between the DelH reference strand of ''D. acidovorans'' and two different exemplary ''E. coli'' clones transformed with the plasmid pHM04 (assembled DelH expression vector). The second line shows the accumulated deletion and the third line shows the clone containing 'only' single base pair substitution. Deletions appeared quite frequently while a substitution was only found in a single clone C5. The substitution changes the corresponding Alanine codon to Threonine.''
+
</html>''Tab.1 DelH 5’ sequence, in which most mutations were observed. The ATG start codon is depicted in bold. The table shows the sequence comparison between the DelH reference strand of <i>D.&nbsp;acidovorans</i> and two different exemplary <i>E.&nbsp;coli</i> clones transformed with the plasmid pHM04 (assembled DelH expression vector). The second line shows the accumulated deletion and the third line shows the clone containing 'only' single base pair substitution. Deletions appeared quite frequently while a substitution was only found in a single clone C5. The substitution changes the corresponding Alanine codon to Threonine.''
<center>
<center>
{| class="wikitable"
{| class="wikitable"
Line 283: Line 314:
! Organism !! Plasmid containing !! DNA -Sequence !!Conclusion
! Organism !! Plasmid containing !! DNA -Sequence !!Conclusion
|-
|-
-
| ''D. acidovorans''|| none ||  ATG GACCGTGGC  CGCCTGCGC  CAAATCGC || correct
+
| <i>D.&nbsp;acidovorans</i>|| none ||  ATG GACCGTGGC  CGCCTGCGC  CAAATCGC || correct
|-
|-
-
| ''E. coli'' DH10ß|| pHM04 || ATG GACCGTG-C  CGCCTGCGC  CAAATCGC || deletion
+
| <i>E.&nbsp;coli</i> DH10ß|| pHM04 || ATG GACCGTG-C  CGCCTGCGC  CAAATCGC || deletion
|-
|-
-
| ''E. coli'' DH10ß C5 || pHM04 || ATG GACCGTGGC  CGCCTGCGC  CAAATCAC || substitution
+
| <i>E.&nbsp;coli</i> DH10ß C5 || pHM04 || ATG GACCGTGGC  CGCCTGCGC  CAAATCAC || substitution
|}
|}
</center>
</center>
<html>
<html>
-
<p>Due to the fact that <i>E. coli</i> seemed to somehow selected for mutated DelH clones, we hypothesize that expression of DelH in absence of the other proteins might be toxic for the cells. This would explain why <i>E. coli</i> selects for mutated, none functional, truncated DelH proteins. The same phenomenon of frequent mutations in presumably positive clones was also observed when we started cloning of the permeability device used in the pIK8 construct. The sequenced plasmids showed an unusually high accumulation of mutations compared to other constructs. In case of the methylmalonyl-CoA plasmid (pIK8), the problem was solved by the usage of a weak promoter and a weak ribosome binding site from the partsregistry for driving the expression of the permeability device. Based on this knowledge, DelH is currently being re-assembled into a new backbone (<a href='http://parts.igem.org/Part:.pSB6A1?title=Part:pSB6A1'>pSB6A1</a>) containing a weak promoter <a href='http://parts.igem.org/Part:BBa_J23114'>BBa_J23114</a> and the ribosome binding site <a href='http://parts.igem.org/Part:BBa_B0032'>BBa_B0032</a>. While the new plasmid is constructed, the following experiments were performed with the C5 clone as we hypothesized, that pHM04 construct #C5 bearing the single nucleotide exchange at position 28 might still show expression of functional DelH when transformed into <i>E. coli</i> (the corresponding amino acid exchange is located at the N-terminus).</p>
+
<p>Due to these observations, we hypothesize that expression of DelH is toxic for <i>E.&nbsp;coli</i>. Therefore natural selection only leads to survival of those clones which incorporate constructs giving rise to unfunctional, truncated DelH proteins or avoiding any expression. This phenomenon of frequent mutations within the primer binding site was also observed when we started cloning of the permeability device used in the pIK8 construct. The sequenced plasmids displayed a high accumulation of mutations compared to other constructs. In case of the methylmalonyl-CoA plasmid (pIK8), the problem was solved by the usage of a weak promoter and a weak ribosome binding site from the parts registry for driving the expression of the permeability device (see above). Based on this knowledge, DelH is currently being assembled into the desired backbone <a href='http://parts.igem.org/Part:pSB6A1'>pSB6A1</a> containing a weak promoter <a href='http://parts.igem.org/Part:BBa_J23114'>BBa_J23114</a> and the ribosome binding site <a href='http://parts.igem.org/Part:BBa_B0032'>BBa_B0032</a>, also reducing expression. While the new plasmid is constructed, the following experiments were performed with the C5 clone as we hypothesized that DelH C5 bearing the single nucleotide exchange at position 28 might still show expression of functional DelH when transformed into <i>E.&nbsp;coli</i> (the corresponding amino acid exchange is located at the N-terminus).</p>
-
<p>Therefore, we transformed all three plasmids, namely the pHM04 #C5 (encoding DelH), the DelRest plasmid (encoding all other del genes despite delH) and the pIK8 construct (bearing the MethylMalonyl-CoA pathway, the Sfp PPTase and the permeability device expression cassette) into <i>E. coli</i> DH10ß and <i>E. coli</i> BL21 DE3 in parallel. This should lead to ''E. coli'' cells producing delftibactin and secreting it into the media.</p>
+
 
                   <h3>Expression of the Delftibactin NRPSs & Associated Genes</h3>
                   <h3>Expression of the Delftibactin NRPSs & Associated Genes</h3>
-
<p>We carried out experiments to test whether our constructs enable expression of the delftibactin NRPS/PKS pathway in <i>E. coli</i>.</p>
+
<p>For further characterization of our constructs, we analyzed expression of the delftibactin NRPS/PKS pathway in <i>E.&nbsp;coli</i>. For expression of DelH and DelRest, SDS-PAGEs were conducted followed by Coomassie staining. Native cells as well as cells transformed with the plasmid backbones obtained from the parts registry containing the used antibiotic resistance markers served as control groups. The proteins DelE, DelG and DelH are significantly larger than any protein that is expressed by our host <i>E.&nbsp;coli</i>. Therefore, the expression of the introduced genes was detectable on the SDS-PAGE
 +
(<a class="fancybox fancyFigure" title="<i>E.&nbsp;coli</i> express the NRPS of the delftibactin production pathway. SDS-PAGE of: a) <i>E.&nbsp;coli</i> DH10ß colony D8w containing the DelRest plasmid pFSN. <i>E.&nbsp;coli</i> DH10ß transformed with pSB4K5 is used as negative control. The bands at the heights of about 190 kDa and 350 kDa (depicted by black arrows) indicate the expression of DelE and DelG. b) <i>E.&nbsp;coli</i> BL21 DE3 colony C5 containing the DelH plasmid. <i>E.&nbsp;coli</i> BL21 is used as negative control. A weak band at a height of more than 260 kDa (largest protein band of the ladder) is present. This could potentially be a band at the height of 670 kDa and thus hint at the production of the NRPS DelH." href="https://static.igem.org/mediawiki/2013/1/18/Heidelberg_SDS-PAGES.png" rel="gallery1">Fig.&nbsp;9</a>)
 +
without specific labeling of the proteins. Although expression of these large proteins was weak, clear distinct bands of the sizes predicted for DelE and DelG were detected. Accordingly a band at the predicted size of DelH for the clone transformed with DelH C5 was visible. As the lac promoter regulating expression of DelE and DelG also drives expression of DelA, DelB, DelC, DelD and DelF, one can conclude simultaneous expression of these del proteins. This is also in accordance with the predicted distribution of promoters within the delfticbatin cluster. </p>
<br/>
<br/>
<center>
<center>
-
<a class="fancybox fancyGraphical" href="https://static.igem.org/mediawiki/2013/1/18/Heidelberg_SDS-PAGES.png" title="SDS-Page of a) E. coli DH10ß DelRest D8w and b) E. coli BL21 DE3 and E. coli BL21 DE3 containing DelH C5.">
+
<a class="fancybox fancyGraphical" href="https://static.igem.org/mediawiki/2013/1/18/Heidelberg_SDS-PAGES.png" title="<i>E.&nbsp;coli</i> express the NRPS of the delftibactin production pathway. SDS-PAGE of: a) <i>E.&nbsp;coli</i> DH10ß colony D8w containing the DelRest plasmid pFSN. <i>E.&nbsp;coli</i> DH10ß transformed with pSB4K5 is used as negative control. The bands at the heights of about 190 kDa and 350 kDa (depicted by black arrows) indicate the expression of DelE and DelG. b) <i>E.&nbsp;coli</i> BL21 DE3 colony C5 containing the DelH plasmid. <i>E.&nbsp;coli</i> BL21 is used as negative control. A weak band at a height of more than 260 kDa (largest protein band of the ladder) is present. This could potentially be a band at the height of 670 kDa and thus hint at the production of the NRPS DelH.">
-
     <img style="width:60%; margin-bottom:10px; padding:1%;border-style:solid;border-width:1px;border-radius: 5px;" src="https://static.igem.org/mediawiki/2013/1/18/Heidelberg_SDS-PAGES.png" ></img>
+
     <img style="width:60%; margin-bottom:10px; padding:1%;border-style:solid;border-width:1px;border-radius: 5px;" src="https://static.igem.org/mediawiki/2013/1/18/Heidelberg_SDS-PAGES.png">
-
     <figcaption style="width:60%;"><b>Fig. 19</b> SDS-Page of a) <i>E. coli</i> DH10ß DelRest D8w and b) <i>E. coli</i> BL21 DE3 and <i>E. coli</i> BL21 DE3 containing DelH C5.</figcaption>
+
     <figcaption style="width:60%;"><b>Figure 9: <i>E.&nbsp;coli</i> express the NRPS of the delftibactin production pathway.</b>SDS-PAGE of: a) <i>E.&nbsp;coli</i> DH10ß colony D8w containing the DelRest plasmid pFSN. <i>E.&nbsp;coli</i> DH10ß transformed with pSB4K5 is used as negative control. The bands at the heights of about 190 kDa and 350 kDa (depicted by black arrows) indicate the expression of DelE and DelG. b) <i>E.&nbsp;coli</i> BL21 DE3 colony C5 containing the DelH plasmid. <i>E.&nbsp;coli</i> BL21 is used as negative control. A weak band at a height of more than 260 kDa (largest protein band of the ladder) is present. This could potentially be a band at the height of 670 kDa and thus hint at the production of the NRPS DelH.</figcaption>
</a>
</a>
</center>
</center>
<br/>
<br/>
-
<p>For expression of DelH and DelRest, we conducted SDS-PAGE followed by Coomassie stainig. As negative controls we used untransformed cells and cells transformed only with the original plasmid backbones. The proteins DelE, DelG and DelH are significantly larger than any protein that is expressed by our host <i>E. coli</i>. Therefore, the expression of the introduced genes was clearly visible on the SDS-PAGE (Fig. 17). Even though the expression was weak, as we have expected for such a large proteins, clear distinct bands at the expected size of DelE and DelG were detected for the clone transformed with the DelRest plasmid and a band at the size of DelH for the clone transformed with the DelH plasmid. As the promoter in front of DelE and DelG controls the expression of DelA, DelB, DelC, DelD and DelF, too, one can assume simultaneous expression of these Del proteins. </p>
+
 
 +
 
 +
<p>Furthermore, the expression of the PPTase was verified by performing an IndC activity assay established by the <a href='https://2013.igem.org/Team:Heidelberg/Project/Tag-Optimization'>indigoidine subproject</a>. The indigoidine synthetase IndC activity is dependent on the presence of a functional PPTase. Upon activation, indC produces the blue pigment indigoidine. Co-transformation of the corresponding plasmid pIK8 construct (enabling PPTase expression) with an IndC indigoidine synthetase expression construct lacking a PPTase expression cassette (<a href='https://static.igem.org/mediawiki/2013/7/7a/Heidelberg_PRB22.png'>pRB22</a>) was conducted. The transformed <i>E.&nbsp;coli</i> displayed inhibited growth and developed the expected blue phenotype. From these results, we conclude that the PPTase on pIK8 is functionally expressed (note: decelerated growth kinetics of <i>E.&nbsp;coli</i> results from the metabolic burden that is caused by the synthesis of the indigoidine).</p>
 +
 
 +
<p>For proving the production of the permeability device which is needed for export of delftibactin, a zone of inhibition test with bactracin was performed. Bacitracin is an antibiotic not able to pass the bacterial cell wall by passive transport [14]. Growth of bacteria containing the permeability device was inhibited upon application of bacitracin confirms the expression of the transporter. Growth of control cells without the device was not impaired by application of bacitracin
 +
(<a class="fancybox fancyFigure" title="Zone of inhibition test proves functionality of the permeability device. Left: TOP10-pIK8.6, right: TOP10-pIK8.1 (negative control); counterclockwise, starting top right: 8 µl, 4 µl, 2 µl, 1 µl bacitracin. It can be seen that the device works as the cells transformed with the construct die upon application of bacitracin." href="https://static.igem.org/mediawiki/2013/4/40/Heidelberg_Bacitracin.png" rel="gallery1">Fig.&nbsp;10</a>).</p>
<br/>
<br/>
<center>
<center>
-
<a class="fancybox fancyGraphical" href="https://static.igem.org/mediawiki/2013/4/40/Heidelberg_Bacitracin.png" title="Left: TOP10-pIK8.6, right: TOP10-pIK8.1 (negative control); counterclockwise, starting top right: 8 µl, 4 µl, 2 µl, 1 µl bacitracin.">
+
<a class="fancybox fancyGraphical" href="https://static.igem.org/mediawiki/2013/4/40/Heidelberg_Bacitracin.png" title="Zone of inhibition test proves functionality of the permeability device. Left: TOP10-pIK8.6, right: TOP10-pIK8.1 (negative control); counterclockwise, starting top right: 8 µl, 4 µl, 2 µl, 1 µl bacitracin. It can be seen that the device works as the cells transformed with the construct die upon application of bacitracin.">
-
     <img style="width:60%; margin-bottom:10px; padding:1%;border-style:solid;border-width:1px;border-radius: 5px;" src="https://static.igem.org/mediawiki/2013/4/40/Heidelberg_Bacitracin.png" ></img>
+
     <img style="width:60%; margin-bottom:10px; padding:1%;border-style:solid;border-width:1px;border-radius: 5px;" src="https://static.igem.org/mediawiki/2013/4/40/Heidelberg_Bacitracin.png">
-
     <figcaption style="width:60%;"><b>Fig. 20</b> Left: TOP10-pIK8.6, right: TOP10-pIK8.1 (negative control); counterclockwise, starting top right: 8 µl, 4 µl, 2 µl, 1 µl bacitracin.</figcaption>
+
     <figcaption style="width:60%;"><b>Figure 10: Zone of inhibition test proves functionality of the permeability device.</b> Left: TOP10-pIK8.6, right: TOP10-pIK8.1 (negative control); counterclockwise, starting top right: 8 µl, 4 µl, 2 µl, 1 µl bacitracin. It can be seen that the device works as the cells transformed with the construct die upon application of bacitracin.</figcaption>
</a>
</a>
</center>
</center>
<br/>
<br/>
-
<p>Furthermore, the expression of the PPTase was verified by performing an IndC activity assay. The indigoidine synthetase IndC activity is dependent on the presence of a functional PPTase which is needed for synthesis of the blue pigment indigoidine. Co-transformation of the corresponding plasmid pIK8 (enabling Sfp PPTase expression) with an IndC indigoidine synthetase expression construct lacking a PPTase expression cassette. The transformed <i>E. coli</i> grew very slowly and developed the expected blue phenotype. From these results, we can clearly conclude that the PPTase on pIK8 is functionally expressed (note: decelerated growth kinetics of <i>E. coli</i> results from the metabolic burden that is caused by the synthesis of the indigoidine).</p>
+
<p style="clear:both">In conclusion, we successfully expressed the recombinant delftibactin NRPS/ PKS pathway as well as the required methylmalonyl-CoA pathway, the PPTase and permeability device in <i>E.&nbsp;coli</i>. </p>
-
 
+
-
<p>For proving that <i>E. coli</i> also produces the permeability device, which is needed for export ofDelftibactin out of the cells, a Hemmhof agar diffusion test with bactracin was performed. Bacitracin is a very large antibiotic which is usually not able to diffuse across the cell membrane passively. Absent growth upon application of bacitracin of bacteria containing the plasmid while in the control cells without the device were not affected by the antibiotic (Fig. 18) confirms expression of the transporter. </p>
+
-
<p>In conclusion, we successfully expressed the recombinant Delftibactin NRPS/ PKS pathway as well as the required Methylmalonyl-CoA pathway, the PPTase and permeability device in <i>E. coli</i>. </p>
+
<p>Furthermore, we showed, that it is not only possible to assemble large plasmids (in sum these were 67 kpb in total size in our case) and transform them into <i>E.&nbsp;coli</i>, but demonstrated successful expression of large NRPS/PKS modules in our host strain.</p>
-
 
+
-
<p>We furthermore showed, that it is not only possible to assemble large plasmids (in sum these were about 64 kpb in total size in our case) and transform them into <i>E. coli</i>, but showed successful expression of large NRPS/PKS modules in our host strain.</p>
+
          
          
                     <h2>Discussion and outlook</h2>
                     <h2>Discussion and outlook</h2>
-
                      </html>{{:Team:Heidelberg/Templates/Delftibactin_Discussion}}<html>
+
                    <h3>Potential of the NRP Delftibactin for the Recovery of Gold from Electronic Waste</h3>
 +
<p>Delftibactin is a secondary metabolite naturally produced by <i>D.&nbsp;acidovorans</i> and has the ability to specifically precipitate gold from solutions [7]. Today, electronic waste has become an immense environmental problem not only in developed nations, but also third-world countries through the increasing export of waste and disposal challenges.</p>
 +
 
 +
<p>Therefore, an efficient method to recycle electronic waste is urgently needed. From our work we concluded, that delftibactin could be used as an efficient substance to recycle gold. In fact, many different electronic scraps contain considerable gold amounts such as PC mainboards (566 ppm in terms of weight) and mobile phones (350 ppm)[15]. Those electronic devices are more and more demanded by society though their average lifetime seems to decrease steadily. To date, existing methods for gold recycling and safe disposal of electronic waste are still very energy-consuming and harmful to the environment[16].</p>
 +
 
 +
<p>Nowadays, harsh substances, such as strong acids, are conventionally used for leaching gold from electronic waste [17]. Applied chemicals pose a potential thread to the environment as they could contaminate the biosphere. Biological agents could become an alternative to chemical clearance of metals from electronic waste. Nature's gold-altering microbes are non-pollutive and they are not genetically modified. While bacteria such as <i>Cupriavidus metallidurans</i> convert the dissolved element to its metallic form inside the cell [18], species like <i>Chromobacterium violaceum</i> [6] or <i>D.&nbsp;acidovorans</i> [7] secrete substances to the surrounding medium for gold precipitation. These microorganisms can therefore be engineered to increase the yield of bioleaching substances. </p>
 +
 
 +
              <h3>Successful Recycling of Gold from Electronic Waste with Delftibactin and Transformation of Delftibactin Gene Cluster in <i>E.&nbsp;coli</i></h3>
 +
<p>In our experiments, we successfully managed to dissolve electronic waste in aqua regia (i.e. nitro-hydrochloric acid) and neutralize the solution to receive gold chloride. When we added the supernatant of a <i>D.&nbsp;acidovorans</i> liquid culture to the gold chloride solution, pure gold nanoparticles were precipitated. Furthermore, we were able to melt the precipitated gold resulting in little gold flakes. The procedure already worked on a small scale in our project. With increased efforts, yields of delftibactin for industrial applications are conceivable. In those dimensions, gold could easily be recycled.  To investigate the question if the recovery of gold is feasible with delftibactin <a href="https://2013.igem.org/Team:Heidelberg/Modelling/Gold_Recovery">modeling</a> of the potential procedure was carried out. With delftibactin there is no further need for chemical reducing reagents to purify gold from solution. Nevertheless, the efficiency of our approach still has to be improved. We were able to apply delftibactin for the extraction of gold from electronic waste but still had to expose the CPUs with aqua regis to bring gold in solution. Ideally, one should get rid of the use of this highly corrosive mixture. </p>
 +
 
 +
<p>To further increase the efficiency of delftibactin production, we aimed at transferring the entire synthesis machinery for delftibactin into <i>E.&nbsp;coli</i>.  As the NRPS producing delftibactin is a very large enzyme complex consisting of many modules, amplification, cloning and transformation of the constructs was very challenging. Nonetheless, we successfully managed to amplify, assemble and transform all of the genes (in sum 59 kbp) required for production of delftibactin. In this process, we established protocols, including Gibson assembly of large fragments, and transformation of those constructs into <i>E.&nbsp;coli</i> via electroporation. Optimized procedures will ease the cloning of large customized NRPSs in future. </p>
 +
 
 +
          <h3>Successful Expression of NRPSs Despite Challenges Concerning the Toxicity of DelH</h3>
 +
<p>A hurdle to overcome is that the DelH displayed an above-average mutation rate when present in <i>E. &nbsp;coli</i> without the rest of the delftibactin pathway genes. DelH could potentially be toxic to the <i>E. &nbsp;coli</i>. Therefore, cells select for mutations that render the clones unable to express a functional DelH protein [19]. In our experiments, the putative selection pressure towards non-functional DelH was manifested in deletions, insertions or substitutions in the respective ribsosome binding side or at the beginning of DelH. However, we managed to obtain a single clone, which only possessed a point mutation that potentially does not interfere with protein expression since no false stop codon was created and no frame shift was caused.  Furthermore when introducing DelH without promotor or ribosome binding site we could obtain clones which did not have the mutations at the beginning of DelH. This is a clear indication that DelH in fact is toxic to <i>E.&nbsp;coli</i> when being expressed. The next step would be to clone the correct DelH into a backbone with weak promoter and ribosome binding site by conventional restriction enzyme based cloning, which would keep the detrimental impact of DelH as low as possible. However due to time limitations we have not been able to take this step yet. Nevertheless this strategy is very promising in obtaining a correct construct.</p>
 +
 
 +
<p>Moreover we proved the expression of the proteins DelE and DelG. Even for the clone with promotor and only one point mutation we were able to show that DelH is successfully expressed in <i>E.&nbsp;coli</i>. The point mutation causes an amino acid exchange (Alanine to Threonine) at the N terminus of the protein. Our prediction of the tertiary structure of the N-terminus of DelH
 +
(<a class="fancybox fancyFigure" title="Predicted tertiary structure formed by the first half of DelH using Phyre2. The N-terminus, which contains the mutation is located at the outer side of the protein (shown in blue)." href="https://static.igem.org/mediawiki/2013/5/53/DelH_Nterm.png" rel="gallery1">Fig.&nbsp;11</a>)
 +
indicated that the substituted amino acid in the mutant DelH is located at the outer side of the protein. Nevertheless it could still have an important function. Therefore it is not possible to foresee whether the mutation will have any effect on the function of DelH. However alanine is an unpolar amino acid whereas threonine is polar. If the mutation was in a functional region, this could have significant effects rendering DelH nonfunctional and keeping <i>E.&nbsp;coli</i> alive.</p>
 +
 
 +
<br/>
 +
<center>
 +
<a class="fancybox fancyGraphical" href="https://static.igem.org/mediawiki/2013/5/53/DelH_Nterm.png" title="Predicted tertiary structure formed by the first half of DelH using Phyre2. The N-terminus, which contains the mutation is located at the outer side of the protein (shown in blue).">
 +
    <img style="width:60%; margin-bottom:10px; padding:1%;border-style:solid;border-width:1px;border-radius: 5px;" src="https://static.igem.org/mediawiki/2013/5/53/DelH_Nterm.png">
 +
    <figcaption style="width:60%;"><b>Figure 11: Predicted tertiary structure formed by the first half of DelH using Phyre2.</b> The N-terminus, which contains the mutation is located at the outer side of the protein (shown in blue).</figcaption>
 +
</a>
 +
</center>
 +
<br/>
 +
 
 +
<p>Furthermore DelH might not be toxic anymore in the context of the complete assembly line, as toxicity most likely derives from conversion of secondary metabolites to harmful compounds. Therefore another strategy would be to directly cotransform the three plasmids, namely  DelH, DelRest (encoding all del genes except for delH) and the pIK8 construct (bearing the MethylMalonyl-CoA pathway, the PPTase sfp as well as the permeability device). Furthermore different host strains could be used as expression levels could potentially be controlled better. In particular, <i>E.Coli</i>  BL21 DE3 cells seem to be suitable for this approach as they overexpress the lac repressor. All in all there are still many promising approaches to obtain a functional DelH construct and therefore enable <i>E. &nbsp;coli</i> to express delftibactin. Consequently the application of recombinantly expressed delftibactin for the recycling of gold from electronic waste becomes conceivable
 +
(<a class="fancybox fancyFigure" title="Industrial application of delftibactin for gold recovery from electronic waste. The electronic waste would be dissolved and the supernatant of a bacteria culture producing delftibactin would be added. This would allow the easy recovery of the precipitated pure gold." href="https://static.igem.org/mediawiki/2013/4/47/Heidelberg_goldrecycling.png" rel="gallery1">Fig.&nbsp;12</a>).</p>
 +
 
 +
<br/>
 +
<center>
 +
<a class="fancybox fancyGraphical" href="https://static.igem.org/mediawiki/2013/4/47/Heidelberg_goldrecycling.png" title="Proposed industrial application of delftibactin for gold recovery from electronic waste. First, the electronic waste is dissolved. Subsequently, the supernatant of a bacteria culture producing delftibactin is added. This procedure allows for the easy recovery of the precipitated pure gold.">
 +
    <img style="width:60%; margin-bottom:10px; padding:1%;border-style:solid;border-width:1px;border-radius: 5px;" src="https://static.igem.org/mediawiki/2013/4/47/Heidelberg_goldrecycling.png">
 +
    <figcaption style="width:60%;"><b>Figure 12: Proposed industrial application of delftibactin for gold recovery from electronic waste.</b> First, the electronic waste is dissolved. Subsequently, the supernatant of a bacteria culture producing delftibactin is added. This procedure allows for the easy recovery of the precipitated pure gold.</figcaption>
 +
</a>
 +
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 +
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<p>1. Eisler R, Wiemeyer SN (2004) Cyanide hazards to plants and animals from gold mining and related water issues. Reviews of environmental contamination and toxicology: 21–54.</p>
+
<p>1. Perrine Chancerel (2010) Substance flow analysis of the recycling of small waste electrical and electronic equipment - An assessment of the recovery of gold and palladium. Technische Universität Berlin, Fakultät III - Prozesswissenschaften</p>
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<p>2. Eisler R (2004) Arsenic hazards to humans, plants, and animals from gold mining. In:. Reviews of environmental contamination and toxicology. Springer. pp. 133–165.</p>
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<p>2. Gesine Kauffmann (08.12.2011) Elektroschrott – die neue Schürfstelle für Gold. Die Welt. http://www.welt.de/wissenschaft/umwelt/article13755992/Elektroschrott-die-neue-Schuerfstelle-fuer-Gold.html</p>
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<p>3. Donato DB, Nichols O, Possingham H, Moore M, Ricci PF, et al. (2007) A critical review of the effects of gold cyanide-bearing tailings solutions on wildlife. Environment international 33: 974–984.</p>
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<p>3. Eisler R, Wiemeyer SN (2004) Cyanide hazards to plants and animals from gold mining and related water issues. Reviews of environmental contamination and toxicology: 21–54.</p>
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<p>4. Tay SB, Natarajan G, bin Abdul Rahim MN, Tan HT, Chung MCM, et al. (2013) Enhancing gold recovery from electronic waste via lixiviant metabolic engineering in Chromobacterium violaceum. Scientific reports 3.</p>
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<p>4. Eisler R (2004) Arsenic hazards to humans, plants, and animals from gold mining. In:. Reviews of environmental contamination and toxicology. Springer. pp. 133–165.</p>
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<p>5. Johnston CW, Wyatt MA, Li X, Ibrahim A, Shuster J, et al. (2013) Gold biomineralization by a metallophore from a gold-associated microbe. Nature chemical biology 9: 241–243.</p>
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<p>5. Donato DB, Nichols O, Possingham H, Moore M, Ricci PF, et al. (2007) A critical review of the effects of gold cyanide-bearing tailings solutions on wildlife. Environment international 33: 974–984.</p>
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<p>6. Strieker M, Tanović A, Marahiel MA (2010) Nonribosomal peptide synthetases: structures and dynamics. Current opinion in structural biology 20: 234–240.</p>
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<p>6. Tay SB, Natarajan G, bin Abdul Rahim MN, Tan HT, Chung MCM, et al. (2013) Enhancing gold recovery from electronic waste via lixiviant metabolic engineering in Chromobacterium violaceum. Scientific reports 3.</p>
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<p>7. Caboche S, Leclère V, Pupin M, Kucherov G, Jacques P (2010) Diversity of monomers in nonribosomal peptides: towards the prediction of origin and biological activity. Journal of bacteriology 192: 5143–5150.</p>
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<p>7. Johnston CW, Wyatt MA, Li X, Ibrahim A, Shuster J, et al. (2013) Gold biomineralization by a metallophore from a gold-associated microbe. Nature chemical biology 9: 241–243.</p>
-
<p>8. Baltz RH (2011) Function of MbtH homologs in nonribosomal peptide biosynthesis and applications in secondary metabolite discovery. Journal of industrial microbiology &amp; biotechnology 38: 1747–1760.</p>
+
<p>8. Strieker M, Tanovic A, Marahiel MA (2010) Nonribosomal peptide synthetases: structures and dynamics. Current opinion in structural biology 20: 234–240.</p>
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<p>9. Quadri LE, Weinreb PH, Lei M, Nakano MM, Zuber P, et al. (1998) Characterization of Sfp, a Bacillus subtilis phosphopantetheinyl transferase for peptidyl carrier protein domains in peptide synthetases. Biochemistry 37: 1585–1595.</p>
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<p>9. Caboche S, Leclère V, Pupin M, Kucherov G, Jacques P (2010) Diversity of monomers in nonribosomal peptides: towards the prediction of origin and biological activity. Journal of bacteriology 192: 5143–5150.</p>
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<p>10. de Jong A, Pietersma H, Cordes M, Kuipers OP, Kok J (2012) PePPER: a webserver for prediction of prokaryote promoter elements and regulons. BMC genomics 13: 299.</p>
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<p>10. Baltz RH (2011) Function of MbtH homologs in nonribosomal peptide biosynthesis and applications in secondary metabolite discovery. Journal of industrial microbiology &amp; biotechnology 38: 1747–1760.</p>
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<p>11. Reith F, Etschmann B, Grosse C, Moors H, Benotmane MA, et al. (2009) Mechanisms of gold biomineralization in the bacterium Cupriavidus metallidurans. Proceedings of the National Academy of Sciences 106: 17757–17762.</p>
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<p>11. Quadri LE, Weinreb PH, Lei M, Nakano MM, Zuber P, et al. (1998) Characterization of Sfp, a <i>Bacillus subtilis</i> phosphopantetheinyl transferase for peptidyl carrier protein domains in peptide synthetases. Biochemistry 37: 1585–1595.</p>
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<p>12. Cárcamo J, Ravera MW, Brissette R, Dedova O, Beasley JR, et al. (1998) Unexpected frameshifts from gene to expressed protein in a phage-displayed peptide library. Proceedings of the National Academy of Sciences 95: 11146–11151.</p>
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<p>12. de Jong A, Pietersma H, Cordes M, Kuipers OP, Kok J (2012) PePPER: a webserver for prediction of prokaryote promoter elements and regulons. BMC genomics 13: 299.</p>
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<p>13. Datsenko KA, Wanner BL. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A. 6;97(12):6640-5.</p>
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<p>14. Barbara A. Sampson, R. M. and S. A. B. (1989). Identification and Characterization of a New Gene of Escherichia coli K-12 Involved in Outer Membrane Permeability. Genetics, 122, 491-501.</p>
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<p>15. Cui J, Zhang L. (2008) Metallurgical recovery of metals from electronic waste: a review. J Hazard Mater. 30;158(2-3):228-56. </p>
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<p>16. Chancerel P, Bolland T, Rotter VS. (2011) Status of pre-processing of waste electrical and electronic equipment in Germany and its influence on the recovery of gold. Waste Manag Res. 2011 Mar;29(3):309-17.</p>
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<p>17. C.Y. Yap, N. Mohamed (2007) An electrogenerative process for the recovery of gold from cyanide solutions. Chemosphere Volume 67, Issue 8, Pages 1502–1510</p>
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<p>18. Reith F, Etschmann B, Grosse C, Moors H, Benotmane MA, et al. (2009) Mechanisms of gold biomineralization in the bacterium Cupriavidus metallidurans. Proceedings of the National Academy of Sciences 106: 17757–17762.</p>
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<p>19. Cárcamo J, Ravera MW, Brissette R, Dedova O, Beasley JR, et al. (1998) Unexpected frameshifts from gene to expressed protein in a phage-displayed peptide library. Proceedings of the National Academy of Sciences 95: 11146–11151.</p>
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Latest revision as of 03:53, 29 October 2013

Gold Recycling. Using Delftibactin to Recycle Gold from Electronic Waste.

Highlights

  • Production and purification of delftibactin, a gold-precipitating NRP, from its native, cultured host Delftia acidovorans.
  • Recovery of pure gold from electronic waste by Delftia acidovorans and purified delftibactin.
  • Optimization of the Gibson assembly method for the creation of large plasmids (> 30 kbp) with high GC content.
  • Amplification and cloning of all components required for recombinant delftibactin production.
  • Transfer of the entire pathway from Delftia acidovorans for the synthesis of delftibactin to Escherichia coli.

Abstract

Undoubtedly, gold is one of the most precious materials on earth. Besides its common use in art and jewelry, gold is also an essential component of our modern computers and cell-phones. Due to the fast turn-over of today’s high-tech equipment, millions of tons of electronic waste accumulate each year containing tons of this valuable metal. The main approach nowadays to recycle gold from electronic waste is by electrolysis. Unfortunately, this is a highly inefficient and expensive procedure, preventing most of the gold from being recovered.

In this subproject we want to demonstrate that the gold-precipitating natural secondary metabolite delftibactin, a non-ribosomal peptide produced by the bacterium Delftia acidovorans, can be used for the efficient recovery of gold from electronic waste.

Moreover we want to show that very large constructs such as the genes needed for the production of delftibactin which are encoded on a 59 kbp long gene cluster can be succesfully inserted into Escherichia coli. Furthermore the aim is to recombinantly express the responsible NRPSs with the promising perspective that delftibactin could readily be produced and used as an efficient way of gold recycling from electronic waste.

We managed to introduce and express the genes needed for delftibactin production. Furthermore the recycling of gold from electronic waste with delftibactin was successful. Consequently the industrial usage of recombinantly expressed delftibactin as an efficient method to recover gold becomes conceivable.

Introduction

The quest for a magical substance to generate gold from inferior metals stirred the imagination of generations. However, this substance, the Philosopher’s Stone, stands for more than just the desire to produce gold. There was a time when the fabled Philosopher’s Stone also represented wisdom, rejuvenation and health. Nowadays, gold is still of great importance for us as it is needed for most of our electronic devices.

In 2007, more than two tons of gold, worth $92 million, were discarded hidden in electronic waste in Germany [1]. Most of the precious element ends up on waste disposal sites as only a minor fraction of 10-15% [2] of the gold is recycled also due to the small amounts per device. Since our planet’s gold supplies are limited, the metal is more and more depleted and the value of gold continuously reaches all-time highs. In order to satisfy our society’s need for gold, we have to develop heavy mining techniques involving strong acids, causing devastating impact on humans and environment [3] [4] [5].

Besides economical usage of the resource gold, one way to reduce global demands for gold is elevation of gold recovery [6]. Intriguingly, nature itself offers a structure that has been reported to efficiently extract pure gold from solutions containing gold ions. This fascinating molecule is called delftibactin and is in fact a small peptide secreted by a gold-ion-tolerant bacterium called Delftia acidovorans [7].

This extremophile has the incredible ability to withstand toxic amounts of gold ions in contaminated soil. If one could culture these bacteria and produce delftibactin in large scales, could one potentially recover gold from electronic waste in a cost- and energy-efficient way? But what is the special feature of delfibactin to precipitate gold that efficiently?

Delftibactin is a non-ribosomal peptide (NRP) [7] [8]. The efficient and non-pollutant large-scale production of this NRP in Escherichia coli could revolutionize the recovery of gold from electronic waste and additionally highlight the plethora of versatile applications for non-ribosomal peptide synthetases (NRPSs). The most striking feature of these non-ribosomal synthetases is their ability to incorporate far more than the 21 common amino acids into peptides. They make use of numerous modified and even non-proteinogenic amino acids to assemble peptides of diverse functions [9].

Delftibactin is a NRP produced by a hybrid NRPS/polyketide synthase (PKS) system. In their recent publication, Johnston and colleagues [7] predicted that the enzymes responsible for producing delftibactin are encoded on a single gene cluster, hereafter referred to as del cluster (Fig. 1). It comprises 59 kbp encoding for 21 genes. DelE, DelF, DelG and DelH constitute the hybrid NRPS/ PKS system producing delftibactin, with DelE, DelG and DelH comprising the NRPS and DelF the PKS. The remaining enzymes involved in the delftibactin synthesis pathway are required for maturation or post-synthesis modification of delftibactin. The predicted activities [7] of the proteins are:

  1. DelA: MbtH-like protein, most likely required for efficient delftibactin synthesis [10]
  2. DelB: thioesterase
  3. DelC: 4’-phosphopanteinyl transferase: required for maturation of ACP/PCP subunits
  4. DelD: taurine dioxygenase
  5. DelL: Ornithine N-monooxygenase
  6. DelP: N5-hydroxyornithine formyltransferase


Figure 1: Cluster of genes responsible for Delftibactin production. The cluster consists of the genes Daci_4754 to Daci_4765. The proteins DelE, DelF, DelG and DelH are directly responsible for the production of delftibactin. Domain architecture of the NRPS-PKS hybrid assembly-line is shown which consists of adenylation (A), thiolation (T), condensation, (C), ketosynthase (KS), acyltransferase (AT), ketoreductase (KR) and thioesterase domains. The predicted structure of delftibactin is shown below. Figure adopted from [7].

We introduced the large del cluster into the commonly used, easy-to-culture model organism E. coli with the aim of recombinant delftibactin expression. Although the del cluster contains the native PPTase of D. acidovorans we additionally introduced the sfp phosphopanteinyl transferase from Bacillus subtilis as this PPTase is able to activate a wide variety of PKSs including those from Saccharomyces cerevisiae. Importantly, it has been proven to work in E. coli by the indigoidine project of our own team [11]. Additionally, DelF, the polyketide synthetase of the del cluster requires methylmalonyl-CoA as substrate. This metabolite, from now on abbreviated as mmCoA is not produced by E. coli. Therefore, we also transfer the mmCoA synthesis pathway from B. subtilis into E. coli. This should allow for efficient production of recombinant delftibactin.

As the del cluster starts with Daci_4760 (DelA; Daci IDs are NCBI Gene gene symbols, Del* gene names as referred to in [12]) and promoters within the Del-cluster were bioinformatically predicted upstream of Daci_4750 (DelK), Daci_4760 (DelA) and Daci_4746 (DelO) we assumed that the entire sequence from Daci_4760 (DelA) to Daci_4753 (DelH) is transcribed as a single polycistronic mRNA of approximately 40 kbp in size [12].

Facing these challenges, we decided to approach the project by cultivation of D. acidovorans and the isolation of native delftibactin to reproduce the findings of Johnston and colleagues [7].

.

In order to achieve recombinant expression of delftibactin, we decided to introduce constructs coding for the delftibactin-cluster the methylmalonyl-CoA pathway and the PPTase sfp. In addition, we transformed the permeability device BBa_I746200 from the parts registry for the export of recombinant delftibactin out of the target organism E. coli. The desired genes from the del cluster were subdivided onto two different plasmids in order to decrease plasmid size and thereby avoid the intricacies expected for cloning of a single 59 kbp plasmid as well as to allow for faster trouble shooting in case issues with the cloning of particular genes occur:

  1. Plasmid: methylmalonyl-CoA pathway, PPTase sfp & permeability device BBa_I746200, transcription regulated by inducible lac promoter, chloramphenicol resistance;
  2. Plasmid: DelH, transcription regulated by inducible lac promoter, ampicillin resistance;
  3. Plasmid: DelA-P, genes of the del cluster required for production of delftibactin, transcription regulated by inducible lac promoter, kanamycin resistance.

Here we show successful amplification, cloning and transformation of plasmids above 30 kbp in size as well as expression of the desired genes of the del cluster from its natural host D. acidovorans. Furthermore, we demonstrate efficient recovery of purified gold from electronic waste using the non-ribosomal peptide delftibactin. Additionally, we proved toxicity of DelH for our target organism E. coli when expressed as the only gene of the del cluster: cloning of DelH into a construct without promoter lead to depletion of DelH from our target system which previously had been selecting for mutated versions of DelH.

Results

Efficient Recycling of Gold from Electronic Waste Using Endogenously-Derived Delftibactin

As a first step into the direction of an environmentally friendly procedure for recycling gold from gold-containing waste, we wanted to show that the non-ribosomal peptide delftibactin can be used to precipitate gold from gold ion-containing solutions.

We obtained D. acidovorans DSM-39 from the DSMZ and successfully reproduced the paper by Johnston and colleagues [7]. What they had been able to show was that delftibactin selectively precipitates gold from gold solution. In our experiments, precipitation on agar plates worked even better than described by Johnston et al. (Fig. 2). D. acidovorans is capable to precipitate solid gold from gold chloride solution as purple-black nanoparticles.


Figure 2: D. acidovorans precipitates elementary gold from gold solution. ACM agar plate with D. acidovorans DSM-39 overlaid with 0.2% HAuCl4 in 0.5% agarose. D. acidovorans had only been growing on the upper part of the shown plate.One can clearly see that gold nanoparticles are exclusively formed on the part of the plate harboring bacteria.

We also showed that another strain, D. acidovorans SPH-1, is also able to precipitate gold ions to gold nanoparticles. When using the supernatant of a culture gold nanoparticles were precipitated at an amount which caused a color change to black already at low concentrations of 0.35 µg/ml of gold chloride. With increasing concentration of gold chloride more nanoparticles formed, even though the process became slower above a certain concentration (Video 1 and Fig. 3a). An optimum is visible at a concentration of about 1.15 µl/ml of gold solution. Johnston et al. [7] described that the optimal ratio of gold ions to delftibactin is 1:1. Therefore it can be concluded that 1.96 nmol of delftibactin was present in 1 ml of D. acidovorans supernatant. Furthermore, we melted the purple-black nanoparticles to shiny, solid gold as shown in Fig. 3b,c,d.


Video 1: Precipitation of elementary gold by delftibactin is dependent on the ratio of peptide to gold ions. Different concentrations of gold solution applied to the supernatant of a D. acidovorans SPH-1 culture. From left to right: 0 µg/ml, 0.15 µg/ml, 0.35 µg/ml, 0.55 µg/ml, 0.75 µg/ml, 0.95 µg/ml, 1.15 µg/ml, 1.35 µg/ml, 1.55 µg/ml, 1.75 µg/ml, 1.95 µg/ml, 2.15 µg/ml, 2.35 µg/ml and 2.55 µg/ml HAuCl4. Black gold nanoparticles form due to the precipitation of solid gold from solution by the NRP delftibactin. According to Johnston and colleages, gold precipitation is most sufficient at peptide to gold ions ration of 1:1 [7], suggesting an amount of peptide of 1.96 nmol. The process is shown in time-lapse and had an actual duration of 8min 23s.

Figure 3: Supernatant of D. acidovorans culture is sufficient for gold precipitation. a) Sequences of movie over time showing gold precipitation in D. acidovorans supernatant using different concentrations of HAuCl4. From left to right: 0 µg/ml, 0.15 µg/ml, 0.35 µg/ml, 0.55 µg/ml, 0.75 µg/ml, 0.95 µg/ml, 1.15 µg/ml, 1.35 µg/ml, 1.55 µg/ml, 1.75 µg/ml, 1.95 µg/ml, 2.15 µg/ml, 2.35 µg/ml and 2.55 µg/ml. b) Sparkling gold appearing in the melting pot after the precipitation of gold ions using the supernatant of D. acidovorans SPH-1. c) Final recovered solid gold with Delftibactin collected in tube. d) Solid gold covering the walls of a 2 ml tube after the application of delftibactin to gold solution.

Next, we established a purification protocol for delftibactin using HP20 resins. Additionally, we proved precipitation of gold by the purified delftibactin (Fig. 4) and detected it by Micro-TOF (Fig. 5).

Figure 4: Purified Delftibactin precipitates gold from gold solution. From left to right: ddH2O, 1:10 ACM media in water, 1:10 supernatant of D. acidovorans SPH-1 in water, 1:10 filtered supernatant of D. acidovorans SPH-1 in water, 1:10 purified delftibactin in water; gold solution: 0.6 µg/ml HAuCl4.


Figure 5: D. acidovorans SPH-1 secretes delftibactin. Micro-TOF results for ACM media (top) compared to purified supernatant of a D. acidovorans SPH-1 liquid culture (middle). Delftibactin is present in D. acidovorans SPH-1 supernatant but not in the pure ACM-media as can be seen as peak of the Micro-TOF spectra at about 1055.5, 1033.5, 517.2 and 539.2 shown in the enlargements (bottom).

Moreover we were interested in the potential of delftibactin for the recycling of gold from electronic waste. An important prerequisite for efficient gold recycling from electronic waste is the feasibility for gold precipitation from gold solutions of low concentration. Undoubtedly, this is likely the case for most gold solutions derived from electronic waste.Therefore, we used an old, broken CPU and established a protocol for dissolving gold from gold-containing metal waste. We incubated gold covered CPU pins in aqua regia resulting in a gold-ion containing solution (Fig. 6a-c). We showed precipitation of dissolved gold recovered from electronic waste by D. acidovorans. Adding this solution to D. acidovorans SPH-1 agar plates resulted in the formation of solid gold nanoparticles (Fig. 6d-f).


Figure 6: D. acidovorans can be used for gold recovery from electronic waste. a) Pins removed from an old CPU. b) Green solution of dissolved pins in aqua regia. c) Gold chloride solution obtained after the dilution of an old CPU. d) "Dissolved electronic waste" and D. acidovorans. e) "Dissolved electronic waste" on D. acidovorans. f) Precipitated "dissolved electronic waste" and D. acidovorans.

Taken together, we have successfully established a method enabling the recycling of pure gold from electronic waste using delftibactin produced by D. acidovorans.

Although the recycling was working efficiently in our hands, the approach of using the natural D. acidovorans bacterial strain for delftibactin production on a larger scale has the following disadvantages:

  1. D. acidovorans are relatively slow in growth (colony formation on plates occurs after 2-3 days)
  2. Efficient production of delftibactin requires the strain to be grown in ACM media, which is moreexpensive, compared to typical E. coli growth media, making the procedure less economic.

Therefore, we wanted to engineer an E. coli strain producing delftibactin in high yields, thereby circumventing the above mentioned limitations.

Expression of Del Cluster Genes in E. coli

We developed a cloning strategy which allowed us to clone all necessary genes encoding the delftibactin-producing non-ribosomal peptide synthetases and polyketide synthetases from the del cluster (about 59 kbp in total) and express them in E. coli. In addition, introducing the methylmalonyl-CoA pathway into E. coli provided one of the basic substrates for the del pathway which is endogenously not present in E. coli.

Therefore, our initial aim was the genomic integration of the genes encoding for the methylmalonyl-CoA pathway into E. coli using the lambda red system established by [13]. This pathway is required for sufficient delftibactin production, as it supplies the substrate methylmalonyl-CoA for DelF, the PKS of the delftibactin cluster. As several genomc integration attempts did not yield positive results, as verified by colony-PCR with the forward primer binding to the genomic region and the reverse primer to the insert (a representative gel image can be seen in the lab journal), a new strategy was developed. Two plasmids were created: pIK2 containing the mm-CoA pathway amplified from Streptomyces coelicolor as well as the PPTase sfp, amplified from Bacillus subtilis in the BioBrick backbone pSB3C5. The permeability device (BBa_I746200) for the outer membrane of E. coli was cloned into another plasmid (pIK1). Since the iGEM Team Cambridge 2007 showed that BBa_I746200 is toxic if produced in higher quantities we inserted it into pIK2 between the two terminators driven by a weak promoter (BBa_J23114) and a weak RBS (BBa_B0030), resulting in the final plasmids pIK8 with a total size of 9,467 bp, which was transformed into the E.coli strains DH10ß and BL21 DE3 via electroporation.

Details of our cloning strategy are shown in Fig. 7. Notably, the three plasmids we wanted to assemble are of large size (23, 32 and 10 kbp in size) and cloning was further complicated due to the high GC content and presence of repetitive elements within the del genes.


Figure 7: Cloning strategy for the production of delftibactin in E. coli. The genes DelA to DelH, DelL, DelO and DelP from the cluster for delftibactin production are introduced in two plasmids (shown in yellow). Additional genes needed for the production and export of delftibactin are located on a third plasmid (shown in blue).

Gibson Assembly & Transformation

Assembly of plasmids above 30 kbp in size composing of multiple fragments is challenging when using conventional restriction enzyme based cloning. Thus, we decided to use Gibson Assembly, a method which was introduced to the iGEM community by Cambridge in iGEM 2010 as a powerful alternative to common cloning procedures. The assembled constructs of up to 32 kbp in size were transformed into E. coli via electroporation. Correct assembly of the fragments was tested by analytical restriction digests. The exemplary restriction digests shown above (Fig. 8) confirmed the correct assembly of the three desired constructs as it displays the expected band pattern. For cloning of the mm-CoA pathway, clones (6 and 9) show the expected restriction pattern (Fig. 8a). Sequencing of the assembly sites of these constructs confirmed the restriction digest results.

Cloning of the DelRest plasmid was validated by different suitable analytic restriction digests (Fig. 8b) and also confirmed by sequencing. The sequence was compared with the available D. acidovorans SPH-1 reference sequence obtained from NCBI (for further information please visit our labjournal). The high quality of the alignment shows that Gibson assembly is a suitable cloning approach for rapid assembly of large NRPS and PKS expression constructs.

Analytical restriction digest of DelH (Fig. 8c) also gave rise to a number of positive clones. However, the sequencing results derived from all DelH showed various mutations, which were exclusively located within the region of the first DelH forward primer. Most of these mutations are deletions present in the first 30 bp of the DelH coding region thereby resulting in frameshifts and formation of a stop codon disposing E. Coli of expression of DelH. As there was no clone without mutations, we proceeded with DelH clone C5, as this clone did not have any bp deletion but only harbored a substitution mutation at bp position 28 of the ORF, leading to the conversion of Alanine at position 10 to Threonine. Two representative sequences compared to D. acidovorans SPH-1 are listed below (Tab. 1) and display two of the observed mutations in different DelH clones.


Figure 8: Restriction digest of plasmids coding for MMCoA, DelH and DelRest which are needed in the NRPS/PKS pathway to generated Delftibactin. a) Digested minipreps of the pIK8-plasmid of four different clones. Clones 6 and 9 show the expected pattern. b) shows the minipreps of one clone of E. coli transformed with the 32 kbp DelRest plasmid which was digested with three different enzymes. All digests show the expected patterns. c) shows the restriction digest of the DelH plasmid with PvuI. The digest of clone 5 shows the anticipated bands and is probably positive.

Tab.1 DelH 5’ sequence, in which most mutations were observed. The ATG start codon is depicted in bold. The table shows the sequence comparison between the DelH reference strand of D. acidovorans and two different exemplary E. coli clones transformed with the plasmid pHM04 (assembled DelH expression vector). The second line shows the accumulated deletion and the third line shows the clone containing 'only' single base pair substitution. Deletions appeared quite frequently while a substitution was only found in a single clone C5. The substitution changes the corresponding Alanine codon to Threonine.

Organism Plasmid containing DNA -Sequence Conclusion
D. acidovorans none ATG GACCGTGGC CGCCTGCGC CAAATCGC correct
E. coli DH10ß pHM04 ATG GACCGTG-C CGCCTGCGC CAAATCGC deletion
E. coli DH10ß C5 pHM04 ATG GACCGTGGC CGCCTGCGC CAAATCAC substitution

Due to these observations, we hypothesize that expression of DelH is toxic for E. coli. Therefore natural selection only leads to survival of those clones which incorporate constructs giving rise to unfunctional, truncated DelH proteins or avoiding any expression. This phenomenon of frequent mutations within the primer binding site was also observed when we started cloning of the permeability device used in the pIK8 construct. The sequenced plasmids displayed a high accumulation of mutations compared to other constructs. In case of the methylmalonyl-CoA plasmid (pIK8), the problem was solved by the usage of a weak promoter and a weak ribosome binding site from the parts registry for driving the expression of the permeability device (see above). Based on this knowledge, DelH is currently being assembled into the desired backbone pSB6A1 containing a weak promoter BBa_J23114 and the ribosome binding site BBa_B0032, also reducing expression. While the new plasmid is constructed, the following experiments were performed with the C5 clone as we hypothesized that DelH C5 bearing the single nucleotide exchange at position 28 might still show expression of functional DelH when transformed into E. coli (the corresponding amino acid exchange is located at the N-terminus).

Expression of the Delftibactin NRPSs & Associated Genes

For further characterization of our constructs, we analyzed expression of the delftibactin NRPS/PKS pathway in E. coli. For expression of DelH and DelRest, SDS-PAGEs were conducted followed by Coomassie staining. Native cells as well as cells transformed with the plasmid backbones obtained from the parts registry containing the used antibiotic resistance markers served as control groups. The proteins DelE, DelG and DelH are significantly larger than any protein that is expressed by our host E. coli. Therefore, the expression of the introduced genes was detectable on the SDS-PAGE (Fig. 9) without specific labeling of the proteins. Although expression of these large proteins was weak, clear distinct bands of the sizes predicted for DelE and DelG were detected. Accordingly a band at the predicted size of DelH for the clone transformed with DelH C5 was visible. As the lac promoter regulating expression of DelE and DelG also drives expression of DelA, DelB, DelC, DelD and DelF, one can conclude simultaneous expression of these del proteins. This is also in accordance with the predicted distribution of promoters within the delfticbatin cluster.


Figure 9: E. coli express the NRPS of the delftibactin production pathway.SDS-PAGE of: a) E. coli DH10ß colony D8w containing the DelRest plasmid pFSN. E. coli DH10ß transformed with pSB4K5 is used as negative control. The bands at the heights of about 190 kDa and 350 kDa (depicted by black arrows) indicate the expression of DelE and DelG. b) E. coli BL21 DE3 colony C5 containing the DelH plasmid. E. coli BL21 is used as negative control. A weak band at a height of more than 260 kDa (largest protein band of the ladder) is present. This could potentially be a band at the height of 670 kDa and thus hint at the production of the NRPS DelH.

Furthermore, the expression of the PPTase was verified by performing an IndC activity assay established by the indigoidine subproject. The indigoidine synthetase IndC activity is dependent on the presence of a functional PPTase. Upon activation, indC produces the blue pigment indigoidine. Co-transformation of the corresponding plasmid pIK8 construct (enabling PPTase expression) with an IndC indigoidine synthetase expression construct lacking a PPTase expression cassette (pRB22) was conducted. The transformed E. coli displayed inhibited growth and developed the expected blue phenotype. From these results, we conclude that the PPTase on pIK8 is functionally expressed (note: decelerated growth kinetics of E. coli results from the metabolic burden that is caused by the synthesis of the indigoidine).

For proving the production of the permeability device which is needed for export of delftibactin, a zone of inhibition test with bactracin was performed. Bacitracin is an antibiotic not able to pass the bacterial cell wall by passive transport [14]. Growth of bacteria containing the permeability device was inhibited upon application of bacitracin confirms the expression of the transporter. Growth of control cells without the device was not impaired by application of bacitracin (Fig. 10).


Figure 10: Zone of inhibition test proves functionality of the permeability device. Left: TOP10-pIK8.6, right: TOP10-pIK8.1 (negative control); counterclockwise, starting top right: 8 µl, 4 µl, 2 µl, 1 µl bacitracin. It can be seen that the device works as the cells transformed with the construct die upon application of bacitracin.

In conclusion, we successfully expressed the recombinant delftibactin NRPS/ PKS pathway as well as the required methylmalonyl-CoA pathway, the PPTase and permeability device in E. coli.

Furthermore, we showed, that it is not only possible to assemble large plasmids (in sum these were 67 kpb in total size in our case) and transform them into E. coli, but demonstrated successful expression of large NRPS/PKS modules in our host strain.

Discussion and outlook

Potential of the NRP Delftibactin for the Recovery of Gold from Electronic Waste

Delftibactin is a secondary metabolite naturally produced by D. acidovorans and has the ability to specifically precipitate gold from solutions [7]. Today, electronic waste has become an immense environmental problem not only in developed nations, but also third-world countries through the increasing export of waste and disposal challenges.

Therefore, an efficient method to recycle electronic waste is urgently needed. From our work we concluded, that delftibactin could be used as an efficient substance to recycle gold. In fact, many different electronic scraps contain considerable gold amounts such as PC mainboards (566 ppm in terms of weight) and mobile phones (350 ppm)[15]. Those electronic devices are more and more demanded by society though their average lifetime seems to decrease steadily. To date, existing methods for gold recycling and safe disposal of electronic waste are still very energy-consuming and harmful to the environment[16].

Nowadays, harsh substances, such as strong acids, are conventionally used for leaching gold from electronic waste [17]. Applied chemicals pose a potential thread to the environment as they could contaminate the biosphere. Biological agents could become an alternative to chemical clearance of metals from electronic waste. Nature's gold-altering microbes are non-pollutive and they are not genetically modified. While bacteria such as Cupriavidus metallidurans convert the dissolved element to its metallic form inside the cell [18], species like Chromobacterium violaceum [6] or D. acidovorans [7] secrete substances to the surrounding medium for gold precipitation. These microorganisms can therefore be engineered to increase the yield of bioleaching substances.

Successful Recycling of Gold from Electronic Waste with Delftibactin and Transformation of Delftibactin Gene Cluster in E. coli

In our experiments, we successfully managed to dissolve electronic waste in aqua regia (i.e. nitro-hydrochloric acid) and neutralize the solution to receive gold chloride. When we added the supernatant of a D. acidovorans liquid culture to the gold chloride solution, pure gold nanoparticles were precipitated. Furthermore, we were able to melt the precipitated gold resulting in little gold flakes. The procedure already worked on a small scale in our project. With increased efforts, yields of delftibactin for industrial applications are conceivable. In those dimensions, gold could easily be recycled. To investigate the question if the recovery of gold is feasible with delftibactin modeling of the potential procedure was carried out. With delftibactin there is no further need for chemical reducing reagents to purify gold from solution. Nevertheless, the efficiency of our approach still has to be improved. We were able to apply delftibactin for the extraction of gold from electronic waste but still had to expose the CPUs with aqua regis to bring gold in solution. Ideally, one should get rid of the use of this highly corrosive mixture.

To further increase the efficiency of delftibactin production, we aimed at transferring the entire synthesis machinery for delftibactin into E. coli. As the NRPS producing delftibactin is a very large enzyme complex consisting of many modules, amplification, cloning and transformation of the constructs was very challenging. Nonetheless, we successfully managed to amplify, assemble and transform all of the genes (in sum 59 kbp) required for production of delftibactin. In this process, we established protocols, including Gibson assembly of large fragments, and transformation of those constructs into E. coli via electroporation. Optimized procedures will ease the cloning of large customized NRPSs in future.

Successful Expression of NRPSs Despite Challenges Concerning the Toxicity of DelH

A hurdle to overcome is that the DelH displayed an above-average mutation rate when present in E.  coli without the rest of the delftibactin pathway genes. DelH could potentially be toxic to the E.  coli. Therefore, cells select for mutations that render the clones unable to express a functional DelH protein [19]. In our experiments, the putative selection pressure towards non-functional DelH was manifested in deletions, insertions or substitutions in the respective ribsosome binding side or at the beginning of DelH. However, we managed to obtain a single clone, which only possessed a point mutation that potentially does not interfere with protein expression since no false stop codon was created and no frame shift was caused. Furthermore when introducing DelH without promotor or ribosome binding site we could obtain clones which did not have the mutations at the beginning of DelH. This is a clear indication that DelH in fact is toxic to E. coli when being expressed. The next step would be to clone the correct DelH into a backbone with weak promoter and ribosome binding site by conventional restriction enzyme based cloning, which would keep the detrimental impact of DelH as low as possible. However due to time limitations we have not been able to take this step yet. Nevertheless this strategy is very promising in obtaining a correct construct.

Moreover we proved the expression of the proteins DelE and DelG. Even for the clone with promotor and only one point mutation we were able to show that DelH is successfully expressed in E. coli. The point mutation causes an amino acid exchange (Alanine to Threonine) at the N terminus of the protein. Our prediction of the tertiary structure of the N-terminus of DelH (Fig. 11) indicated that the substituted amino acid in the mutant DelH is located at the outer side of the protein. Nevertheless it could still have an important function. Therefore it is not possible to foresee whether the mutation will have any effect on the function of DelH. However alanine is an unpolar amino acid whereas threonine is polar. If the mutation was in a functional region, this could have significant effects rendering DelH nonfunctional and keeping E. coli alive.


Figure 11: Predicted tertiary structure formed by the first half of DelH using Phyre2. The N-terminus, which contains the mutation is located at the outer side of the protein (shown in blue).

Furthermore DelH might not be toxic anymore in the context of the complete assembly line, as toxicity most likely derives from conversion of secondary metabolites to harmful compounds. Therefore another strategy would be to directly cotransform the three plasmids, namely DelH, DelRest (encoding all del genes except for delH) and the pIK8 construct (bearing the MethylMalonyl-CoA pathway, the PPTase sfp as well as the permeability device). Furthermore different host strains could be used as expression levels could potentially be controlled better. In particular, E.Coli BL21 DE3 cells seem to be suitable for this approach as they overexpress the lac repressor. All in all there are still many promising approaches to obtain a functional DelH construct and therefore enable E.  coli to express delftibactin. Consequently the application of recombinantly expressed delftibactin for the recycling of gold from electronic waste becomes conceivable (Fig. 12).


Figure 12: Proposed industrial application of delftibactin for gold recovery from electronic waste. First, the electronic waste is dissolved. Subsequently, the supernatant of a bacteria culture producing delftibactin is added. This procedure allows for the easy recovery of the precipitated pure gold.

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