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| | <div class="abstract"> | | <div class="abstract"> |
| | <p>ABSTRACT<br/></p> | | <p>ABSTRACT<br/></p> |
| − | <p>In order to detect the presence of the specific non-natural amino acid (nnAA) <i>in vivo</i> the concept of <b>amber suppression</b> is used [1]. This means that the occurrence of the amber stop codon (UAG) in an ORF does not stop the protein translation but codes for a specific nnAA, in our case <i>O</i>-methyl-<span style="font-variant:small-caps">l</span>-tyrosine (OMT). However, the incorporation requires the presence of the nnAA in the medium, otherwise the translation stops. The mechanism requires a <b>tRNA</b> with an anticodon complementary to the amber stop codon and an aminoacyl RNA synthetase (aaRS) loading the tRNA with the specific nnAA. The tRNA and aaRS combination is called an 'orthogonal pair'.</p> | + | <p>In order to detect the presence of a specific non-natural amino acid (nnAA) <i>in vivo</i> the concept of <b>amber suppression</b> is used [1]. This means that the occurrence of the amber stop codon (UAG) in an ORF does not stop the protein translation but codes for a specific nnAA, in our case <i>O</i>-methyl-<span style="font-variant:small-caps">l</span>-tyrosine (OMT). However, the incorporation requires the presence of the nnAA in the medium, otherwise the translation stops. The mechanism requires a <b>tRNA</b> with an anticodon complementary to the amber stop codon and an aminoacyl RNA synthetase (aaRS) loading the tRNA with the specific nnAA. The tRNA and aaRS combination is called an 'orthogonal pair'.</p> |
| | <a href="https://2016.igem.org/Team:TU_Darmstadt/Lab/OrthogonalPair"><button class="read_more" id="lab1b">Interested? Read more</button></a> | | <a href="https://2016.igem.org/Team:TU_Darmstadt/Lab/OrthogonalPair"><button class="read_more" id="lab1b">Interested? Read more</button></a> |
| | </div> | | </div> |
| − | <!-- <div class="content" id="lab1c" style="display:none"> | + | |
| − | <p><h6>Orthogonal Pair</h6></p>
| + | |
| − | <p>The recognition of the amber stop codon requires a tRNA with an anticodon complementary to the amber stop codon and an aaRS specifically loading the tRNA with the nnAA. In order to ensure the nnAA is not incorporated for other codons except the amber stop codon, the tRNA and the aaRS have to be orthogonal to the natural aaRS's and tRNAs. This means the aaRS must not load any other tRNA and the tRNA must not be loaded by any other aaRS. Therefore, <i>Wang et. al</i> originally used the tyrosyl-tRNA and tyrosyl-RS from the methanogenic archaeon <i>Methanocaldococcus jannaschii</i> : The anticodon of the tRNA was replaced by the amber anticodon and the aaRS was optimized for the recognition of OMT in place of tyrosine via directed evolution. Introduced into <i>Escherichia coli</i>, this pair is orthogonal to every natural pair due to the genetic distance between <i>E. coli</i> and <i>M. jannaschii</i>. Nowadays, over 70 different aaRS <b>[3]</b> have been designed, each one capable of incorporating a specific amino acid, many of them with special chemical characteristics, allowing e.g. 'click' chemistry or photoactivation.</p>
| + | |
| − |
| + | |
| − | <p> In our project, we use an orthogonal pair from the <a>"Expanded Genetic Code Measurement Kit"</a> as template, specifically the one used for incorporation of ONBY <a>(BBa_SomeBrick)</a>, and replaced the ORF with an <i>E. coli</i> codon optimized ORF for OMT-RS. Furthermore we placed the OMT-RS coding region behind a RBS <a>(BBa_B0034)</a> and a strong constitutive Anderson promotor <a>(BBa_J23101)</a>. A successful expression of the OMT-RS gene in this construct was observed (Fig. 1).</p>
| + | |
| − | | + | |
| − | <p><h6>Usage of amber codon</h6></p>
| + | |
| − | <p>The incorporation of an amber codon causes the complete translation of the respective protein in presence of the nnAA and cancels the translation in absence. In our implementation the amber codon is replacing a codon in the beginning of the ORFs of the <a href="bla">Colicin E2 Immunity protein</a> (Y8OMT) and the <a href="bla">Zif23-GCN4 repressor</a> (F4OMT). In consequence, both proteins are functionally produced only if the nnAA is available in sufficient concentration in the medium.</p>
| + | |
| − | | + | |
| − | <p><h6>The non-natural amino acid</h6></p>
| + | |
| − | <p>We decided to use <i>O</i>-methyl-<span style="font-variant:small-caps">l</span>-tyrosine for our nnAA due to its multiple advantageous properties:</p>
| + | |
| − | <ul style="list-style-type:disc">
| + | |
| − | <li>Low costs</li>
| + | |
| − | <li>Nontoxic</li>
| + | |
| − | <li>Unproblematic import into cells</li>
| + | |
| − | <li>No further biochemical activity</li>
| + | |
| − | <li>Feasible chemical synthesis</li>
| + | |
| − | <li>Stable in water</li>
| + | |
| − | <li>Unavailable in nature</li>
| + | |
| − | <li>Well documented</li>
| + | |
| − | <li>Low interference with protein activity</li>
| + | |
| − | </ul>
| + | |
| − | <p>An institute or company could choose its own specific nnAA with the corresponding orthogonal pair. This enables a reliable protection against corporate espionage or bioterrorism, since the opposing party does normally not know which nnAA is used in the respective application. However, using the same nnAA like OMT in every application should prevent the biological and genetic spread of the respective microorganism in the environment.</p>
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| − |
| + | |
| − | </div> -->
| + | |
| | <div class="verlinked" id="repo"><h5>REPORTER</h5></div> | | <div class="verlinked" id="repo"><h5>REPORTER</h5></div> |
| | <div class="abstract"> | | <div class="abstract"> |
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| | <a href="https://2016.igem.org/Team:TU_Darmstadt/Lab/Reporter"><button class="read_more" id="lab2b">Interested? Read more</button></a> | | <a href="https://2016.igem.org/Team:TU_Darmstadt/Lab/Reporter"><button class="read_more" id="lab2b">Interested? Read more</button></a> |
| | </div> | | </div> |
| − | <!-- <div class="content" id="lab2c" style="display:none">
| |
| − | <p><h6> The Reporter System</h6></p>
| |
| − | <p>For the detection of a low non-natural amino acid concentration, which in this case is <i>O</i>-methyl-L-tyrosine, we designed a reporter system that includes the reporter protein <b>mVenus</b>. In order to make sure that the expression of mVenus does only start at a low OMT concentration, we use a dimeric repressor. An <i>amber</i> mutation was introduced to the DNA sequence of the repressor. This <i>amber</i> mutation --><!--LINK: zu amber suppression von colicin--><!-- leads to OMT being integrated in the dimeric repressor protein. However, the repression of the mVenus promoter can only be executed if there is a sufficient amount of OMT in the medium. If the OMT concentration drops below a threshold, the expression of mVenus is induced. As a result, we can detect a yellow fluorescence signal. <br/>
| |
| − | The reason why we utilize a dimeric repressor was that this kind of repressor binds strongly to the respective promotor. Moreover, this dimeric repressor creates a sigmoidal repression curve (x‑axis = concentration of OMT; y‑axis = repressor molecule concentration). Once the concentration of OMT drops, we get a signal quickly.</p><p>To make sure that the repression does not take place even if the concentration of OMT is low, an LVA degradation tag is expressed with the dimeric repressor. To ensure that there is no permanent fluorescent signal caused by mVenus, it is marked with an LVA degradation tag as well. So, both proteins degrade quite fast after their translation. To connect this system to the expression of colicin, we can use different Anderson promoters for test purposes (BBa_J23104, BBa_J23113, BBa_J23107, BBa_J23100 and BBa_J23114). By doing so, we take care that the fluorescent signal of mVenus appears before the expression of the DNase that degrades the DNA and makes the genomic information inaccessible.</p>
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| − |
| |
| − | <p><h6> mVenus</h6></p>
| |
| − | <p>The fluorescent reporter protein mVenus is a mutant of the green fluorescent protein GFP which is often used for fluorescence assays. Due to mutagenesis (F46L/F64L/M153T/V163A/S175G), the maturation time is decreased compared to GFP. In general, the maturation process can be divided in the folding step and formation of the chromophore. During the maturation process, the chromophore formation is the rate-limiting step. After the folding, a torsional rearrangement effects the formation of the chromophore. This results from the involved residues being in close proximity. After cyclization of two amino acids has taken place, oxidation is the final step. Molecular oxygen is necessary for the reaction that generates the delocalized pi electron system, resulting in the fluorophore being maturated and fluorescent. It is protected by the Beta-barrel protein from interfering influences. All the processes are influenced by the general cell- and cell-cycle processes and can be delayed or accelerated. In vitro, the maturation time of mVenus is in average 40 min (Lizuka et al., 2011).
| |
| − | Another effect of the mutation F46L is the lowered sensitivity to the pH and chloride ion concentration which is one of the drawbacks of wild‑type GFP.
| |
| − | </p>
| |
| − | <p>mVenus is expressed with a LVA degradation tag to decrease the protein half‑life. Moreover, the reporter is not regulated by any proteins, cofactors or substrates. The lack of disulfide bonds supports the choice of mVenus in our model microorganism <i>E. coli</i>. Its absorption maximum is at 512 nm and its emission maximum at 528 nm. The atomic mass is approximately 27 kDa. </p><center>
| |
| − | <div class="bild" style="width:40vw"><img src="https://static.igem.org/mediawiki/2016/5/54/T--TU_Darmstadt--mvenus.png" style="width:40vw"><p>The figure shows the mVenus reporter protein (without LVA degradation tag). The typical Beta-barrel fold is highlighted in yellow. The fluorophore is hidden inside the barrel structure. PDB ID 1MYW, created with Pymol.</p></div></center>
| |
| − |
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| − |
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| − |
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| − |
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| − | <p><h6> Rational Design of the <i>Amber</i> Mutant of the Dimeric Zif23-GCN4 Repressor </h6></p>
| |
| − | <p>The regulation of the reporter protein mVenus is carried out by a dimeric zinc finger protein. It binds cooperatively to DNA (a specific promoter region), connecting with the major groove of the DNA. The dimeric Cys2His2 zinc finger protein is the DNA binding domain and attached to a leucine zipper dimerization domain. Therefore, the targeted gene is controlled by the specific DNA binding. The monomers bind the DNA specifically and dimerization happens upon binding.<br/></p>
| |
| − | <p>In order to control expression of the repressor on a translational level, an <i>amber</i> stop codon is introduced to the sequence of the repressor. First, the mutation site had to be determined. A position was chosen in which the non-natural amino acid should not interfere with the protein structure. A localization close to the N-terminus was selected as the protein expression will stop early once the non-natural amino acid concentration decreases. Phenylalanine was replaced by <i>O</i>-methyl-L-tyrosine (F4OMT) in order to retain stacking interactions. All nearby side chains as well as the helix (starting from R15) were considered and destabilizing mutations were avoided. Additionally, it is important to choose a residue that is not involved in DNA binding. Otherwise, the repressor may lose its function. The residue of the <i>amber</i> mutation is highlighted in yellow in the picture.</p>
| |
| − |
| |
| − | <center>
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| − | <div class="bild" style="width:40vw"><img src="https://static.igem.org/mediawiki/2016/6/66/T--TU_Darmstadt--reporter1.png" style="width:40vw">Overview of the <i>amber</i> mutation site in the repressor protein that binds DNA (shown in black). The phenylalanine residue is mutated to <i>O</i>-methyl-L-tyrosine (F4OMT). The residue is located close to the N-terminus of the repressor protein in order to interrupt protein expression early when the non-natural amino acid concentration decreases. Created with Pymol software, PDB ID <i>1LLM</i></div></center>
| |
| − | </div> -->
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| | | | |
| | <div class="verlinked" id="kill"><h5>KILL(switch)</h5></div> | | <div class="verlinked" id="kill"><h5>KILL(switch)</h5></div> |
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| | <a href="https://2016.igem.org/Team:TU_Darmstadt/Lab/MetabolicBurden"><button class="read_more" id="lab4b">Interested? Read more</button></a> | | <a href="https://2016.igem.org/Team:TU_Darmstadt/Lab/MetabolicBurden"><button class="read_more" id="lab4b">Interested? Read more</button></a> |
| | </div> | | </div> |
| − | <!-- <div class="content" id="lab4c" style="display:none"> | + | |
| − | <p><h6>Metabolic burden</h6></p>
| + | |
| − | <p> .......</p>
| + | |
| − | <p><h6>Genomic integration</h6></p>
| + | |
| − | | + | |
| − | <p>The <i>λ</i>‑integrase, originally derived from the <i>λ</i>‑Phage, catalyzes in combination with several assisting proteins the excessive and integrative recombination of the phage's genome with the chromosomal genome of a host. For this, two attachment sites are needed: one located on the bacterial genome (<i>attB</i>) and the other located on the <i>λ</i>‑genome (<i>attP</i>), which also contains several binding sites for regulatory proteins. The attachment sites contain homologous recognition sequences, called BOB' Region (<i>attB</i>) and COC' Region (<i>attP</i>). These can be connected by the <i>λ</i>‑integrase and the bacterial <i>integration host factor</i> (IHF) via <i>Holliday junction</i> forming an intasome, a DNA‑protein‑complex, producing hybrid attachment sites <i>attL</i> and <i>attR</i>. <br>
| + | |
| − | For the integration of a gene of interest (GOI) into the chromosomal genome of <i>E. coli</i> there are two plasmids needed.
| + | |
| − | The integration plasmid contains the constitutively expressed GOI GFP, which, as previously mentioned, is also the reporter that is necessary for the measurement of the metabolic burden and should be integrated into the <i>E. coli</i> genome. To measure only the temporary fluorescence a LVA degradation tag is added to the GFP. The plasmid also contains the <i>attP </i>site that enables the integration. Additionally, two bidirectional terminators are located on each side of the <i>attP</i> to protect the GFP operon from the transcription of the other neighbouring genes.
| + | |
| − | To create the integration plasmid E0240 (RBS(BB0032+GFP)) was put on J61002 to locate the GFP behind the promoter J23101. The construct J23101+E0240 was then transformed on the high copy vector pSB1C3 to increase the yield of the prep after a Quick Change PCR, which was necessary for the optimization of BBa_I11023, mutating attp2 to <i>λ</i>‑attP. The final construct was then transformed on the backbone pSB4A5, which possesses a <i>low copy ori</i> and eases the later performed plasmid curing.
| + | |
| − | The second plasmid is a helper plasmid, that is necessary for transposing the GFP into the chromosomal genome as it contains the synthesized protein <i>λ</i>‑integrase with a ribosomal binding site (RBS). For the registry the construct was transformed on a pSB1C3, then we again chose a <i>low copy vector</i> pSB4K5 to ease the later transformed plasmid curing via sustained lack of selection pressure.<br>
| + | |
| − | To verify whether the recombination was successful one can perform a PCR with primers binding to the <i>attB</i> site of the <i>E. coli</i> and the VR Primer, which binds on every BioBrick compliant plasmid. As the one primer binds on the genome and the other on a plasmid, there can only be a PCR amplicon if the integration has succeeded. </p>
| + | |
| − | | + | |
| − | <p><h6>Integration strains</h6></p>
| + | |
| − | <p>A suitable genomic integration strain needs to carry the <i>attB</i> sequence needed for <i>λ</i>‑integrase mediated recombination, which can be troublesome because many commonly used <i>E. Coli</i> strains already have the <i>λ</i>‑phage integrated into their genome. Also, the <i>attB site</i> needed for the integration is blocked in <i>λ</i> (DE3) phages.<br>
| + | |
| − | For our integration strain we chose the <i>E. Coli</i> JM109 strain because it matched all our demands and was also freely and easily available to us.</p>
| + | |
| − | </div> -->
| + | |
| | <div class="verlinked" id="chem"><h5>CHEMICAL SYNTHESIS</h5></div> | | <div class="verlinked" id="chem"><h5>CHEMICAL SYNTHESIS</h5></div> |
| | <div class="abstract"> | | <div class="abstract"> |
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| | <button class="read_more" id="lab5b">Interested? Read more</button> | | <button class="read_more" id="lab5b">Interested? Read more</button> |
| | </div> | | </div> |
| − | <!-- <div class="content" id="lab5c" style="display:none">
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| − |
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| − | <h5>Theoretical background</h5><br>
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| − | <p>In order to methylate the <i>p</i>‑hydroxy group at the benzene ring of tyrosine, the amino group which is more reactive, has to be protected. As protective group acetic anhydrite is used to acetylate the amino group <i>(figure 1)</i>.</p>
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| − | <br>
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| − | <center><img src="https://static.igem.org/mediawiki/2016/9/98/T--TU_Darmstadt--ChemTheo1.png" width="750"></center>
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| − | <br>
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| − | <p>In the second step, a Williamson ether synthesis, sodium hydroxide (NaOH) was added in order to deprotonate both hydroxyl groups of the amino acid. Dimethyl sulfate (DMS) transfers its methyl group to the now hydrolisated hydroxyl group at the benzene ring of tyrosine. Simultaneously the other methyl group is added to the carboxyl group as a methoxyl group <i>(figure 2)</i>.</p>
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| − | <br>
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| − | <center><img src="https://static.igem.org/mediawiki/2016/1/19/T--TU_Darmstadt--ChemTheo2.png" width="750"></center>
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| − | <br>
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| − | <p>In order to eliminate the protecting groups hydrochloric acid (HCl) was added <i>(ficture 3)</i>, both protective groups can be easily removed by hydrolysation to achieve <i>o</i>ૹMethyl tyrosine.</p>
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| − | <br>
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| − | <center><img src="https://static.igem.org/mediawiki/2016/d/d3/T--TU_Darmstadt--ChemTheo3.png" width="750"></center>
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| − | <br>
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| − |
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| − | <h5>Implementation</h5><br>
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| − | <p> The chemical synthesis of <i>O</i>‑Methyl‑tyrosine (OMT) includes 4 steps which are listed in the scheme below.</p>
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| − | <br>
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| − | <center><img src="https://static.igem.org/mediawiki/2016/1/1a/T--TU_Darmstadt--ChemImp1.png" width="800"></center>
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| − | <br>
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| − | <br>
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| − | <center><img src="https://static.igem.org/mediawiki/2016/0/0b/T--TU_Darmstadt--ChemImp2.png" width="300"><p>Equipment consisting of a three-necked flask, a Dimroth condensor, a dropping funnel and a thermometer.</p></center>
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| − | <br>
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| − | <h6>Sample A</h6><br>
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| − |
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| − | <p>5 g tyrosine was dissolved in 100 ml H<sub>2</sub>O. The solution was heated up to 65 °C. Acetic anhydrite was added to the solution and then the solution was heated up for 2 hours, afterwards the heating process was stopped. There was a milky precipitation.
| |
| − | The pH value was adjusted to pH 12 by the addition of NaOH. The solution was heated up to reach 42 °C and then 5,22 ml DMS was added. Following that, the solution was heated up to 100 °C for 30 min and set to cool down. The solution was used for a TLC to prove if primary amino groups (→ results) were present.</p>
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| − | <br>
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| − |
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| − | <h6>Sample B</h6><br>
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| − |
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| − | <p>5 g tyrosine was dissolved in 100 ml H<sub>2</sub>O. The solution was heated up to 65 °C. Acetic anhydrite and sodium hydroxide (NaOH) were were added simultaneously to the solution so the pH value was constantly between slightly acidic and neutral. The pH value needs to be neutral for the reaction of tyrosine and acetic anhydrite because of the weak solubility of tyrosine in H<sub>2</sub>O, so that's ensured, that acetic anhydrite works as a protective group at the amino group of tyrosine. The solution was heated up for 2 hours. As soon as the solution turned into a clear green colored solution, the heating process was stopped.
| |
| − | The pH value was adjusted to pH 12 with addition of NaOH. The solution was heated up to reach 42 °C and then 5,22 ml DMS was added. Following that the solution was heated up to 100 °C for 30 min and set to cool down.</p>
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| − | <br>
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| − |
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| − | <h6>Sample B1</h6><br>
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| − |
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| − | <p>MTBE (Methyl <i>tert</i>‑butyl ether) was added to the now cooled down solution which was filled into a separating funnel for the extraction. This step guarantees that the acetylated amino acid stays in the organic phase while the by-products remain in the inorganic phase
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| − | <br>
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| − | The organic phase was set into a rotary evaporator with the help of negative pressure to distillate the solution in order to acquaintance the intermediate product (step 3; scheme). The product which was left in the evaporator, was added to a three-necked flask with H<sub>2</sub>O and a small amount of hydrochloric acid (pH value = 1) then heated up to 100 °C for 30 min. In Order to increase the yield of the product, NaOH was added so that the solution is saponified (Fisher Reaction) shifting the balance towards the product. During the reaction, a yellow colored substance appeared on the solution surface which was aspirated. The by-product was dried and the melting point was determined to 92 °C.
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| − | <br>
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| − | Further Hydrochloric acid was added to the solution and heated up for 30 min. While cooling off, NaOH was added to the solution to adjust the pH value to 5.66 which is the isoelectric point of tyrosine.
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| − | <br>
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| − | Subsequently the solution was recrystallized with the help of activated carbon and cooled down in an ice bath in order to precipitate the product. The sediment was filtered and dried to measure the melting point (thiele tube).</p>
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| − | <br>
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| − |
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| − | <h6>Sample B2</h6><br>
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| − |
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| − | <p>MTBE (Methyl <i>tert</i>‑butyl ether) was added to the now cooled down solution which was filled into a separating funnel for the extraction. This step guarantees that the acetylated amino acid stays in the organic phase while the by-products remain in the inorganic phase
| |
| − | <br>
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| − | The organic phase was set into a rotary evaporator with the help of negative pressure to distillate the solution in order to acquaintance the intermediate product (step 3; scheme). The product, which was left in the evaporator, was added to a three-necked flask with H<sub>2</sub>O and a small amount of hydrochloric acid (pH value = 1) then heated up to 100 °C for 30 min.
| |
| − | While cooling off hydrochloric acid (HCl) was added to the solution to adjust the pH value to 5.66, which is the isoelectric point of tyrosine. Subsequentlyubsequently, the solution was recrystallized with the help of activated carbon and cooled down in an ice bath in order to precipitate the product. The sediment was filtered and dried to measure the melting point.</p><br>
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| − |
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| − | <h5>Results & Conclusion</h5>
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| − | <br>
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| − | <h6>Run of sample A</h6><br>
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| − |
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| − | <table>
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| − | <table style="text-align="center" height="50" width="50" align="center">
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| − | <thead>
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| − | <tr>
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| − | <th>Sample</th>
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| − | <th>name</th>
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| − | </tr>
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| − | </thead>
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| − | <tbody>
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| − | <tr>
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| − | <th>E</th>
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| − | <th>educt (tyrosine)</th>
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| − | </tr>
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| − | <tr>
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| − | <td>P</td>
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| − | <td>product (Sample A)</td>
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| − | </tr>
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| − | </tbody>
| |
| − | </table>
| |
| − | <right><img src="https://static.igem.org/mediawiki/2016/f/f9/T--TU_Darmstadt--ChemRes1.png" width="250"></right><p>Sample A was taken after reacting with Dimethyl sulfate (DMS) (scheme; Step 3). Instead of a solution, a suspension had formed, therefore the inorganic phase was analyzed.
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| − | <br>
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| − | A ninhydrin-test was done after a Thin Layer Chromatography (TLC), to test whether primary amino groups were present.
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| − | <br>
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| − | As a result, the test shows that no primary amino groups had formed from the acetylation after the addition of acetic anhydrite and after addition of DMS.
| |
| − | Therefore, Sample A was considered as a failure.</p>
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| − | <br>
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| | | | |
| − | <h6>Run of sample B1</h6><br>
| |
| − |
| |
| − | <table>
| |
| − | <table style="text-align="center" height="50" width="50" align="center">
| |
| − | <thead>
| |
| − | <tr>
| |
| − | <th>Sample</th>
| |
| − | <th>name</th>
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| − | </tr>
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| − | </thead>
| |
| − | <tbody>
| |
| − | <tr>
| |
| − | <th>O</th>
| |
| − | <th>product (sample B1)</th>
| |
| − | </tr>
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| − | <tr>
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| − | <td>T</td>
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| − | <td>educt (tyrosine)</td>
| |
| − | </tr>
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| − | </tbody>
| |
| − | </table>
| |
| − | <right><img src="https://static.igem.org/mediawiki/2016/0/01/T--TU_Darmstadt--ChemRes2.png" width="250"></right><p>Sample B1 was taken after a full synthesis (scheme; Step 4) and a recrystallization.
| |
| − | Tyrosine, which was used as a control sample, and the sample B1 were solutionized and applied on the Thin Layer Plate (TLP). On the right side of the TLP, tyrosine was applied, and on the left side of the TLP sample B1 was applied.
| |
| − | <br>
| |
| − | A ninhydrin-test was done after a Thin Layer Chromatography (TLC) to test whether primary amino groups were present.
| |
| − | <br>
| |
| − | The semicircles at the top of the bands are a sign for using an unsuitable solvent (propanol, ammoniac, H<sub>0</sub>2O). Both samples have migrated the same distance at the end of the screening, which proves a relation of the chemical structure between the two samples.
| |
| − | <br>
| |
| − | Additionally a melting point determination was performed. The melting point of sample B1 was determined to be 268 °C. The determination of the product’s melting point showed a significant deviation to the educt’s (tyrosine) melting point of 342 °C. The melting point of sample B1 was also far from the melting point of <i>O</i>ૹMethyl tyrosine which is 258 °C (<a href="scifinder.cas.org">CAS: 6230-11-1</a>).
| |
| − | <br>
| |
| − | As a result Sample B1 contains residues of the educt (tyrosine) which increases the melting point of the product. Furthermore, the melting point apparatus has a deviation of 0,1 °C-2,5 °C <a href="www.de.vwr.com/store/product/591659/schmelzpunktbestimmungsgeraet-digital-smp10-und-smp20">Melting point apparatus</a>)</p>
| |
| − | <br>
| |
| − |
| |
| − | <h6>Run of sample B2</h6><br>
| |
| − |
| |
| − | <table>
| |
| − | <table style=text-align="center" height="50" width="50" align="center">
| |
| − | <thead>
| |
| − | <tr>
| |
| − | <th>Sample</th>
| |
| − | <th>name</th>
| |
| − | </tr>
| |
| − | </thead>
| |
| − | <tbody>
| |
| − | <tr>
| |
| − | <th>T</th>
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| − | <th>educt (tyrosine)</th>
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| − | </tr>
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| − | <tr>
| |
| − | <td>2</td>
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| − | <td>Sample B1</td>
| |
| − | </tr>
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| − | <tr>
| |
| − | <td>5</td>
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| − | <td>Sample C (not finished; Step 2)</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>6</td>
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| − | <td>Sample C (not finisched; Step 3)</td>
| |
| − | </tr>
| |
| − | </tbody>
| |
| − | </table>
| |
| − | <br>
| |
| − |
| |
| − | <right><img src="https://static.igem.org/mediawiki/2016/6/62/T--TU_Darmstadt--ChemRes3.png" width="250"</right><p>Sample B2 was taken after a full synthesis (figure x; scheme Step 4) and a recrystallization.<br>
| |
| − | Tyrosine which was used as a control sample, and the sample B2 (22) were solutionized and applied on the Thin Layer Plate (TLP). Also sample B1 (2) and samples from a not finished Run C (5,6) were applied as a final test.
| |
| − | <br>
| |
| − | A ninhydrin-test was done after a Thin Layer Chromatography (TLC) to test whether primary amino groups were present.
| |
| − | <br>
| |
| − | Sample B1 (22) and sample B2 (2) have migrated the same distance as tyrosine after the run of the TLC which proves a relation of the chemical structure between the samples.
| |
| − | <br>
| |
| − | Additionally a melting point determination was performed. The melting point of sample B2 was determined to be 270 °C. The determination of the product’s melting point showed a significant deviation to the educt’s (tyrosine) melting point of 342 °C. The melting point of sample B2 was also far from the melting point of <i>O</i>ૹMethyl L-tyrosine which is 258 °C (<a href="scifinder.cas.org">CAS: 6230-11-1</a>).
| |
| − | <br>
| |
| − | As a result Sample B2 contains residues of the educt (tyrosine) which increases the melting point of the product. Furthermore, the melting point apparatus has a deviation of 0,1 °C-2,5 °C <a href="www.de.vwr.com/store/product/591659/schmelzpunktbestimmungsgeraet-digital-smp10-und-smp20">(Melting point apparatus</a>)</p></p>
| |
| − | <br>
| |
| − | <h5>Conclusion</h5>
| |
| − |
| |
| − | <p>The melting point of the samples B1 and B2 are respectively 268 °C and 270 °C, even considering the uncertainty of the melting point apparatus to be 0,1-2,5 °C.
| |
| − | <br>
| |
| − | To be absolutely sure that the targeted molecule was synthesized, further analysis of the samples should be following. Scientific methods like Nuclear Magnetic Resonance (NMR), Gas Chromatography (GC), IR spectroscopy, or Mass Spectrometry (MS) are capable of identifying the structure of the molecules represented by sample B1 and B2. Sadly the department of chemistry sciences gave us only 8 days to use their faculties, which was a way too short to successfully run all the experiments and therefore to run the final tests.
| |
| − | <br>
| |
| − | However it is a great success to have achieved very positive results after running the very first experiment. For sure we can proudly display our exertions as a co-working team that has learned a lot and achieved results beyond the initial expectations. In coming iGEM projects, more in-depth cooperations with the chemistry department are conceivable. Also projects with a heavier chemistry part are of potential future interest, so the established connections could be of high value to the next generations of iGEM teams in Darmstadt.</p>
| |
| − | <br>
| |
| − | <h5>Methods</h5>
| |
| − | <br>
| |
| − | <h6>Fisher reaction</h6><br>
| |
| − |
| |
| − | <p>Saponification is a mechanism which involves a series of equilibria by a base (e.g. hydrochloric acid).</p>
| |
| − | <br>
| |
| − | <center><img src="https://static.igem.org/mediawiki/2016/f/f4/T--TU_Darmstadt--ChemMeth1.png" width="750"></center>
| |
| − | <br>
| |
| − |
| |
| − | <h6>Recrystallization</h6><br>
| |
| − |
| |
| − | <p>Recrystallization is a process for purifying a solid or crystalline substance.
| |
| − | <br>
| |
| − | The substance is dissolved and filtered to remove impurities and to regain the crystallized product. Recrystallization depends on temperature and solubility of the purified substance and its impurities. The substance is required to be soluble under high temperatures and less soluble if the temperature is lowered.
| |
| − | Solvent and substance should have similar polarities and the solvent should be inert towards the recrystallized substance. Expected impurities should be highly or unremarkably soluble in the chosen solvent. The optimal solvent and its volume is usually determined in several experiments before the recrystallization process can be started.
| |
| − | <br>
| |
| − | During the implementation the isoelectric point of tyrosine was used for precipitation. The pH value of tyrosine is 5.66 <a href="www.chemie-fu-berlin.de/chemistry/bio/aminoacid/tyrosin_en.html">amino acid tyrosine</a> where tyrosine possess the slightest solubility in H<sub>2</sub>O.</p>
| |
| − | <br>
| |
| − |
| |
| − | <h6>Thin Layer Chromatography (TLC)</h6>
| |
| − |
| |
| − | <p>Thin Layer Chromatography (TLC) is based on two principles called adsorption and distribution. Adsorption means, dissolved substances in leveling agents adsorb on stationary phase (e.g. silica gel) and distribution describes different solubility of substances in different, not mixed, liquid phases. For separation, both effects play a role.
| |
| − | <br>
| |
| − | The stationary phase has to be three characteristics: Chemical structure (Cellulose, silica gel, poly amide, etc.), grain size (smaller and more uniform grains for higher separating efficiency) and film thickness (100-250 µm).
| |
| − | Leveling agents in the mobile phase also have influence on separation, they allow polarity groups of the substance to be separated. Often a mixture of different leveling agents is used.
| |
| − | <br>
| |
| − | The main concepts of Thin Layer Chromatography (TLC) are separating efficiency, selectivity and saturation of the separating chamber.
| |
| − | Separating efficiency can be observed by spot widening with increasing separation height. The general spot widening decreases when separating efficiency increases.
| |
| − | Separating efficiency is influenced by selectivity, how well two similar substances can be separated from each other. In the Separating chamber, the movement has to be horizontal, the saturation with fumes in the separating chamber should be optimal for best results.
| |
| − | Leveling agents flow across the stationary phase because of capillary forces. The solvent front must not reach the upper edge of the Thin Layer. After removing the Thin Layer, the solvent front has to be marked and dried in a horizontal position.</p>
| |
| − | <br>
| |
| − | <b>Implementation and Development of TLC</b>
| |
| − | <p><ol>
| |
| − | <li><b>Sample preparation</b></li>
| |
| − | The sample has to be a homogeneous liquid. Typically purified samples are used. A comparative solution of known concentrations are used as a control sample.
| |
| − | <br>
| |
| − | <li><b>Sample apply</b></li>
| |
| − | The samples should be placed on a line, which is 1.5cm from the edge. The distance between the samples should be 1cm. Typical sample volume ranges between 1-10µl.
| |
| − | <br>
| |
| − | <li><b>Chromatogram development</b></li>
| |
| − | The liquid level in the chamber must not touch the placed samples at any time. Furthermore the TLC plate must not stand towards the wall with its coated surface because of capillary effects. The mobile phase moves across the TLC plate. The solvent front should not reach the end of the TLC plate.
| |
| − | </ol>
| |
| − | <br>
| |
| − | <p>After development, the solvent front is immediately marked by a pencil.
| |
| − | The chamber shouldn't be put into the air stream of a deduction.
| |
| − | For Detection often fluorescence or fluorescence-discharge are used. Fluorescence can help, to visualize the sample via UV-Light, fluorescence-discharge have some Thin-Layer-plates integrating fluorescing Layer. Some substances prevent fluorescence and don't appear under UV-Light. Also coloring chemicals (p.e. ninhydrin) which sprayed on Thin-Layer-plates after chromatography are used.</p>
| |
| − | <br>
| |
| − | <p><b>Quantitative analysis</b>
| |
| − | Amount of substances can be estimated of the size of the spot. TLC-Scanner (densitometric evaluation) measure the intensity reflected light with certain wavelength. </p>
| |
| − | <br>
| |
| − |
| |
| − | <h6>Development of a TLC with the ninhydrin-test</h6>
| |
| − | <br>
| |
| − | <p>ninhydrin targets primary amino groups. These amino groups are colored yellow on the TLC with the reaction in the scheme below.</p>
| |
| − |
| |
| − | <center><img src="https://static.igem.org/mediawiki/2016/2/2e/T--TU_Darmstadt--ChemMeth3.png" width="500"></center>
| |
| − | <br>
| |
| − |
| |
| − | <h6>Melting point determination</h6>
| |
| − | <br>
| |
| − |
| |
| − | <p>Melting point determination is a standard identification method in chemistry for solid products. A substance’s melting point depends on its crystal construction. If there is a contamination, the melting point decreases.
| |
| − | eviations from literature values up to 1-2 centigrade are tolerable. The substance is placed in a capillary tube and heated up until the substance starts to melt. </p>
| |
| − | <br>
| |
| − |
| |
| − | <h5>References</h5><br>
| |
| − |
| |
| − | <p>
| |
| − | <ul>
| |
| − | <li>Organikum (<a href="https://books.google.de/books?isbn=3527322922">ISBN: 3527322922</a>)</li>
| |
| − | <li>Schmelzpunkttabellen organischer Verbindungen; Berlin; Akad.-Verl., 1951</li>
| |
| − | </p>
| |
| − | </ul>
| |
| − |
| |
| − | </div> -->
| |
| − | <!-- <div class="references"><h6>References</h6>
| |
| − | <ul><li>[1]</li><li>[2]</li><li>[3]</li></ul></div> -->
| |
| | </div> | | </div> |
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