Difference between revisions of "Team:TU Darmstadt/Lab"
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<div class="verlinked" id="repo"><h5>REPORTER</h5></div> | <div class="verlinked" id="repo"><h5>REPORTER</h5></div> | ||
<div class="abstract"> | <div class="abstract"> | ||
| − | <p | + | <p>ABSTRACT<br/></p> |
| − | <b><p>Glow before you go- What does this actually mean? The aim of our project is to make biology safer by introducing a suicide system to <i>E. coli</i>. Before the suicide is triggered, a <b>reporter protein</b> is expressed to indicate the release of <i>E. coli</i> or to show a deficiency of the non-natural amino acid in the surrounding medium which is necessary for the bacteria to survive. As a reporter protein, we chose <b>mVenus</b> which is a mutant of eYFP. mVenus is located downstream of a promoter which is repressed by a dimeric protein, the <b><i>Zif23-GCN4</i> repressor</b>. This repressor carries an <i>amber</i> mutation at position 4 (F4OMT). As a result, the non-natural amino acid <b><i>O</i>-methyl-L-tyrosine (OMT)</b> is integrated into the protein sequence as long as there is enough OMT in the medium. With decreasing OMT concentration, the translation of the repressor stops due to the early <i>amber</i> stop codon and the repressor cannot bind to the promoter. This leads to expression of the reporter protein mVenus which can be detected by fluorescence measurements. | + | <b><p>Glow before you go- What does this actually mean? The aim of our project is to make biology safer by introducing a suicide system to <i>E. coli</i>. Before the suicide is triggered, a <b>reporter protein</b> is expressed to indicate the release of <i>E. coli</i> or to show a deficiency of the non-natural amino acid in the surrounding medium which is necessary for the bacteria to survive. As a reporter protein, we chose <b>mVenus</b> which is a mutant of eYFP. mVenus is located downstream of a promoter which is repressed by a dimeric protein, the <b><i>Zif23-GCN4</i> repressor</b>. This repressor carries an <i>amber</i> mutation at position 4 (F4OMT). As a result, the non-natural amino acid <b><i>O</i>-methyl-L-tyrosine (OMT)</b> is integrated into the protein sequence as long as there is enough OMT in the medium. With decreasing OMT concentration, the translation of the repressor stops due to the early <i>amber</i> stop codon and the repressor cannot bind to the promoter. This leads to expression of the reporter protein mVenus which can be detected by fluorescence measurements.</p> |
<button class="read_more" id="lab2b">interested? read more</button> | <button class="read_more" id="lab2b">interested? read more</button> | ||
</div> | </div> | ||
<div class="content" id="lab2c" style="display:none"> | <div class="content" id="lab2c" style="display:none"> | ||
| − | <p> <h6> The Reporter System</h6> | + | <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> | + | <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> | 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> | ||
| − | <h6> mVenus</h6> | + | <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). | <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. | 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. | ||
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| − | <h6> Rational Design of the <i>Amber</i> Mutant of the Dimeric Zif23-GCN4 Repressor </h6> | + | <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>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> | <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> | ||
Revision as of 13:11, 3 October 2016
IN THE LAB...
THE PROJECT
[...]
ORTHOGONAL PAIR
ABSTRACT
In order to detect the presence of the specific non-natural amino acid (nnAA) in vivo the concecpt of amber suppression 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 O-methyl-l-tyrosine (OMT). However, the incorporation requires the presence of the nnAA in the medium, otherwise the translation stops. The mechanism requires a tRNA 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'.
REPORTER
ABSTRACT
Glow before you go- What does this actually mean? The aim of our project is to make biology safer by introducing a suicide system to E. coli. Before the suicide is triggered, a reporter protein is expressed to indicate the release of E. coli or to show a deficiency of the non-natural amino acid in the surrounding medium which is necessary for the bacteria to survive. As a reporter protein, we chose mVenus which is a mutant of eYFP. mVenus is located downstream of a promoter which is repressed by a dimeric protein, the Zif23-GCN4 repressor. This repressor carries an amber mutation at position 4 (F4OMT). As a result, the non-natural amino acid O-methyl-L-tyrosine (OMT) is integrated into the protein sequence as long as there is enough OMT in the medium. With decreasing OMT concentration, the translation of the repressor stops due to the early amber stop codon and the repressor cannot bind to the promoter. This leads to expression of the reporter protein mVenus which can be detected by fluorescence measurements.
Overview of the amber mutation site in the repressor protein that binds DNA (shown in black). The phenylalanine residue is mutated to O-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 1LLMKILL(switch)
ABSTRACT
[...]
GENOMIC INTEGRATION
ABSTRACT
Abstract
Artificial plasmids, which we transform into cells during the year on iGEM all the time, are a significant burden to the host. The design of our pathways, for example the combination of a promoter and RBS, result in different amounts of product. The measurement of the metabolic burden is the key for a quantitative optimization in metabolic engineering. Hereby, the measurement of the hosts' optical density, which should give you a feedback on the growth rate, shows you a very inaccurate value of the metabolic burden and even that just after a long time. F. Ceronie, R. Algar, G.B. Stan, T. Ellis thought about the need of a highly quantitative accurate measurement and found a solution in the measurement of a fluorophore, which the host expresses constitutively. They demonstrate, that the measurement of GFP has great advantages over the measurement of OD, because it is much faster and more precise. Using this method, it is now possible to measure the impact of transformed plasmid live and with high accuracy. This new approach is of economical interest, because it enables scientist to test a lot of different pathways at once in a short time, just by using a fluoreader. Our main project aims on developing a safety plasmid. To measure the metabolic burden caused by the safety plasmid, but also every plasmid that we design now and in the future, we want to build a meausurement strain based on the model of F. Ceronie et al. To achieve the most sensitive results, we used the λ‑Integrase Site‑specific Recombination Pathway, described by A. Landy in 2015, to integrate exactly one copy of GFP into E. coli K12 JM109. Therfore we designed two plasmids, based on BBa_I11020 and BBa_I11023. We measured our strain using single cell measurement as well as measurements with a fluoreader.
CHEMICAL SYNTHESIS
ABSTRACT
[...]
