Difference between revisions of "Team:TU Darmstadt/Lab"

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     <p>mVenus is expressed with a LVA degradation tag to decrease the protein half&#8209;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.&nbsp;coli</i>. Its absorption maximum is at 512&nbsp;nm and its emission maximum at 528&nbsp;nm. The atomic mass is approximately 27 kDa.  </p>
 
     <p>mVenus is expressed with a LVA degradation tag to decrease the protein half&#8209;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.&nbsp;coli</i>. Its absorption maximum is at 512&nbsp;nm and its emission maximum at 528&nbsp;nm. The atomic mass is approximately 27 kDa.  </p>
     <div class="bild" style="width:40vw"><img src="https://static.igem.org/mediawiki/2016/0/0b/T--TU_Darmstadt--mVenusinCD.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>
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     <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>
 
 
 
      
 
      

Revision as of 11:07, 3 October 2016

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iGEM TU Darmstadt 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.

KILL(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
[...]