Team:TU Darmstadt/Lab
IN THE LAB...
THE PROJECT
[...]
Incorporation of OMT
ABSTRACT
In order to detect the presence of a specific non-natural amino acid (nnAA) in vivo the concept of amber suppression is used. This means the occurrence of the amber stop codon (UAG) in an ORF does not cancel 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
Synthetic suicide systems have been choice safeguards in synthetic biology for as long as the field exists. There are different kinds of designs, often based on a regulating mechanism and a toxin such as host killing proteins or different kinds of metabolism inhibiting pathways. However, these most often don't tackle the problem of synthetic DNA surviving the death of the host cell.
Here we show a possible design for a simple synthetic killswitch based on an endonuclease called colicin E2 and its corresponding suppressing protein, Im2. It is regulated by amber suppression, the usage of an amber stop codon to code for a non-natural amino acid O-methyl-L-tyrosine. The aim of the system is to not only kill its host, but also to destroy all DNA within the cell and its surroundings, preventing the escape of transgenic DNA.
METABOLIC BURDEN
ABSTRACT
Artificial plasmids are a significant burden to the host. The design of our pathways, for example the combination of a promoter and RBS, results in different amounts of product. The measurement of the metabolic burden is the key for a quantitative optimization in metabolic engineering. We want to establish a new approach to iGEM by providing a measurement strain to the community. As described by F. Ceroni et al., we genomically integrated one copy of GFP into E. coli, which offers us a highly accurate and instantaneous measurement of the impact of our plasmids on the host. This is of economical interest because it enables academic and industrial researchers to test a lot of different pathways at once in a short time just by using a microplate reader. For the integration we used the λ‑Integrase site‑specific recombination pathway, described by A. Landy in 2015. Therefore, we designed two plasmids (BBa_K1976000 and BBa_K1976001) and measured them using single cell measurement and via microplate reader.
CHEMICAL SYNTHESIS
ABSTRACT
Since non‑natural amino acids are expensive in comparison to natural amino acids we searched for a high yield synthesis method for O‑methyl‑L‑tyrosine. Problems with chemical alterations of amino acids to form non‑natural derivates often lie in the higher reactivity of amino and carboxyl groups compared to other reactive groups. For this reason both groups need to be kept in mind while searching for a possible reaction for the desired synthesis.
For the protection of the amino group an acetylation reaction was carried out to form N‑acetyl‑L‑tyrosine. The tested method used N‑acetyl‑L‑tyrosine as a reagent which was then methylated at the carboxyl group and at the hydroxyl group using dimethyl sulfate by Williamson ether synthesis. To finally form the non‑natural amino acid, an acidic hydrolysis using hydrochloric acid was performed.
