REPORTING LOW nnAA LEVELS
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.
For the detection of a low non-natural amino acid concentration, in this case O-methyl-l-tyrosine, we designed a reporter system that includes the reporter protein mVenus. In order to make sure that the expression of mVenus does only start at a low OMT concentration, we use a dimeric repressor in which an amber mutation was introduced to the encoding DNA sequence. This amber mutation 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 concentration 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. We utilize a dimeric repressor because 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). If the concentration of OMT drops, we quickly obtain a signal.
To make sure that the repression does not take place when the concentration drops below the treshold, an LVA degradation tag is expressed with the dimeric repressor. In addition, to ensure that no permanent fluorescent signal is caused by mVenus, it is fused to a LVA degradation tag as well. In consequence, both proteins shall be short-living after their translation . To fit this system to the expression of colicin E2, we can use different Anderson promoters (BBa_J23100, BBa_J23104, BBa_J23107, BBa_J23113, and BBa_J23114). We aimed to find an expression rate ensuring that mVenus is translated before the DNase degrades mVenus encoding DNA and therefore inhibits the protein biosynthesis of mVenus.
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, triggered by the close proximity of involved residues. 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 π−electron system, resulting in the fluorophore being maturated and fluorescent. It is protected by the surrounding β−barrel 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 minutes. Another effect of the mutation F46L is the lowered sensitivity to the pH and the chloride ion concentration, one of the drawbacks of wild‑type GFP .
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 E. coli. Its absorption maximum is at 512 nm and its emission maximum at 528 nm. The atomic mass is approximately 27 kDa. [3, 4]
Rational Design of an amber mutant of the Zif23-GCN4 repressor
The regulation of the reporter protein mVenus is carried out by a dimeric zinc finger protein binding cooperatively to a specific promoter region by interfering with its major groove of the DNA. The dimeric Cys2His2 zinc finger protein is the DNA binding domain and attached to a leucine zipper dimerization domain. Hence, the transcription of the targeted gene is repressed by the specific DNA binding. The monomers bind the DNA specifically and dimerization happens upon binding.
In order to control expression of the repressor on a translational level, an amber stop codon is introduced into 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 to stop protein expression early, if the non-natural amino acid concentration drops below a treshold. Phenylalanine was replaced by O-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 amber mutation is highlighted in figure 2.
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