The Reporter System

For the detection of a low non-natural amino acid concentration, which in this case is 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. An amber mutation was introduced to the DNA sequence of the repressor. 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 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.
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.

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[1]. 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.

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. 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. 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[2].

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]

Figure 1: 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 the PyMOL Molecular Graphic System.

Rational Design of the Amber Mutant of the Dimeric Zif23-GCN4 Repressor

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.[4]

In order to control expression of the repressor on a translational level, an amber 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 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 yellow in the picture.

Figure 2: 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 1LLM