Orthogonal Pair

The recognition of the amber stop codon requires a tRNA with an anticodon complementary to the amber stop codon and an aaRS specifically loading the tRNA with the nnAA. In order to ensure the nnAA is not incorporated for other codons except the amber stop codon, the tRNA and the aaRS have to be orthogonal to the natural aaRS's and tRNAs. This means the aaRS must not load any other tRNA and the tRNA must not be loaded by any other aaRS. Therefore, Wang et. al [1] originally used the tyrosyl-tRNA and tyrosyl-RS from the methanogenic archaeon Methanocaldococcus jannaschii : The anticodon of the tRNA was replaced by the amber anticodon and the aaRS was optimized for the recognition of OMT (see figure 1) in place of tyrosine via directed evolution. Introduced into Escherichia coli, this pair is orthogonal to every natural pair due to the genetic distance between E. coli and M. jannaschii. Nowadays, over 70 different aaRS [2] have been designed, each one capable of incorporating a specific amino acid, many of them with special chemical characteristics, allowing e.g. 'click' chemistry or photoactivation.

In our project, we use an orthogonal pair from the "Expanded Genetic Code Measurement Kit" by the iGEM team Austin Texas 2014 as template, specifically the one used for incorporation of ONBY (BBa_SomeBrick), and replaced the ORF with an E. coli codon optimized ORF for OMT-RS. Furthermore we placed the OMT-RS coding region behind a RBS (BBa_B0034) and a strong constitutive Anderson promotor (BBa_J23101).

Figure 1: Dimer of the Methanocaldococcus jannaschii tyrosyl-tRNA synthetase specific for O-methyl-tyrosine (RCSB PDB entrance 1U7X)

Usage of Amber Codon

The incorporation of an amber codon causes the complete translation of the respective protein in presence of the nnAA and cancels the translation in absence. In our implementation the amber codon is replacing a codon in the beginning of the ORFs of the Colicin E2 Immunity protein (Y8OMT) and the Zif23-GCN4 repressor (F4OMT). In consequence, both proteins are functionally produced only if the nnAA is available in sufficient concentration in the medium.

The Non-Natural Amino Acid

We decided to use O-methyl-l-tyrosine for our nnAA due to its multiple advantageous properties:

• Low costs
• Nontoxic
• Unproblematic import into cells
• No further biochemical activity
• Feasible chemical synthesis
• Stable in water
• Unavailable in nature
• Well documented
• Low interference with protein activity

An institute or company could choose its own specific nnAA with the corresponding orthogonal pair. This enables a reliable protection against corporate espionage or bioterrorism, since the opposing party does normally not know which nnAA is used in the respective application. However, using the same nnAA like OMT in every application should prevent the biological and genetic spread of the respective microorganism in the environment.

Results

The OMT-RS was successfully expressed under control of the strong constitutive Anderson promoter BBa_J23101 with the RBS BBa_B0034 as showed in Figure 2. The expression was conducted in TOP10 cells. Furthermore the OMT-RS was epxressed in control of the combined T7 promoter and RBS brick BBa_K525998 in BL21. In this case no expression was detected via SDS_PAGE up to 6 h after induction with 10 mm IPTG. This negative result might be caused by BBa_K525998 itself, since the brick does not contain a spacer sequence between the T7 promoter and the RBS. Similar negative results were also observed in the expression of mVenus.

Figure 2: SDS-PAGE of TOP10 culture lysate with and without OMT tRNA synthatase. The left column shows a lysate from a TOP10 control culture with no plasmid, the right column shows the lysate from a TOP10 culture containing the J23101-B0034-Synthetase (BBa_blabla). The blue arrow indicates the additional band corresponding the OMT tRNA synthatase at ~35 kDa.