Orthogonal Pair

The recognition of the amber stop codon requires a tRNA with an anticodon complementary to the amber stop codon and an aminoacyl RNA synthetase (aaRS) specifically loading the tRNA with the non-natural amino acid (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. [2] 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 [3] 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 of protein function.

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, and replaced the ORF with an E. coli codon optimized ORF for O-methyl-l-tyrosine-tRNA synthetase (OMT-RS) (BBa_K1976025). 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-l-tyrosine [4].

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 as non-natural amino acid due to its multiple advantageous properties:

• Low costs
• Nontoxic
• Sufficient import into cells
• No further biochemical activity
• Feasible chemical synthesis
• High stabilility 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 shown in Figure 2. The expression was conducted in E. coli TOP10. Furthermore, the OMT-RS was expressed under control of a T7 promoter in E. coli BL21. The generator was cloned by using BBa_K525998 by iGEM Bielefeld 2011. In this case no expression was detected via SDS_PAGE 6 hours after induction with 10 mm IPTG. This result might be caused by BBa_K525998 itself, since the brick does not contain the 6 basepair counting BioBrick scar as an additional spacer sequence between the T7 promoter and the RBS. Similar negative results were observed in the expression of the reporter mVenus.

Figure 2: SDS-PAGE of E. coli TOP10 culture lysate after 6 hours of constitutive expression of OMT-RS. Left: Cell lysate from E. coli TOP10 not transformed with any plasmid. Right: Cell lysate from E. coli TOP10 transformed with the constitutive OMT generator J23101-B0034-OMT-RS (BBa_K1976022). The OMT-RS holds a molar mass of ~35 kDa.