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 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 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.

In our project, we use an orthogonal pair from the "Expanded Genetic Code Measurement Kit" 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). A successful expression of the OMT-RS gene in this construct was observed (Fig. 1).

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

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bildunterschrift: igem team darmstadt 2016

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Metabolic burden

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Genomic integration

The λ‑integrase, originally derived from the λ‑Phage, catalyzes in combination with several assisting proteins the excessive and integrative recombination of the phage's genome with the chromosomal genome of a host. For this, two attachment sites are needed: one located on the bacterial genome (attB) and the other located on the λ‑genome, which also contains several binding sites for regulatory proteins. The attachment sites contain homologous recognition sequences, called BOB' Region (attB) and COC' Region (attP). These can be connected by the λ‑integrase and the bacterial integration host factor (IHF) via Holliday junction forming an intasome, a DNA‑protein‑complex, producing hybrid attachment sites attL and attR.
For the integration of a gene of interest (GOI) into the chromosomal genome of E. coli there are two plasmids needed. One, called integration plasmid, contains the constitutively expressed GOI GFP with a LVA degradation tag, which, as previously mentioned, is also the reporter that is necessary for the measurement of the metabolic burden and should be integrated into the E. coli genome. It also contains the attP site that enables the integration. There are two bidirectional terminators located on each side of the attP to protect the GFP Operon from the transcription of the other neighbouring genes. The antibiotic resistance will also be integrated into the genome if the genomical integration succeeds, so we decided to use a Kanamycin resistance, as it is less commonly used in iGEM than Ampicillin or Chloramphenicol. Therefore, we chose the backbone pSB3K3, which also possesses a low copy ori and eases the later performed plasmid curing. The second plasmid is a helper plasmid and is necessary for transposing the GFP into the chromosomal genome as it contains the protein λ‑integrase with a ribosomal binding site (RBS).
To verify whether the recombination was successful one can perform a PCR with primers binding to the attB site of the E. coli and the VR Primer, which binds on every BioBrick compliant plasmid. As the one primer binds on the genome and the other on a plasmid, there can only be a PCR amplicon if the integration has succeeded.

Integration strains

A suitable genomic integration strain needs to carry the attB sequence needed for λ‑integrase mediated recombination, which can be troublesome because many commonly used E. Coli strains already have the λ‑phage integrated into its genome. Also, the attB site needed for the integration is blocked in λ (DE3) phage.
For our integration strain we chose the E. Coli JM109 strain because it matched all our demands and was also freely and easily available to us.

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