Part:BBa_K2201400

Tyrosyl synthetase, library plasmid from Methanococcus jannashii

Tyrosyl synthetase from wild type Methanococcus jannashii in pSB1C3, designed as library plasmid for the randomerization of certain positions and following selection cycles for the incorporation of non canonical amino acids or unnatural bases. Tyrosyl tRNA synthetase from Methanococcus jannashii in pSB1C3, designed as library plasmid for the randomerization of certain positions and following selection cycles for the incorporation of non-canonical amino acids or unnatural bases. In addition BBa_K2201400, the part BBa_K2201411 contains an mRFP (BBa_J04450) in the position of the synthetases binding pocket.

Usage and Biology

We designed and cloned this part to generate a tRNA/synthetase based on the Methanococcus jannashii wild type tyrosyl-tRNA/aminoacyl synthetase, which is able to incorporate 2-Nitro-L-phenylalanine, used for the photocleaving of the polypeptide backbone. Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Functional Parameters

The tyrosyl-tRNA/aminoacyl synthetase (TyrRS) is inserted in pSB1C3. The TyrRS library was generated by using two primers, one with nine randomized position (NNK), which are designed to form a dimer. This dimer is completed to a dsDNA by the Klenow fragment.

Figure 2: pSB1C3 backbone, TyrRS (based on Methanocoocs jannashii TyrRS) library plasmid.

We cloned the library using Gibson Assembly, after transformation we platet them out on LB-plates with chloramphenicol. Altogether, we received more than 130,000 colonies. We received approximately 125,236.64 library plasmids out of 130,000 colonies, showing a cloning efficiency of 96,34 %, offering a wide diversity of different TyrRS variants.

Figure 3: Example of our TyrRS library containing the optical control, platet out on LB plates with chloramphenicol.

Figure 4: Sanger sequencing of our TyrRS library, showing nine positions, randomized by the use of the NNK scheme. All nucleotides are presented at these positions.

We cloned a library of more than 130,000 clones, including more than 27,672 different TyrRS variants out of 32,768 possible sequence variants, analyzed with MiSeq, Illumina next generation sequencing. These sequences code for more than 8,787 different peptides.

We used this TyrRS library as a basis to select an tRNA/aminoacyl synthetase pair, able to incorporate 2-Nitrop-L-phenylalanine. Therefore we used our selection system, consisting of a positive (BBa_K2201900) and negative selection plasmid (BBa_K2201901) .