Tyrosyl synthetase library plasmid from M. jannashii with mRFP as optical controle for a selection
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 to BBa_K2201400, the synthetase contains an mRFP (BBa_J04450)in the position of the synthetases binding pocket. When generating the library with randomerized short double stranded DNA, the mRFP serves as optical controle if the randomerized DNA sequence incorporateded or not.
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-phnylalanine, used for the photocleaving of the polypeptide backbone.
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. As optical control, a mRFP is incorporated in this certain position to be ranomized, which is then replaced by the dsDNA.
Figure 1: Generating the library by using randomized primers, forming a dimer, in combination with an optical control
Figure 2: pSB1C3 backbone, TyrRS (based on
Methanocoocs jannashii TyrRS) library plasmid. Depicted without the optical control.
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. In evidence due to th optical controle of the template used for the not randomized TyrRS plasmid backbone, we could easily determine the negative colonies. As depicted in Figure 1, 48 of 1310 colonies approximately did not contain the randomized TyrRS library plasmid. Extrapolating this data, 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) .
</html>
