Part:BBa_K2201201

Pyrrolysyl tRNA/aminoacyl-synthetase for the incorporation of propargyllysine

Pyrrolysyl aminaoacyl-tRNA synthetase for the incorporation of propargyllysine.

Usage and Biology

For our toolkit we decided to use the non-canonical amino acid propargyllysine (PrK) with a propargyl group. Propargyl groups can be used to form a covalent bond to azide groups in a click-chemistry reaction. Our aim was to provide an orthogonal tRNA/aminoacyl-synthetase (aaRS) which could incorporate thie amino acid through the amber codon. We received a plasmid from the Lemke group from EMBL in Heidelberg containing an evolved pyrrolysyl synthetase from Methanosarcina mazei for the incorporation of PrK in response to the amber codon. We used Gibson assembly to clone the tRNA/aaRS and the tRNA from this plasmids into pSB1C3 and replaced cutting sites for EcoRI and SpeI with site directed mutagenesis to provide this synthetases for the iGEM community.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 585
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1992
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 573
  Illegal AgeI site found at 1667
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 247
  

Functional Parameters

The alignment of the wildtype PylRS from methanosarzia mazei and the evolved synthetase for the incorporations of PrK shows only one amino acid exchange at position 124. Although this is the aaRS with the least amino acid exchanges of our toolkit, it turned out to be the most specific. We proved the incorporation of PrK through this synthetase with our screening system for the incorporation of ncAAs. The results from our screening system, which compares the incorporation efficiency and specify are shown in figure 2. For more details of our screening method, please refer to the part improvement page.

<img class="figure image" src="">

Figure 2: Comparison of the incorporation rate of PrK and native amino acids through the evolved PrK-aaRS.