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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 585
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1992
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 573
Illegal AgeI site found at 1667 - 1000INCOMPATIBLE 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, shown in Figure 1, 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.
Figure 1:Alignment of the amino acid sequence of PrK-aaRS and the wildtype methanosarzia mazei Pyl-RS.
Figure 2: Comparison of the incorporation rate of PrK and native amino acids through the evolved PrK-aaRS.
To demonstrate the tool analysing we wanted to express a protein called Sup35, a saccaromyces cerevisiae protein with prionic abilities.The expression plasmid of Sup35 containing one amber stop codon at position 21 (BBa_K2201331) was cotransformed with the evolved PylRS synthetase for the incorporation of PrK (BBa_K2201201). The cells containing both plasmids were cultivated like described in expression of recombinant proteins, without PrK in the media and with 1 mM PrK in the media. Sup35 was purified through NiNTA chromatography. As positive control Sup35 without stop codons was expressed and purified the same way. All three samples were analyzed on the SDS-PAGE shown in figure 3.
Figure 3: SDS-PAGE of the eluates of the three cultivations. The positive control (PC: BBa_K220302) left and BBa_K2201301 without PrK (-PrK) in the media in the middle and with 1 mM PrK (+PrK) in the media on the right side.</p>
The SDS-PAGE confirmed the results from our screening system. The positive control showed a high expression of the recombinant Sup35 and both samples with BBa_K2201301 showed a band on the same height as the positive control. This proved that the PrK-aaRS incorporates endogenous amino acids if PrK is not supplemented in the media. But if PrK is present the expression of Sup35 is stronger. The affinity of PrK-aaRS to PrK seems to be stronger than to the endogenous amino acids, so the test protein is expressed with the non-canonical amino acid as planned.
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