Evolved Synthetases for the Incorporation of Propargyllysine and p-Acetophenylalanine
We received plasmids from the Lemke group from EMBL in Heidelberg containing an evolved tyrosyl tRNA/ synthtase pair (tRNA/TyrRS) from Methanococcus jannaschii for the incorporation of AcF and an evolved pyrrolysyl synthetase from Methanosarcina mazei for the incorporation of PrK, both in response to the amber codon. We used Gibson Assembly to clone the tRNA/aaRS and the tRNA from these plasmids into pSB1C3 and replaced cutting sites for EcoRI and SpeI with site directed mutagenesis to provide these synthetases for the iGEM community. Furthermore, we changed the anticodon in the tRNA of the TyrRS tRNA to the anticodon for the less used leucine codon, so the new aaRS incorporates AcF in response to the codon CTA. An alignment of both evolved synthetases with the wildtypes is shown in Figure 1 (PrK-aaRS) and Figure 3(AcF-aaRS).
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. The negative score results from the emission quotient CFP(475 nm)/YFP(525 nm) when cultivated without the specific ncAA. The positive score results from the emission quotient YFP(525 nm)/CFP(475 nm) when cultivated with the specific ncAA. The mean rank allows the combination of the negative and the positive score to compare the efficiency of synthetases among each other.
Figure 3: Alignment of the amino acid sequence of the evolved AcF-aaRS and the wildtype TyrRS from methancoccus jannaschii.
Construction of the Expression Plasmid for the Sup35 Test Protein
According to our expert Iker Valle Aramburu, the incorporation of two noncanonical amino acids lowers the yield. He recommended us to use only one non-canonical amino acid and one cysteine, which could be labeled in a maleimide coupling reaction. Mukhopadayay et al. 2006 showed that mutants of Sup35 containing no cysteines are still able to form prions. So, we decided to order a gene synthesis of sup35 containing no cysteines.
For our experiments we cloned the gene synthesis of the NM region of Sup35 into pSB1C3 and used site-directed mutagenizes to construct the part BBa_K2201231 containing a His6tag, one amber codon at position 21 and a cysteine codon at position 121. The control BBa_K2201232 contains cysteine codons at positions 21 and 121. In addition, we constructed these three parts also with a T7-promoter and RBS (B0034) (BBa_K2201331 and BBa_K2201332), for the inducible expression of the Sup35 variants.
Expression and Analysis of the Sup35 Test Protein
Labeling of the Sup35 Test Protein
Furthermore, we wanted to detect the labeling efficiency through UV-VIS spectralphotometer, but the results indicate that a high excess of free Cy5 remains in the sample and the background was too high to perform FRET-measurements. To investigate the labeling efficiency and to get a fluorescence signal the labeling reaction and especially the last washing step needs to be improved to generate suitable test protein for the prion detection assay.