Team:Bielefeld-CeBiTec/Results/toolbox/analysing

Analyzing

Short summary

The analysis tool gives the opportunity to measure intermolecular distances with the help of Foerster resonance energy transfer (FRET) in the target protein. We provide three tRNA/aminoacyl-synthetases (aaRS) for the incorporation of non-canonical amino acids (ncAAs) with additional functional groups to the canonical amino acids. The functional groups could form a covalent bond to a fluorophore in chemical reactions. The fluorescence of the fluorophores indicates how far the distance to each other is. With this tool, target proteins could be labeled at specific positions to find out if the folding of the protein changes, give information about the protein structure or if two different proteins interact. To demonstrate this tool, we labeled the yeast prion sup35 at specific positions. During translation of Sup35, two different orthogonal tRNA/aaRS incorporate two different ncAAs which can be labeled specific with fluorophores. With this test protein, applications like a quick prion detection assay are possible.

Evolved synthetases for the incorporation of propargyllysine and p-acetophenylalanine

For our toolkit we decided to use the non-canonical amino acids p-acetophenylalanine (AcF) with a ketone group and propargyllysine (PrK) with a propargyl group. Propargyl groups can be used to form a covalent bond to azide groups in a click-chemistry reaction and ketone groups could form a covalent bond to hydrazide groups. Our aim was to provide two orthogonal tRNA/aminoacyl-synthetases (aaRS) which could incorporate these amino acids through the amber codon. Furthermore, we want to provide an aaRS which incorporates AcF through the less used leucine codon (CTA). This way, both amino acids could be incorporated simultaneously and at a specific position during translation.
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 are shown in figure 1 (PrK-aaRS) and 3(AcF-aaRS).

Figure 1:Alignment of the amino acid sequence of PrK-aaRS and the wildtype methanosarzia mazei Pyl-RS.

Construction of the expression plasmid for the Sup35 test protein

For our test protein, we decided to order a gene synthesis of the NM-region of sup35. The NM region is 250 amino acids long and responsible for the prion forming function of Sup35. The test protein contains an amber codon at position 21 and the less used leucine codon CUA at position 121 for the incorporation of PrK and AcF.
According to our expert Iker Valle Aramburu, the incorporation of two noncanonical amino acids lowers the yield. He recommended us to use only one noncanonical amino acid and one cysteine, which could be labeled in a maleimide coupling reaction. Mukhopadhyay 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 histag, 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-promotor and RBS (BBa_K2201331 and BBa_K2201332), for the inducible expression of the Sup35 variants.

References

Mukhopadhyay, S., Krishnan, R., Lembke, E. A., Lindquist, S., Deniz, A. A.(2007)A natively unfolded yeast prion monomer adopts an ensemble of collapsed and rapidly fluctuating structures.PNAS.104(8):2649-2654.