Composite

Part:BBa_K2201202

Designed by: Svenja Vinke   Group: iGEM17_Bielefeld-CeBiTec   (2017-10-20)


Tyrosyl tRNA/aminoacyl-synthetase for the incorporation of p-acetophenylalanine

Tyrosyl aminaoacyl-tRNA synthetase for the incorporation of p-acetophenylalanine in response to the amber stop codon.


Usage and Biology

For our toolkit we decided to use the non-canonical amino acid p-acetophenylalanine (AcF) with a ketone group. Ketone groups could form a specific covalent bond to hydrazide groups. Thus, this amino acid could be used for specific, terminus independent labeling of a target protein. Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1553
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1223
  • 1000
    COMPATIBLE WITH RFC[1000]


Functional Parameters

We received a plasmid 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 in response to the amer stop 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 this synthetases for the iGEM community.

An alignement of the wildtype methanococcus jannaschii tyrosine aaRS and the evolved AcF-aaRS are shown in figure 1.

T--Bielefeld-CeBiTec--SVI-Analysing-results-3.png

Figure 1: Alignment of the amino acid sequence of the evolved AcF-aaRS and the wildtype TyrRS from methancoccus jannaschii.

Furthermore we wanted to compare the growth of different aaRS and the wildtype. The results of the growth experiment are shown in Figure 2.

Figure 2:Results of the growth experiment. The cultivations were incubated at 37 °C in 1 mL LB in a 12 well plate and shaken with 600 rpm.
[edit]
Categories
Parameters
None