Difference between revisions of "Part:BBa K2201202"
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Tyrosyl aminaoacyl-tRNA synthetase for the incorporation of p-acetophenylalanine in response to the amber stop codon. | Tyrosyl aminaoacyl-tRNA synthetase for the incorporation of p-acetophenylalanine in response to the amber stop codon. | ||
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| − | + | ===Usage and Biology=== | |
| + | For our toolkit we decided to use the non-canonical amino acids p-acetophenylalanine (AcF) with a ketone group. | ||
| + | Ketone groups could form a specific covalent bond to hydrazide groups. Thus, this synthetase could be used for specific, terminus independent labeling of a target protein. | ||
<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
<partinfo>BBa_K2201202 SequenceAndFeatures</partinfo> | <partinfo>BBa_K2201202 SequenceAndFeatures</partinfo> | ||
| − | + | ||
===Functional Parameters=== | ===Functional Parameters=== | ||
| + | We received a plasmid from the Lemke group from EMBL in Heidelberg containing an evolved tyrosyl tRNA/ synthtase pair (tRNA/TyrRS) from <i>Methanococcus jannaschii</i> 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 <i>Eco</i>RI and <i>Spe</i>I with site directed mutagenesis to provide this synthetases for the iGEM community. | ||
| + | |||
| + | An alignement of the wildtype <i>methanococcus jannaschii</i> tyrosine aaRS and the evolved AcF-aaRS are shown in figure 1. | ||
| + | |||
| + | <div class="figure large"> | ||
| + | https://static.igem.org/mediawiki/2017/2/27/T--Bielefeld-CeBiTec--SVI-Analysing-results-3.png <br> | ||
| + | <p class="figure subtitle"><b>Figure 1:</b> Alignment of the amino acid sequence of the evolved AcF-aaRS and the wildtype TyrRS from <i>methancoccus jannaschii</i>.</p> | ||
| + | </div> | ||
| + | |||
| + | Furthermore we wanted to compare the growth of different aaRS and the wildtype. The results of the growth experiment are shown in Figure 2. | ||
| + | |||
| + | <div class="figure large"> | ||
| + | https://static.igem.org/mediawiki/2017/b/bd/T--Bielefeld-CeBiTec--SVI-growth-experiment.png <br> | ||
| + | <p class="figure subtitle"><b>Figure 2:</b>Results oof the growth experiment of different aaRS. The cultivations were in 1 mL volumne in 12 well plates, 37 °C with 600 rpm</p> | ||
| + | </div> | ||
| + | |||
<partinfo>BBa_K2201202 parameters</partinfo> | <partinfo>BBa_K2201202 parameters</partinfo> | ||
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Revision as of 09:16, 30 October 2017
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 acids p-acetophenylalanine (AcF) with a ketone group. Ketone groups could form a specific covalent bond to hydrazide groups. Thus, this synthetase could be used for specific, terminus independent labeling of a target protein. Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1553
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1223
- 1000COMPATIBLE 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.
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 oof the growth experiment of different aaRS. The cultivations were in 1 mL volumne in 12 well plates, 37 °C with 600 rpm