Difference between revisions of "Team:Bielefeld-CeBiTec/Results/translational system/translation mechanism"
| Line 44: | Line 44: | ||
<article> | <article> | ||
| − | For generating our | + | For generating our <a href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/toolbox">Toolbox</a>), we used variants of two different aminoacyl tRNA/synthetases (aminoacyl tRNA/RS), <a href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/translational_system/library_and_selection">tyrosyl and pyrrosylyl</a>. When working with our toolbox, we recognized that the <i>Escherichia coli</i> cells containing these different aminoacyl tRNA/RS plasmids had different characteristics. We could confirm this assumption by performing growth experiments with <i>E.coli</i> transformed with our different aminoacyl tRNA/synthetases. |
| − | Therefore, we transformed the certain aminoacyl tRNA/RS plasmid and two controls (<a href="http://parts.igem.org/Part:pSB1C3">pSB1C3</a> and <a href="http://parts.igem.org/Part:pSB3T5">pSB3T5</a>) in <i>E. coli</i> BL21 (DE3). We performed the cultivation in 1 mL LB-media with the matching non | + | Therefore, we transformed the certain aminoacyl tRNA/RS plasmid and two controls (<a href="http://parts.igem.org/Part:pSB1C3">pSB1C3</a> and <a href="http://parts.igem.org/Part:pSB3T5">pSB3T5</a>) in <i>E. coli</i> BL21 (DE3). We performed the cultivation in 1 mL LB-media with the matching non canonical amino acid (ncAA) in 12‑well microtiter plates by 600 rpm at 37 °C. We used two biological replicates, each with three technical replicates of the optical density of each cultivation by NanoDrop. |
| − | When depicting the growth rate of the cells, containing the aminoacyl tRNA/RS on the high copy plasmid pSB1C3 (Figure 1), the difference in growth is striking. The growth experiments show that the incorporation of ncAAs through the amber codon are a major metabolic pressure for the cells, resulting in a 0.25 to 0.5 % slighter growth rate than of the control. Only the | + | When depicting the growth rate of the cells, containing the aminoacyl tRNA/RS on the high copy plasmid pSB1C3 (Figure 1), the difference in growth is striking. The growth experiments show that the incorporation of ncAAs through the amber codon are a major metabolic pressure for the cells, resulting in a 0.25 to 0.5 % slighter growth rate than of the control. Only the <a href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/toolbox/analysing">PrK</a>)‑tRNA/synthetase showed a better growth of up to 17 % better than the control. This can be explained by the metabolic pressure of pSB1C3 by coding mRFP. In addition, the very specific incorporation of the ncAA by the PrK (Figure 3) also supports a faster growth of the culture, containing the PrK aminoacyl tRNA/synthetase. That is due to the similarity of the used ncAA Prk to the original amino acid pyrrolysyl, requiring only one point mutation of the aminoacyl synthetase (aaRS). This could be demonstrated by us with our CFP‑YFP system(<a href="http://parts.igem.org/Part:BBa_K2201343">BBa_K2201343</a>)for the efficiency of the incorporation of ncAA. |
| Line 72: | Line 72: | ||
<br> | <br> | ||
</br> | </br> | ||
| − | Given that the incorporation of an ncAA through the amber codon implies a major metabolic pressure for the organism, we also investigated the influence of the recoding of the leucine codon. Therefore we used variants of the AcF tRNA/syntheatase pair, adapted to the amber codon (AcF‑TAG) and to the leucine codon (AcF‑Leu). | + | Given that the incorporation of an ncAA through the amber codon implies a major metabolic pressure for the organism, we also investigated the influence of the recoding of the leucine codon. Therefore we used variants of the <a href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/toolbox/analysing">AcF</a>)‑tRNA/syntheatase pair, adapted to the amber codon (AcF‑TAG) and to the leucine codon (AcF‑Leu). |
When comparing the growth of the different variants, a lower growth rate of the AcF-TAG variant is in evidence. When coding the ncAA through amber codon, the low specificity of the AcF‑TAG synthetase induces an extension of the translated proteins, which results in the lower growth. In contrary to the AcF-TAG, the incorporation of ncAA through the leucine codon is much more specific and therefore causes less disadvantage for the cell growth. | When comparing the growth of the different variants, a lower growth rate of the AcF-TAG variant is in evidence. When coding the ncAA through amber codon, the low specificity of the AcF‑TAG synthetase induces an extension of the translated proteins, which results in the lower growth. In contrary to the AcF-TAG, the incorporation of ncAA through the leucine codon is much more specific and therefore causes less disadvantage for the cell growth. | ||
| Line 78: | Line 78: | ||
<div class="figure medium"> | <div class="figure medium"> | ||
<img class="figure image" src="https://static.igem.org/mediawiki/2017/a/ad/T--Bielefeld-CeBiTec--growth_experiment_AcF-TAG_AcF-Leu.jpg"> | <img class="figure image" src="https://static.igem.org/mediawiki/2017/a/ad/T--Bielefeld-CeBiTec--growth_experiment_AcF-TAG_AcF-Leu.jpg"> | ||
| − | <p class="figure subtitle"><b>Figure 3: Optical density of the cultivation of AcF | + | <p class="figure subtitle"><b>Figure 3: Optical density of the cultivation of AcF‑tRNA/synthetase variants adapted to the amber codon and the leucine codon. </b><br> The AcF-tRNA/synthetases are integrated in pSB1C3 and are cultivated in <i>E.coli</i> BL21 (DE3) with the matching non canonical amino acid.</p> |
</div> | </div> | ||
Revision as of 11:10, 31 October 2017
Translation Mechanism
Short Summary
Comparison of different aminoacyl tRNA/synthetases
Figure 1: Optical density of the cultivation of five different aminoacyl tRNA/synthetases.
The Agilent BioAnalyzer High Sensitivity DNA Assay is used for the measurement. The aminoacyl tRNA/synthetases are integrated in pSB1C3 and are cultivated in E.coli BL21(DE3) with the matching non canonical amino acid.
Figure 2: Optical density of the cultivation of two different aminoacyl tRNA/synthetases. The aminoacyl tRNA/synthetases are integrated in pSB3T5 and are cultivated in E.coli BL21(DE3) with the matching non canonical amino acid.
Given that the incorporation of an ncAA through the amber codon implies a major metabolic pressure for the organism, we also investigated the influence of the recoding of the leucine codon. Therefore we used variants of the AcF)‑tRNA/syntheatase pair, adapted to the amber codon (AcF‑TAG) and to the leucine codon (AcF‑Leu). When comparing the growth of the different variants, a lower growth rate of the AcF-TAG variant is in evidence. When coding the ncAA through amber codon, the low specificity of the AcF‑TAG synthetase induces an extension of the translated proteins, which results in the lower growth. In contrary to the AcF-TAG, the incorporation of ncAA through the leucine codon is much more specific and therefore causes less disadvantage for the cell growth.
Figure 3: Optical density of the cultivation of AcF‑tRNA/synthetase variants adapted to the amber codon and the leucine codon.
The AcF-tRNA/synthetases are integrated in pSB1C3 and are cultivated in E.coli BL21 (DE3) with the matching non canonical amino acid.
Efficiency in incorporation of non canonical amino acids
Unfortunately the specificity of an orthogonal tRNA aminoacyl synthetase pair and the amber stop codon is not optimal. Endogenous amino acids are also incorporated, so the screening system is not completely accurate. In addition to that problem, some orthogonal tRNA synthetases own a low efficiency and resulting in a poorly incorporation of the ncAA are. Although the screening system enables to compare the efficiency, a low sfGFP signal in some cases can be difficult to detect. With this construction, the incorporation of an ncAA is detectable throughout the fluorescence of the mRFP or sfGFP. If the ncAA is incorporated within the linker sequence, a red and green fluorescence is detectable. If the ncAA is not incorporated, only, the mRFP is expressed, so only the red fluorescence is going to be detected.
For our Toolkit, we used six different ncAA and therefore six different tRNA‑aminoacyl synthetases. We characterized the efficiency of each synthetase for the incorporation of the matching ncAA.
Figure 11: Scores resulting from the synthetase-test system. 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.
To receive a completely specific aaRS, a selection system can be used to select the most specific aaRS out of a library of different aaRS variants. After the selection for the specificity of the aaRS, it can be analyzed on their efficiency with our improved measurement kit. Therefore, we generated a 27,672 different sequence variants containing tyrosyl tRNA‑synthetase library, based on pSB1C3.
References
Wang, Magliery TJ, Liu DR, Shcultz PG. (2000). A new functional suppressor tRNA/aminoacyl-tRNA synthetase pair for the in vivo incorporation of unnatural amino acids into proteins. J. Am. Chem. Soc.122,5010-5011Wankg L, Brock A, Herberich B, Schultz PG.(2001). Expanding the Genetic Code of Escherichia coli. sciencemag
