Difference between revisions of "Team:Bielefeld-CeBiTec/Results/translational system/translation mechanism"

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Generating our Toolkit, we used mutated variants of two different aminoacyl tRNA/synthetases. We proved that the incorporation of non-canonical amino acids through the amber codon is a major metabolic pressure for the cells. In addition to that, we demonstrated that the aminoacyl tRNA/synthetases we used in our Toolkit are very different in specificity, and therefore some are not very efficient in incorporating those. This led us to generating our own aminoacyl tRNA/synthetase variants and selecting the most efficient aminoacyl tRNA/synthetase candidates.
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For generating our Toolkit, we used variants of two different aminoacyl tRNA/synthetases (aminoacyl tRNA/RS), tyrosyl and pyrrosylyl. When working with our toolkit, 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.
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Therefore, we transformed the certain aminoacyl tRNA/RS plasmid and two controls (pSB1C3 with mRFP and pSB3T5 with mRFP) 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 600rpm at 37 °C. We used two biological replicates, each with three technical replicates of the optical density of each cultivation by NanoDrop.
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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 PrK-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 (BBa_K2201343)for the efficiency of the incorporation of ncAA.
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<p class="figure subtitle"><b>Figure 1: Optical density of the cultivation of five different aminoacyl tRNA/synthetases. </b><br> The Agilent BioAnalyzer High Sensitivity DNA Assay is used for the measurement. The aminoacyl tRNA/synthetases are integrated in pSB1C3 and are cultivated in <i>E.coli</i> BL21(DE3) with the matching non canonical amino acid. </p>
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<p class="figure subtitle"><b>Figure 2: Optical density of the cultivation of two different aminoacyl tRNA/synthetases. </b> The aminoacyl tRNA/synthetases are integrated in pSB3T5 and are cultivated in <i>E.coli</i> BL21(DE3) with the matching non canonical amino acid.</p>
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Revision as of 23:18, 30 October 2017

Translation Mechanism

Short Summary

Generating our Toolkit, we used mutated variants of two different aminoacyl tRNA/synthetases. We proved that the incorporation of non-canonical amino acids through the amber codon is a major metabolic pressure for the cells. In addition to that, we demonstrated that the aminoacyl tRNA/synthetases we used in our Toolkit are very different in specificity, and therefore some are not very efficient in incorporating those. This led us to generating our own aminoacyl tRNA/synthetase variants and selecting the most efficient aminoacyl tRNA/synthetase candidates.

Comparison of different aminoacyl tRNA/synthetases

For generating our Toolkit, we used variants of two different aminoacyl tRNA/synthetases (aminoacyl tRNA/RS), tyrosyl and pyrrosylyl. When working with our toolkit, we recognized that the Escherichia coli cells containing these different aminoacyl tRNA/RS plasmids had different characteristics. We could confirm this assumption by performing growth experiments with E.coli transformed with our different aminoacyl tRNA/synthetases. Therefore, we transformed the certain aminoacyl tRNA/RS plasmid and two controls (pSB1C3 with mRFP and pSB3T5 with mRFP) in E. coli 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 600rpm 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 PrK-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 (BBa_K2201343)for the efficiency of the incorporation of ncAA.

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.