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

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Generating our <a href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/toolbox">Toolbox</a>, 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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In order to generate our <a href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/toolbox">toolbox</a>, we used mutated variants of two different aminoacyl tRNA/synthetases complex pair. We proved that the incorporation of non-canonical amino acids through the amber codon poses as a major metabolic burden for the cells. In addition, we demonstrated that the aminoacyl tRNA/synthetases we used in our toolkit are very different in specificity, leading to inefficient incorporation of non canonical amino acids (ncAA). Therefore, we decides to generate  our own aminoacyl tRNA/synthetase variants and enabling so the most efficient selection of aminoacyl tRNA/synthetase candidates.
  
 
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For generating our Toolbox, 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.  
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Generating our Toolbox, we used variants of two different aminoacyl tRNA/synthetases (aminoacyl tRNA/RS), namely <a href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/translational_system/library_and_selection">tyrosyl and pyrrosylyl</a>. While apllying our toolbox, we recognized that the <i>Escherichia coli</i> cells containing different aminoacyl tRNA/RS plasmids had different characteristics. We could further confirm this assumption by performing growth experiments with <i>E.coli</i> that prior to the cultivation were transformed with our different aminoacyl tRNA/synthetases.  
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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>&nbsp;BL21&nbsp;(DE3). We performed the cultivation in 1&nbsp;mL LB-media with the matching non canonical amino acid (ncAA) in 12&#x2011;well microtiter plates by 600&nbsp;rpm at 37&nbsp;°C. We used two biological replicates, each with three technical replicates of the optical density of each cultivation by NanoDrop.
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For this purpose, we transformed chosen aminoacyl tRNA/RS plasmid and two control vectors, <a href="http://parts.igem.org/Part:pSB1C3">pSB1C3</a> and <a href="http://parts.igem.org/Part:pSB3T5">pSB3T5</a>, into <i>E. coli</i>&nbsp;BL21&nbsp;(DE3). We performed the cultivation in 1&nbsp;mL LB-media with the matching non canonical amino acid (ncAA) in 12&#x2011;well microtiter plates by 600&nbsp;rpm at 37&nbsp;°C. We used two biological replicates, each with three technical replicates. The optical density of each cultivation was measured with NanoDrop at 600 nm. The comparison of the growth rates of each cultivation for the cells containing the aminoacyl tRNA/RS in high copy plasmid pSB1C3, as depicted in <b>Figure 1</b>, showed significant  differences between different the cultures.
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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&nbsp;% slighter growth rate than of the control. Only the <a href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/toolbox/analysing">PrK</a>&#x2011;tRNA/synthetase showed a better growth of up to 17&nbsp;% 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&#x2011;YFP system(<a href="http://parts.igem.org/Part:BBa_K2201343">BBa_K2201343</a>)for the efficiency of the incorporation of ncAA.  
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Additionally, the growth experiments show that the incorporation of ncAAs through the amber codon cause a major metabolic burden for the cells, resulting in a growth rate decreased by 0.25 and 0.5 %, as compared to control cultures. Only the <a href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/toolbox/analysing">Propargyllysine (PrK)</a>&#x2011;tRNA/synthetase showed growth of up to 17&nbsp;% higher than the growth of the control cultures. This can be explained by the metabolic pressure, exerted by the pSB1C3, due to coding the mRFP. In addition, the highly specific incorporation of the ncAA by the PrK <b>(Figure 3)</b> also supports faster growth of the culture, containing the PrK aminoacyl tRNA/synthetase. This is due to the similarity of the used ncAA Prk-couple to the wild type amino acid pyrrolysyl, requiring only one point mutation of the aminoacyl synthetase (aaRS). We could demonstrate this with our CFP&#x2011;YFP system(<a href="http://parts.igem.org/Part:BBa_K2201343">BBa_K2201343</a>)for the efficiency of the incorporation of ncAA.  
  
  
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<p class="figure subtitle"><b>Figure 4:</b> Scores resulting from the synthetase-test system. The negative score results from the emission quotient CFP(475&nbsp;nm)/YFP(525&nbsp;nm) when cultivated without the specific ncAA. The positive score results from the emission quotient YFP(525&nbsp;nm)/CFP(475&nbsp;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.</p>
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<p class="figure subtitle"><b>Figure 11:</b> Scores resulting from the synthetase-test system. The negative score results from the emission quotient CFP(475&nbsp;nm)/YFP(525&nbsp;nm) when cultivated without the specific ncAA. The positive score results from the emission quotient YFP(525&nbsp;nm)/CFP(475&nbsp;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.</p>
 
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As shown in Figure 4, the efficiency of the different synthetases to incorporate their specific ncAAs vary in a wide range and are not completely specific. Meaning, that the used aaRS also incorporates endogenous amino acids. In addition to the lacking specificity, the characterization of many different tRNA/aaRS imply much effort, so it is inapplicable in a higher throughput.
 
As shown in Figure 4, the efficiency of the different synthetases to incorporate their specific ncAAs vary in a wide range and are not completely specific. Meaning, that the used aaRS also incorporates endogenous amino acids. In addition to the lacking specificity, the characterization of many different tRNA/aaRS imply much effort, so it is inapplicable in a higher throughput.

Revision as of 20:03, 1 November 2017

Translation Mechanism

Short Summary

In order to generate our toolbox, we used mutated variants of two different aminoacyl tRNA/synthetases complex pair. We proved that the incorporation of non-canonical amino acids through the amber codon poses as a major metabolic burden for the cells. In addition, we demonstrated that the aminoacyl tRNA/synthetases we used in our toolkit are very different in specificity, leading to inefficient incorporation of non canonical amino acids (ncAA). Therefore, we decides to generate our own aminoacyl tRNA/synthetase variants and enabling so the most efficient selection of aminoacyl tRNA/synthetase candidates.

Comparison of Different Aminoacyl tRNA/Synthetases

Generating our Toolbox, we used variants of two different aminoacyl tRNA/synthetases (aminoacyl tRNA/RS), namely tyrosyl and pyrrosylyl. While apllying our toolbox, we recognized that the Escherichia coli cells containing different aminoacyl tRNA/RS plasmids had different characteristics. We could further confirm this assumption by performing growth experiments with E.coli that prior to the cultivation were transformed with our different aminoacyl tRNA/synthetases. For this purpose, we transformed chosen aminoacyl tRNA/RS plasmid and two control vectors, pSB1C3 and pSB3T5, into 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 600 rpm at 37 °C. We used two biological replicates, each with three technical replicates. The optical density of each cultivation was measured with NanoDrop at 600 nm. The comparison of the growth rates of each cultivation for the cells containing the aminoacyl tRNA/RS in high copy plasmid pSB1C3, as depicted in Figure 1, showed significant differences between different the cultures. Additionally, the growth experiments show that the incorporation of ncAAs through the amber codon cause a major metabolic burden for the cells, resulting in a growth rate decreased by 0.25 and 0.5 %, as compared to control cultures. Only the Propargyllysine (PrK)‑tRNA/synthetase showed growth of up to 17 % higher than the growth of the control cultures. This can be explained by the metabolic pressure, exerted by the pSB1C3, due to coding the mRFP. In addition, the highly specific incorporation of the ncAA by the PrK (Figure 3) also supports faster growth of the culture, containing the PrK aminoacyl tRNA/synthetase. This is due to the similarity of the used ncAA Prk-couple to the wild type amino acid pyrrolysyl, requiring only one point mutation of the aminoacyl synthetase (aaRS). We could demonstrate this 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.

When investigating the influence of a low or high copy plasmid, the low copy plasmid turned out to be the best choice for the growth when expressing aminoacyl tRNA/synthetases to incorporate ncAA. The tendency, depicted in figure 1 and 2, is caused by the different antibiotic resistance The comparison of the low and high copy plasmid backbone, depicted in figure 1 and 2, did not lead to the same conclusion, due to different antibiotic resistances. In context of a better growth when using low copy plasmids (Wang et. al., 2001, Wang et. al. , 2000), the chloramphenicole resistance turned out to be the better choice for the expression of aminoacyl tRNA/synthetases

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

The incorporation of a non canonical amino acid or an endogenous amino acid through an amber codon, can be tested in different ways. A safe method to prove the incorporation is the detection by mass spectrometry. That is a very accurate, but also complex and expensive method. A much simpler method is the Measurement Kit, created by iGEM Austen-Texas 2014 und used by Aachen 2016. The Measurement Kit is based on a reporter plasmid combined with a screening plasmid containing the tRNA and corresponding amino acyl‑synthetase (aaRS). The screening plasmid and the reporter plasmid have to be cotransformed. The reporter plasmid is constructed with an mRFP domain. This domain is connected through a linker sequence (containing a recoded amber stop codon) with a sfGFP domain.

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.

As shown in Figure 4, the efficiency of the different synthetases to incorporate their specific ncAAs vary in a wide range and are not completely specific. Meaning, that the used aaRS also incorporates endogenous amino acids. In addition to the lacking specificity, the characterization of many different tRNA/aaRS imply much effort, so it is inapplicable in a higher throughput.

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.122,5010-5011
Wankg L, Brock A, Herberich B, Schultz PG.(2001). Expanding the Genetic Code of Escherichia coli. sciencemag