Difference between revisions of "Team:Bielefeld-CeBiTec/Project/translational system/library and selection"
m (small fixes) |
(article to div article) |
||
Line 19: | Line 19: | ||
<!-- Normaler Text --> | <!-- Normaler Text --> | ||
− | <article> | + | <div class="article"> |
The incorporation of a non-canonical amino acid (ncAA) requires a <a href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/translational_system/library_and_selection">tRNA/aminoacyl-tRNA synthetase(tRNA/aaRS)</a> pair which is able to accept and bind the ncAA (to charge the tRNA with the ncAA). The tRNA/aaRS pair should be very specific in the incorporation of the ncAA. Therefore a library of the mutated <a href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/translational_system/translation_mechanism">orthogonal tRNA/aaRS</a> is generated and undergoes numerous rounds of positive and negative selection for the adaption of the codon recognition and the amino acid binding. The selection results in a synthetase which can be expressed efficiently in E.coli and is able to reliable incorporate an unnatural amino acid. | The incorporation of a non-canonical amino acid (ncAA) requires a <a href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/translational_system/library_and_selection">tRNA/aminoacyl-tRNA synthetase(tRNA/aaRS)</a> pair which is able to accept and bind the ncAA (to charge the tRNA with the ncAA). The tRNA/aaRS pair should be very specific in the incorporation of the ncAA. Therefore a library of the mutated <a href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/translational_system/translation_mechanism">orthogonal tRNA/aaRS</a> is generated and undergoes numerous rounds of positive and negative selection for the adaption of the codon recognition and the amino acid binding. The selection results in a synthetase which can be expressed efficiently in E.coli and is able to reliable incorporate an unnatural amino acid. | ||
− | </ | + | </div> |
Line 39: | Line 39: | ||
<!-- Normaler Text --> | <!-- Normaler Text --> | ||
− | <article> | + | <div class="article"> |
There are several amino acids which have been incorporated into peptides and proteins in <i> Escherichia coli </i>. Among others, it was possible to incorporate ncAA with heavy atoms, keto and alkaline side chains, photo crosslinking and so on (Zhang<i> et al.</i>2005). | There are several amino acids which have been incorporated into peptides and proteins in <i> Escherichia coli </i>. Among others, it was possible to incorporate ncAA with heavy atoms, keto and alkaline side chains, photo crosslinking and so on (Zhang<i> et al.</i>2005). | ||
For the binding of the ncAA, an adaption of the synthetase is necessary. This is done by certain mutations of several regions of the synthetase, leading to a conformational change of the binding pocket and resulting in the binding of a new amino acid. | For the binding of the ncAA, an adaption of the synthetase is necessary. This is done by certain mutations of several regions of the synthetase, leading to a conformational change of the binding pocket and resulting in the binding of a new amino acid. | ||
Line 45: | Line 45: | ||
The TyrRS binding pocket is highly hydrophilic, preventing the binding of phenylalanine, whose structure is similar to the one of tyrosine (Goldgur<i> et al.</i>1997). Changing the characteristic of the binding pocket into a hydrophobic kind, could favor the binding of phenylalanine derivates (Goldgur<i> et al.</i>1997). In the middle of the negative charged binding pocket (Zhang<i> et al.</i>2005), there is an aspartic acid on position 158. The entrance of the binding pocket is positioned between two glutamic acids at positions 36 and 172. Upon tRNA binding, the aspartic amino acid Asp158 forms two hydrogen bonds with the glycine at position 34 (Zhang<i> et al.</i>2005). | The TyrRS binding pocket is highly hydrophilic, preventing the binding of phenylalanine, whose structure is similar to the one of tyrosine (Goldgur<i> et al.</i>1997). Changing the characteristic of the binding pocket into a hydrophobic kind, could favor the binding of phenylalanine derivates (Goldgur<i> et al.</i>1997). In the middle of the negative charged binding pocket (Zhang<i> et al.</i>2005), there is an aspartic acid on position 158. The entrance of the binding pocket is positioned between two glutamic acids at positions 36 and 172. Upon tRNA binding, the aspartic amino acid Asp158 forms two hydrogen bonds with the glycine at position 34 (Zhang<i> et al.</i>2005). | ||
− | </ | + | </div> |
Line 57: | Line 57: | ||
</div> | </div> | ||
− | <article> | + | <div class="article"> |
When the ligand tyrosine is bound, hydrogen bonds to the Tyr32, Asp158, Glu36, Gln173, Tyr151 and Gln155 are formed, resulting in subtle movements of the side chains within the tyrosine-binding pocket (Zhang<i> et al.</i>2005). Beside the binding pocket, also other domains are affected by the binding of the tyrosine. For example, the loop 73-83, positioned at the entrance of the binding pocket, which due to the conformational changes then provides a hydrophobic lid over the binding pocket. This is assumed to have the effect of separating the activated tyrosine from water during the catalytic reaction (Zhang<i> et al.</i>2005). | When the ligand tyrosine is bound, hydrogen bonds to the Tyr32, Asp158, Glu36, Gln173, Tyr151 and Gln155 are formed, resulting in subtle movements of the side chains within the tyrosine-binding pocket (Zhang<i> et al.</i>2005). Beside the binding pocket, also other domains are affected by the binding of the tyrosine. For example, the loop 73-83, positioned at the entrance of the binding pocket, which due to the conformational changes then provides a hydrophobic lid over the binding pocket. This is assumed to have the effect of separating the activated tyrosine from water during the catalytic reaction (Zhang<i> et al.</i>2005). | ||
− | </ | + | </div> |
<h4> Pyrosyl-Lysine tRNA/aminoacyl-synthetase </h> | <h4> Pyrosyl-Lysine tRNA/aminoacyl-synthetase </h> | ||
Line 67: | Line 67: | ||
</div> | </div> | ||
− | |||
</div> | </div> | ||
<div class="contentbox"> | <div class="contentbox"> | ||
− | |||
<div class="content"> | <div class="content"> | ||
Line 78: | Line 76: | ||
<!-- Normaler Text --> | <!-- Normaler Text --> | ||
− | <article> | + | <div class="article"> |
Being able to pass as many as possible mutated synthetases through the cycle of the two selections for the adaption of the specific ncAA, a large library of different synthetase versions has to be created. The large library of random variants of sequences can be generated by side specific mutagenesis using randomized primers with the NNK scheme to avoid the incorporation of stop codons. The randomized N codes for the bases A,C,G,T and the randomized K codes for the bases G,T. This leads to the use of 32 codons, so every codon except the three stop codons and the one start codon is encoded through the NNK scheme (Yuval<i> et al.</i>2011). By comparison to other randomized schemes, the NNK has a relatively low ratio between the most common and rarest coded amino acid (3/32 vs. 1/32) (Yuval<i> et al.</i>2011). | Being able to pass as many as possible mutated synthetases through the cycle of the two selections for the adaption of the specific ncAA, a large library of different synthetase versions has to be created. The large library of random variants of sequences can be generated by side specific mutagenesis using randomized primers with the NNK scheme to avoid the incorporation of stop codons. The randomized N codes for the bases A,C,G,T and the randomized K codes for the bases G,T. This leads to the use of 32 codons, so every codon except the three stop codons and the one start codon is encoded through the NNK scheme (Yuval<i> et al.</i>2011). By comparison to other randomized schemes, the NNK has a relatively low ratio between the most common and rarest coded amino acid (3/32 vs. 1/32) (Yuval<i> et al.</i>2011). | ||
By the randomization of more than one position, numerous more sequence variants occur. If one position is randomized, 130 variants of the sequences are needed to attain a 0.99 probability of discovering the best variant (Yuval<i> et al.</i>2011). At the same time, for the randomization of three positions, there are statistically 102,478 variants needed to discover the best variant. In this context, the probability of full codon coverage changes from 0.82 % for one randomizes position to 3.25 • 10<sup>-36</sup> % for three randomized position (Yuval<i> et al.</i>2011). Regarding, that not the full coverage is essential, but rather the discovering of the best variant, the NNK is a solid method for the generation of a synthetase library for the selection of the best ncAA binding site. | By the randomization of more than one position, numerous more sequence variants occur. If one position is randomized, 130 variants of the sequences are needed to attain a 0.99 probability of discovering the best variant (Yuval<i> et al.</i>2011). At the same time, for the randomization of three positions, there are statistically 102,478 variants needed to discover the best variant. In this context, the probability of full codon coverage changes from 0.82 % for one randomizes position to 3.25 • 10<sup>-36</sup> % for three randomized position (Yuval<i> et al.</i>2011). Regarding, that not the full coverage is essential, but rather the discovering of the best variant, the NNK is a solid method for the generation of a synthetase library for the selection of the best ncAA binding site. | ||
− | </ | + | </div> |
</div> | </div> | ||
− | |||
</div> | </div> | ||
<div class="contentbox"> | <div class="contentbox"> | ||
− | |||
<div class="content"> | <div class="content"> | ||
Revision as of 17:43, 30 October 2017
Library and Selection
The incorporation of a non-canonical amino acid (ncAA) requires a tRNA/aminoacyl-tRNA synthetase(tRNA/aaRS) pair which is able to accept and bind the ncAA (to charge the tRNA with the ncAA). The tRNA/aaRS pair should be very specific in the incorporation of the ncAA. Therefore a library of the mutated orthogonal tRNA/aaRS is generated and undergoes numerous rounds of positive and negative selection for the adaption of the codon recognition and the amino acid binding. The selection results in a synthetase which can be expressed efficiently in E.coli and is able to reliable incorporate an unnatural amino acid.
Tyrosine and Pyrosyl-Lysine tRNA/aminoacyl-synthetase
Tyrosine tRNA/aminoacyl-synthetase
There are several amino acids which have been incorporated into peptides and proteins in Escherichia coli . Among others, it was possible to incorporate ncAA with heavy atoms, keto and alkaline side chains, photo crosslinking and so on (Zhang et al.2005).
For the binding of the ncAA, an adaption of the synthetase is necessary. This is done by certain mutations of several regions of the synthetase, leading to a conformational change of the binding pocket and resulting in the binding of a new amino acid.
The tyrosine synthetase is the first orthogonal E. coli tRNA/aaRS pair generated from archaea and the best known so far (Wang et al.2001). This tyrosine synthetase (TyrRS) has a small anticodon loop binding domain (Steer et al.1999), so the change of the recognition in order to change the anticodon loop of its cognate tRNA to CUA is not as difficult as with a larger anticodon loop binding domain (Steer et al.1999). The binding side is located deep inside a small pocket of the TyrRS (Tian et al.2004). This leads to assume, that no large conformational changes are necessary for the incorporation of the ncAA. Rather very small conformational changes result in a larger or smaller binding pocket and an altered hydrogen-binding interaction with the ligand (Zhang et al.2005).
The TyrRS binding pocket is highly hydrophilic, preventing the binding of phenylalanine, whose structure is similar to the one of tyrosine (Goldgur et al.1997). Changing the characteristic of the binding pocket into a hydrophobic kind, could favor the binding of phenylalanine derivates (Goldgur et al.1997). In the middle of the negative charged binding pocket (Zhang et al.2005), there is an aspartic acid on position 158. The entrance of the binding pocket is positioned between two glutamic acids at positions 36 and 172. Upon tRNA binding, the aspartic amino acid Asp158 forms two hydrogen bonds with the glycine at position 34 (Zhang et al.2005).
Figure 1: Tyrosine-binding site in apo M. jannaschii TyrRS
The electrostatic distribution around the tyrosine-binding site is shown. Positive potential is blue (10 mV), neutral potential (0 mV) is white, and negative potential (−10 mV) is red (Zhang et al.2005).
When the ligand tyrosine is bound, hydrogen bonds to the Tyr32, Asp158, Glu36, Gln173, Tyr151 and Gln155 are formed, resulting in subtle movements of the side chains within the tyrosine-binding pocket (Zhang et al.2005). Beside the binding pocket, also other domains are affected by the binding of the tyrosine. For example, the loop 73-83, positioned at the entrance of the binding pocket, which due to the conformational changes then provides a hydrophobic lid over the binding pocket. This is assumed to have the effect of separating the activated tyrosine from water during the catalytic reaction (Zhang et al.2005).
Pyrosyl-Lysine tRNA/aminoacyl-synthetase
Generating the Library
Being able to pass as many as possible mutated synthetases through the cycle of the two selections for the adaption of the specific ncAA, a large library of different synthetase versions has to be created. The large library of random variants of sequences can be generated by side specific mutagenesis using randomized primers with the NNK scheme to avoid the incorporation of stop codons. The randomized N codes for the bases A,C,G,T and the randomized K codes for the bases G,T. This leads to the use of 32 codons, so every codon except the three stop codons and the one start codon is encoded through the NNK scheme (Yuval et al.2011). By comparison to other randomized schemes, the NNK has a relatively low ratio between the most common and rarest coded amino acid (3/32 vs. 1/32) (Yuval et al.2011).
By the randomization of more than one position, numerous more sequence variants occur. If one position is randomized, 130 variants of the sequences are needed to attain a 0.99 probability of discovering the best variant (Yuval et al.2011). At the same time, for the randomization of three positions, there are statistically 102,478 variants needed to discover the best variant. In this context, the probability of full codon coverage changes from 0.82 % for one randomizes position to 3.25 • 10-36 % for three randomized position (Yuval et al.2011). Regarding, that not the full coverage is essential, but rather the discovering of the best variant, the NNK is a solid method for the generation of a synthetase library for the selection of the best ncAA binding site.
Selection
Modification of the aaRS
Figure 2: Adaption of the aminoacyl-synthetase.
Positive and negative selection for the specificity of the orthogonal synthetase for the ncAA in E. coli.