Difference between revisions of "Team:Bielefeld-CeBiTec/Project/translational system/library and selection"

(added title image)
Line 17: Line 17:
 
 
 
<!-- Ueberschriften -->
 
<!-- Ueberschriften -->
−
<h2> Translational System </h2>
+
<h2> Library and Selection </h2>
−
<h4> Transational Mechanism</h4>
+
 
 
 
 
−
</div>
+
<!-- Normaler Text -->
−
<div class="bevel bl"></div>
+
−
</div>
+
−
<div class="contentbox">
+
−
<div class="bevel tr"></div>
+
−
<div class="content">
+
−
+
−
<!-- Ueberschriften -->
+
−
<h2> Amber-codon</h2>
+
 
<article>
 
<article>
−
The genetic code, consisting of the four bases adenine, guanine, thymidine, and cytosine, provides the framework for the building of peptides and proteins, and thus builds the foundation for all living organisms. Three bases each code for an amino acid, and multiple amino acids in turn form a polypeptide. The genetic code itself is fixed, meaning that every possible base triplet codes for an amino acids or a translation stop, such that there are no “unused” combinations of three bases. In order for a non-canonical amino acid to be incorporated into a polypeptide, it is necessary that a certain base triplet codes for this amino acid specifically. As there are no unused triplets available, an existing codon needs to be repurposed. The amber codon (UAG/TAG) has been a prominent candidate, as it is one of the three codons signaling translation stop and occurs relatively rarely (roughly 8% of all stop codons in E. coli K-12 genome [1]). An amber suppressor system, consisting of a mutated tRNA and its corresponding tRNA-synthetase, is then used to replace the stop-function of the codon with the incorporation of the novel amino acid. More specifically, the tRNA has been evolved to suppress the stop-function. This is most commonly achieved by exchanging a base at the anticodon part, such that the amber-codon is read as a codon for an amino acid. Such systems are common in research ([3], [4], [5]). Especially the studies by Liu & Schultz (2010) [3] serve as a guideline for our own ncAA incorporation, as they provide us with comprehensive information on the suppressor system.
+
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-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/library_and_selection"> 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.
−
<br>
+
−
<br> <h3> Sources </h3>
+
−
<br> https://www.ncbi.nlm.nih.gov/nuccore/U00096
+
−
<br> http://www.spektrum.de/lexikon/biologie/amber-suppressor/2650
+
−
<br> Liu, Chang C., and Peter G. Schultz. "Adding new chemistries to the genetic code." Annual review of biochemistry 79 (2010): 413-444.
+
−
<br> Dumas, Anaëlle, et al. "Designing logical codon reassignment–expanding the chemistry in biology." Chemical Science 6.1 (2015): 50-69.
+
−
<br> Santoro, Stephen W., et al. "An efficient system for the evolution of aminoacyl-tRNA synthetase specificity." Nature biotechnology 20.10 (2002.
+
−
 
+
 
</article>
 
</article>
 +
 +
 +
 
 
 
 
Line 46: Line 32:
 
<div class="bevel bl"></div>
 
<div class="bevel bl"></div>
 
</div>
 
</div>
 +
 
<div class="contentbox">
 
<div class="contentbox">
 
<div class="bevel tr"></div>
 
<div class="bevel tr"></div>
Line 51: Line 38:
 
 
 
<!-- Ueberschriften -->
 
<!-- Ueberschriften -->
−
<h2> tRNA/aminoacyl-synthetase </h2>
+
<h2> Tyrosine and Pyrosyl-Lysine tRNA/aminoacyl-synthetase </h2>
−
+
<h4> Tyrosine tRNA/aminoacyl-synthetase </h4>
 
 
 
<!-- Normaler Text -->
 
<!-- Normaler Text -->
 
<article>
 
<article>
−
Synthetases form a family of enzymes which attach amino acids to the tRNA in a two-step reaction. Therefore, each tRNA synthetase (aaRS) aminoacylates all the tRNAs in an isoaccepting group, representing a particular amino acid. ‘Isoaccepting’ implies that each amino acid can be transported to the ribosome by more than one tRNA.
+
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).
−
The tRNA recognition is based on particular nucleotides, labelled as identity set. This identity set is often concentrated in the acceptor stem and anticodon loop region of the tRNA. However, the anticodon sequence of the tRNA is not necessary for a specific tRNA synthetase recognition due identity determinants which vary in their importance. <br>
+
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 <i> E. coli </i> tRNA/aaRS pair generated from archaea and the best known so far (Wang<i>  et al.</i>2001). This tyrosine synthetase (TyrRS) has a small anticodon loop binding domain (Steer<i>  et al.</i>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<i>  et al.</i>1999). The binding side is located deep inside a small pocket of the TyrRS (Tian<i>  et al.</i>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<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).
  
−
The first step of the two-step reaction is the formation of an aminoacylated intermediate by the reaction of the amino acid and ATP. This is possible through the Rossmann fold. This domain describes a six-stranded parallel β-sheet with connecting helices. The Rossmann fold acts homologous to the active site domains of hydrogenases and is responsible for binding ATP, the amino acid and the 3’-terminus of the tRNA.
 
 
</article>
 
</article>
 
 
Line 66: Line 53:
 
 
 
 
−
<article>
+
−
The second step is the nucleophilic attack of the carbonyl group of the anhydride from either the 2’-OH or the 3’-OH group on the 3’-terminus of the tRNA. This leads to the delivery of the amino acid to the tRNA.
+
<!-- Mittleres zentriertes Bild -->
−
 
+
<div class="figure medium">
−
The synthetase can either recognize directly a base specific functional group or the enzyme directly binds nonspecific portions of the tRNA. Both readouts function within the context of mutual induced fit, so conformational changes in both tRNA and synthetase occur while forming a catalytic complex.
+
<img class="figure image" src="https://static.igem.org/mediawiki/2017/c/c5/T--Bielefeld-CeBiTec--TyrRS_Ladungen_Zhang_2005_2017_08_26_.png">
−
The recognition requires determinants in the tRNA which are often located in the acceptor stem and the anticodon loop, but the identity signal can be in the tertiary core region as well (Krebs<i>  et al.</i></i>, 2014).
+
<p class="figure subtitle"><b>Figure 1: Tyrosine-binding site in apo <i> M. jannaschii TyrRS</i> </b><br> 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<i>  et al.</i>2005).</p>
−
 
+
−
aaRS can be categorized into two classes, based on mutually exclusive sets of sequence motifs and properties of structural domains. The synthetases of these two classes are functionally different. While the class I synthetases aminoacylate tRNAs at the 2’-OH position of A76, the class II synthetases aminoacylate tRNAs at the 3’-OH position. Nevertheless, the position of the initial aminoacylation is related to the binding orientation of the tRNA to the enzyme.
+
−
</article>
+
−
<div class="contentline">
+
−
<div class="half left">
+
−
The class I synthetases are monomeric synthetases and feature all structurally similar active site Rossmann fold domains in the region of the N-terminal. Beside this region, there are no significant structural or sequence similarities among the class I enzymes.
+
−
There is an acceptor binding site inserted into the Rossmann-fold domain at a common location which binds the single stranded terminal end of the tRNA, while its C-terminal domain binds in the minor groove of the L-shaped tRNA and the anticodon arm. That is the point for the discrimination among the different tRNAs. The binding requires the formation of a hairpin structure of the single stranded 3’-terminus with the amino acid and the ATP in the active site (Chang<i>  et al.</i></i>, 2010, Krebs<i>  et al.</i></i>, 2014).
+
−
</div>
+
−
<div class="half right">
+
−
<img class="figure large" src="https://static.igem.org/mediawiki/2017/thumb/5/59/T--Bielefeld-CeBiTec--classI_synthetases_Buch_Krebs_2014_2017_08_26.png/800px-T--Bielefeld-CeBiTec--classI_synthetases_Buch_Krebs_2014_2017_08_26.png">
+
−
<p class="figure subtitle"><b>Figure 1: Crystal structure of a class I tRNA/aminoacyl synthetase. </b><br> The tRNA is shown in red and the protein i n blue (Krebs<i>  et al.</i></i>, 2014).</p>
+
−
</div>
+
 
</div>
 
</div>
−
 
+
 
<article>
 
<article>
−
The class II synthetases build a quaternary structure, which in general consists of dimers. They own a structurally conserved active side domain, located towards the C-terminal end of the synthetase. This domain is mixed out of antiparallel β-sheed folds, flanked by α-helices, dissimilar to the Rossmann-fold (see class I synthetase) (Wang<i>  et al.</i></i>, 2000). In this domain, there are three conserved short sequences. One of these sequences is important for the multimerization and the other two are essential for the enzymatic activity. The class II synthetases bind to the major groove side of the tRNA acceptor stem, whereby unlike the class I synthetases no hairpin structure of the single stranded 3’-terminus into the active site is required (Chang<i>  et al.</i></i>, 2010, Krebs<i>  et al.</i></i>, 2014).
+
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&nbsp;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).
−
 
+
 
</article>
 
</article>
 
 
 +
<h4> Pyrosyl-Lysine tRNA/aminoacyl-synthetase </h>
 +
<article>
 +
 +
</article>
 
 
 
</div>
 
</div>
 
<div class="bevel bl"></div>
 
<div class="bevel bl"></div>
 
</div>
 
</div>
−
 
−
 
 
<div class="contentbox">
 
<div class="contentbox">
 
<div class="bevel tr"></div>
 
<div class="bevel tr"></div>
Line 101: Line 77:
 
 
 
<!-- Ueberschriften -->
 
<!-- Ueberschriften -->
−
<h2> An orthogonal tRNA/aminoacyl-synthetase pair </h2>
+
<h2> Generating the Library </h2>
 +
 
 
 
<!-- Normaler Text -->
 
<!-- Normaler Text -->
 
<article>
 
<article>
−
The incorporation of a non-canonical amino acid (ncAA) requires a tRNA/synthetase (tRNA/aaRS) pair which can accept and bind the ncAA. The tRNA/aaRS pair should be very specific in the incorporation of the ncAA and most importantly does not cross-react with endogenous amino acids (Ryu<i>  et al.</i></i>, 2006).
+
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).
−
This is achieved by the use of an orthogonal tRNA/aaRS pair, which does not cross-react with the endogenous components of the translational system. The cognate tRNA is not recognized by the endogenous synthetase and the cognate synthetase does not incorporate the endogenous amino acids due to differences in tRNA identity elements, especially in the acceptor stem (Kwok<i>  et al.</i></i>, 1980). At the same time, the orthogonal tRNA/synthetase pair has a good translational function (Wang<i>  et al.</i></i>, 2001, Chang<i>  et al.</i></i>, 2010).
+
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&nbsp;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.
−
<br>
+
−
Since the tRNA recognition by the aaRS can be domain or species specific (Kwok<i>  et al.</i></i>, 1980) a heterologous aaRS/tRNA pair from a different organism is used. A possible source for an orthogonal tRNA/synthetase pair to be applied in bacterial cells can be eukaryotic. However, the adaption of these tRNA/aaRS pair which aminoacylates in <i>Escherichia coli</i> is very difficult (Liu<i>  et al.</i></i>, 1999). In contrast, evolved synthetases from archaea can be expressed efficiently in <i>E. coli</i> (Wang<i>  et al.</i></i>, 2001, Wang<i>  et al.</i></i>, 2000) and at the same time are more similar to eukaryotic organisms than to the prokaryotic (Kwok<i>  et al.</i></i>, 1980, Zhang<i>  et al.</i></i>, 2005). The reason are significant differences in the acceptor stem and anticodon binding recognition domain between prokaryotic and archaea tRNA/aaRS (Wang<i>  et al.</i></i>, 2000).
+
−
<br>
+
−
One possible orthogonal tRNA/aaRS pair is the tyrosine tRNA/aaRS pair of <i>Methanococcus jannaschii</i>, where the first base pair of the acceptor stem CG as a tRNA<sup>Tyr</sup>identity element differs from those of <i>E. coli</i> tRNA<sup>Tyr</sup> containing a GC (Wang<i>  et al.</i></i>, 2001).
+
−
The orthogonal tRNA/aaRS pair is incorporated as a response of the amber stop-codon, which does not incorporate any of the 20 canonical amino acids. We use the amber stop-codon (UAG), because this codon is rare in <i>E.coli</i> and incorporation of random amino acids has been shown before.
+
−
 
+
 
</article>
 
</article>
−
+
−
+
−
+
 
 
 
</div>
 
</div>
Line 126: Line 96:
 
 
 
<!-- Ueberschriften -->
 
<!-- Ueberschriften -->
−
+
<h2> Selection </h2>
 +
 
 
 
<!-- Normaler Text -->
 
<!-- Normaler Text -->
 
<article>
 
<article>
 +
For the translational incorporation of ncAAs, a corresponding tRNA/aminoacyl-tRNA synthetase (tRNA/aaRS) pair is essential. The tRNA/aaRS pair must recognize and deliver the novel amino acid in response to a unique codon that does not encode any of the 20 canonical amino acids. This leads to a modification of the tRNA for the recognition of the amber-codon and to a modification of the synthetase for the specific binding of a new amino acid.
 +
To ensure specificity and orthogonality, a tRNA/aaRS pair from a distinct microbial organism is used to generate tRNA and aaRS libraries which are used to evolve a suitable tRNA/aaRS pair in iterative negative and positive selection steps (Wang<i>  et al.</i>2006). Therefore the tRNA and the aaRS each have to be separately mutated and selected.
 +
 +
</article>
 
 
 +
<h4> Modification of the aaRS </h4>
 
 
 +
<article>
 +
In the negative selection step, the synthetase (aaRS) library is tested for orthogonality of the exogenous tRNAs towards the endogenous translation system. The expression of a barnase gene is prevented by an amber stop codon in its coding region. If the exogenous synthetase binds an endogenous amino acid, the amber stop codon preventing the expression of barnase is suppressed, which leads to cells death. Therefore, only cells with orthogonal or non-functional synthetases survive (Wang<i>  et al.</i>2006, Liu<i>  et al.</i>2010).
 +
The orthogonal aaRS are then subjected to positive selection, in which the tRNA/aaRS pairs are tested for their functionality. The expression of an antibiotic resistance gene is prevented by an amber stop codon. Only if the exogenous aaRS incorporates any amino acid, the amber stop codon is suppressed and the cell attains antibiotic resistance. Resulting in the death of cells who do not own a functional aaRS (Wang<i>  et al.</i>2006, Liu<i>  et al.</i>2010).
 +
 
</article>
 
</article>
 +
 +
 +
<!-- Grosses zentriertes Bild -->
 +
<div class="figure large">
 +
<img class="figure image" src="https://static.igem.org/mediawiki/2017/thumb/4/4b/T--Bielefeld-CeBiTec--poster_aaRS_adaption_neu_2017_08_26.png/800px-T--Bielefeld-CeBiTec--poster_aaRS_adaption_neu_2017_08_26.png">
 +
<p class="figure subtitle"><b>Figure 2: Adaption of the aminoacyl-synthetase.</b><br> Positive and negative selection for the specificity of the orthogonal synthetase for the ncAA in <i> E. coli</i>. </p>
 +
</div>
 +
 +
<h4> Modification of the tRNA </h4>
 +
<article>
 +
Similar to the adaption by selection of the aaRS is the selection of a proper variant of the tRNA. Regarding the unnatural base pairs (UBP), the recognition of the unnatural bases through the tRNA for the incorporation of the non-canonical amino acids requires significant modifications of the tRNA. The recognition of the amber stop codon has already been achieved in other laboratories (Wang<i>  et al.</i>2006, Liu<i>  et al.</i>2010). Therefore, a selection cycle of negative and positive selections similar to the one of the aaRS, mentioned above, was performed. The main difference is the exchange of the tRNA by the aaRS on the plasmids used for the selection (Wang<i>  et al.</i>2006, Liu<i>  et al.</i>2010). The outcome is a tRNA, only able to recognize the amber codon and at the same time recognizes its orthogonal synthetase.
 +
 +
The combination of the tRNA and aaRS modification results in an orthogonal tRNA/aaRS pair capable of specifically incorporating a certain ncAA if the amber codon is present.
 +
</article>
 +
 +
 
 
 
 
Line 144: Line 140:
 
$("#project").addClass("active");
 
$("#project").addClass("active");
 
$("#project-translation").addClass("active");
 
$("#project-translation").addClass("active");
−
$("#project-translation-library").addClass("active");
+
$("#project-translation-mechanism").addClass("active");
 
</script>
 
</script>
 
</html>
 
</html>
 
{{Team:Bielefeld-CeBiTec/Footer}}
 
{{Team:Bielefeld-CeBiTec/Footer}}

Revision as of 03:34, 4 October 2017

#

Library and Selection

The incorporation of a non-canonical amino acid (ncAA) requires a tRNA/aminoacyl-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

For the translational incorporation of ncAAs, a corresponding tRNA/aminoacyl-tRNA synthetase (tRNA/aaRS) pair is essential. The tRNA/aaRS pair must recognize and deliver the novel amino acid in response to a unique codon that does not encode any of the 20 canonical amino acids. This leads to a modification of the tRNA for the recognition of the amber-codon and to a modification of the synthetase for the specific binding of a new amino acid. To ensure specificity and orthogonality, a tRNA/aaRS pair from a distinct microbial organism is used to generate tRNA and aaRS libraries which are used to evolve a suitable tRNA/aaRS pair in iterative negative and positive selection steps (Wang et al.2006). Therefore the tRNA and the aaRS each have to be separately mutated and selected.

Modification of the aaRS

In the negative selection step, the synthetase (aaRS) library is tested for orthogonality of the exogenous tRNAs towards the endogenous translation system. The expression of a barnase gene is prevented by an amber stop codon in its coding region. If the exogenous synthetase binds an endogenous amino acid, the amber stop codon preventing the expression of barnase is suppressed, which leads to cells death. Therefore, only cells with orthogonal or non-functional synthetases survive (Wang et al.2006, Liu et al.2010). The orthogonal aaRS are then subjected to positive selection, in which the tRNA/aaRS pairs are tested for their functionality. The expression of an antibiotic resistance gene is prevented by an amber stop codon. Only if the exogenous aaRS incorporates any amino acid, the amber stop codon is suppressed and the cell attains antibiotic resistance. Resulting in the death of cells who do not own a functional aaRS (Wang et al.2006, Liu et al.2010).

Figure 2: Adaption of the aminoacyl-synthetase.
Positive and negative selection for the specificity of the orthogonal synthetase for the ncAA in E. coli.

Modification of the tRNA

Similar to the adaption by selection of the aaRS is the selection of a proper variant of the tRNA. Regarding the unnatural base pairs (UBP), the recognition of the unnatural bases through the tRNA for the incorporation of the non-canonical amino acids requires significant modifications of the tRNA. The recognition of the amber stop codon has already been achieved in other laboratories (Wang et al.2006, Liu et al.2010). Therefore, a selection cycle of negative and positive selections similar to the one of the aaRS, mentioned above, was performed. The main difference is the exchange of the tRNA by the aaRS on the plasmids used for the selection (Wang et al.2006, Liu et al.2010). The outcome is a tRNA, only able to recognize the amber codon and at the same time recognizes its orthogonal synthetase. The combination of the tRNA and aaRS modification results in an orthogonal tRNA/aaRS pair capable of specifically incorporating a certain ncAA if the amber codon is present.