Difference between revisions of "Team:Bielefeld-CeBiTec/Model"

Revision as of 01:12, 2 November 2017

Modeling

Organization of our modeling projects

On this page, we describe our main modeling project, which was integral for our whole project. However, besides this complex modeling, we also conducted and applied several straight-forward stochastic and statistical models to support and guide numerous steps of our laboratory work. Some of these modeling projects are briefly described in this box; however, we recommend reading the linked pages for further information.

Discriminant function model for the ICG prediction:

We conducted a discriminant function analysis for the classification of nucleotides in Oxford Nanopore sequencing reads at a specific position. This model is part of our iCG software module and enabled the successful detection of unnatural bases.

Calculation of the required library size for the selection system:

We applied combinatorics and statistics to calculate the optimal library size for the tRNA synthetase selection process. This was a trade off between putting lots of efforts into constructing a very huge library and missing diversity in a too small library. Therefore, we predicted the optimal library size. Experimental validation of this prediction was done via MiSeq analysis of the diversity of a subset of this library.

Strength prediction for a transcription signal amplification system (BBa_K2201373):

We modeled and visually compared the mRFP production over time for a normal mRFP reporter system and compared it to our enhanced signaling.. Validity was done as part of the positive selection process for an adapted tRNA synthetase.

Short Summary

As our project explores possibilities of an expanded genetic code via unnatural bases and non-canonical amino acids, we set out to complement and improve our lab work via modeling of novel amino acyl tRNA synthetases (aaRS) for a non-canonical amino acids, which were synthetized in our lab. In order to incorporate non-canonical amino acids into proteins via the translational process, the aaRS has to attach the amino acid to the respective tRNA. Thus, we designed aaRS sequences which were adjusted to link our own non-canonical amino acid to a fitting tRNA. Candidates were evaluated and selected, based on a ROSETTA score. Most promising sequences were ordered via gene synthesis for the experimental validation. Figure 1 provides a rough overview of our modeling project. Table 1 below summarizes the realization in practice.

Figure 1: Modeling Project Overview
A stylized overview of our modeling project, containing both in silico and in vivo components.

Step	Software/Method	Meaning
1. Ligand preparation	Manually via Avogadro	Due to the novelty of our amino acid, no information on the ligand is available in databases. Therefore, manual generation of a conformer ensemble, containing for example all energetically useful arrangements of atoms within the molecule, was required.
2. Scaffold categorization	ROSETTA protocol	The scaffold describes the rough layout of the synthetase. We downloaded the scaffold 1J1U , the aaRS of Methalonococcus janischii as a template, and then relaxed its structure to improve the outcome of the ROSETTA algorithm.
3. Set simulation constrains	Manually via ROSETTA	Constrains with regards to possible mutations of the synthetase ensure that the generated sequences fit to the amino acid. For example, we constrained the distance between certain atoms and their angle to a range optimal for hydrogen bonds.
4. Enzyme matching	ROSETTA protocol	ROSETTA combines information about the ligand and constrains to find possible hydrogen bonding partners and propose the shape of the scaffold within the set constraints.
5. Enzyme design	ROSETTA protocol	An algorithm uses the information from the previous step and information on the ligand to simulate the mutation process and generate sequences for optimized scaffolds with corresponding scores as measures of fit.
6. Evaluate results in silico	Manually	Based on the score values, we ordered the synthesis of the most promising sequences.
7. Evaluate results in vivo	Manually	The synthetases are validated in the lab with the corresponding ncAA via a positive-negative selection system.

Table 1: Steps of our modeling project
Our modeling project consists of seven main steps, combining in silico and in vivo components.

Introduction

Overview

Figure 2: Tyrosyl-tRNA-synthetase
3D-structure based on "1J1U" from PDB edited with pymol

As part of our iGEM project, we faced the challenge of adapting the tRNA synthetase to non-canonical amino acids. For this purpose, we modeled possible candidates for synthetases as an alternative to carrying out a positive-negative selection according to (Liu et al., 2007) in the laboratory. Due to the rapid development in the field of protein and molecular structure analysis, there has been an increase in the availability of molecular 3D structure data. These data are organized in publicly available databases which provide a foundation for the modeling and simulation of chemical-biological processes in bioinformatics. As our ncAA has been synthetized in our lab, no such comprehensive information is available, yet. However, information of similarly structured amino acids can potentially serve as a basis for our modeling. Specifically, we focused on simulation to design an aaRS for the new ncAA CBT-Asparagine.

Method

We used the open-source software "ROSETTA" for the main part of our modeling project, which was introduced at the University of Washington by David Baker in 1997 (Simon et al.,1997), initially in the context of protein structure prediction. ROSETTA has grown through the addition of numerous modules and is currently widely used in research. In our application, we focus on the Rosetta module called the "Rosetta Enzyme Design Protocol"

ROSETTA Enzyme Design

Overview

Since the non-canonical amino acid synthesized in the laboratory is completely novel, there is no corresponding tRNA synthetase which can load the tRNA, yet. For this reason, we use the enzyme design protocol to design the binding pocket in a way that allows it to form an effective and specific enzyme. The protocol consists of two main steps: matching and designing (Richter et al., 2011) The enzyme design algorithm is briefly summarized in Fig. 2

Figure (2): Flowchart Enzym Design Protocol

Matching Step

Figure (NUMMER ANGEBEN!): ABBILDUNGSTITEL
BILDUNTERSCHRIFT

The meaning of the matching step is to match the amino acids which constrains to the ligand, following specific constrains which ensure that the result is sensible and feasible. For this, ROSETTA analyzes the structural formula of the non- canonical amino acid and offers the possible hydrogen binding partners.

Matching step inputs

For the matching step, the following input-files are needed:

a “.params”-file specifying information about the ligand
a “.pdb”-file providing a rough scaffold layout
a “.cst”-file to define the bindings between ligand and scaffold
a “.pos”-file to define the positions of the amino acids in the scaffold
a “.flags” file to control all inputs. These files are necessary, as they describe the ligand and backbone and specify the parameters of the algorithm

To read about each file in further detail, please click the technical detail button below:
SHOW TECHNICAL DETAILS

“.params”-file:
A conformer ensemble has to be generated using information about the ligand, as the non-canonical amino acids are not generally available in databases like PDB, making it necessary to build them manually using tools like pymol, Avogadro or Chemdraw. Using these tools, files can be saved in the desired format. The ligand needs to be specified in the “.sdf”, “.mol” or “.mol2” file format. Such a file can be obtained automatically by converting the relevant information from a “.pdb” file, if available. This conversion process usually also involves augmenting the data with hydrogen atoms in case they are missing from the “.pdb” file. Alternatively, the ligand can be designed using "Simplified Molecular Input Line Entry Specification" (SMILES) or manually using tools such as Avogadro, as we did. In the next step, the ligand file is used to create a conformer ensemble that is in turn used to create a Rosetta parameter (“.params”) file. In addition to the specific names of all atoms present in the ligand, this parameter file also stores all bonds between the individual atoms, including the binding angles and binding distances. Rosetta cannot generate the conformer ensemble by itself, so an additional tool is needed. Different tools are capable of creating the conformer ensemble automatically, but it is best to manually define constraints for the chi1, chi2 and backbone psi torsion angles that define the orientation of the ligand in the binding pocket. For this, we know of three tools: The first is OpenEye Omega, but the full license is very costly and the academic free version is hard to obtain. The second tool is Accelrys Discovery Studio, but Accerlys does not provide a free license. The third tool is TINKER, which is free, but poorly documented and depends on a specific keyfile, which requires a high amount of chemical expertise to generate. Conformers might also be generated without constrains, for which different tools are available, in our case, we used ConFlex. Conformers need to be stored in one file (“.sdf”, “.mol”, or “.mol2”).
“.pdb”-file:
The input-file for the scaffold, in our case the tRNA synthetase, can be downloaded in PDB format from Protein Data Bank (PDB). It is then necessary to delete the natural ligand from the PDB-file, as we need to incorporate our own aaRS. Additionally, it is advised to relax the preferably, the structure should be relaxed in order to allow for flexibility with regards to the simulation outcomes. For further details, see the ROSETTA Relaxing documentation.
“.cst”-file:
The .cst-file defines the potential hydrogen bonds between the ligand and the amino acid. For example, the code block characterized by the tags “CST::BEGIN” and “CST::END”, specifies the orientation or catalytic function of the enzyme.
More specifically, the first record of the block begins with “TEMPLATE::ATOM_MAP”, followed by either “atom_name” or “atom_type”, depending on whether a specific residue or a specific type of residue is provided. In the latter case, it is not important to choose specific atoms. Instead, a catalytic residue of the amino acid such as “OH” or “Nhis” is specified. The next lines of the TEMPLATE::ATOM_MAP record define the residues using one-letter or three-letter-codes that are prefixed by “residue1” or “residue3”, respectively. The second record, beginning with the tag “CONSTRAINT”, contains all relevant distance, angle and torsion constraints for the matching. Each constraint is described with five parameters. In the case of the distance constraint, the first parameter describes the optimal distance “x0” between the chosen residues, the second parameter describes the tolerance “xtol”, the third parameter defines the strength “k” and the fourth parameter specifies the type of bond (1 for a covalent bond, 0 otherwise). If the modulus of the difference between the actual distance “x” and the specified optimal distance is smaller than the tolerance, then the penality score is zero. Otherwise, the constraint consists of the term
k*(|x-x₀|-x_tol)
to the penality score. For the angle and torsion constraints, the description is similar. If necessary, additional hydrogen bonds to other atoms of the ligand are specified in terms of additional blocks, using the tag “VARIABLE::CST”. Finally, most of the blocks described above can be optionally followed by an “ALGORITHM_INFO” record that stores details of the matching algorithm by parameter values. We refer to the Rosetta documentation for further details.
”.pos”-file:
The “.pos” file contains the allowed locations in the scaffold for the chosen catalytic residues in each constraint block of the “.cst” file.

Matching step outputs

Figure (NUMMER ANGEBEN!): ABBILDUNGSTITEL
BILDUNTERSCHRIFT

The output generated in the matching step is the layout of the scaffold as well as one or more states of the amino acid which enable interaction with the ligand. This information is stored as a “.pdb” file and becomes part of the input for the design step.

Our results for this step

We used the “1J1U”-scaffold from PDB for our matching step. The “1J1U.pdb”-file contains the tyrosyl-tRNA-synthetase, which is labeld under “Chain A”, the orthogonol tRNA under “Chain B” and the natural ligand tyrosyl. For our project, we deleted the natural ligand and “Chain B”, because it was not necessary to change their structure or sequence and it was a way to save compute time. We designed the ligands manually via Avogadro based on a default matching algorithm for both amino acids, thus creating useful .cst-files.

Design Step

The design step applies an algorithm such that the binding pocket and the near environment are mutated and the remaining scaffold is repacked. Additionally, a badness-of-fit score is generated which indicates how well the mutation fits the amino acid. For every file from the matching step, a model with a score and a “.pdb-file” was generated, specifying where the sequence can be located. Additionally, the ".pdb-file" makes visual analysis of the 3D-structure possible. Notably, the amino acid structure can be extracted separately. The following section describes the structure of the design step. Further details on each step can be obtaind by showing the Technical Details Section.
1. Optimizing the catalytic interactions
For the first alternative, the file can be generated either by the Rosetta standard or a manually created .”res”- file. For more details, we refer to the Rosetta documentation.
For the latter alternative, residues are automatically categorized by their location of the Cα;lpha;.

TECHNICAL DETAILS

Residues are catagorized as follows:

residues that have their Cα within a distance cut1 angstroms of any ligand heavyatom will be set to designable
res that have Cα within a distance cut2 of any ligand heavyatom and the Cβ closer to that ligand atom than the Calpha will be set to designable. cut2 has to be larger than cut1
res that have Cα within a certain distance cut3 of any ligand heavyatom will be set to repackable. cut3 has to be larger than cut2
res that have Cα within a distance cut4 of any ligand heavy atom and the Cβ closer to that ligand atom will be set to repackable. cut4 has to be larger than cut3
all residues not in any of the above 4 groups are kept static.

2. Cycles of sequence design and minimazation within constrains
To optimize the structure we applied an iterative optimization algorithm. This algorithm mutates all residues from the backbone, which are not part of the catalytic center, to alanine, and a small energy function refraction will place the ligand in an optimal position to the backbone.

TECHNICAL DETAILS

For this approach, bb_min and chi_min allow for backbone flexibility and the rotation of the torsions. An alternative for this minimization step is the Monte Carlo rigid body ligand sampling. For further information on this method, we refer to the ROSETTA documentation.

Design step inputs

The following input files are relevant for the design procedure:

“.pdb”-file generated in the matching step
“.cst”-file for the ligand
“.params”-file for the ligand and the scaffold
“.flags” to coordinate the inputs

For further information on these files, please refer to step 2 above.

Figure (NUMMER ANGEBEN!): ABBILDUNGSTITEL
BILDUNTERSCHRIFT

Design step outputs

The output for the design step is a “.pdb”-file containing the mutated scaffold and a “.score”-file. For every .pdb-file, a line in the score-file is generated, so it is easy to evaluate the given structure. The first score in the file is the total score of the model. After that, the number of hydrogen bonds in the protein as a whole and in the constraints is listed, followed by the number of dismissed solutions in the catalytic residues as well in the whole protein and in the constraints. See the technical details below for a full overview of the output information
TECHNICAL DETAILS

total_score: energy (excluding the constraint energy)
fa_rep: full atom repulsive energy
hbond_sc: hbond sidechain energy
all_cst: all constraint energy
tot_pstat_pm: pack statistics, 0-1, 1 = fully packed
total_nlpstat_pm: pack statistics withouth the ligand present
tot_burunsat_pm: buried unsatisfied polar residues, higher = more buried unsat polars (just a count)
tot_hbond_pm: total number of hbonds
tot_NLconst_pm: total number of non-local contacts ( two residues form a nonlocal contact if they are farther than 8 residues apart in sequence but interact with a Rosetta score of lower than -1.0 )

Results

Results in silico

We choose our synthetases based on a good total score and a good ligand score. We checked the corresponding PDB-files, and rated the ligand and the binding pocket as satisfying, so that the ligand assumedly does not collide with residues in the near environment. The total scores for CBT are not as good as the scores for NPA. However, the ligand scores are acceptable in both cases. A visual evaluation confirms that the ligand fits into the binding pocket.
Our results for this step
We used this algorithm to simulate the evolution of the tyrosyl-tRNA with the amino acids Nitrophenylalanine(NPA) and N^γ‑2‑cyanobenzothiazol‑6‑yl‑L‑asparagine (CBT-asparagine).
NPA simulation:
The .cst-file contained two blocks for the nitrogroup of NPA. Since there are two oxygen-atoms in the nitrogroup, we defined two atom nametags. As several possibilities are useful, we defined two possible constraint partners for the hydrogen bonds. The first is asparagine (N) or glutamine (Q) and the second is glycine (G). We set the possible distance to 2.8 Å, as it is the optimal distance for hydrogenbonds, and a tolerance level of 0.5 Å. We set the angles to 120° with a tolerance of 40°, as recommended by Florian Richter during our discussion in cologne. The torsion angles were set to 180° with a tolerance of 180° and a penalty of 0, such that the torsion angles can rotate completely freely.(Richter, unpublished data)
CBT-Asparagine simulation:
CBT-Asparagine can build hydrogen bonds in two ways. The first is a weak hydrogen bond on the sulfur atom and the other possibility is a normal hydrogen bond on the nitrogen (N₂) after the Cγ. We wrote three cst-files, one for a possible bond with sulfur, one for a possible bond with nitrogen, and one for both bonds. As possible corresponding amino acids, we chose serine, threonine, tyrosine, asparagine, glutamine, and glycine.
It is recommended to write a “.flags”-file, because there are several input parameters to be defined, but it is also possible to define them via console user interface.
For the categorization of the scaffold, we chose the automatic determination and set the following cuts: cut1: 6 Å,
cut2: 8 Å,
cut3: 10 Å
and cut4: 12 Å,
like the Baker-lab commonly used. We used this algrithm to simulate the evolution of the tyrosyl-tRNA with the amino acids Nitrophenylalanine and CBT-ASP We obtained 13 synthetase sequences for CBT-ASP, and 43 sequences for NPA, which fit well into the binding site according to the ROSETTA score. The sequences for the best synthetases for NPA is available here

Sequence Number	Total Score	Ligand Score	ncAA
15	124.88	-3.77	NPA
19	23.55	-3.93	NPA
31	-3.40	-2.47	NPA
32	-1.57	-3.82	NPA
40	11.67	-4.33	NPA
41	11.55	-2.98	NPA
43	66.36	-5.05	NPA
2	38.01	-6.56	CBT-Asparagine
4	58.45	-4.37	CBT-Asparagine
5	109.13	-4.25	CBT-Asparagine

Table 2: ROSETTA Enzyme Design Protocol Results
ROSETTA scores of the best modeled synthetases for CBT-Asparagine and NPA.

Position	Synthetase Number	Original Amino Acid	Mutation Amino Acid
30	5	Serine	Asparagine
32	5	Tyrosine	Threonine
34	2, 4	Glycine	Alanine
36	2	Glutamine acid	Isoleucine
61	5	Asparagine acid	Arginine
63	5	Isoleucine	Alanine
65	4	Leucine	Glycine
65	5	Leucine	Threonine
68	4	Asparagine acid	Alanine
69	4	Leucine	Alanine
70	2	Histidine	Asparagine acid
70	4	Histidine	Glycine
72	4	Tyrosine	Glutamine acid
73	2	Leucine	Alanine
73	4	Leucine	Methionine
74	2	Asparagine	Asparagine acid
76	2	Lysine	Serine
79	4	Leucine	Arginine
101	5	Lysine	Glutamine acid
103	5	Valine	Triptophane
104	5	Tyrosine	Valine
105	4, 5	Glycine	Serine
107	5	Glutamine acid	Lysine
108	4	Phenylalanine	Lysine
108	5	Phenylalanine	Arginine
109	4, 5	Glutamine	Alanine
114	4	Tyrosine	Alanine
115	4	Threonine	Triptophane
118	4	Valine	Serine
134	2	Methionine	Asparagine
137	2	Isoleucine	Alanine
139	2	Arginine	Serine
147	2, 4	Alanine	Serine
148	2	Glutamine	Lysine
149	2	Valine	Threonine
150	2, 4	Isoleucine	Leucine
151	2	Tyrosine	Serine
152	2	Proline	Threonine
153	2	Isoleucine	Leucine
153	4	Isoleucine	Threonine
154	2	Methionine	Asparagine
154	4, 5	Methionine	Serine
155	2	Glutamine	Glycine
155	4, 5	Glutamine	Alanine
156	4	Valine	Alanine
157	4	Asparagine	Alanine
158	4, 5	Asparagine	Tyrosine
159	4	Isoleucine	Glycine
161	4	Tyrosine	Glutamine acid
161	5	Tyrosine	Asparagine acid
162	4	Leucine	Methionine
162	5	Leucine	Alanine
164	5	Valine	Alanine
172	2	Glutamine acid	Lysine
173	2	Glutamine	Serine
176	2	Isoleucine	Serine
204-210	2, 4, 5	varying	-
307	2	-	Alanine
307	4, 5	-	Tyrosine

in vivo validation of predicted tRNA synthetase structures

In order to test the functionality and specificity of our modeled aaRS, we selected the most promising 11 amino acid sequences and ordered seven synthetase sequences of NPA via IDT, and four synthetase sequences of CBT-Asparagine by courtesy of Genscript, where we had previously won a grant of 500€. Since the DNA synthesis by GenScript and IDT were delayed by several weeks, we could not perform the best practice characterization of these parts. Finally, we received only three of the ordered syntheses in sufficient quality for further experiments. All of them encode predicted CBT-tRNA-synthetases. We subjected the sequences to a positive selection as initial characterization. Plasmids encoding the predicted best candidates were cotransformed with our positive selection plasmid into E. coli(BL21 DE3). Due to the IPTG induced promoter, we tested different IPTG concentrations, including 0 mM, 5 mM, 10 mM, and 15 mM. In addition, we tried different concentration of the antibiotics: kan15, cm15/kan15, and cm15/kan15/tet5. The number of resulting colonies for each sample is summarized in figure X. Our in vivo results show that our in silico designed enzymes kept their native function and are able to integrate amino acids through an amber codon matching tRNA. Since these results only indicate the acceptance and transfer of the non-canonical amino acid, additional experiment are required to demonstrate a high specificity of these enzymes.
We offer the predicted sequences to the community for further characterization via the parts-reg (LINKS).

Figure (NUMMER ANGEBEN!): ABBILDUNGSTITEL
BILDUNTERSCHRIFT

References

Liu, W., Brock, A., Chen, S., Chen, S., Schultz, P. G. ,(2007). Genetic incorporation of unnatural amino acids into proteins in mammalian cells. Nature methods, 4(3), 239-244.
Richter, F., Leaver-Fay, A., Khare, S. D., Bjelic, S., Baker, D. (2011). De novo enzyme design using Rosetta3. PloS one, 6(5): e19230.
Simons, K. T., Kooperberg, C., Huang, E., Baker, D. (1997). Assembly of protein tertiary structures from fragments with similar local sequences using simulated annealing and Bayesian scoring functions. Journal of molecular biology, 268(1), 209-225.

@@ Line 25: / Line 25: @@
 				However, besides this complex modeling, we also conducted and applied several straight-forward stochastic and statistical models
-				to support and guide our laboratory work. These modeling projects are briefly described here; for further information, please check
+				to support and guide numerous steps of our laboratory work. Some of these modeling projects are briefly described in this box; however,
+				we recommend reading the linked pages for further information. </br>
-				the corresponding link leading to the part of our project the model has been an element of. </br>
 				<h4>Discriminant function model for the ICG prediction: </h4>
-				We conducted a discriminant function analysis for the recognition of which base – natural or unnatural – is present at a specified position of a base sequence. This model is part of our ICG model software, found
+				We conducted a discriminant function analysis for the classification of nucleotides in Oxford Nanopore sequencing reads
+				at a specific position. This model is part of our
+				<a target="_blank"href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Software#iCG">iCG</a> software module and enabled the successful detection of unnatural bases.
-				<a target="_blank"href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Software#iCG">here. </a>
+				<h4>Calculation of the required library size for the selection system: </h4>
-				<h4>Calculation of an effective library size for the selection system: </h4>
+				We applied combinatorics and statistics to calculate the optimal library size for the <a target="_blank"href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/translational_system/library_and_selection">tRNA synthetase selection process.</a>
-				We used a combination of combinatorics and statistics to calculate the optimal library size for the selection process,
+				This was a trade off between putting lots of efforts into constructing a very huge library and missing diversity in a too small library.
+				Therefore, we predicted the optimal library size. Experimental validation of this prediction was done via MiSeq analysis of the diversity
+				of a subset of this library.
-				such that it is expected to contain all possible sequence mutations, and therefore easily all possible resulting amino acids, at least once.
+				<h4>Strength prediction for a transcription signal amplification system <a target="_blank"href="http://parts.igem.org/Part:BBa_K2201373">(BBa_K2201373)</a>: <!--den part gibts nicht --> </h4>
-				This calculation is part of the translational system, found <a target="_blank"href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/translational_system/library_and_selection">here</a>.
+				We modeled and visually compared the mRFP production over time for a normal mRFP reporter system and compared it to our <a target="_blank"href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Composite_Part">enhanced signaling.</a>.
+				Validity was done as part of the positive selection process for an adapted tRNA synthetase.
-				<h4>Comparison of mRFP production for the positive selection system <a target="_blank"href="http://parts.igem.org/Part:BBa_K2201373">(BBa_K2201373)</a>: <!--den part gibts nicht --> </h4>
-				We modeled and visually compared the mRFP production over time for the normal signaling and the enhanced signaling circuit of the positive selection system.
-				The system and plot can be found <a target="_blank"href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Composite_Part">here</a>.
 			</div class="article">
@@ Line 61: / Line 62: @@
 		<div class="article">
 			As our project explores possibilities of an expanded genetic code via unnatural bases and non-canonical amino acids,
-			we set out to complement our lab work via modeling of novel amino acyl tRNA synthetases (aaRS) for a non-canonical amino
+			we set out to complement and improve our lab work via modeling of novel amino acyl tRNA synthetases (aaRS) for a non-canonical amino
-			acids we synthetized in the lab. In order to incorporate non-canonical amino acids into proteins via the translational
+			acids, which were synthetized in our lab. In order to incorporate non-canonical amino acids into proteins via the translational
-			process, the aaRS has to attach the amino acid to the respective tRNA. Thus, we designed aaRS sequences which were meant
+			process, the aaRS has to attach the amino acid to the respective tRNA. Thus, we designed aaRS sequences which were adjusted
-			to link our own non-canonical amino acid to a fitting tRNA. As a result, we obtained a couple of sequences of possible aaRS
+			to link our own non-canonical amino acid to a fitting tRNA. Candidates were evaluated and selected, based on a ROSETTA score.
-			candidates, which we evaluated, based on a ROSETTA score, and ordered via gene synthesis.
+			Most promising sequences were ordered via gene synthesis for the experimental validation.
-			The following figure provides a rough overview of our modeling project. The table below summarizes the realization in practice.
+			Figure 1 provides a rough overview of our modeling project. Table 1 below summarizes the realization in practice.
 		</div>
@@ Line 102: / Line 103: @@
    </tr>
    <tr>
-     <td>1. Ligand Preparation</td>
+     <td>1. Ligand preparation</td>
      <td>Manually via <a href="https://avogadro.cc/">Avogadro</a></td>
      <td>Due to the novelty of our amino acid, no information on the ligand is available in databases.
-	Therefore, all information has to be provided manually and then generate a conformer ensemble, containing
+	Therefore, manual generation of a conformer ensemble, containing
-	for example all energetically useful arrangements of atoms within the molecule.</td>
+	for example all energetically useful arrangements of atoms within the molecule, was required.</td>
    </tr>
    <tr>
@@ Line 122: / Line 123: @@
    </tr>
    <tr>
-     <td>4. <a href="https://www.rosettacommons.org/manuals/archive/rosetta3.5_user_guide/d7/dfc/match.html">Enzyme Matching</a></td>
+     <td>4. <a href="https://www.rosettacommons.org/manuals/archive/rosetta3.5_user_guide/d7/dfc/match.html">Enzyme matching</a></td>
      <td>ROSETTA protocol</td>
      <td>ROSETTA combines information about the ligand and constrains to find possible hydrogen bonding partners and propose the shape
@@ Line 128: / Line 129: @@
    </tr>
    <tr>
-     <td>5. <a href="https://www.rosettacommons.org/manuals/archive/rosetta3.5_user_guide/d6/dbc/enzyme_design.html">Enzyme Design</a></td>
+     <td>5. <a href="https://www.rosettacommons.org/manuals/archive/rosetta3.5_user_guide/d6/dbc/enzyme_design.html">Enzyme design</a></td>
      <td>ROSETTA protocol</td>
      <td>An algorithm uses the information from the previous step and information on the ligand to simulate the mutation process and
@@ Line 134: / Line 135: @@
    </tr>
    <tr>
-     <td>6. Evaluate results in silico</td>
+     <td>6. Evaluate results <i>in silico</i></td>
      <td>Manually</td>
-     <td>We evaluate the visual output and the score values and order the sequences with the most promising results via gene synthesis. </td>
+     <td>Based on the score values, we ordered the synthesis of the most promising sequences. </td>
    </tr>
    <tr>
-   <td>7. Evaluate results in vivo</td>
+   <td>7. Evaluate results <i>in vivo</i></td>
      <td>Manually</td>
      <td>The synthetases are validated in the lab with the corresponding ncAA via a
@@ Line 166: / Line 167: @@
 			<div class="third double">
 				<div class ="article">
-				As part of our iGEM project, we are faced with the challenge of adapting the tRNA synthetase
+				As part of our iGEM project, we faced the challenge of adapting the tRNA synthetase
-				to non-canonical amino acids. For this purpose, we modeled possible candidates for synthetases as a
+				to non-canonical amino acids. For this purpose, we modeled possible candidates for synthetases as an
-				preparation for carrying out a positive-negative selection according to (Liu <i>et al.</i>, 2007) in the laboratory.
+				alternative to carrying out a positive-negative selection according to (Liu <i>et al.</i>, 2007) in the laboratory.
 				Due to the rapid development in the field of protein and molecular structure analysis, there has been an
 				increase in the availability of molecular 3D structure data. These data are organized in publicly available
 				databases which provide a foundation for the modeling and simulation of chemical-biological processes in bioinformatics.
-				As our ncAA has been synthetized by ourselves, no such comprehensive information is available, yet.
+				As our ncAA has been synthetized in our lab, no such comprehensive information is available, yet.
 				However, information of similarly structured amino acids can potentially serve as a basis for our modeling.
-				As evaluating an expanded genetic code is a complex task, the practical laboratory work of our project is supplemented by a
+				Specifically, we focused on simulation to
-				theoretical approach, involving modeling, simulation, and evaluation on the computer in silico. Specifically, we focused on simulation to
+				design an aaRS for the new ncAA CBT-Asparagine.
-				design an aaRS for the new ncAA CBT-Asparagine. Additionally to CBT-Asparagine, we also generated aaRS sequences for the ncAA Nitrophenylalanine (NPA) as
-				a validation of our modeling procedure; as synthases for this ncAA are known and
-				thus comparable to our in silico result, we can evaluate our modeling procedure.
-				Our core challenge was to evolve the binding pocket in a manner which effectively charges the tRNA with the amino acid, thus also recognizing this amino acid specifically.
 				</div>
 			</div>
@@ Line 190: / Line 187: @@
 <h4>Method</h4>
 		<div class ="article">
-			We used the open-source software <a href="https://www.rosettacommons.org/docs/latest/getting_started/Getting-Started">"Rosetta"</a> for the main part of our modeling project, which was introduced at the University of Washington by David Baker in 1997 (Simon <i>et al.</i>,1997), initially in the context of protein structure prediction. Since then, Rosetta has grown to include numerous modules and is currently widely used in research. In our application, we focus on the Rosetta module called the "Rosetta Enzyme Design Protocol"
+			We used the open-source software <a href="https://www.rosettacommons.org/docs/latest/getting_started/Getting-Started">"ROSETTA"</a>
+			for the main part of our modeling project, which was introduced at the University of Washington by David Baker in 1997
+			(Simon <i>et al.</i>,1997), initially in the context of protein structure prediction. ROSETTA has grown through the addition of numerous modules and is currently widely used in research. In our application, we focus on the Rosetta module called the "Rosetta Enzyme Design Protocol"
           </div>
@@ Line 204: / Line 203: @@
 		<div class ="article">
 					Since the non-canonical amino acid synthesized in the laboratory is completely novel, there is no corresponding tRNA synthetase which can load the tRNA, yet. For this reason, we use the enzyme design protocol to design the binding pocket in a way that allows it to form an effective and specific enzyme. The protocol consists of two main steps: matching and designing (Richter <i>et al.</i>, 2011)
-					The enzyme design algorithm basically is summarized in Fig. B
+					The enzyme design algorithm is briefly summarized in Fig. 2
 		</div>
@@ Line 238: / Line 237: @@
 				<li>a “.pdb”-file providing a rough scaffold layout</li>
 				<li>a “.cst”-file to define the bindings between ligand and scaffold</li>
-				<li>a “.pos”-file to define the positions of the amino acids of the scaffold </li>
+				<li>a “.pos”-file to define the positions of the amino acids in the scaffold </li>
 				<li>a “.flags” file to control all inputs. These files are necessary, as they describe the ligand and backbone and specify the parameters of the algorithm </li>
 				</ul>
@@ Line 297: / Line 296: @@
 						The output generated in the matching step is the layout of the scaffold as well as one or more states of the amino acid which enable interaction with the ligand. This information is stored as a “.pdb” file and becomes part of the input for the design step. </br>
 							<h4>Our results for this step </h4>
-						We used the <a href="http://www.rcsb.org/pdb/explore.do?structureId=1j1u">“1J1U”</a>-scaffold from PDB for our matching step. The “1J1U.pdb”-file contains the Tyrosyl-tRNA-synthetase, which is labeld under “Chain A”, the orthogonol tRNA under “Chain B” and the natural ligand Tyrosyl. For our project, we deleted the natural ligand and “Chain B”, because it was not neccerary to change their structure or sequence and it was a way to save computer time and power.
+						We used the <a href="http://www.rcsb.org/pdb/explore.do?structureId=1j1u">“1J1U”</a>-scaffold from PDB for our matching step.
+						The “1J1U.pdb”-file contains the tyrosyl-tRNA-synthetase, which is labeld under “Chain A”, the orthogonol tRNA under “Chain B”
+						and the natural ligand tyrosyl. For our project, we deleted the natural ligand and “Chain B”, because it was not necessary to
+						change their structure or sequence and it was a way to save compute time.
-						We designed the ligands manually by using <a href="https://avogadro.cc/">Avogadro</a>, and for the .cst-file, we choose the default matching algorithm for simulations of both amino acids.
+						We designed the ligands manually via <a href="https://avogadro.cc/">Avogadro</a> based on a default matching algorithm for both
+						amino acids, thus creating useful .cst-files.
 				</article>
 			</div>
@@ Line 315: / Line 318: @@
 							<p>The design step applies an algorithm such that the binding pocket and the near environment are mutated and the remaining scaffold is
 							 repacked. Additionally, a badness-of-fit score is generated which indicates how well the mutation fits the amino acid. For every file from the
-							 matching step, a model with a score and a “.pdb-file” will be generated, specifying where the sequence can be located, and the 3D-structure can be
+							 matching step, a model with a score and a “.pdb-file” was generated, specifying where the sequence can be located. Additionally, the ".pdb-file"
-							 analyzed. Notably, the amino acid structure can be extracted separately.
+							 makes visual analysis of the 3D-structure possible. Notably, the amino acid structure can be extracted separately.
-							The following section describes the structure of the design step. For further details on each step, click the technical details button. </br>
+							The following section describes the structure of the design step. Further details on each step can be obtaind by showing the Technical Details Section. </br>
 . Optimizing the catalytic interactions </br>
 							For the first alternative, the file can be generated either by the Rosetta standard or a manually created .”res”- file. For more details, we refer to the
@@ Line 334: / Line 337: @@
 							</article>
 							<article> <p>2. Cycles of sequence design and minimazation within constrains </br>
-							To optimize the structure we used applied an iterative optimization algorithm. This algorithm mutates all residues from the backbone, which are not part
+							To optimize the structure we applied an iterative optimization algorithm. This algorithm mutates all residues from the backbone, which are not part
-							#of the catalytic center, to alanine, and a small energy function refraction will place the ligand in an optimal position to the backbone. </br>
+							of the catalytic center, to alanine, and a small energy function refraction will place the ligand in an optimal position to the backbone. </br>
 							</p> <a class="hidden-expand">TECHNICAL DETAILS</a></article>
 							<article class="hidden-block">
@@ Line 362: / Line 365: @@
 							<p><b>Design step outputs </b></p>
 									<article>The output for the design step is a “.pdb”-file containing the mutated scaffold and a “.score”-file.
-									For every PDB-file, a line in the score-file is generated, so it is easy to evaluate the given structure.
+									For every .pdb-file, a line in the score-file is generated, so it is easy to evaluate the given structure.
 									The first score in the file is the total score of the model. After that, the number of hydrogen bonds
-									in the protein as a whole and in the constraints is listed, followed by the number of dismissed polars in the catalytic residues as well in the whole protein and in the constraints.
+									in the protein as a whole and in the constraints is listed, followed by the number of dismissed solutions in the
+									catalytic residues as well in the whole protein and in the constraints.
 									See the technical details below for a full overview of the output information </br>
 									<a class="hidden-expand">TECHNICAL DETAILS</a></article>
@@ Line 396: / Line 400: @@
 		<div class="content">
 			<h3> Results </h3>
-			<h4> Results in silico </h4>
+			<h4> Results <i>in silico</i> </h4>
 			<div class="article">
-				We choose our synthetases because of a good total score and a good ligand score. We checked the corresponding PDB-files, and rated the ligand and the binding pocket
+				We choose our synthetases based on a good total score and a good ligand score. We checked the corresponding PDB-files, and rated the ligand and the binding pocket
 							 as satisfying, so that the ligand assumedly does not collide with residues in the near environment.
 							The total scores for CBT are not as good as the scores for NPA. However, the ligand scores are acceptable in both cases. A visual evaluation confirms that the ligand
@@ Line 405: / Line 409: @@
 							We used this algorithm to simulate the evolution of the tyrosyl-tRNA with the amino acids Nitrophenylalanine(NPA) and N<sup>γ</sup>&#x2011;2&#x2011;cyanobenzothiazol&#x2011;6&#x2011;yl&#x2011;L&#x2011;asparagine (CBT-asparagine). </br>
 							NPA simulation: </br>
-							We created one .cst-file-block for the nitrogroup of NPA. Since there are two oxygen-atoms in the nitrogroup,
+							The .cst-file contained two blocks for the nitrogroup of NPA. Since there are two oxygen-atoms in the nitrogroup,
 							 we defined two atom nametags. As several possibilities are useful, we defined two possible constraint partners
 							  for the hydrogen bonds. The first is asparagine (N) or glutamine (Q) and the second is glycine (G). We set the
 							   possible distance to 2.8 &#8491;, as it is the optimal distance for hydrogenbonds, and a tolerance level of 0.5 &#8491;.
-							   We set the angles to 120° with a tolerance of 40°, as recommended by Florian Richter during our talk in cologne.
+							   We set the angles to 120° with a tolerance of 40°, as recommended by Florian Richter during our discussion in cologne.
 							   The torsion angles were set to 180° with a tolerance of 180° and a penalty of 0, such that the torsion angles can rotate
 							   completely freely.(Richter, unpublished data) </br>
-							CBT-ASP simulation: </br>
+							CBT-Asparagine simulation: </br>
-							CBT-ASP can build hydrogen bonds in two ways. The first is a weak hydrogen bond on the
+							CBT-Asparagine can build hydrogen bonds in two ways. The first is a weak hydrogen bond on the
-							sulphur atom and the other possibility is a normal hydrogen bond on the nitrogen (N<sub>2</sub>)
+							sulfur atom and the other possibility is a normal hydrogen bond on the nitrogen (N<sub>2</sub>)
-							after the C&gamma;. We wrote three cst-files, one for a possible bond with sulpur, one for a
+							after the C&gamma;. We wrote three cst-files, one for a possible bond with sulfur, one for a
-							 possible bond with nitrogen, and one for both bonds. As possible corresponding amino acids, we chose serine, threonine, tyrosine, asparagine, glutamine, and glycine. </br>
+							 possible bond with nitrogen, and one for both bonds. As possible corresponding amino acids, we chose serine,
-							It is recommended to write a “.flags”-file, because there are several input- parameters to be defined, but it is also possible to define them via console user interface. </br>
+							 threonine, tyrosine, asparagine, glutamine, and glycine. </br>
-							For the categorization of the scaffold, we chose the automatic determination and set the following cuts: cut1: 6 &#8491;, cut2: 8 &#8491;, cut3: 10 &#8491; and cut4: 12 &#8491;, like the Baker-lab commonly used.
+							It is recommended to write a “.flags”-file, because there are several input parameters to be defined,
+							but it is also possible to define them via console user interface. </br>
+							For the categorization of the scaffold, we chose the automatic determination and set the following cuts: cut1: 6 &#8491;,</br> cut2: 8 &#8491;,</br> cut3: 10 &#8491;</br> and cut4: 12 &#8491;,</br> like the Baker-lab commonly used.
 					We used this algrithm to simulate the evolution of the tyrosyl-tRNA with the amino acids Nitrophenylalanine and CBT-ASP
 					We obtained 13 synthetase sequences for CBT-ASP, and 43 sequences for NPA, which fit well into the binding site according to the ROSETTA score.
-					The sequences for the best synthetases for NPA is avaible
+					The sequences for the best synthetases for NPA is available
 					<a target="_blank"href="https://static.igem.org/mediawiki/2017/1/12/T--Bielefeld-CeBiTec--DKE_NPAseq.pdf">here</a>
 </style>
@@ Line 621: / Line 627: @@
      <td>36</td>
      <td>2</td>
-     <td>Glutamine Acid</td>
+     <td>Glutamine acid</td>
 	<td>Isoleucine</td>
    </tr>
@@ Line 627: / Line 633: @@
      <td>61</td>
      <td>5</td>
-     <td>Asparagine Acid</td>
+     <td>Asparagine acid</td>
 	<td>Arginine</td>
    </tr>
@@ Line 651: / Line 657: @@
      <td>68</td>
      <td>4</td>
-     <td>Asparagine Acid</td>
+     <td>Asparagine acid</td>
 	<td>Alanine</td>
    </tr>
@@ Line 664: / Line 670: @@
      <td>2</td>
      <td>Histidine</td>
-	<td>Asparagine Acid</td>
+	<td>Asparagine acid</td>
    </tr>
      <tr>
@@ Line 676: / Line 682: @@
      <td>4</td>
      <td>Tyrosine</td>
-	<td>Glutamine Acid</td>
+	<td>Glutamine acid</td>
    </tr>
      <tr>
@@ Line 694: / Line 700: @@
      <td>2</td>
      <td>Asparagine</td>
-	<td>Asparagine Acid</td>
+	<td>Asparagine acid</td>
    </tr>
      <tr>
@@ Line 712: / Line 718: @@
      <td>5</td>
      <td>Lysine</td>
-	<td>Glutamine Acid</td>
+	<td>Glutamine acid</td>
    </tr>
      <tr>
@@ Line 735: / Line 741: @@
      <td>107</td>
      <td>5</td>
-     <td>Glutamine Acid</td>
+     <td>Glutamine acid</td>
 	<td>Lysine</td>
    </tr>
@@ Line 892: / Line 898: @@
      <td>4</td>
      <td>Tyrosine</td>
-	<td>Glutamine Acid</td>
+	<td>Glutamine acid</td>
    </tr>
          <tr>
@@ Line 898: / Line 904: @@
      <td>5</td>
      <td>Tyrosine</td>
-	<td>Asparagine Acid</td>
+	<td>Asparagine acid</td>
    </tr>
          <tr>
@@ Line 921: / Line 927: @@
      <td>172</td>
      <td>2</td>
-     <td>Glutamine Acid</td>
+     <td>Glutamine acid</td>
 	<td>Lysine</td>
    </tr>
@@ Line 956: / Line 962: @@
 </table>
 			</div>
-			<h4> Results in vivo </h4>
+			<h4> <i>in vivo</i> validation of predicted tRNA synthetase structures </h4>
 			<div class="article">
-				In order to test the functionality and specificity of our modeled aaRS, we translated a selection of the most promising amino acid sequences into DNA sequences optimized for E.coli and ordered them via gene synthesis. We then used a positive-negative selection system for characterization. The experiment proceeds as follows:
+				In order to test the functionality and specificity of our modeled aaRS, we selected the most promising 11 amino acid sequences and ordered seven synthetase sequences of NPA via IDT, and four
-				Due to problems with regards to the protein- and salt-concentration, we retransformed the gensyntheses which had been cloned into pSB1C3. In a next step, these syntheses were cotransformed in E.coli(BL21) with our positive selection plasmid.
+				synthetase sequences of CBT-Asparagine by courtesy of Genscript, where we had previously won a grant of 500€.
-				With regards to CBT2, only the original colony could be transformed. From CBT4 and CBT5, were used each originally isolated clone and its retransformed counterpart.
+				Since the DNA synthesis by GenScript and IDT were delayed by several weeks, we could not perform the best practice characterization of these parts.
-				Due to the IPTC-induced promoter, we used variants without IPTG, and with 5 mM, 10 mM, and 15 mM added IPTS for all plasmids for the kanamycine resistance.
+				Finally, we received only three of the ordered syntheses in sufficient quality for further experiments. All of them encode predicted CBT-tRNA-synthetases.
-				We chose additional variants with regards to the antibiotics; one variant each of kanamycine, kanamycine and chloramphenicole, and kanamycine, chloramphenicole and tetracycline. The number of resulting colonies for each variant are summarized in figure X. Our in vivo results show that our in silico designed enzymes did not lead to a loss of functioning.
+				We subjected the sequences to a positive selection as initial characterization. Plasmids encoding the predicted best candidates were cotransformed with our
-					We ordered seven synthetase sequences of NPA via IDT, and four synthetase sequences of CBT-Asparagine by courtesy of Genscript, where we had previously
+				positive selection plasmid into <i>E. coli(BL21 DE3)</i>. Due to the IPTG induced promoter, we tested different IPTG concentrations, including 0 mM, 5 mM,
-					won a grant of 500€. Due to problems on the part of IDT, the sequences for NPA could not be synthetized. We still provide the sequences for further use below.
+mM, and 15 mM. In addition, we tried different concentration of the antibiotics: kan15, cm15/kan15, and cm15/kan15/tet5. The number of resulting colonies for
-					All further descriptions therefore refer to the synthetase sequences for CBT-Asparagine. </br>
+				each sample is summarized in figure X. Our <i>in vivo</i> results show that our <i>in silico</i> designed enzymes kept their native function and are
-					Unfortunately, we were not able to amplify the four sequences or clone them into the detection system pSB3T5. A test digestion revealed that the length of the
+				able to integrate amino acids through an amber codon matching tRNA. Since these results only indicate the acceptance and transfer of the non-canonical
-					sequence in the plasmid pUC57, which was the plasmid delivered by Genscript, did not correspond to the sequence length ordered. Therefore, we disregarded synthetase candidate number 1
+				amino acid, additional experiment are required to demonstrate a high specificity of these enzymes. </br>
-					for further tests. </br>
+				We offer the predicted sequences to the community for further characterization via the parts-reg (LINKS).
-					For the remaining three sequences, we instead utilized the positive-negative selection plasmids for validation of our syntheses.
-					After the first, positive selection cycle, colonies formed only if the non-canonical amino acid was present.
-					Thus, through the positive selection, we could show that the synthetase did not lead to the loss of functioning of the enzyme.
-					To show specificity, we conducted a negative selection as well. We managed to clone the synthetases into the negative selection plasmid,
-					but were not able to verify this selection cycle. Therefore, further tests are needed to validate the specificity of the synthetases.
 					<div class="contentline">
 			<div class="third">