Difference between revisions of "Team:Bielefeld-CeBiTec/Part Collection"

Latest revision as of 01:54, 2 November 2017

Part Collection

Short Summary

Our aim was to expand the possibilities in protein design and with our part collection we were able to realize this plan. With the parts listed below we enable every iGEM team to incorporate any non-canonical amino acid (ncAA). These ncAAs provide additional chemical functions, that canonical amino acids lack. Our toolbox contains five different tRNA/aminoacyl-synthetases (aaRS) that could incorporate different ncAAs. All these ncAAs have chemical abilities that enable to design proteins with new functions. This applications are only a proof of concept. With our selection system, every iGEM Team could evolve their own aaRS for their amino acids and expand the possibilities for protein design themselves.

Part Collection for the Incorperation of Non-canonical Amino Acids

Currently, protein design is limited to the chemical functions of the 20 to 22 canonical amino acids. Our part collection allows to add novel amino acids with other chemical abilities than those of the canonical, to expand the possibilities of advanced protein design. It contains five different tRNA/aminoacyl-synthetases (tRNA/aaRS) to incorporate non-canonical amino acids (ncAAs) during translation. These aaRS are parts of our toolbox and provide the translational incorporation of four different ncAAs as new functionality to the iGEM community. All amino acids add additional functional groups, thus functions to enable advanced protein design. The amino acids that can be incorporated with the aaRS in our toolkit are:

2-nitrophenylalanine (2-NPA), which cleaves the backbone when irradiated by light of a certain wavelength. Therefore, it could be used for inactivation and activation of proteins.
Propargyllysine (PrK), which provides a propargyl group. Propargyl groups form a highly specific bond to azides in click chemistry reaction. Thus, this amino acid could be used for specific, terminus independent labeling.
p-acetophenylalanine (AcF), which provides a ketone group. Ketones form a highly specific covalent bond to hydroxylamines. Like PrK, this amino acid could be used for specific, terminus independent labeling.
7-hydoxy-L-coumaryl-ethylglcine (CouAA), a fluorescent amino acid, which could be used for in vivo and in vitro localization and labeling.

In addition to parts for the translational incorporation of hese ncAAs, our part collection also provides parts for a selection system to evolve novel tRNA synthetases. Moreover, instructions for the generation of a library of plasmids, that contain an aaRS encoding sequence with randmly mutated amino acids in the appropriate tRNA-binding sites. Additionally, a positive and negative selection system enables every iGEM team to expand the possibilities of protein design. The library and the positive selection plasmid (BBa_K2201900 in pSB3T5) are cotransformed to select sells, which survive due to a synthetase from the library that incorporates an any amino acid in response to the amber codon. Thus, all clones that survive the positive selection contain an aaRS ceapable of incorporating the target ncAA or any endogenous amino acid. To select only the clones that incorporate only the target ncAA, an additional round of negative selection is necessary.
The negative selection plasmid (BBa_K2201901 in pSB3T5) is used to select for high specificity of enzymes. During this negative selection, the target ncAA is not supplemented in the media. The negative selection plasmid encodes the barnase, a cell toxic protein. Since the coding sequence contains two amber codons at permissive sites, the gene product is only generated if the ncAA is present or if the aaRS is unspecifically integrated a canonical amino acid.
To determine the most effective and specific aaRS for the incorporation of the desired ncAAs, an evaluation system of the synthetasesis crucial. Therefore, we optimized the synthetase test system “pFRY” initially developed by iGEM Texas 2014 leading to our new screening part BBa_K2201343. Our screening system contains the CDS of the cyan fluorescent protein and yellow fluorescent protein connected with a linker containing an amber stop codon. This part is placed placed under control of a T7-promotor to ensure high product amounts on demand. The amount and ratio of both fluorescent proteins could indicate how efficient and specific the investigated aaRS is working. Thoroughly characterization of all parts (Table 1) can be found on the respective part pages.
Our constructed parts are listed below. For the complete characterization please refer to the linked part collection pages.

Figure 1: Plasmid map of BBa_K2201200
Plasmid map of the 2-NPA-aaRS (BBa_K2201200).

BBa_K2201200 2-NPA-aaRS in response to UAG
Evolved tyrosine tRNA/aminoacyl-synthetase for the incorporation of 2-NPA in response to the amber stop codon (CUA).

Figure 2: Plasmid map of BBa_K2201201
Plasmid map of the PrK-aaRS (BBa_K2201201).

BBa_K2201201 PrK-aaRS in response to UAG
Evolved Pyrrolysyl tRNA/aminoacyl-synthetase for the incorporation of PrK in response to the amber stop codon (UAG).

Figure 3: Plasmid map of BBa_K2201202
Plasmid map of the AcF-aaRS in response to the amber stop codon.

BBa_K2201202 AcF-aaRS in response to UAG
Evolved tyrosine tRNA/aminoacyl-synthetase for the incorporation of AcF-aaRS in response to the amber stop codon (UAG).

Figure 4: Plasmid map of BBa_K2201203 Plasmid map of AcF-aaRS in response to the less used leucine codon CUA.

BBa_K2201203 AcF-aaRS in response to CUA Evolved tyrosine tRNA/aminoacyl-synthetase in response to the less used leucine codon in E. coli (CUA).

Figure 5: Plasmid map of BBa_K2201204
Plasmid map of the CouAA-aaRS in response to the amber stop codon UAG.

BBa_K2204 CouAA-aaRS in response to UAG
Evolved tyrosine tRNA/aminoacyl-synthetase for the incorporation of the fluorescent amino acid CouAA in response to the amber stop codon (UAG).

Figure 6: Plasmid map of BBa_K2201900
Plasmid map of the positive selection plasmid.

BBa_K2201900 Positive selection plasmid for the incorporation of ncAAs
The positive selection plasmid contains a tRNA and a kanamycin resistance with two amber codons. Cotransformed with the library of tRNA/aminoacyl-synthetase with random mutated binding sites, on kanamycin containing plates only the clones survive that could charge any amino acid to the tRNA in response to the UAG codon.

Figure 7: Plasmid map of BBa_K2201901
Plasmid map of the negative selection plasmid for the selection of aaRS that specifically inocporate ncAAs.

Ba_K2201901 Negative selection plasmid against the incorporation of endogenous amino acids
The negative selection plasmid contains an tRNA with the anticodon for the amber codon and a barnase containing amber codons at permissive sites. In the negative selection, the target amino acid is not supplemented to the media. If the cotransformend clones from the positive selection charge endogenous amino acids to the tRNA, the cells die. This provides a selection method for high specific aaRS.

Figure 8: Plasmid map of BBa_K2201343
Plasmid map of the screening plasmid for the incorporation of ncAAs.

BBa_K2201343 Screening system for the incorporation of ncAA Our screening system contains the CDS of cyan fluorescent protein and yellow fluorescent protein connected with a linker containing the amber stop codon, under control of a T7-promotor. The amount and relation of the fluorescent proteins could indicate how efficient and specific the cotransformed aaRS is.

@@ Line 16: / Line 16: @@
 <div class="contentbox">
 <div class="content">
-<h2>Short summary</h2>
+<h2>Short Summary</h2>
 <article>
-Our aim was to expand the possibilities in protein design and with our part collection we were able to realize this plan. With the parts listed below we enable every iGEM team to incorporate any non-canonical amino acid (ncAA) and thus we provide the opportunity to use additional chemical functions to the canonical amino acids in protein design. Our toolbox contains five different tRNA/aminoacyl-synthetases (aaRS) that could incorporate different ncAAs. These ncAAs all have chemical abilities that enable to design proteins with new functions. But this is only the beginning, with our selection system every iGEM Team could evolve their own aaRS for their amino acids and expand the possibilities for protein design themselves.
+Our aim was to expand the possibilities in protein design and with our part collection we were able to realize this plan. With the parts listed below we enable every iGEM team to incorporate any non-canonical amino acid (ncAA). These ncAAs provide additional chemical functions, that canonical amino acids lack. Our toolbox contains five different tRNA/aminoacyl-synthetases (aaRS) that could incorporate different ncAAs. All these ncAAs have chemical abilities that enable to design proteins with new functions. This applications are only a proof of concept. With our selection system, every iGEM Team could evolve their own aaRS for their amino acids and expand the possibilities for protein design themselves.
 </article>
 </div>
@@ Line 25: / Line 25: @@
 <div class="contentbox">
 <div class="content">
-<h2>Part collection for the incorperation of non-canonical amino acids</h2>
+<h2>Part Collection for the Incorperation of Non-canonical Amino Acids</h2>
 <article>
-At the moment protein design is limited to the chemical functions of the 20 to 22 canonical amino acids. Our part collection allows to add novel amino acids with other chemical abilities than the canonical, to expand the possibilities of advanced protein design. It contains five different tRNA/aminoacyl-synthetases (tRNA/aaRS) to incorporate noncanonical amino acids (ncAAs) during translation. These aaRS are from our toolbox to provide the incorporation of four different ncAAs to the iGEM community. All amino acids add additional functional groups, thus functions to enable advanced protein design. The amino acids that could be incorporated with the aaRS  in our toolkit are:
+Currently, protein design is limited to the chemical functions of the 20 to 22 canonical amino acids. Our part collection allows to add novel amino acids with other chemical abilities than those of the canonical, to expand the possibilities of advanced protein design. It contains five different tRNA/aminoacyl-synthetases (tRNA/aaRS) to incorporate non-canonical amino acids (ncAAs) during translation. These aaRS are parts of our toolbox and provide the translational incorporation of four different ncAAs as new functionality to the iGEM community. All amino acids add additional functional groups, thus functions to enable advanced protein design. The amino acids that can be incorporated with the aaRS  in our toolkit are:
 </article>
 <ul>
-<li><b>2-nitrophenylalanine (2-NPA)</b>, which cleaves the backbone when irradiated by light of a certain wavelength. Thus it could be used for inactivation and activation of proteins.</li>
+<li><b>2-nitrophenylalanine (2-NPA)</b>, which cleaves the backbone when irradiated by light of a certain wavelength. Therefore, it could be used for inactivation and activation of proteins.</li>
 <li><b>Propargyllysine (PrK)</b>, which provides a propargyl group. Propargyl groups form a highly specific bond to azides in click chemistry reaction. Thus, this amino acid could be used for specific, terminus independent labeling.</li>
 <li><b>p-acetophenylalanine (AcF)</b>, which provides a ketone group. Ketones form a highly specific covalent bond to hydroxylamines. Like PrK, this amino acid could be used for specific, terminus independent labeling.</li>
@@ Line 36: / Line 36: @@
 </ul>
-<article>
+<div class="article">
-Furthermore, these four ncAAs are only the beginning, our part collection contains also the parts for a selection system. With the help of a library plasmid, that contains an aaRS with random mutated amino acid binding sites and a positive and negative selection system, every iGEM team could evolve their own aaRS to incorporate ncAAs with new functions and expand the possibilities of protein design.<br>
+In addition to parts for the translational incorporation of hese ncAAs, our part collection also provides parts for a selection system to evolve novel tRNA synthetases. Moreover, instructions for the generation of a library of plasmids, that contain an aaRS encoding sequence with randmly mutated amino acids in the appropriate tRNA-binding sites. Additionally, a positive and negative selection system enables every iGEM team to expand the possibilities of protein design.
-In the positive selection, our positive-selection plasmid (BBa_K2201900 in pSB3T5), which contains a kanamycin resistance with amber stop codons, is cotransformed with the library plasmid .After cotransformation only the cells survive which contain a synthetase from the library that incorporates any amino acid in response to the amber codon. Thus, all clones that survive the positive selection contain an aaRS that incorporate the target ncAA or any endogenous amino acid. To select only the clones that incorporate only the target ncAA a further round of negative selection is necessary.<br>
+The library and the positive selection plasmid (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2201900">BBa_K2201900</a> in pSB3T5) are cotransformed to select sells, which survive due to a synthetase from the library that incorporates an any amino acid in response to the amber codon. Thus, all clones that survive the positive selection contain an aaRS ceapable of incorporating the target ncAA or any endogenous amino acid. To select only the clones that incorporate only the target ncAA, an additional round of negative selection is necessary.<br>
-The negative selection plasmid (BBa_K2201901 in pSB3T5) is used for the selection for the specificity of the clones that survive the positive selection. For the negative selection the target ncAA is not supplemented in the media. The negative selection plasmid contains the barnase, a cell toxin, with amber codon at permissive sites. If the aaRS are able to incorporate any endogenous amino acids, the cell dies and only the cells survive that incorporate specific the target ncAA in the positive selection, thus incorporate no amino acid in the negative selection.<br>
+The negative selection plasmid (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2201901">BBa_K2201901</a> in pSB3T5) is used to select for high specificity of enzymes. During this negative selection, the target ncAA is not supplemented in the media. The negative selection plasmid encodes the barnase, a cell toxic protein. Since the coding sequence contains two amber codons at permissive sites, the gene product is only generated if the ncAA is present or if the aaRS is unspecifically integrated a canonical amino acid. <br>
-To determine the most effective and specific aaRS for the incorporation of the desired ncAAs, an evaluation system of the synthetases seemed useful to us. Thus, we got aware of the synthetase test system “pFRY” (iGEM Texas 2014) and optimized this part. Our new screening part is BBa_K2201343. Our screening system contains the CDS of cyan fluorescent protein and yellow fluorescent protein connected with a linker containing the amber stop codon, under control of a T7-promotor. The amount and relation of the fluorescent proteins could indicate how efficient and specific the cotransformed aaRS is.<br>
+To determine the most effective and specific aaRS for the incorporation of the desired ncAAs, an evaluation system of the synthetasesis crucial. Therefore, we optimized the synthetase test system “pFRY” initially developed by iGEM Texas 2014 leading to our new screening part <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2201343">BBa_K2201343</a>. Our screening system contains the CDS of the cyan fluorescent protein and yellow fluorescent protein connected with a linker containing an amber stop codon. This part is placed placed under control of a T7-promotor to ensure high product amounts on demand. The amount and ratio of both fluorescent proteins could indicate how efficient and specific the investigated aaRS is working. Thoroughly characterization of all parts (Table 1) can be found on the respective part pages.<br>
 Our constructed parts are listed below. For the complete characterization please refer to the linked part collection pages.
-</article>
+</div>
 </div>
 </div>
@@ Line 52: / Line 52: @@
 <div class="figure large">
 <img class="figure image" src="https://static.igem.org/mediawiki/2017/c/c8/T--Bielefeld-CeBiTec--SVI-Collection-2.png">
-<p class="figure subtitle"><b>Figure 1: Plasmid chart of BBa_K2201200</b><br> Plasmid chart of the 2-NPA-aaRS (BBa_K2201200).</p>
+<p class="figure subtitle"><b>Figure 1: Plasmid map of BBa_K2201200</b><br> Plasmid map of the 2-NPA-aaRS (BBa_K2201200).</p>
 </div>
 </div>
 <div class="half right">
-<b>BBa_K2201200 2-NPA-aaRS in response to UAG</b><br>
+<b><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2201200">BBa_K2201200</a> 2-NPA-aaRS in response to UAG</b><br>
 Evolved tyrosine tRNA/aminoacyl-synthetase for the incorporation of 2-NPA in response to the amber stop codon (CUA).
@@ Line 70: / Line 70: @@
 <div class="figure large">
 <img class="figure image" src="https://static.igem.org/mediawiki/2017/f/f0/T--Bielefeld-CeBiTec--SVI-Collection-1.png">
-<p class="figure subtitle"><b>Figure 2: Plasmid chart of BBa_K2201201</b><br> Plasmid chart of the PrK-aaRS (BBa_K2201201).</p>
+<p class="figure subtitle"><b>Figure 2: Plasmid map of BBa_K2201201</b><br> Plasmid map of the PrK-aaRS (BBa_K2201201).</p>
 </div>
 </div>
 <div class="half right">
-<b>BBa_K2201201 PrK-aaRS in response to UAG</b><br>
+<b><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2201201">BBa_K2201201</a> PrK-aaRS in response to UAG</b><br>
 Evolved Pyrrolysyl tRNA/aminoacyl-synthetase for the incorporation of PrK in response to the amber stop codon (UAG).
 </div>
@@ Line 89: / Line 89: @@
 <div class="figure large">
 <img class="figure image" src="https://static.igem.org/mediawiki/2017/7/79/T--Bielefeld-CeBiTec--SVI-Collection-3.png">
-<p class="figure subtitle"><b>Figure 3: Plasmid chart of BBa_K2201202</b><br> Plasmid chart of the AcF-aaRS in response to the amber stop codon.</p>
+<p class="figure subtitle"><b>Figure 3: Plasmid map of BBa_K2201202</b><br> Plasmid map of the AcF-aaRS in response to the amber stop codon.</p>
 </div>
 </div>
 <div class="half right">
-<b>BBa_K2201202 AcF-aaRS in response to UAG</b><br>
+<b><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2201202">BBa_K2201202</a> AcF-aaRS in response to UAG</b><br>
 Evolved tyrosine tRNA/aminoacyl-synthetase for the incorporation of AcF-aaRS in response to the amber stop codon (UAG).
 </div>
@@ Line 106: / Line 106: @@
 <div class="figure large">
 <img class="figure image" src="https://static.igem.org/mediawiki/2017/6/67/T--Bielefeld-CeBiTec--SVI-Collection-4.png">
-<p class="figure subtitle"><b>Figure 4: Plasmid chart of BBa_K2201203</b> Plasmid chart of AcF-aaRS in response to the less used leucine codon CUA.</p>
+<p class="figure subtitle"><b>Figure 4: Plasmid map of BBa_K2201203</b> Plasmid map of AcF-aaRS in response to the less used leucine codon CUA.</p>
 </div>
 </div>
 <div class="half right">
-<b>BBa_K2201203 AcF-aaRS in response to CUA</b>
+<b><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2201203">BBa_K2201203</a> AcF-aaRS in response to CUA</b>
 Evolved tyrosine tRNA/aminoacyl-synthetase in response to the less used leucine codon in <i>E. coli</i> (CUA).
 </div>
@@ Line 123: / Line 123: @@
 <div class="figure large">
 <img class="figure image" src="https://static.igem.org/mediawiki/2017/b/ba/T--Bielefeld-CeBiTec--SVI-Collection-5.png">
-<p class="figure subtitle"><b>Figure 5: Plasmid chart of BBa_K2201204</b><br> Plasmid chart of the CouAA-aaRS in response to the amber stop codon UAG.</p>
+<p class="figure subtitle"><b>Figure 5: Plasmid map of BBa_K2201204</b><br> Plasmid map of the CouAA-aaRS in response to the amber stop codon UAG.</p>
 </div>
 </div>
 <div class="half right">
-<b>BBa_K2204 CouAA-aaRS in response to UAG</b><br>
+<b><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2201204">BBa_K2204</a> CouAA-aaRS in response to UAG</b><br>
 Evolved tyrosine tRNA/aminoacyl-synthetase for the incorporation of the fluorescent amino acid CouAA in response to the amber stop codon (UAG).
 </div>
@@ Line 140: / Line 140: @@
 <div class="figure large">
 <img class="figure image" src="https://static.igem.org/mediawiki/2017/6/68/T--Bielefeld-CeBiTec--SVI-Collection-6.png">
-<p class="figure subtitle"><b>Figure 6: Plasmid chart of BBa_K2201900</b><br>Plasmid chart of the positive selection plasmid. </p>
+<p class="figure subtitle"><b>Figure 6: Plasmid map of BBa_K2201900</b><br>Plasmid map of the positive selection plasmid. </p>
 </div>
 </div>
 <div class="half right">
-<b>BBa_K2201900 Positive-selection plasmid for the incorporation of ncAAs</b><br>
+<b><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2201900">BBa_K2201900</a> Positive selection plasmid for the incorporation of ncAAs</b><br>
-The positive selection plasmid contains a tRNA and a kanamycin resistance with two amber codons. Cotransformed with the library of tRNA/aminoacyl-synthetase with random mutated binding sites, on kanamycin only the clones survive that could charge any amino acid to the tRNA in response to the UAG codon.
+The positive selection plasmid contains a tRNA and a kanamycin resistance with two amber codons. Cotransformed with the library of tRNA/aminoacyl-synthetase with random mutated binding sites, on kanamycin containing plates only the clones survive that could charge any amino acid to the tRNA in response to the UAG codon.
 </div>
 </div>
@@ Line 157: / Line 157: @@
 <div class="figure large">
 <img class="figure image" src="https://static.igem.org/mediawiki/2017/2/2c/T--Bielefeld-CeBiTec--SVI-Collection-7.png">
-<p class="figure subtitle"><b>Figure 7: Plasmid chart of BBa_K2201901</b><br> Plasmid chart of the negative selection plasmid for the selection of aaRS that specifically inocporate ncAAs.</p>
+<p class="figure subtitle"><b>Figure 7: Plasmid map of BBa_K2201901</b><br> Plasmid map of the negative selection plasmid for the selection of aaRS that specifically inocporate ncAAs.</p>
 </div>
 </div>
 <div class="half right">
-<b>Ba_K2201901 Negative selection plasmid against the incorporation of endogenous amino acids</b><br>
+<b><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2201901">Ba_K2201901</a> Negative selection plasmid against the incorporation of endogenous amino acids</b><br>
-The negative selection contains an tRNA with the anticodon for the amber codon and a barnase containing amber codons at permissive sites. In the negative selection the target amino acid is not supplemented to the media. If the cotransformend clones from the positive selection charge endogenous amino acids to the tRNA, the cells die. This provides a selection method for high specific aaRS.
+The negative selection plasmid contains an tRNA with the anticodon for the amber codon and a barnase containing amber codons at permissive sites. In the negative selection, the target amino acid is not supplemented to the media. If the cotransformend clones from the positive selection charge endogenous amino acids to the tRNA, the cells die. This provides a selection method for high specific aaRS.
 </div>
@@ Line 175: / Line 175: @@
 <div class="figure large">
 <img class="figure image" src="https://static.igem.org/mediawiki/2017/d/df/T--Bielefeld-CeBiTec--SVI-Collection-8.png">
-<p class="figure subtitle"><b>Figure 8: Plasmid chart of BBa_K2201343</b><br> Plasmid chart of the screening plasmid for the incorporation of ncAAs.</p>
+<p class="figure subtitle"><b>Figure 8: Plasmid map of BBa_K2201343</b><br> Plasmid map of the screening plasmid for the incorporation of ncAAs.</p>
 </div>
 </div>
 <div class="half right">
-<b>BBa_K2201343 Screening system for the incorporation of ncAA</b>
+<b><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2201343">BBa_K2201343</a> Screening system for the incorporation of ncAA</b>
 Our screening system contains the CDS of cyan fluorescent protein and yellow fluorescent protein connected with a linker containing the amber stop codon, under control of a T7-promotor. The amount and relation of the fluorescent proteins could indicate how efficient and specific the cotransformed aaRS is.
 </div>