Team:Bielefeld-CeBiTec/Part Collection

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) 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.

Part collection for the incorperation of non-canonical amino acids

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:
  • 2-nitrophenylalanine (2-NPA), which cleaves the backbone when irradiated by light of a certain wavelength. Thus 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.
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.
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.
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.
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.
Our constructed parts are listed below. For the complete characterization please refer to the linked part collection pages.

Figure 1: Plasmid chart of BBa_K2201200
Plasmid chart 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 chart of BBa_K2201201
Plasmid chart 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 chart of BBa_K2201202
Plasmid chart of the AcF-aaRS in response to the amber stop codon.

BBa_K2201202 AcF-aaRS is 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 chart of BBa_K2201203 Plasmid chart of AcF-aaRS in response to the less used leucine codon CUA.
BILDUNTERSCHRIFT

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 chart of BBa_K2201204
Plasmid chart of the CouAA-aaRS iin 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 chart of BBa_K2201900
Plasmid chart 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 only the clones survive that could charge any amino acid to the tRNA in response to the UAG codon.

Figure 7: Plasmid chart of BBa_K2201901
Plasmid chart 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 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 chart of BBa_K2201343
Plasmid chart 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.