Part Collection for the Incorperation of Non-canonical Amino Acids
- 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.
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).
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).
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.
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.
Figure 5: Plasmid map of BBa_K2201204
Plasmid map of the CouAA-aaRS in response to the amber stop codon 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.
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.
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.