Part:BBa_K2201200

Tyrosyl tRNA/aminoacyl-synthetase for the incorporation of 2-nitrophenylalanine

To get the noncanonical amino acid 2-nitrophenylalanine (2-NPA) incorporated into a protein auch as the fusion protein of K2201321, a sequence with a TAG is needed along with an additional tRNA synthetase (RS) that is able to load the ncAA to the tRNA. The ribosome is then able to incorporate this amino acid at an amber codon if the target protein with the amber codon and a plasmid with the 2NPA-RS and tRNA are co-transformed. This part contains a promotor for the 2-NPA-RS and the coding sequence for the RS. Additionally, it contains the promoter for the CUA-tRNA and the tRNA sequence itself followed by a terminator.

Figure 1: Plasmid map of the BioBrick K2201200 coding for a 2-NPA-tRNA-synthetasis system based on the ONBY-Part (K1416000). The ONBY-RS was removed by PCR and then a gene synthesis containing the 2-NPA-RS and matching overlaps sides was integrated through Gibson assembly.

We created this specific aaRS by ordering a gene synthesis at Integrated DNA Technologies (IDT) with a codon optimized sequence of an aaRS able to incorporate 2-NPA based on an evolution experiment of Peters et al [3]. This gene synthesis was integrated into the ONBY-Part K1416000 replacing the ONBY-RS. This approach is applicable, as the 2-NPA-RS and the ONBY-RS are mutated versions of the M. jannaschii TyrRS, such that both recognize the same tRNA and only the sequence of the aaRS itself has to be changed to get a new synthetase binding other amino acids to the tRNA. The gene synthesis we ordered had 30 base pairs overlap to a linearized vector of the ONBY-part K1416000, where only the gene sequence of the ONBY-RS was removed. The fragment of the NPA-RS was integrated through Gibson assembly.

Figure 2: History of the plasmid construction of K2201200.

In that specific evolution experiment, ten amino acids in the TyrRS were randomly mutated, resulting in six clones with good incorporation properties for 2-NPA. The best one displayed fidelity rates of approximately 95%. Therefore, its sequence was used for our gene synthesis [3]. Below is a comparison of the mutated amino acid sequences of the M. jannaschii TyrRS, the ONBY-RS and the 2-NPA-RS (table 1).

Table 1: Comparison of the mutated amino acids of the ONBY-RS and 2-NPA-RS to the native Tyr-RS of M. jannaschii.

Tyr-RS Tyr32 Leu65 Ala67 His70 Phe108 Gln109 Tyr114 Asp158 Ile159 Leu162 ONBY-RS Gly Gly Ala His Glu Gln Tyr Ser Ile Glu 2-NPA-RS Gly His Gly Gly Leu Ser Ser Thr Tyr Asp

To proof that the 2-NPA-RS is expressed when E.coli is transformed with this BioBrick, we made a SDS-Page with the cell lysate of a preculture containing this part. The expected molecular weight of our synthetase is 34.76 kDa with an isoelectric point of 6.23. As seen in Figure 3, the SDS-Page shows that the 2-NPA-RS is well expressed in the E.coli strain BL21(DE3) when transformed with the BioBrick.

Figure 3: SDS-Page of the protein extract of three E.coli precultures. Left Standard Ruler, next to it samples of BL21(DE3) with K2201200, then positive control of BL21(DE3) with K1416000, and at last negative control of BL21(DE3) with K525998 which is not expressed.

To proof that our new synthetase is also functional and loads 2-NPA onto the CUA-tRNA, we expressed the fusion proteins of K2201320 and K2201321 separately, and additionally K2201321 co-transformed with K2201200. K2201320 encodes a fusion protein of GFP and streptavidin just like K2201321. The fusion protein of K2201321 contains an amber-codon in the linker between GFP and streptavidin, such that the expression will stop after the GFP sequence. A western blot with GFP antibodies was performed to show the filling of the amber-codon in K2201301 through our synthetase (Figure 4).

Figure 4: Western blot with GFP-antibodies of the whole fusion protein of K2201320 (-TAG), the partial expressed fusion protein of K2201321 (+TAG) and the whole fusion protein of K2201321 if co-transformed with K2201200 (+TAG, +aaRS, +2NPA).

To test if we are even able to change the structure of 2-NPA by irradiating with our LED panel we made an in vitro test. We supplemented LB media with 1 mM of 2-NPA and did absorption measurements over 4 hours while irradiating with UV-light in a microwell plate. We noticed that there was a high absorption in the UV spectrum (< 300 nm), which makes sense hinge 2-NPA absorbs the UV-light to do the self-cyclization reaction (Figure 5). Over time we noticed a constant increase in the absorption spectrum at approximately 340 nm which indicates the emergence of a chemical component due to the irradiation process. We are pretty sure that this compound is the self-cyclezised 2-NPA, proposed by Peters et al. which leads to a shift in the absorption spectrum.

Figure 5: Changes in the absorption spectrum of 2-NPA in LB media while irradiated at 367 nm for 240 minutes. The emerging peak at ~ 340 nm indicates the change in the structure of 2-NPA from its native form, to the self-cyclized form.

The western blot confirmed our expectations. The bands of the whole fusion protein, of the GFP-units and the cleaved streptavidin-Tags are clear to see. We also see that the GFP-bands are very thick compared to the others, what cannot be explained by the photolysis itself. It seems like the 2-NPA-RS is not very effective in loading 2-NPA to the amber tRNA, which leads to a high amount of incomplete expression of the fusion protein. The low efficiency of our 2-NPA-RS was confirmed by our synthetase-test system (K2201343).

Figure 6: Western Blot of an SDS-Page marked with anti-GFP antibodies. It proves that the low bands are indeed cleaved streptavidin-Tags.

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Figure 7: Shift of the emission spectrum of a the CFP-YFP aaRS-test system (), when cotransformed with the 2-NPA-RS and cultivated with and without 2-NPA in the media.

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Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1153
  Illegal BamHI site found at 1159
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 404
  Illegal NgoMIV site found at 1185
  Illegal NgoMIV site found at 1645
  
• 1000
  COMPATIBLE WITH RFC[1000]