Part:BBa_K3143000
Cl2Tyr aminoacyl tRNA synthetase
Cl2Tyr aminoacyl tRNA synthetase is an aminoacyl-tRNA synthetase that attaches 3,5-dichlorotyrosine non-canonical amino acid onto its cresponding tRNA(TAG). Once the tRNA is charged, a ribosome can transfer 3,5-dichlorotyrosine from the tRNA(TAG) onto a growing peptide where there are UAG codon on mRNA. It is an mutated TyrRS version originated from Methanococcus jannaschii tRNATyr-TyrRS system[1].
Figure 1: 3,5-dichlorotyrosine non-canonical amino acid chemical structure.
Usage and Biology
This enzyme is a key component of the ncAA system. Together with several other parts (see BBa_K3143006.), Cl2Tyr ncAA system could introduce 3,5-dichlorotyrosine specificity into the proteins.
In the 2019 BEAS_China design, we used this ncAA system to build a ncAA-dependent toxin-antitoxin system, see our Design.
Characterization
In the non-canonical amino acid system, the aminoacyl tRNA synthetase is under the control of the arabinose promoter, while the tRNA recognizing the TAG codon is regulated by a constitutively expressed promoter.
Figure 2: Schematic illustration of genetic circuits for incorporation of 2 ncAAs into GFP(39TAG & 151TAG).
As can be seen from Fig 3, when arabinose or ncAA was not added to the system, the expression level of GFP was almost zero. When arabinose and ncAA was not added to the system, GFP was fully synthesized thus fluorescence signal can be detected.
Figure 3: Figure 10: Characterization of Cl2Y RS system.
Reference
1. Liu X, Jiang L, Li J, et al. Significant expansion of fluorescent protein sensing ability through the genetic incorporation of superior photo-induced electron-transfer quenchers[J]. Journal of the American Chemical Society, 2014, 136(38): 13094-13097.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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