Part:BBa_K2201207

Expression plasmid of an aminoacyl-tRNA synthetase for AzoPhe

Aminoacyl-tRNA synthetase for incorporating phenylalanine-4‘-azobenzene (AzoF) at the amber codon.

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Figure 1: Plasmid map of the BioBrick K2201207 coding for a AzoF-tRNA-synthetase system based on the ONBY-Part (K1416000). The ONBY-RS was removed by PCR and then a gene containing the AzoF-RS and matching overlaps sides was integrated through Gibson assembly.

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Figure 2: History of the plasmid construction of K2201207.

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Figure 3: Alignment of the protein sequence of the AzoF-RS and the M. jannaschii TyrRS.

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Figure 4: Structure of AzoF in cis- and trans-conformation. Switching is possible via irradiation.

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Figure 5: Absorption spectrum of AzoF in LB media after irradiation with light of 367 nm and 465 nm wavelength. The black line shows the typical absorption of AzoF in a mixed state, the trans-conformation is blue and green and the other lines show the absorption spectrum in the cis-conformation.

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Figure 6: Absorption at 476 nm (indicator for lycopene) normalized to the OD600 (indication for the cell density) to calculate the relative lycopene production of each crtI variant cultivated with AzoF in cis- and trans-conformation.

The pEVOLpAzF is available at request at CU Boulder, Bielefeld CeBiTec or addgene. For further use we recommend to clone the CDS of the AzoF-RS into a low copy plasmid like pSB3T5 or in a high copy plasmid like pSB1C3 as shown in Figure 2. The characterization was done in p15A.

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 1164
  Illegal BamHI site found at 1170
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1196
  Illegal NgoMIV site found at 1656
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 295
  Illegal SapI.rc site found at 327
  Illegal SapI.rc site found at 1120