Design of the AzoF-RS
Figure 1: Sequence alignment of the M. jannaschii TyrRS and the AzoF-RS of the Schultz lab. The alignment shows six differences in the protein sequences, at position partizipating in the binding process.
Figure 2: Cell pellets of the three crtI-variants vortexed in 400 µL acetone. Functional CrtI-variant (left), amber-codon at position 318 (middle) and amber-codon at position 353 (right).
Figure 3: Absorption spectrum of the positive lycopene sample from 400 to 550 nm normalized with the measurement of a 1:1 acetone water sample. It shows the typical absorption spectrum of lycopene.
Figure 4: Absorbance at 476 nm of the extracted lycopene of the samples. The functional crtI (left: LP), the crtI with an amber codon at position 318 (middle: TAG318) and with an amber codon at position 353 (right: TAG353). The absorbance at 476 nm was normalized using a 1:1 aceton water solution.
Basic Lycopene Production of the Cotransformants
Figure 5: Absorption spectrum of the extracted lycopene of the three samples. LP is the lycopene producing strain with an intact crtI, TAG318 has the amber-codon at position 318 in crtI and TAG353 has an amber-codon at position 353 in crtI.
Figure 6: Mean and standard deviation of the absorption spectrum of the three samples from 400 to 550 nm.
Irradiation, Switching and Stability of AzoF
Figure 7: Absorption spectrum of AzoF in LB media after irradiation with light of 367 nm wavelength. The black line shows the typical absorption of AzoF in the trans-conformation while the other lines show the absorption spectrum in the cis-conformation. The spectrum was measured directly after the irradiation, then after 2, 4, 17 and 20 hours. The sample was incubated at 30°C.
Influence of Photoswitching on the Lycopene Production
Figure 8: OD600 of three biological and three technical replicates of two crtI variants after cultivation.
Figure 9: Absorption spectrum of the four samples of the crtI variants. Cultivated with AzoF supplemented to the media, photoswitched to cis- or trans-conformation.
Figure 10: 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.