Team:Bielefeld-CeBiTec/Results/toolbox/photoswitching

Photoswitching

Design of AzoF-RS

The AzoF-RS was provided by CU Boulder and deposited in the Registry of Standard Biological Parts as BBa_X. They got it from the Schultz lab, which performed a selection experiment on the M. jannaschii TyrRS to evolve a new aaRS capable of incorporating the photoisomerizable phenylalanine-4‘-azobenzene (AzoF). Figure 1 shows a sequence alignment of the protein sequences of the M. jannaschii TyrRS and the AzoF-RS after the selection process.

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.

Two Amber-CrtI-Variants

We created two variants in which the crtI protein in the lycopene pathway has an amber-codon incorporated; one at the position 318 and the other at position 353. We cultivated E.coli BL21(DE3) transformed with the two amber-variants and a functional crtI for 24 hours at 37°C and centrifuged the culture. The pellet of the strain with the functional crtI showed a visible orange color, typical for lycopene (Figure 2). The two amber-variants showed no color due to the absence of lycopene caused by the non-functional crtI in the lycopene pathway.

Figure 2: Cell pellets of the functional CrtI-variant (left), the amber318 (middle) and the amber353 (right) variants vortexed in 500 µl acetone.

We then extracted the lycopene from the pellet to quantify the amount of lycopene produced by the three cultures. For that, we resuspended the pellet in 400 µl acetone and vortexed it to solve the lycopene. We then added 400 µl water and made and absorbance measurement. We first made an absorbance spectrum to identify the best wavelength for the quantification (Figure 3).

Figure 3: Absorbance spectrum of the positive lycopene sample from 400 to 550 nm normalized with the measurement of a 1:1 acetone water sample.