Difference between revisions of "Team:Bielefeld-CeBiTec/Results/toolbox/photoswitching"

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<h2> Two Amber-CrtI-Variants </h2>
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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.
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<p class="figure subtitle"><b>Figure 2: Cell pellets of the functional CrtI-variant (left), the amber318 (middle) and the amber353 (right) variants vortexed in 500 µl acetone.  </b><p>
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Revision as of 10:50, 4 October 2017

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.