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

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To showcase the possibility of enzyme activity regulation on protein level, we designed a photoswitching experiment in which we controlled the lycopene production of an E. coli strain. This was achieved by incorporation of the non-canonical amino acid (ncAA) phenylalanine-4'-azobenzene (AzoF) into pytoene desaturase, encoded by <i>crtI</i>. The lycopene production can be completely terminated by introduction of amber codons into <i>crtI</i>. The enzyme activity can be partially recovered by cotransformation with an aminoacyl-tRNA synthetase (aaRS). Even without supplementation of the media with the desired ncAA, this will lead to some enzyme activity recovery. We also showed that we are able to switch the conformation of AzoF from a mixed state to trans and cis with our LED panel and that the amino acids are stable in their specific conformation over several hours. When cultivated with AzoF in cis- or trans-conformation we detected a significant difference in the lycopene production. Therefore, we proved that photoswitching of enzyme activity on protein level  can be achieved using our system.
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Revision as of 17:13, 26 October 2017

Photoswitching

Short summary

To showcase the possibility of enzyme activity regulation on protein level, we designed a photoswitching experiment in which we controlled the lycopene production of an E. coli strain. This was achieved by incorporation of the non-canonical amino acid (ncAA) phenylalanine-4'-azobenzene (AzoF) into pytoene desaturase, encoded by crtI. The lycopene production can be completely terminated by introduction of amber codons into crtI. The enzyme activity can be partially recovered by cotransformation with an aminoacyl-tRNA synthetase (aaRS). Even without supplementation of the media with the desired ncAA, this will lead to some enzyme activity recovery. We also showed that we are able to switch the conformation of AzoF from a mixed state to trans and cis with our LED panel and that the amino acids are stable in their specific conformation over several hours. When cultivated with AzoF in cis- or trans-conformation we detected a significant difference in the lycopene production. Therefore, we proved that photoswitching of enzyme activity on protein level can be achieved using our system.

Design of AzoF-RS

The AzoF-RS (BBa_K2201207) was based on an part exchange with CU Boulder 2017. 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.

Figure 3 shows that the absorbance maximum of the lycopene lays at 476 nm, which correlates with the data of the references. We then performed the quantification of the lycopene production with five biological and three technical replicates (Figure 4). It verifies the implications of Figure 2 that the cells containing the amber-variants are not able to produce lycopene. That makes them suitable for the incorporation process of AzoF, such that an increase of the lycopene production is to be expected after cotransformation with the AzoF-RS and feeding with AzoF.

Figure 4: Absorbance at 476 nm of the samples with extracted lycopene of the transformants with the functional crtI (left), the crtI with an amber codon at position 318 (middle) and with an amber codon at position 353 (right). The absorbance at 476 nm of a 1:1 aceton water solution was subtracted from the samples.

Cultivation and irradiation of the cotransformants

The incorporation of different amino acids influences the lycopene production...

Figure 5: tbc

Figure 6: tbc