Photocaging of Subtilisin E

To prove the principle of our project idea, we performed a site-directed mutagenesis on each of our targeted sites to simulate the integration of a photo-labile, non-canonical amino acid (ncAA). At first, we exchanged serine²²¹ in the active site of subtilisin E against tyrosine. In addition to this, we substituted tyrosine⁷⁷ in the pro-peptide cleavage site against tryptophan. Then, the cells with a modified version of the expression system for subtilisin E in E. coli that had been proven to work, which can be seen in the characterization of the BioBrick K2020002, were streaked on skim milk agar plates containing the inducer IPTG and the needed antibiotics.

Neither the empty backbone nor the expression system with the modified catalytic triade seems to cause a proteolytic activity. A clearance of the skim milk plates and therefore a proteolytic activity can only be observed for the native protease (as it has been demostrated before) and the Y77W-mutated enzyme.
Through this experiment, we are now able to prove that serine is essential for the proteolytic activity of the protease and that exchanging it would inactivate the enzyme.
As seen on the pictures above, a clearance had occurred for the cells modified to express subtilisin with tryptophan instead of tyrosine⁷⁷. As a result, a proteolytic activity can be assumed. Contrary to our former beliefs, it can now be deduced that exchanging tyrosine doesn’t result in a change of activity. Consequently, tyrosine in the pro-peptide cleavage site is not essential for the activity of subtilisin E.

Conclusion
Unfortunately, tyrosine⁷⁷ seems to be not essential for the proteolytic activity. Thus, exchanging tyrosine against ONB-tyrosine would not influence the activity of the enzyme.
On the other hand, we are able to proof that exchanging serine²²¹ in the active site will result in a loss of activity. Hence, we have demonstrated that substituting serine against a photo-labile, non-canonical amino acid like DMNBS will inactivate subtilisin E. Thus, we can now provide a valid proof of the principle of our project.

Evolving a New tRNA-Synthetase

Evolving a new tRNA/synthetase pair for incorporation of a non-canonical amino acid via amber termination suppression takes time and effort due to the extensive screening process. To prove the preliminary computational modelling, an already established two plasmid screening system based on fluorescence protein formation was used. Only if the cell can incorporate an amino acid at an amber stop codon, green fluorescence is visible. As various synthetases yielded a high fluorescence value in positive screening but a low one in negative, incorporation of DMNBS into the fusion-protein has been proven. Evaluation of screening data implies the presence of clones which incorporate the ncAA more selectively than others, thus a parts collection was created with different as well as properly working synthetases.

Dark Bench

In order to prove that our Dark Bench is light proof, we needed to characterize our optical window and safelight. As it is already known that the emission of red LED is well below 500 nm, we have only analyzed the absorption spectrum of our windows specimen. From the graph below, we can confirm that, in the wavelength below 500 nm, the specimen blocks about 99% of incident radiation in UV-A region and 99% in blue region

Conclusion
In short, we demonstrated that our device, Dark Bench, can provide a light proof workspace.

LIPs-Stick

To validate that our UV-exposure device called LIPs-Stick, activates the caged protease, we studied the photo-cleavage reaction of photo-caged amino acid by visible spectrophotometry and found that our device can effectively activate the caged amino acid. From the graph below, we can see not only the formation of cleavage products but also the amount of the cleavage product depends on UV intensity and exposure time.

Conclusion
To sum up, we proved that, the activation of caged enzymes is possible and effective by using our tool LIPs-Stick.