Recombinant Expression of Subtilisin E

Before photocaging, we needed to express subtilisin E recombinantly in Escherichia coli or Saccharomyes cerevisiae as we had to use an already existing tRNA/synthetase pair for our first attempts.

In S. cerevisiae
Unfortunately, we were not able to express Subtilisin E in S. cerevisiae. That is not greatly surprising, as it originates from a prokaryote. If we would have been able to, the possibility of glycosylation causing the enzyme to be inactive, would still be quite high.

In E. coli
In the course of our project we were able to express native subtilisin E in E. coli. But as the production of the protease interfered with the well-being of the organism it took a long time to see proteolytic activity. But we are positive that inhibiting the enzyme would improve the growth conditions and therefore yield faster results and a higher production rate.
For the varification of our results, we performed a skim milk assay on agar plates. Therefore, we poured LB skim milk agar plates containing IPTG and the needed antibiotic and streaked out the E. coli BL21 cells containing the plasmid with the expression system.

Comparing the clearance of the skim milk plates, a proteolytic activity could be proven for the cells containing the expression system for native subtilisin E. As a result, we concluded that within three days these cells are able to produce the native protease, which will then digest the skim milk in the agar plates, resulting in a clearance.
In conclusion, we were able to express subtilisin E in E. coli and to prove its proteolytic activity via skim milk assay.

Photocaging of Subtilisin E

To avoid the immoderate use of boric acid for liquid laundry detergents, we aimed to develop a subtilisin E variant which is inactivated via introduction of a photocaged amino acid into the protein in vivo.

In S. cerevisiae
Targeting the serine²²¹ in the active center could not be tested in Saccharomyes cerevisiae, as expression of the protease was unsuccessful.

In E. coli
With E. coli we were able to simulate the integration of a photo-labile, non-canonical amino acid both in the catalytic triad and the pro-peptide cleavage site by exchanging the targeted amino acids against larger amino acids. The results of these experiments showed that incorporating DMNB-serine would definitely lead to a reversible inhibition. Due to a lack of time, it was not possible to perform further investigations.
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. 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 beforehand 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.

Development of a New Synthetase

Furthermore, for making introduction of a photocaged serine in a prokaryot possible, which would be an ideal approach to our goal, and for improving the photocaging method in general by enlarging its applications, we intended to extend the genetic code in Escherichia coli.

Summary of achievements
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Synthetase Text
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HERE E. COLI (expression was successful but targeting tyrosine did not in activete)

Making Light Isolated Work More Comfortable

A lot of chemicals are extremely sensitive to light and therefore require a dark work area for scientists. Working in dark rooms is very inconvenient and can also affect health. We wanted to build a tool that allows you to stay in the daylight while your chemicals can remain in a protective box. We were able to build a device called Dark Bench, which is affordable and convenient. Also the possibility to assemble and disassemble the device, makes it portable. Use of opaque Plexiglas as a building material and UV foil as a protective cover for the viewing window, makes Dark Bench light proof Thus, the Dark Bench is light proof device and can provide a suitable environment to work with light sensitive materials.

Activation of inhibited Protease Before Washing

As an important step for the development of the light inducible proteases we were in need to find a solution for the activation of the photocaged proteases. For that, we built a device which can cleave the protection group off the caged protease, thus activating the protease. The inclusion of inexpensive and simple components makes the LIPs-Stick highly economical and compact, which in the near future will facilitate the installation of the device in washing machines

New Biobrick Parts

Click here to see all the Biobricks we created in the course of our project