Team:Aachen/Results
Results
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.
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.
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
