Difference between revisions of "Team:Aachen/Proof"

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<h2 style="border-bottom: 5px solid #005b04;padding-left: 1.0cm;">Photocaging subtilisin E in <i>E. coli </i></h2>
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<h2 style="border-bottom: 5px solid #005b04;padding-left: 1.0cm;">Photocaging of Subtilisin E </h2>
 
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Revision as of 11:30, 19 October 2016

Welcome to iGEM Aachen 2016

Proof of Concept

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. 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.

Figure 1: Skim milk plates assay. Cells containing the empty backbone (1) in comparison to either cells producing the native (2), S221Y-mutated (3) and Y77W-mutated subtilisin E (4) after incubation for 3 days at 30°C.

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 did 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.