Line 200: | Line 200: | ||
</table> | </table> | ||
− | <table 1>: Table of Incorporation rates of DMNBS synthetase mutants with and without supplementation of DMNBS. <b>Incorporation rate (%)</b> Incorporation value of DMNBS normalized to incorporation value of Mj tyr-RS of tyrosine, <b>neg (%)</b> Incorporation value of tyrosine normalized to Mj tyr-RS, | + | |
+ | <p align="justify" style="padding-left: 1.0cm; padding-right: 1.0cm; font-size:16px;"><table 1>: Table of Incorporation rates of DMNBS synthetase mutants with and without supplementation of DMNBS. <b>Incorporation rate (%)</b> Incorporation value of DMNBS normalized to incorporation value of Mj tyr-RS of tyrosine, <b>neg (%)</b> Incorporation value of tyrosine normalized to Mj tyr-RS, | ||
Line 215: | Line 216: | ||
Though good fluorescent signals are obtained via plate reader, an online measurement is significantly increasing the accuracy of collected data, as the exact end-point detection is possible. | Though good fluorescent signals are obtained via plate reader, an online measurement is significantly increasing the accuracy of collected data, as the exact end-point detection is possible. | ||
Furthermore the influence of mutating position 176 and 177 is to be evaluated in detail, as well as probing various combinations of here identified beneficial residues at all sites. According to the project idea the incorporation of DMNBS in Subtilisin E to replace serine within the active site at position 221 will follow. | Furthermore the influence of mutating position 176 and 177 is to be evaluated in detail, as well as probing various combinations of here identified beneficial residues at all sites. According to the project idea the incorporation of DMNBS in Subtilisin E to replace serine within the active site at position 221 will follow. | ||
− | <b style="color:#005b04;"> Conclusion </b> | + | <br/><br/><b style="color:#005b04;"> Conclusion </b> |
<p align="justify" style="padding-left: 1.0cm; padding-right: 1.0cm; font-size:16px;"> Wild type <i>Methanococcus janaschii </i> tyrosyl synthetase is successfully muted to incorporate DMNBS in <i>E.coli</i> with reasonable efficiency by using a computational engineering method as well as an already established fluorescence based screening system. | <p align="justify" style="padding-left: 1.0cm; padding-right: 1.0cm; font-size:16px;"> Wild type <i>Methanococcus janaschii </i> tyrosyl synthetase is successfully muted to incorporate DMNBS in <i>E.coli</i> with reasonable efficiency by using a computational engineering method as well as an already established fluorescence based screening system. | ||
This is the first tRNA/synthetase pair to incorporate a photocaged serine in E.coli by using amber termination suppression. | This is the first tRNA/synthetase pair to incorporate a photocaged serine in E.coli by using amber termination suppression. | ||
End read more<br/> | End read more<br/> | ||
− | + | ||
</p> | </p> | ||
Revision as of 00:03, 20 October 2016
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.
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
Start read more(will add when i have text)
Synthetase Text
DMNBS-RS variant | Incorporation rate with DMNBS (%) | Incorportion rate without DMNBS (%) | Incorporation ratio | Mutation site 65 | Mutation site 158 | Mutation site 159 | Mutation site 176 | Mutation site 177 |
WT | 100 | 100 | 1 | L | D | I | I | H |
1 | 85 | 28 | 3,04 | H | G | P | A | A |
2 | 90 | 42 | 2,14 | G | R | T | V | A |
3 | 85 | 42 | 2,02 | H | P | P | - | - |
4 | 87 | 41 | 2,12 | G | S | P | V | A |
5 | 93 | 40 | 2,33 | L | P | P | - | - |
6 | 59 | 27 | 2,19 | R | G | S | F | A |
7 | 19 | 3 | 6,33 | G | A | S | V | A |
8 | 60 | 24 | 2,5 | H | T | P | A | V |
9 | 86 | 32 | 2,69 | H | P | P | P | F |