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.
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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| 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 |
The two plasmid screening system for testing incorporation efficiency and fidelity of ncAA herein is used to get a first approximation of properties of DMNBS synthetase clones for E.coli, and moreover offers a good control with RFP always formed if the reporter plasmid is present and induced, on the contrary to other screening systems.
As basis for comparison we chose to normalize fluorescence levels with wild type tyrosyl synthetase. This is possible due to equal optical densities. Furthermore, the biogenic background fluorescence levels are eliminated.
As shown in table 1 mutants occur which incorporate DMNBS more selectively than others. By comparing evaluated sequencing with ncAA incorporation values, the following statements can be made:
Position L65 is in most cases mutated to histidine or glycine. Arginine is also occurring. The previous modeling with Yasara 16.4.6 shows, that nitro group of DMNBS will most likely be in the vicinity of this residue possibly resulting in positively charged histidine or arginine stabilizing the nitro group. This is in accordance with previously reported evolved p-Nitrophenylalanine synthetase which also requires a histidine residue at this site [8].
Preferred residues at position 158 and 159 are in most cases either one or two adjecent proline. As proline has been determined prior in other synthetases [5,6,7] for ncAA, the occurance in the DMNBS synthetase might have a beneficial spacial or functional aspect e.g. enlargement of the binding pocket.
Residues 176 and 177 are in most cases mutated to an amino acid with no sterical hindrance, giving also space to the bulky photoprotection group. As shown, additional mutations on postion XY is quite small, as there are 2 clones identified among the most efficient ones, who show wild type amino acids at these sites.
If there is no tiny amino acid at last two positions to be found, then two adjecent prolines in 158 and 159 occur, as seen in table 1 (mutants no. 3,5 and 9).
Mutant 7 (table 1, L65G, D158A, I159S, I176V, H177A) shows a very low incorporation value, also a low incorporation efficiency at all. Sequencing results confirm the exchange of amino acids in a way , that reduces specificity for the original substrate way more than for the desired one. This means incorporation of DMNB-S still exceeds the one of tyrosine, while being very low overall.
In general can be said, that a positively charged amino acid is necessary either in position 65 or 158 due to the nitrogroup of DMNBS. Furthermore an amino acid showing no sterical hindrance is required to give space to the photoprotection group. Otherwise one or two proline residues might bend the protein structure to enlarge the binding pocket.
The general approach herein is used to get an approximation of the DMNBS synthetase mutants’ efficiency and fidelity. In order to improve one of the now existing tRNA/synthetase pairs, determinination of impact factors on incorporation values and cell growth rate will be made.
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.
Conclusion
Wild type Methanococcus janaschii tyrosyl synthetase is successfully muted to incorporate DMNBS in E.coli 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.
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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
