Escherichia coli is widely used in synthetic biology. It offers the advantage of being a comparatively simple and well-understood model organism while being easy to handle in the laboratory environment. Also, an expansion of the genetic code has already been successfully implemented in E. coli multiple times [9-12] by introducing an orthogonal tRNA/synthetase pair.
Therefore, working with E. coli is an obvious choice.

Due to a limited range of tRNA/synthetase pairs for non-canonical amino acids in general and especially for those that act orthogonally in E. coli, photocaging serine in the active site of subtilisin E with DMNB-serine is currently not possible. Hence, another strategy is needed to produce temporarily inactive proteases. This part of the project focuses on utilizing the maturation process of subtilisin E.

Subtilisin E is an alkaline serine protease found in Bacillus subtilis that has to autoprocess itself to become functional. At first, the enzyme exists as a precursor, namely the pre-pro-subtilisin. The pre-sequence serves as a recognition sequence for secretion across the cytoplasmic membrane and is cleaved off in the course of the process. The pro-peptide acts as an intramolecular chaperone (IMC) and facilitates the folding of the protease. Folding is essential for the activity of an enzyme. Still, the maturation process of Subtilisin E is not completed, as the pro-peptide covers the substrate binding site and inhibits activity. However, enough proteolytic activity is achieved to autoprocess the IMC-domain and therefore cleave off the pro-peptide. Yet, the C-terminal end of the pro-peptide continues to block the substrate binding site. After the degradation of the pro-peptide, the substrate-binding site is cleared and the protease becomes effectively active. [13]

This mechanism can be used to implement a novel inactivation method.
O-(2-Nitrobenzyl)-L-tyrosine (ONBY) is a derivative of the canonical amino acid tyrosine. It carries a photo-labile protection group that can be cleaved off by irradiation with UV-light (365nm, [14]).

By adding ONBY to the genetic code of E. coli and incorporating said amino acid in the pro-peptide cleavage-site of subtilisin E the maturation process is disturbed. Due to its size ONBY sterically hinders the protease [15]. The pro-peptide cannot be cleaved from the enzyme and subtilisin E is not able to achieve its full proteolytic activity. A temporarily inactive protease is produced.
After removal of the protection group the maturation process can be completed and subtilisin E acquires its full proteolytic activity.

Introduction
Photocleavable non-canonical amino acids offer the opportunity to control protein function on a non-invasive basis. Working with non-canonical amino acids requires an additional, orthogonal pair of a tRNA and a cognate synthetase which does not crossreact with the endogenous tRNA/synthetase pairs [5].
A previously reported tRNA/synthetase pair for O-(4,5-dimethoxy-2-nitrobenzyl)-L-serine (DMNBS) derived from Escherichia coli and was used in Saccharomyces cerevisiae [5]. However, this tRNA/synthetase pair can not be used in E. coli due to orthogonality. Nevertheless, the shape of the binding pocket which is adapted to this specific non-canonical serine can be used as template to create an orthogonal tRNA/synthetase pair for the use in E. coli based on computational modeling, a synthetase for DMNBS in E. coli is designed, mainly because of three reasons:

1. Serine is a crucial amino acid for enzyme catalysis and therefore an obvious target for pathway analysis and control of protein activity via photocaging [5]
2. E. coli is the most used organism in the field of biotechnology.
3. DMNBS has been proven to work very well as a non-canonical amino acid in yeast and is superior to oNBY regarding the physicochemical properties, e.g. a higher quantum yield for photocleavage and an absorption wavelength apart from the UV spectrum. [5]

For that purpose, the well described tyrosyl-tRNA/synthetase pair of Methanococcus janaschii is mutated to selectively incorporate DMNBS into proteins in E. coli.

The cognate tRNA's anticodon contains an amber stop anticodon. Hence, it is possible to incorporate an amino acid at a chosen position in a protein via amber codon suppression.

Design of DMNBS-tRNA/synthetase pair

As the basis for designing a DMNBS specific synthetase the wild type tyrosyl-synthetase of M. janaschii (Mj-Tyr-RS) is chosen due to its well characterized properties, its resemblance to the E. coli-leucyl-synthetase (Ec-Leu-RS) derived DMNBS-synthetase used in yeast and its availability through the iGEM-Team Austin, Texas, 2014.

It was also previously shown, that this pair is orthogonal to endogenous E. coli synthetases [16].

The determination of the alterations to be made for changing the amino acid specificity are based on the comparison of the crystal structures of Mj-tyrosyl- and Ec-Leu-tRNA/synthetase pairs. Highly conserved amino acids Y³², L⁶⁵ and D¹⁵⁸ are known to build H-bonds with the natural ligand tyrosine [17]. Furthermore, these sites resemble the mutated sites within E. coli-leucyl-synthetase which was already changed to a DMNBS-specificity and is successfully used in yeast to incorporate DMNBS [16,18]. Some more positions are chosen to be mutated due to their location within the 2Å-radius of bound DMNBS.

For creating a mutant library, sites to mutate were determined and partially randomized codons for site saturated mutagenesis, also with respect to the codon usage of E. coli, were chosen.

Characterization of Synthetase Plasmids

A low copy number plasmid (p15A, 10-12 copies per cell) is used to express the DMNBS-synthetase variants and Methanococcus janaschiiwild type tyrosyl-tRNA in order to test incorporation values of DMNBS via amber codon suppression. Expression is used to be constitutive. A gentamicin resistance reporter gene is used for selection.


Characterization of pRXG (Part:BBa_K1416004 called pFRY)

Once the screening method for measuring the fidelity and incorporation efficiency of the created tRNA/synthetase pairs, a fluorescence-based method as established by the the iGEM-Team Austin, Texas, 2014 is used: a two-plasmid system - one plasmid (pRXG) contains an IPTG-inducible RFP-sfGFP reporter protein construct and the additional plasmid the tRNA/synthetase pair.
The pRXG plasmid consists of a RFP domain which is connected by a linker sequence containing an amber stop codon with a sfGFP domain. The expression of the plasmid results either in red fluorescence, or - if the ncAA is incorporated at the amber stop codon within the linker site - in both: red and green fluorescence.
By comparison of fluorescence levels it is possible to determine incorporation efficiency of the generated synthetase variants.
A kanamycin resistance reporter gene is used for selection of the reporter plasmid. Furthermore, the expression of the fluorescence proteins is kept under lac-operon control for IPTG induction.
The reporter plasmid, as described above, has been created by iGEM Team Austin, Texas, 2014. A BioBrick has been built from it and characterized for further use: by iGEM-Team Austin, Texas, 2014

Screening
For both, positive and negative screening, M9 minimal medium is used, because it has a lower optical density itself and contains buffer to stabilize the fluorescence proteins.
As a pretest, the excitation and emission wavelengths of the fluorescence proteins are measured, along with the OD₆₀₀. Thus, the wavelengths for the screening process are identified.

To transform the mutation library into E. coli, the following strategy is applied: The reporter plasmid is transformed into E.coli BL21 DE3 gold from which subsequently chemical competent cells are made. The library then is transformed into those cells and onto M9 minimal solid medium with gentamycin and kanamycin for selection.
A preculture is inoculated each well with one colony in 96-well microtiter plates in M9 minimal medium. Microtiter plates are sealed with microporous tape sheets, because fluorescence proteins mature under the influence of oxygen.

As an intermediate step the precultures are replicated onto a next microtiter plate for the purpose of homogenizing the cell density.

Thereof, two separate screening microtiter plates are inoculated: Firstly, a positive screening plate with M9 minimal medium, addition of 2mM DMNBS as well as 100µM IPTG for expression. Secondly a negative screening plate without DMNBS. All microtiter plates are measured after the same amount of time. Incubation was performed at 900rpm, 30°C and pH 7.6.

OD₆₀₀ and both, red and green fluorescence levels are measured as an endpoint measurement. Thus, the most efficient clones can be determined to selectively incorporate DMNBS at an amber stop codon.

To obtain final results, the new-found, working DMNBS tRNA/synthetase pair clones undergo an online measurement rescreening with each nine replicates and the previously mentioned growth and screening conditions.

Results can be found here

Characterization of Existing Parts

Characterization of pYES2 Galactose Inducible Vector (Part:BBa_K555009)

To get an overview of the growth of S. cerevisiae CENPK2-1D cells, we compared the growth of the yeast cells carrying the empty vector in the induced and the non-induced conditions. We used the growth profiler GP960 (EnzyScreen, Heemstede, The Netherlands), which is a temperature controlled shaker for microtiterplates with a scanner for growth detection.

The logarithmic growth phase was determined to be from 4 to 7 hours after induction. Comparing the induced and non-induced cultures, it can be seen that they grow much better with additional galactose, since S. cerevisiae can metabolize it as a carbon source. In order to improve the significance of these measurements, addition of glucose to the non-induced cells can be an outlook for further work. Furthermore, the cells carrying the MFalpha+mCherry inserts (Part:BBa_K2020026 and Part:BBa_E2060) are not affected in their growth after induction as the blue and yellow curves show.

Characterization of pRXG (Part:BBa_K1416004 called pFRY)

How the Reporter Plasmid works
This reporter plasmid is one of the two plasmids containing screening system for determining efficiency and fidelity of non-canonical amino acids´ (ncAA) incorporation via amber termination suppression. One plasmid contains tRNA and its corresponding aminoacylation-synthetase. The other one is this plasmid presented herein. It consists of a mRFP1 domain, which is connected through a linker sequence containing a recoded amber stop codon with a sfGFP domain. The expression of the plasmid gives either a red fluorescence, or - if the ncAA is incorporated at the recoded amber stop codon within the linker site - both a red and a green fluorescence.
Synthetase and tRNA are constitutively expressed in a plasmid with low copy replicon p15A under metabolic stress, but are not under IPTG control for the purpose of avoiding abrupt and unpredictable effects considering the time and energy required for their assembly. Whereas the reporter plasmid containing two fluorescence proteins, is kept under operon control for IPTG induction like a low copy plasmid with ColE1.
Cultivation Conditions with High Throughput Measurement
In order to evolve a new aminoacylation synthetase for DMNBS in E.coli and transforming a mutant library into competent cells by using the following order of events to get a maximum output and equal optical densities:

1. Transform into (BL21 DE3 gold + pRXG) on M9 solid, growth: 2 days, 37°C
2. Pick into M9 liquid: Masterplate, growth: 2 days at 30°C, 900rpm, shaking diameter: 50 mm
3. Replicate into M9 liquid, growth 2 days at 30°C, 900 rpm, shaking diameter: 50 mm
4. Replicate into M9 liquid Screening plate, growth 2 days at 30°C, 900 rpm, shaking diameter: 50 mm

It was shown previously, that sufficient GFP expression was achieved by supplementing IPTG and ncAA, but causes a decelerated growth (1). In fact, cultivation of BL21 DE3 gold containing two different plasmids show reduced growth rates, when adding 100µM IPTG and 2 mM DMNBS to M9 minimal medium results in a growth phase of 42-48 h at 30°C to reach maximum cell density.

Host Organism
This reporter plasmid and the measurement of its corresponding proteins expressed are previously used both in an amberless E.coli strain and BL21 DE3 gold. The latter results in competition of the suppressor tRNA with RF1 at the amber stop codon.

Measurement: Wavelength
As a pre-screening setup, an endpoint detection of OD and fluorescence intensities with microtiter-plate reader is chosen. Excitation and emission spectra of mRFP1 and sfGFP were obtained from a previously conducted measurement (fig 8). For screening with Tecan Plate Reader, the following settings were used:

1. OD: 600 nm
2. sfGFP 480/508 nm
3. mRFP1 584/605 nm
4. Slid: 8 nm

While rescreening for the detection of endpoint, the same plate reader setup was used. Additionally an online measurement monitoring sfGFP and mRFP1 formation as well as scattered light measurement at 650 nm, is used. This is achieved by a screening platform based on the established Biolector setup. [19]

Measurement: Evaluation
If the levels of optical density of the synthetase to be evaluated and a working synthetase are equal, a first approximation of efficiency and fidelity can be made by normalizing GFP levels. Thus one can eliminate the biogenic background fluorescence levels and compare the clones with each other. This mutant corresponds to No.: 1

With the reporter plasmid evaluated synthetases

1. Y-RS, canonical amino acid
2. oNBY-RS
3. AzF-synthetase
4. CN-F synthetase
5. Iodo-Y synthetase
6. Nitro-Y synthetase
7. OMe-Y-RS
8. 5HT-P synthetase

DMNBS mutants:

1. DMNBS-RS Clone 1
2. DMNBS-RS Clone 2
3. DMNBS-RS Clone 3
4. DMNBS-RS Clone 4
5. DMNBS-RS Clone 5
6. DMNBS-RS Clone 6
7. DMNBS-RS Clone 7
8. DMNBS-RS Clone 8
9. DMNBS-RS Clone 9
10. DMNBS-RS Clone 10

Links

1. Fluorescence reporter for measurement of incorporation of ncAA Whereas you find hereunder the whole plasmid as a biobrick, we provide the two flourescent proteins connected by the linker sequence as a biobrick for self-assembly in a low copy plasmid.
2. DMNBS-RS Clone 10 - Team Austin, Texas 2014 built this measurement kit and probed various synthetases.
3. Team Aachen 2016 evolved a new synthetase for incorporation of DMNBS in E. coli.

Attributions
iGEM Team Austin, Texas: Thanks for making the measurement kit available to us.

One of the things which we need to consider when we choose to work with light-sensitive materials is an apt environment. Light sensitive materials on exposure to stray light experience changes in properties on exposure to stray light what makes them problematic to work with under normal conditions. It requires a dedicated room where the lighting is controlled, usually by using filtered red or amber safelight. But due to economic, time and spatial limitations, not every school, university and institute can have this dark room. Hence, we have developed an affordable, convenient and portable workspace to handle these light-sensitive materials, called “Dark-bench”. It can be assembled and disassembled at any place with little effort.
After resolving a safe place to work with, we required a device with which we can study the photo cleavage reaction by activating the caged amino acid by irradiating at a specific wavelength. Since the real-world application of our project is to activate the caged subtilisin in the liquid detergent, the uncaging device, the so called LIPS stick, should be cheap and easy-to-use. That is made possible by incorporating pumps and a control circuit for optimal light exposure by adjusting various parameters automatically. With further miniaturization it can also be integrated into washing machines in the future.
Both of the devices were developed not only to satisfy our project needs but also were made with affordable and readily available components so as to make it accessible to all. More information about the hardware aspect of our project can be found here.

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