Team:TU Kaiserslautern/Design

Design

Introduction
The first hurdle we encountered at the beginning of this year’s team was the selection of a project. We looked at the project of last year’s iGEM-Team of Kaiserslautern, and at various ongoing concerns in the Environment Track. Because of recent events and the deteriorating state of our planet, our goal was to design a project that can help the environment. We noticed that there are basically no measures to lessen the micropollution in smaller municipal wastewater treatment plants. Only in big treatment centers are there very expensive methods like for example ozoning. We focused on deactivating the micropollutants in wastewater by implementing synthetic biology. With our enzymes we can make these micropollutants less toxic to microorganisms and thus less dangerous to our dear planet.

Many of these micropollutants are not covered by legal restrictions, and interactions between compounds are not taken into consideration. One of the most problematic micropollutants in Germany is Diclofenac, a common anti-inflammatory drug in use since the 1960’s which has been linked to the near extinction of Indian vultures. But this is not only a problem in Germany. All over the world there are micropollutants that are not sufficiently broken down and accumulate in the environment, e.g. in Europe Paracetamol and Aspirin, the latter also in North America and Ofloxazin in Asia.1

Laccases are multicopper-oxidases that oxidize substrates like polyphenols, methoxylated phenols, aromatic amines, and inorganic materials. It results in a direct one-electron substrate oxidation, an electron transported to other copper domains where up to four electrons can be stored, and finally a four-electron reduction of O2 to H2O. This allows it to inactivate substrates like Diclofenac.

Chassi Chlamydomonas reinhardtii
Since the previous Kaiserslautern iGEM team also worked with C. reinhardtii and we read many papers utilizing this organism, we wanted to follow the footsteps of their project. It is highly studied optional phototroph, making it an unbelievably valuable and versatile research organism, with a collection of more than 300 plasmids and over 3000 strains. Laccase, our enzyme, originates from two different organisms; from Botrytis aclada, an anamorphic fungus which is a parasite on agricultural and forest trees, and from an uncultured marine bacterium.2 Since posttranslational modifications (PTMs) only occur in eukaryotes, C. reinhardtii was the perfect choice to produce the laccases. Also, the green algae are already an established model organism in our work group, the group of Michael Schroda.



Fig. 1: Green algae mutant under light microscope


Chassi Escherichia coli
We´re using E. coli as an attempt to see if our proteins, the Laccases, work if they´re produced by a prokaryotic organism. It's been used in previous studies and is a common and easily replicated organism. Also, E. coli is an established organism in the work group of Nicole Frankenberg-Dinkel, which means that we received a great deal of assistance in any troubleshooting that was needed.

Modular Cloning (MoClo)
The MoClo system is based on the Golden Gate cloning system but works with type IIS restriction enzymes. These enzymes cut DNA outside their recognition side. This allows for the design of defined overhangs. During the ligation process, the recognition site gets excised, which results in a scarless construct. The MoClo system is a standardized system for C. reinhardtii, so the exchange between different labs is very easy. The MoClo system is established in our research group, therefore we have access to a lot of expertise and support. The greatest advantages of the MoClo system are the possibility to design different parts efficiently while being able to swap gene parts easily. With the help of this fast ligation system, we able to obtain our working part collection. One thing that also must be mentioned is the efficiency of the MoClo system. It allows a fast and highly efficient working process which results in a faster workflow in comparison to standard cloning methods.3 (Similar to last year’s team)



Fig. 2: The MoClo System3


Our Parts
E. coli

For E. coli we only used two parts. We cloned the genes for our laccases (BaLac and marLac) into the cloning vector pGEX-6P-1. On this vector, there are already a resistance gene for Ampicillin with its own promoter for selection, an ori, a lac promoter, the lacZ gene, and a gst-tag gene with a tac promoter, with a multiple cloning site after. The promoter combines the strong sigma-factor binding site from trp promotor with the controllability with IPTG from lac promotor. The GST-tag is used to isolate the protein.



Fig. 3: pGEX-6P-1 Vector with the gene for the protein BaLac (a) and marLac (b)


Chlamydomonas reinhardtii

During the course of the competition we designed 14 Level 1 constructs and 14 Level 2 constructs. For our two enzymes, the marine laccase, and the mutated laccase from Botrytis aclada, the pAR promoter and the RPL23 terminator were used in all constructs.  The pAR promotor is a fusion promotor which has shown to be efficient for expression in Chlamydomonas reinhardtii. For the antibiotic cassette the PSAD promotor was used in combination with the PSAD terminator. 

 For selection, the Spectinomycin resistance gene aadA was added. To detect our protein, we used a HA-tag which allowed us to detect via immunoblot with the primary antibody (anti-HA, mouse) and a secondary antibody (anti-mouse, rabbit) which has a conjugated horseradish peroxidase. This allows detection with chemiluminescence. Furthermore, the HA-tag was combined with a 8His-tag which can be used for protein purification. 
The first construct we designed without any secretion signals.

Figure 4: shows the first Level 2 constructs without secretions signals and only a 3HA-tag part numbers BBa_K3589207 (BaLac) and BBa_3589208 (marLac)


These constructs were tested first to verify the cytosolic expression. After we could detect our protein via Western Blot, the cCA secretion signal was added. 



Figure 5: shows part numbers BBa-K3589209 (BaLac-sp20cca), BBa_K3589210 (marLac-sp20cca), BBa_K3589211 (BaLac-cca), BBa_K3589212 (marLac-cca). Level 2 constructs consisting of the resistance marker, the coding sequence, the cCA secretion signal and a (SP20)-HA-RGS-8His-tag


The results showed that neither of the two laccases is secreted. New constructs were made, but the growth of organisms with this construct was too slow as to properly screen them.

Diclofenac as an Example of Micropollutants
Diclofenac is a member of the acidic antipyretic analgesics group (pain agents). Like all representatives of this class, Diclofenac inhibits cyclooxygenases (COX) and thus reduces prostaglandin synthesis, which influences peripheral pain formation.4 Because it is an active ingredient in pain-relieving ointment, Diclofenac is highly consumed among the German population, having been amongst the most popularly sold drugs in Germany for many years.5



Fig. 6: Chemical structure of Diclofenac


In addition, Diclofenac has an antiphlogistic antirheumatic effect and is counted among the non-selective non-steroidal anti-inflammatory drugs (non-selective NSAID) due to its inhibitory effect on both COX isoenzymes (COX-1 and COX-2). In Germany, Diclofenac is the most common administered active ingredient used to treat rheumatic arthritis.4

The wide application, primarily within Germany, generates a consumption of about 90 tons per year there. This high utilization has costs however, as only about 30 % of the active substance is metabolized by the human body, the rest being excreted unchanged through urine. This leads to an estimated 63 tons of Diclofenac per year that enter the Germany water systems.6

Due to its high stability and water solubility, Diclofenac is poorly filtered out in conventional wastewater treatment plants. Although Diclofenac does not pose an acute danger to humans in the concentrations for which it is found in German waters, it is highly toxic to aquatic organisms. More modern sewage treatment plants are trying to solve this problem by additional purification (e.g. nanofiltration, ozone or activated carbon). In addition to the high costs, the lack of experimental data on emerging products in the application of non-specific degradation methods (e.g. ozone) make these far from ideal. Recent work, including our project, have set themselves the objective of investigating the use of enzymes as catalysts for the degradation of micropollutants such as Diclofenac.7,8

We hope to establish a self-sustaining, enzyme-based system, which is characterized by high enzymatic production, low maintenance, and low costs. In addition, the use of enzymes and the specificity associated with them should make it predictable which reaction products are produced during the degradation of Diclofenac, so that a disruption of environmental homeostasis by possibly toxic products can be avoided.

Laccase Reaction
Laccases (p-benzenediol: oxygen oxidoreductase EC 1.10.3.2) are enzymes which catalyze the oxidation of a wide variety of aromatic and non-aromatic molecules.9 They contain multiple copper atoms in their active site to store and transport electrons.10

The laccases have three copper types which have multiple differing arrangements. The mononuclear copper-binding site found in Type 1 copper is located near the protein´s surface. Here the substrate one-electron oxidation takes place. In addition, a trinuclear copper-binding site (TNC) which is built by one T2 copper and two T3 copper atoms are located here. The TNC can bind oxygen in the fully reduced state of the protein and is responsible for reducing O2 to H2O. Therefore, it takes the electrons from the substrate oxidation after a cycle of four of the previously detailed reactions.9,10

The reaction cycle consists of 4 single-electron transfers from a reducing substrate to the copper atoms in the active site. This forms a radical cation from the substrate (Fig. 6). In its reduced form, the laccase is then able to reduce molecular oxygen into two-electron transfer reactions to form water.9,10



Fig. 7: Overview for the direct reaction mediated by a laccase. One electron is transferred from the substrate to the copper cluster, turning the substrate into a radical cation.9 Some toxic substances and pharmaceuticals are shown as example substrates.11,12 After four electrons have been transferred to the enzymes copper cluster, it binds molecular oxygen. Two consecutive two-electron reductions then form two water molecules, putting the enzyme back into its oxidized form.9,10


Diclofenac Oxidation



Fig. 8: Oxidation of diclofenac by laccase.


It is believed, that the radical cation (b) of Diclofenac (a), which is generated by a laccase-mediated oxidation reaction, reacts with water to form the para-hydroxy substituted intermediate (c). This can undergo further oxidation to form the para-benzoquinone imine derivate (d). Formation of 4‘-Hydroxydiclofenac, where the hydroxylation takes place in para-position to the nitrogen atom on the chlorinated benzene ring has also been described.

Our Plan
Laccases (p-benzenediol: oxygen oxidoreductase EC 1.10.3.2) are enzymes which catalyze the oxidation of a wide variety of aromatic and non-aromatic molecules.9 In there active site they contain multiple copper atoms.10



Fig. 9: Our Workflow.


Our project involves the modification of the green algae Chlamydomonas reinhardtii, enabling the chemical modification of diclofenac which results in its functional degradation. We will integrate genes for the enzyme laccase into the genome of our green algae, as well as perform a control by cloning the genes into the bacterium Escherichia coli. This will facilitate the production of these enzymes and secretion into medium (the wastewater from wastewater treatment plants), where they can then break down diclofenac. We split into 3 teams to best implement our experimental process, one team focused on producing proteins with our model organism called C. reinhatdii Team, one focused on doing the same with our control organism called E. Coli Team, and our Assay Team who performed the confirmation activity assays with both previous teams’ produced laccases.

C. reinhardtii Screening

In the course of our project we used and developed various screening methods for intra- and extracellular proteins. For the intracellular proteins we first used glass-beads and sonication as lysis methods. With these methods we were able to detect both enzymes marLac and BaLac via Western Blot in the lysates. The next step was to establish activity. Here we ran into the problem that the lysates from these two methods were of a very dark green colour due to the high amount of chlorophyll. Therefore, it was impossible to detect any activity using the standard ABTS-assay. Thus we switched to the more gentle freeze and thaw method.

After we demonstrated that C. reinhardtii does in fact produce both proteins, we made new constructs containing the secretion signal cCA. The previous iGEM-Team, who also worked with C. reinhardtii, demonstrated that the secretion signal cCA is best suitable for that organism. Moreover, we included one of two different C-terminal tags: SP20-HA-RGS-8His and HA-RGS-8His. We screened all constructs using the screening protocol from last year’s TUK’s iGEM-Team which involves a lyophilization of the samples. In the meantime, the Schroda research group made improvements to that protocol which we also tried. We screened 50 random colonies for each construct via SDS-PAGE and Western Blot. A laccase-producing colony was not detected using this method, so made new constructs. Because of the pandemic we could not screen them.

E. coli

The vector was obtained through in vitro synthesis and purchased from BioCat GmbH. First, two vectors which include the gene for one of the laccases (BaLac or marLac), a tac promoter and a gene for ampicillin resistance were transformed into E.coli. The transformed E.coli was grown on LB-plates with ampicillin to select for the colonies that took up the vector. One colony was chosen and produced in a fluid culture with which proteins were expressed. The production was induced with IPTG. After expression, proteins were purified with GST-Sepharose columns. The concentrated protein was then ready to be tested with ABTS or HPLC. As a visible proof that laccases were produced, SDS-PAGE and Western Blot screening was performed.
To visualize the enzymatic activity, we ran ABTS assays with produced lacasses. To complete the assay, we also did several positive controls with T. versicolor laccase, and negative controls were composed identically without enzyme. The assay ran 4 hours at 30°C. The wells were measured every five minutes.

 Assay Team

Because ABTS interacts with laccase in a similar way to the laccase-diclofenac interaction (Fig.8), a series of ABTS assays were performed to determine reaction strength. To begin, the optimal wavelength for ABTS was confirmed via spectrometer. An assay was then performed to visualize the positive control laccase from Trametes versicolor at the two optimal pH’s documented for BaLac (pH 4) and marLac (pH 7), as well as its own optimal pH 5. In a 96 well plate, varying concentrations of the positive control laccase at different pH levels were added to the substrate. Once produced, we ran the same assay with the manufactured BaLac and marLac laccases (both intracellular and secreted, depending what was produced) at their optimal pH and compared it against the positive control to confirm activity.
HPLC was run first with Diclofenac to verify standard retention time, then a positive control reaction with T. versicolor to determine the products to compare them against the produced enzymes. Finally, the manufactured laccase products were analyzed.

Integrated human practices
To achieve a broader spectrum of the field, we collaborated with iGEM Teams from Stuttgart and Darmstadt. We also did a lot of expert interviews with a variety of subject matter professionals, which you can find in our integrated human practices tab.

Outlook: The Bioreactor
To make sure the genetically modified organisms will not contaminate the environment, we want to restrict them to a bioreactor. There they can grow under optimal conditions and can be directed into the wastewater via a filtration system, so that only the proteins reach it. The GMOs cannot pass into the wastewater system this way, allowing laccase to be released into the wastewater from the bioreactor. The produced lacasses are influenced by e.g. the pH conditions, temperature and salt concentrations. Read more about it on our implementation page
References
(1) Stephen R. Hughes, Paul Kay, and Lee E. Brown Global Synthesis and Critical Evaluation of Pharmaceutical Data Sets Collected from River Systems Environmental Science & Technology 2013 47 (2), 661-677 DOI: 10.1021/es3030148

(2) Scheiblbrandner, S., Breslmayr, E., Csarman, F., Paukner, R., Führer, J., Herzog, P. L., Shleev, S. V., Osipov, E. M., Tikhonova, T. V., Popov, V. O., Haltrich, D., Ludwig, R., & Kittl, R. (2017). Evolving stability and pH-dependent activity of the high redox potential Botrytis aclada laccase for enzymatic fuel cells. Scientific Reports, 7(1), 13688. https://doi.org/10.1038/s41598-017-13734-0

(3) Weber, E.; Engler, C.; Gruetzner, R.; Werner, S.; Marillonnet, S. A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLoS One 2011, 6 (2). https://doi.org/10.1371/journal.pone.0016765.

(4) Aktories, K., Förstermann, U., Hofmann, F., & Starke, K. (2005). Allgemeine und spezielle Pharmakologie und Toxikologie (9. Auflage). Urban & Fischer Verlag.

(5) Glaeske, G. (2017). Medikamente 2015 – Psychotrope und andere Arzneimittel mit Missbrauchs- und Abhängigkeitspotential. Jahrbuch Sucht.

(6) Meißner, M. (2008b). Arzneimittel in der Umwelt: Natur als Medikamentendeponie. Dtsch Arztebl International, 105(24), A-1324.

(7) Bilal, M., Adeel, M., Rasheed, T., Zhao, Y., & Iqbal, H. M. N. (2019). Emerging contaminants of high concern and their enzyme-assisted biodegradation – A review. Environment International, 124, 336–353. https://doi.org/10.1016/j.envint.2019.01.011

(8) Meißner, M. (2008a). Arzneimittel in der Umwelt: Natur als Medikamentendeponie. Dtsch Arztebl International, 105(24), A-1324.

(9) Agrawal, K., Chaturvedi, V., & Verma, P. (2018). Fungal laccase discovered but yet undiscovered. Bioresources and Bioprocessing, 5(1), 4. https://doi.org/10.1186/s40643-018-0190-z

(10) Zerva, A., Simić, S., Topakas, E., & Nikodinovic-Runic, J. (2019). Applications of Microbial Laccases: Patent Review of the Past Decade (2009–2019). Catalysts, 9(12), 1023. https://doi.org/10.3390/catal9121023

(11) Hahn, V. Enhanced Laccase-Mediated Transformation of Diclofenac and Flufenamic Acid in the Presence of Bisphenol A and Testing of an Enzymatic Membrane Reactor. 2018, 11.

(12) Kittl, R.; Mueangtoom, K.; Gonaus, C.; Khazaneh, S. T.; Sygmund, C.; Haltrich, D.; Ludwig, R. A Chloride Tolerant Laccase from the Plant Pathogen Ascomycete Botrytis Aclada Expressed at High Levels in Pichia Pastoris. Journal of Biotechnology 2012, 157 (2), 304–314. https://doi.org/10.1016/j.jbiotec.2011.11.021.