Team:TU Kaiserslautern/Results

Results

After the pandemic became a large scale concern, we were concerned we wouldn't have access to the lab this year. Fortunately, through good safety practices and our supervisors who moved mountains to give us the chance at a real iGEM experience, we worked dedicatedly from May until the last weeks in October. Below you will find the results of our teams' hard work.

Results for C. reinhardtii

Parts and Project Design

For iGEM 2020, we chose to work with two organisms, Escherichia coli and Chlamydomonas reinhardtii. We were able to build upon the work and expertise of the 2019 iGEM team from TU Kaiserslautern, which genetically engineered the C. reinhardtii via the Modular Cloning (MoClo) technology^1,2 for its use in bioremediation. MoClo is based on Golden Gate assembly and follows a well-defined syntax that allows quick assembly of genetic elements for Synthetic Biology.¹ Several genetic elements could be reused from their Kaiser Collection but we also introduced new MoClo genetic parts. This includes 6 level 0, 6 level 1 and 6 level 2 constructs. Figure 1 shows all level 2 constructs that we generated.

Fig. 1: All the Level 2 MoClo constructs we have designed for C. reinhardtii. Transformants from the constructs on the left were screened for laccase-expression and activity. Constructs on the right were transformed in C. Reinhardtii but have not yet been screened.

We worked with the C. reinhardtii strains UVM4³ and its sister strain in the CC-4533 background, referred to as ‘CLiP’.

To achieve oxidation of micropollutants, not just under laboratory conditions but also under the rather harsh conditions of a wastewater treatment plant, we chose to work with two different laccases which have only recently been described. The first one, marLac, was first cloned from a marine metagenomic library by Yang et al. in 2018. It is cold adapted, thermostable and shows a high tolerance for organic solvents and salt, but has allow redox potential.⁴ The second one is called BaLac. It is a laccase from the ascomycete Botrytis aclada with the point mutation L499F which was first described by Schreiblbrandner et al. in 2017. This laccase has a high redox potential and shows residual activity at pH 6.5.⁵

The properties of these two laccases fit the requirements for usage in a wastewater treatment plant very well and therefore show a good potential for bioremediation. We designed different MoClo constructs with and without secretion signals which encode the enzymes marLac and BaLac. While our final goal was to have the proteins secreted, we designed the constructs without the secretion signals to make sure that the alga can produce our laccases.

Cytosolic Expression

Proof of Expression

We first designed constructs without any secretion signal and transformed them into C. reinhardtii to make sure that the green alga is capable of producing both enzymes. We chose the strain UVM4³ as a recipient.

Fig. 2: Proof for the expression of BaLac in C. reinhardtii.

a) Level 2 MoClo construct containing the coding sequence for BaLac and a 3xHA-tag for detection as well as the pAR-promotor and the tRPL23-terminator. Spec^R is a spectinomycin resistance cassette built from the level 0 parts P_PSAD, aadA and PSAD_ter introduced by team TU Kaiserslautern 2019 as part of the Kaiser Collection.
b) Immunoblot of 12 randomly picked spectinomycin-resistant colonies transformed with construct a). They were inoculated in TAP medium under mixotrophic conditions. Total protein samples were analyzed via SDS page and immunoblotting using anti-HA antibody. 2 µg of chlorophyll were loaded onto the gel. Expression of BaLac (~ 70kDa) is visible in transformants 5, 9 and 11. The recipient strain served as a negative control, a 3xHA-tagged protein as a positive control.

Fig. 3: Proof for the expression of marLac in C. reinhardtii.

a) Level 2 MoClo construct containing the coding sequence for marLac and a 3xHA-tag for detection as well as the pAR-promotor and the tRPL23-terminator. Spec^R is a spectinomycin resistance cassette built from the level 0 parts P_PSAD, aadA and PSAD_ter introduced by team TU Kaiserslautern 2019 as part of the Kaiser collection.
b) Immunoblot of 12 randomly picked spectinomycin-resistant colonies transformed with construct a). They were inoculated in TAP medium. Total protein samples were analyzed via SDS page and immunoblotting using an anti-HA antibody. 2 µg of chlorophyll were loaded onto the gel. Expression of marLac (~ 60kDa) is visible in transformant 12. The recipient strain served as a negative control, a HA-tagged psaD as a positive control. MW: molecular weight.

Both enzymes were expressed and accumulated in the cytosol when fused to a 3xHA-tag, proving that the green alga is in fact capable of producing them. Both proteins showed a higher molecular mass than their theoretical calculated value (62 kDa for BaLac and 45 kDa for marLac). On the immunoblot, BaLac’s signal appears at around 70 kDa and marLac at around 60 kDa. These shifts are due to the 3xHA-tag.

Measuring Activity of the Intracellular Laccases

To measure the activity of the expressed cytosolic laccases, we performed an ABTS-assay. The assay is based on the change in color which appears when ABTS is oxidized to its corresponding radical cation (more details on this reaction can be found below). We performed this assay in a 96-well plate. A commercially available laccase from the fungus Trametes Versicolor served as a positive control, supernatant of the lysate from the recipient strain as a negative control. First, we tried to lyse the cells by vortexing them with glass beads. The supernatant was then used for the assay.

Fig. 4: Activity assay for the cytosolically expressed enzymes BaLac and marLac.

a) Level 2 constructs of marLac and BaLac as described in Fig. 1. b) ABTS activity assay performed in a 96-well plate. Absorption was measured at 415 nm. The error bars represent the standard deviation of four independent replicates for each sample. The concentration in the parentheses represents the final concentration of the whole protein in the 96-well plate. For the positive control, 75 µg of commercially available Trametes Versicolor laccase was added to the 375 µg/ml of whole cell protein in 200 µl of lysate from the recipient strain. Only one sample was measured for the negative control. Cells were lysed using glass beads. The assay was performed at pH 7. Normalization of the data was done by dividing each absorption value by the initially measured value for that replicate.

The assay allowed to monitor the activity of the commercially available laccase, as documented by the blue line in Figure 4 which indicates that the assay worked. Unfortunately, no activity could be detected for the recombinant proteins BaLac and marLac when they are localized in the cytosol. We therefore tried different lysis methods, i.e. sonication and freeze and thaw to test if it impacted the amount of protein in the lysate or its activity. With the sonication method, we were able to load up to 500 µg of whole protein into the assay. At this concentration, chlorophyll hindered the measurement because of its own absorption at 415 nm, rendering the method useless. We therefore tried the freeze and thaw method to decrease the amount of chlorophyll in the lysate. The method was successful at reducing chlorophyll levels, giving us the ability to load 1,365 µg of whole protein into the assay. Unfortunately, we were not able to detect any activity with this method, either.



From these results, we concluded that either the concentration of the expressed laccases was too low or that the enzymes are not active when expressed in the cytosol. The immunoblots in Fig. 2 and Fig. 3 indicate that the expression levels of both enzymes, especially that of marLac, are very low when compared to the positive control. It is also possible that, when the laccase is expressed in the cytosol, it is inactive because of the lack of essential post-translational modifications, which usually occur during the secretion process, i.e. glycosylation. Laccases are usually heavily glycosylated. They typically contain 10 – 45 % carbohydrates, depending on their origin.⁶ The carbohydrates are believed to play a key role in the stability of the laccases and protect them from proteolysis and inactivation by radicals.⁶ Since both laccases are not produced by C. reinhardtii natively, it is also possible that chaperones required for the insertion of the copper ions are missing. To check the validity of one of these explanations, the assays could be repeated with concentrated enzymes, e.g., via affinity chromatography. When the results are still negative with a much higher laccase concentration, one can assume that the enzymes are in fact not active when expressed in Chlamydomonas.

Protein Secretion

The Screening Process

For these experiments, we switched to the CC-4533 strain. TU Kaiserslautern’s 2019 iGEM team was able to produce good results with that strain for secreted proteins. It still has parts of its cell wall and is therefore more robust and better suited for its usage in a photobioreactor than the UVM4 strain.

First, we designed four different constructs: two for each laccase. Each construct contained a N-terminal secretion signal from the enzyme carbonic anhydrase (cCA) and an HA-8xHis tag for purification. The constructs pAR-BaLac/marLac-SP20-HA-RGS-8His additionally harbor a module consisting of a repeat of 20 serine and proline residues (SP20). This module was reported to strongly increase the secretion of proteins.⁷ TU Kaiserslautern’s 2019 iGEM team was able to increase the secretion of their proteins significantly when using this module. The other construct, pAR-BaLac/marLac-HA-RGS-8His, was designed to verify the results of TU Kaiserslautern’s 2019 iGEM team regarding the positive effect of the SP20 portion on secretion efficiency.

Fig. 5: Both laccases cannot be detected in the supernatant when fused to an N-terminal cCA secretion signal and a C-terminal (SP20)-HA-RGS-8His-tag.

a), b), e) and f) shows the constructs consisting of a spectinomycin-resistance cassette (Spec^R), the pAR-promotor, the coding sequence for the respective laccase, and the tRPL23 terminator. c), d), g) and h) shows secreted proteins precipitated from the medium of twelve independent transformants generated with each of the four constructs. The respective constructs are shown above each immunoblot. 6 mL of supernatant were harvested, lyophilized and resuspended in SDS sample buffer. 15 µL of sample were loaded onto the gel and analyzed via immunoblotting using an HA-antibody. Secreted proteins precipitated from medium of the recipient CC-4533 strain served as a negative control, that of a transformant expressing cCA and SP20-HA-8His-tagged brazil nut 2S albumin as a positive control. Controls were treated like the samples.

Fig 5 shows that neither BaLac nor marLac could be detected in the supernatant. We therefore lysed the cell pellets of the transformants to see if the proteins are produced but not secreted or if the cells do not produce the proteins at all.

Fig. 6: Neither laccase could be detected in the whole cell body when fused to an N-terminal cCA secretion signal and a C-terminal (SP20)-HA-RGS-8His-tag.

a) and b) show the constructs as described in Figure 1. c) and d) proteins of the lysed cell pellets were analyzed by immunoblotting. Cell cultures were lysed by boiling them with SDS sample buffer. For the samples and controls, proteins corresponding to 2 µg of chlorophyll were loaded onto the gel and analyzed via immunoblotting using an HA-antibody. In panel c), lysate from a culture expressing BaLac served as a positive control. Note that no dedicated signal for the positive control could be detected, likely due to a weak signal. Since we observed a signal derived from a cross-reaction of the antibody with a protein of around 30 kDa in all samples including the negative control, we can conclude that there was no problem with the detection itself. For blot d) we switched the positive control to a transformant expressing cCA and SP20-HA-8His-tagged brazil nut 2S albumin (see Fig. xyy). The cross-reaction that can be seen in picture c) can also be seen in picture d) but with a weaker signal because the detection time was shorter. The recipient CC-4533 strain served as a negative control in both cases.

In the course of the project we screened in total the supernatants of around 200 randomly picked spectinomycin-resistant colonies transformed with the four different constructs for BaLac or maLac secretion. Neither of the two laccases could be detected in any case.
Since we were able show that both BaLac and marLac are expressed in the cytosol, we have three possible explanations for our inability to detect them in the supernatant: (i) the expression levels of the enzymes are so low that they cannot be detected via immunoblotting. (ii) the N-terminal cCA-tag does not efficiently drive secretion of the enzymes. (iii) Laccases are not compatible with the secretion pathway in C. reinhardtii, for example because chaperones required for the insertion of the copper ions are missing. The apoproteins lacking these copper ions might be rapidly degraded in the ER.
To test the validity of the first explanation, we designed new constructs with the secretion signals ARS and GLE, introduced by TU Kaiserslautern‘s 2019 iGEM team. We also tried going back to a 3xHA-tag instead of the one containing an 8xHis portion. Because expression could not be observed, neither with nor without the SP20-portion of the C-terminal tag, we reasoned that it is not causing the problem in expression. We therefore chose the SP20-3xHA tag. As TU Kaiserslautern’s 2019 iGEM team has shown, the SP20 repeat greatly enhanced the secretion of their extracellular proteins. That’s why we did not want to waive it. Unfortunately, we were not able to screen transformants with these new constructs because of time restrictions.

Verifying Media Copper Concentration was not a Limiting Factor

Meanwhile, we contacted Dr. Dietmar Schlosser, an expert on fungal laccases. Because laccases are multi-copper enzymes, he suggested that we increase the copper concentration in the medium to identify whether copper is a limiting factor for the expression of our laccases. The medium we used before was the revised TAP medium by Kropat et al.⁸. The copper concentration in this medium is 2 µM. Dr. Schlosser suggested that we should try a concentration of around 20 µM copper. Back in the laboratory, we made a modified version of the TAP medium containing 20 µM copper.

Fig. 7: Immunoblots of the supernatant from 4x12 randomly picked spectinomycin-resistant colonies transformed with the respective constructs and grown in TAP medium with 20 µM copper.

a) and b) shows the constructs, c) and d) shows secreted proteins precipitated form medium for each transformant. The respective constructs are shown above each immunoblot. 6 mL of supernatant were harvested, lyophilized and resuspended in SDS sample buffer. 15 µL of sample were loaded onto the gel and analyzed via immunoblotting using an HA-antibody. Secreted proteins precipitated from medium of the recipient CC-4533 strain served as a negative control, that of a transformant expressing cCA and SP20-HA-8His-tagged brazil nut 2S albumin as a positive control. Controls were treated equally to the samples. For the positive control, only 5 µL were loaded onto the gel.

Filter Tests

Meanwhile, we had an interview with our local authorities to gather information on the legal restrictions, our project would face when implemented in the real world. They told us that the legal barriers are not so high in Germany as long as we could make sure that no GMO could escape our photobioreactor. We therefore started a run of preliminary filter tests to see whether a FP 30 CA-S 0.22 µm filter would be enough to hold C. reinhardtii back. We performed these tests under 21 °C and 4 °C to simulate different seasons. This setup was suggested to us by our local authorities.

Fig. 8: No growth of C. reinhardtii could be observed after 5 or 9 days respectively.

a) and b) show TAP-agar plates after 5 days, c) and d) after 9 days. For each replicate, an aliquot of an exponentially growing culture was pressed through a FP 30 CA-S 0.22 µm filter. 1 ml of filtrate was incubated on TAP-agar plates. Tests a) and b) where performed under 4 °C with a transgene C. reinhardtii from the CLiP strain, c) and d) under 21 °C with a transgene C. reinhardtii from the UVM4 strain.

As can be seen in Fig. 8, no growth of C. reinhardtii can be observed. This suggests that there are no living C. reinhardtii cells present in the filtrate. To see, if our proposed photobioreactor is implementable in the real world under current law, these preliminary tests should be repeated with more replicates and the plates need to be observed for a longer period of time.

Summary and Outlook

Summary

Over the course of iGEM, we were able to produce both laccases, i.e. marLac and BaLac in the cytosol of the green alga Chlamydomonas reinhardtii. We were not able to detect any activity for either of those enzymes using an ABTS assay. Neither of the enzymes was expressed when fused to a N-terminal cCA-secretion signal and a C-terminal (SP20)-HA-RGS-8His-tag. An increased copper concentration did not change these results.

Outlook

To test whether C. reinhardtii can express and secrete laccases, more experiments are required. Transformants of the designed constructs with different secretion signals, i.e. GLE and ARS and/or different tags, i.e. an SP20-3xHA-tag should be screened. Another large obstacle could be the activity of the enzymes. Expression of the laccases does not automatically mean that they show any activity. Prof. Dr. Antonio Pierik, an expert on iron-sulfur proteins explained to us that the incorporation of metallic ions into a protein is not a trivial process. As laccases are multi-copper enzymes, it could be that secreted laccases expressed by C. reinhardtii would not be active at all. A screening for activity should therefore always follow a proof of expression.

Results for E. coli

Design of the constructs

For the recombinant expression of the laccase genes from Botrytis aclada (baLac) and from an uncultured marine bacterium (marLac) the E. coli vector pGEX-6P-1 was used (Fig. 8). This expression vector is used to construct a translation fusion protein of Glutathione S-transferase (GST) and our two different laccases. The expression is regulated by a tac promotor. This promotor combines the strong expression rate from the tryptophan promotor and can be induced with IPTG like the lac-operon. To make sure the promotor is inhibited if there is no induction with IPTG the vector also includes the genetic code for the lac-inhibitor that can bind the lac-operon. Because GST has a high affinity for glutathione, the fusion of the laccases with GST allows the purification by affinity chromatography using glutathione agarose. In addition, a protease cleavage site is incorporated between the GST and our fusion proteins (BaLac-GST and MarLac-GST). This allows the separation of GST and the laccases using PreScission Protease. BaLac has a size of 89.3 kDa with GST and a size of 61.6 kDa without GST. MarLac has a size of 75.8 kDa with and 48.2 kDa without GST. For selection of plasmid containing cells the expression vector carries an ampicillin resistance gene.
In order to be able to do further enzyme assays with the laccases the vectors pGEX-6P-1-marlac and pGEX-6P-1-balac have to be transformed in the E. coli expression strain BL21(DE3). Additionally, we transformed them in E. coli DH5α for isolation the vectors. The strain has an endA1 mutation. This leads to the inactivity of an intracellular endonuclease, which degrades plasmid DNA. Therefore, plasmid DNA isolation is more efficient.

Fig. 9: pGEX-6P-1_baLac (a) and pGEX-6P-1_marLac (b).

It includes a tac promotor that can be regulated with the lac-inhibitor (expressed with lacI) which can be inactivated with IPTG. The protein is fused to GST, with a PreScission protease cleavage site in between. The vector has an ampicillin resistance gene, which is used for selection.

Growth test

First, we wanted to find out the best conditions for the plasmid containing E. coli BL21(DE3) cells to produce BaLac and marLac. We did a test expression where the cells grow at different temperatures (37°C, 30 °C and 17°C; Fig. 10). After every hour we took a sample for an SDS-PAGE (Fig. 11) and measured the optical density (OD) at 600 nm. We designed the experiment based on Kittl et al., 2012 where they used copper sulfate in the media for the protein production. So, we tested the growth at all the temperatures, one time with CuSO₄ and one time without, to be sure that the copper sulfate doesn’t influence the growth (Fig. 10).
To see if our protein is soluble or insoluble, we lysed the cells and separated the pellet and the soluble fraction with SDS-PAGE (Fig. 11).

Fig. 10: Growth curve from E. coli BL21(DE3) pGEX-6P-1_baLac and E. coli BL21(DE3) pGEX 6P 1_marLac producing cells at different temperatures.

The expression was done over 19 hours, the cells were induced at x=0. In (a) the growth of E. coli BL21(DE3) pGEX-6P-1_baLac is shown. In (b) the growth of E. coli BL21(DE3) pGEX-6P-1_marLac can be seen. Both were tested at 37°C, 30°C and 17°C and all temperatures both with [w] and without CuSO₄.

Fig. 11: SDS-PAGE of the test expression with different temperatures.

Samples of E. coli BL21(DE3) pGEX-6P-1_baLac and E. coli BL21(DE3) pGEX-6P-1_ marLac were taken before induction and after inducing with IPTG after every hour for every temperature. The LB medium contains CuSO₄. The cells were disrupted by sonication and insoluble and soluble fraction were separated. The red boxes show the produced marLac (a) and BaLac (b). Marker: New England BioLabs ® Blue Protein Standard Broad Range. The proteins sought are at the level of a relative molecular mass of the marker between 75 and 100 kDa (BaLac, size 89.3 kDa) and 75 kDa (marLac, size 75.8 kDa). The positive control is a GST fusion protein with a size of 26 kDa .

Furthermore, we wanted to test in which medium the cells grow best. We compared LB-Medium with 2YT-Medium in two different temperatures (37°C, 30°C; Fig. 4)

Fig. 12: SDS-PAGE and immunoblot of the test expression with LB medium and 2YT medium.

(a) shows BaLac producing cells growing in LB medium, (b) shows BaLac producing cells growing in 2YT medium. marLac producing cells growing in LB medium is shown in (c) and 2YT medium in (d). Each medium contains CuSO₄. After induction, the cells grow at 37°C and 30°C for 3 h and 5 h. The cells were disrupted by sonification and insoluble and soluble fraction were separated. The red boxes show the produced BaLac. The fusion protein (BaLac and GST) was detected by anti-GST-antibodies (first antibody) and anti-Goat alkaline phosphatase conjugated antibodies (second antibody). Marker: New England BioLabs ® Blue Protein Standard Broad Range.

As it is shown in Figure 3 and 4 the protein seems to be insoluble. So, we took the conditions where we had the biggest amount of soluble protein. Because of that we decided to produce BaLac at 30°C for 5 hours (Fig. 10) in 2YT-Medium (Fig. 11) and marLac at 37 °C for 3 h (Fig. 11) in 2YT-Medium (Fig. 12). Because the laccases are enzymes with copper-centers and the cells growth isn’t inhibited by the copper sulfate (Fig. 10), we decided to use CuSO₄ in our production medium every time.

Production and Purification

With the knowledge about the best conditions we started the production of our protein in the transformed E. coli BL21(DE3) strain. The expression was induced with IPTG. After the respective times (BaLac 30 °C for 5 h, marLac 37 °C for 3 h) we harvested the cells. To purify the protein, we lysed the cells and worked on with the soluble proteins in the cytoplasm. The first step of purification is to separate the translation fusion protein from other soluble proteins using affinity chromatography (Fig. 13a, 14a). After dialysis with PreScission Protease a second affinity chromatography removes the GST from the solution (Fig. 13b, 14b). Then our purified protein was ready to be tested for activity. For BaLac we got around 0.15 mg protein per gram cell wet weight and for marLac it was around 0.33 mg/g cell wet weight.

Fig. 13: Purification of BaLac with affinity chromatography.

The cells grew at 30°C and were harvested after 5 h then lysed with sonification and centrifuged to receive the soluble fraction. The BaLac in the soluble fraction was purified with glutathione-agarose affinity chromatography. The SDS-PAGE shows the samples taken after every step. The fusion protein (BaLac and GST) was detected by anti-GST-antibodies (first antibody) and anti-Goat alkaline phosphatase conjugated antibodies (second antibody). (a) shows the first step of the purification before dialysis with PreScission Protease. The cell lysate was applied to the column. The fusion protein with GST-tag was able to bind to the column, the remaining lysate passed the column (flow through). This was followed by a washing step with washing buffer. Finally, the fusion protein was eluted with elution buffer containing glutathione (eluates 1-6). This was followed by dialysis with the PreScission protease to separate the laccase from the GST-tag. (b) shows the second step after dialysis. The GST binds to the glutathione agarose due to its affinity. The laccase flows through the column (D1-3). After a washing step with PBS (D4), the GST is eluted using an elution buffer containing glutathione (elution). The produced BaLac is shown in the red boxes. Marker: New England BioLabs ® Blue Protein Standard Broad Range.

Fig. 14: Purification of marLac with affinity chromatography.

The cells grew at 37°C and were harvested after 3 h then lysed with ultrasonic and centrifuged to receive the soluble fraction. The marLac in the soluble fraction was purified with glutathione-agarose affinity chromatography. The SDS-PAGE shows the samples taken after every step. The fusion protein (marLac and GST) was detected by anti-GST-antibodies (first antibody) and anti-Goat alkaline phosphatase conjugated antibodies (second antibody). (A) shows the first step of the purification before dialysis with PreScission Protease. The cell lysate was applied to the column. The fusion protein with GST-tag was able to bind to the column, the remaining lysate passed the column (flow through). This was followed by a washing step with washing buffer. Finally, the fusion protein was eluted with elution buffer containing glutathione (eluates 1-6). This was followed by dialysis with the PreScission protease to separate the laccase from the GST-tag. (b) shows the second step after dialysis. The GST binds to the glutathione agarose due to its affinity. The laccase flows through the column (D1-3). After a washing step with PBS (D4), the GST is eluted using a washing buffer containing glutathione (elution). The produced marLac is shown in the red boxes. Marker: New England BioLabs ® Blue Protein Standard Broad Range.

Fig. 15: SDS-PAGE and immunoblot of the test expression with different E. coli strains.

The following strains are shown: (a) E. coli Rosetta gami (b) E. coli BL21 Codon Plus RIL (c) E. coli DH5α (d) E. coli BL21(DE3) (e) E. coli Origami (f) E. coli AD494(DE3). After induction the cells are grown at 17°C for 3 h and for 19 h. The samples taken were lysed with sonication. The fusion protein (BaLac and GST) was detected by anti-GST-antibodies (first antibody) and anti-Goat alkaline phosphatase conjugated antibodies (second antibody). Marker: Promega GmbH © Broad Range Protein Molecular Markers

On the SDS-PAGE showed in Figure 15 we could see, that compared to E. coli BL21(DE3) we have much more soluble protein in the strains E. coli AD494(DE3) and E. coli Rosetta gami. We started a new production and purification with these two strains (Fig. 8) and got a higher protein yield in the end (E. coli Rosetta gami pGEX-6P-1_baLac: 0.21 mg/g cell wet weight; E.coli AD494 (DE3) pGEX-6P-1_baLac: 0.18 mg/g cell wet weight). With this we started a new ABTS assay.

Fig. 16: SDS-PAGE of the BaLac production in E.coli AD494(DE3) and E.coli Rosetta gami.

The cells grew at 17°C and were harvested the next day, then lysed with ultrasonic and centrifuged to receive the soluble fraction. The BaLac in the soluble fraction is purified with glutathione-agarose affinity chromatography. The SDS-PAGE shows the samples taken after every step. The fusion protein (marLac and GST) was detected by anti-GST-antibodies (first antibody) and anti-Goat alkaline phosphatase conjugated antibodies (second antibody). (a) shows E. coli AD494(DE3), (b) shows E. coli Rosetta gami. Marker: Promega GmbH © Broad Range Protein Molecular Markers

Results of Assay Team

ABTS Assays

ABTS is an important substrate used to measure oxidizing reactions and track the rate and strength of ongoing enzymatic activity. ABTS interacts with laccase in a similar way to the laccase-diclofenac interaction while producing a colored substrate which can be measured as a surrogate for the reaction progression.

Fig. 17: Oxidation of ABTS by laccase.

In the course of the reaction, 4 moles of ABTS are oxidized to their corresponding radical cation form, turning the solution from colorless to green. This change can be measured via UV/Vis-spectroscopy at 420nm. ^9,10 The electrons from the substrate are stored in the laccase, turning it from its oxidized form (Laccaseox,. relative oxidation number: +IV) into its reduced form (Laccasered., relative oxidation number: ±0). In its reduced form, the laccase is able to reduce 1 mole of oxygen to 2 moles of water, via two two-electron transfers.¹¹

We performed a series of ABTS assays to determine reaction strength of the proteins produced by the E.coli and Chlamy teams. The laccase derived from the fungus Trametes versicolor (bought from Sigma-Aldrich Co.) acted as a positive control. The standards were established by both μg/mL and μM to compare against Bradford results and most easily prepare dilutions of positive control enzymes at differing concentrations.

Table 1: Concentrations of standardized laccase (T. versicolor).

All dilutions were prepared from a 20 mg/mL stock and followed the tiers of dilution to create a positive control standard graph.

The lowest concentration (40 μM) was used as a standard in the pH 4 assays with BaLac (where T. versicolor was able to effectively react), while both the lowest concentration and an additional 3570 μM positive control was added for MarLac because of the low activity observed in T. versicolor at pH 7 established in subsequent positive control standard verification assays.

Preliminary ABTS Assay

We performed an assay in different pH Phosphate-Citrate buffers to visualize the positive control laccase T. versicolor at the two optimal pH’s documented for BaLac (pH 4) ¹² and marLac (pH 7).¹³ In addition, we ran the assay at the established optimal for T. versicolor (pH 5) to compare our methods against the documented optimal. ¹⁴ The preliminary tests were used to reconfirm whether ABTS (2,2'-azino-di (3-ethylbenzthiazoline-6-sulfonic acid)) is suitable as a stand-in for the laccase reaction with micropollutants such as diclofenac, as well as establishing the best equilibrium of laccase to ABTS reaction ratio.
In a 96 well plate, varying concentrations of T. versicolor laccase at different pH levels were added to the substrate ABTS (250 μM). Enzyme activity for the different pH’s were calculated from 4 technical replicates with 2 biological replicates for each concentration.

Fig. 18: ABTS assay plate set up.

For the positive control assays, each well held 250 μM with varying concentrations of T. versicolor. The negative controls contained the corresponding pH buffer in lieu of ABTS.

The assay was performed for 4 hours on a TECAN plate reader and the final well was photographed to reference final color shifts. From the data collected absorption graphs were created to visualize our positive control activity at each of our created enzyme’s optimal pH levels for later comparison.

Fig. 19: Analysis of the ABTS assay.

a) Samples are prepared on 96 well plate and inserted into the TECAN plate reader for 4 hours at 30 °C. b) As the reaction occurs, TECAN i-control 1.11 software records the absorbance at 420 nm every 5 minutes; data is analyzed, normalized, and compared to standards and controls. c) A photograph of the final plate is taken to visualize the strength and reactivity of the enzyme.

With the large number of replications, standard deviations were obtained and showed pH 5 to be the most consistent and quickly reacting pH level for T. versicolor. Noting the intensity of color in each well after the assays, pH 5 performed the best while pH 7 was not as active.
The assay enabled the determination of saturation levels for certain enzyme concentrations, allowing us to see the optimal concentrations for displaying our eventual reactions with our produced enzymes. These graphs were later compared to our positive controls to verify the reaction occurred at the correct rates and absorbances, especially when troubleshooting unreactive samples.
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Fig. 20: Positive Control ABTS assay of T. versicolor laccase at differing pH.

The absorption was measured at 420nm over a time period of 4 hours. Seven tiers of enzyme concentration were sampled for each pH listed and the ABTS concentration was 250 μM . a) Assay at pH 4. b) Assay at pH 5. c) Assay at pH 7.

As concentration of protein increases, absorption increases and eventually saturates over the course of the 4 hour assay. Fig. 20a demonstrates that pH 4 samples had quick reaction curves and saturation for higher concentrations of laccase. At pH 5 (Fig. 20b), the optimal pH level for T. versicolor laccase oxidation,⁶ demonstrated lower standard deviations of reactions between concentration levels, however pH 4 performed nearly as well with less consistency. Samples at pH 7 (Fig. 20c) exhibit the lowest absorbance of all buffers, even at the normally saturated 3570 μM concentration.
Due to the lower activity at pH 7, it was established in the tests involving produced marLac an additional positive control at the highest concentration available to still have a control reaction in the buffer used.

ABTS Assay with Produced Enzymes

With the standards established, it could be used to verify both the activity compared against baselines determine if produced laccase efficiency was equivalent to the bought positive control, as well as confirm our stock solutions were not defective. The proteins which were produced and concentrated from E. coli team were tested in an equivalent ABTS assay. (A comparable assay was also done with Chlamy intracellular lysate, which is described before. Three different E. coli strains were used for the BaLac laccase (from the ascomycete fungi Botrytis aclada) and one of marLac (an unknown marine bacteria) to increase stability and production in E. coli cultures.

E.coli BaLac Strain BL21(DE3)

BaLac produced in E. coli BL21(DE3), henceforth BL21, was the primary sample we ran assays with, due to it being produced as the initial construct from the E. coli team. The assay was performed with the T. versicolor positive control at 40 μM and a negative control without enzyme, with all samples receiving 250 μM ABTS (Fig. 21). The assay was run identically to the positive controls, 4 hours at 30˚C. All following assays were performed this way.
There was activity shown, however because initially concentration of the enzyme (30 μM for the sample documented in Fig. 20) was determined using spectroscopy, an error prone method to determine the protein concentration, it was uncertain if this was the actual concentration due to imprecision in the method. In later assays a Bradford assay analysis was performed to confirm concentration more accurately. ABTS activity appears very similar to the T. versicolor positive control at a similar concentration run in pH 4 buffer, suggesting it could be, however concentration produced by this strain was never confirmed with the Bradford due to time constraints. Due to this and low concentration issues, only one positive result was achieved in ABTS assays, though later analysis suggests it had activity higher or similar to T. versicolor (see HPLC results below).

Fig. 21: BL21 ABTS assay performed in pH 4 Phosphate-Citrate buffer.

1: Positive (+) control is 40 μM T. versicolor 2: BaLac BL21 sample containing 30 μM. 3: Negative (-) control contains no enzyme. Each well in row A contains 250 μM ABTS, while all B rows have ABTS substituted with buffer.

Fig. 22: BL21 ABTS assay analysis.

a) Raw data: Positive (+) control is 40 μM T. versicolor. Negative (-) control contains no enzyme. BaLac (30 μM determined by spectra) shows similar activity to positive control. b) Normalized data (Raw absorbance/Initial absorbance) demonstrates a change from initial to visualize the reaction more clearly, showing the BaLac and T. versicolor sample reacting at similar rates over the course of 4 hours.

E. coli marLac

The marine laccase marLac was an alternative that we were excited to try due to its optimal pH being at 7, closer to the average wastewater pH and more thermodynamically stable.5 In the assays, we noted that there appeared to be no significant activity however, even when adjusting parameters such as growing conditions, salt levels, and temperature.
We noted that when pipetting the samples in, often the solution would be extremely foamy, an indicator of denatured proteins. This would lead to less sample in each well or delays in beginning the assay, which could have been a source of error in our measurements. The samples would also often elute proteins during the course of the assay, causing any increase in the actual measurement to likely be a result of this rather than actual ABTS oxidation occurring.
The final assay done with marLac (23.85 μM obtained in less than 200 μL solution, so a negative ABTS control was not possible, see Fig. 23) was a replication of our initial protocol to verify concentration obtained by Bradford and check if too many factors had been adjusted. The positive control was T. versicolor at our highest tested concentration (3570 μM) due to the poor activity this laccase had at pH 7.

Fig. 23: ABTS assay for both marLac and BaLac (strain AD494).

The assay was performed in pH 7 and 4 Phosphate-Citrate buffer respectively to provide the manufactured enzymes optimal conditions. Positive (+) control is both 3570 μM in pH 7 buffer (C1) for marLac and 40 μM T. versicolor in pH 4 buffer (A1) for BaLac. All wells except D1, B3, and B4 contained 250 μM ABTS to act as an ABTS negative control. There was not enough produced enzyme of marLac to run an ABTS negative control. Negative (-) controls contained no enzyme, and tested ABTS against both pH 4 (A4) and pH 7 (B4). A light blue tint in the BaLac well (A3) indicates a reaction over the 4 hour assay.

Fig. 24: Negative (-) control contains no enzyme. The marLac sample had its concentration (23.85 μM) determined by Bradford assay and shows similar activity to negative control, beginning at a slightly higher absorbance likely due to eluting proteins. b) Normalized data (Raw absorbance/Initial absorbance) demonstrates the change from initial to visualize the reaction more clearly, showing the marLac sample reacting at similar rate as the negative sample over the course of 4 hours.
No activity was found in any tested marLac sample due to the complications with insoluble proteins and low concentrations, so further investigation is required to implement this construct.

E.coli BaLac strain AD494(DE3)

In later assays, it became apparent that the concentration provided for the assays was a large issue, as yield was low and much of the protein was insoluble. As a result, E. coli team decided to try two new strains of chassis. The first was E. coli AD494(DE3), referred to as AD494, and while the yield and concentration provided for the assay after purification was slightly better than previous reactions, now being verified with the Bradford assay (26.8 μM per around 500 μL obtained), the reaction appeared less dramatic.

Fig. 25: AD494 ABTS assay analysis.

a) a) Raw data: Positive (+) control is 40 μM T. versicolor. Negative (-) control contains no enzyme. BaLac (26.8 μM determined by Bradford assay) shows some activity, however much lower compared to positive control, which is about 1.5 times more concentrated. b) Normalized data (Raw absorbance/Initial absorbance) demonstrates a change from initial to visualize the reaction more clearly, showing the BaLac sample reacting more linearly over the course of 4 hours when zoomed in and compared to the stable negative control without enzyme.

However, the sample did react, as shown in Fig. 25b and in well A3 in Fig. 23, demonstrating another successful construct. It is important to note that due to time constraints because of COVID19, we were unable to perform additional replications or an HPLC with these samples, so more study into this construct would be necessary, however our positive results through assay and photographic evidence are indicative of a valid construct and a promising direction.

E.coli BaLac Strain Rosetta gami

The final enzyme was produced in E. coli Rosetta gami, referred to as Rosetta gami. This construct had a yield of 27.5 μM for around 200 μL of purified protein, preventing any additional replications due to time constraints and logistics. This variant did appear to demonstrate some oxidation, however it was on par with what was seen in AD494 in reaction strength, and did not produce a blue coloration in the well in Fig. 25 (though this could have faded as there was a delay in photographing the wells after the experiment). Again, the results are promising, however more replications are needed to confirm and perfect the construct.

Fig. 26: Rosetta gami ABTS assay performed in pH 4 Phosphate-Citrate buffer.

Positive (+) control is 40 μM T. versicolor with wells A1-A3 containing 250 μM ABTS. Negative (-) control contains no enzyme, while wells C1 and B3 had ABTS replaced with buffer. There was not enough enzyme to have an ABTS negative control for the BaLac sample.

Fig. 27: Rosetta ABTS assay analysis.

a) Raw data: Positive (+) control is 40 μM T. versicolor . Negative (-) control contains no enzyme. BaLac (27.5 μM determined by Bradford assay) shows some activity, however much lower compared to positive control, which is about 1.5 times more concentrated. b) Normalized data (Raw absorbance/Initial absorbance) demonstrates the change from initial to visualize the reaction more clearly, showing the BaLac sample reacting more linearly over the course of 4 hours when zoomed in and compared to the stable negative control.

Sources of Error

One source of error was likely the length of time the concentration and plating steps took, which possibly denatured some of the proteins before they could react in the assay. We also experienced very low yield, which made technical replicates very difficult to run. Due to the nature of the assay, some runs were performed delayed when the software froze on the TECAN plate reader, causing the initial values, which were used to determine the normalized reaction curves, to be elevated.
Elution of protein did occur for some of the produced proteins after time passed, especially in warmer temperatures, and was noted with marLac especially, though occasionally BaLac did demonstrate this tendency, which obviously would lead to a skewed result when reading absorbance.
Bubbles and foam due to denatured proteins was also another very large issue, especially when resuspending or simply adding sample to the plate, and so after several runs with BL21 and marLac, we realized it was important to inject the ABTS into the well to begin the reaction rather than inject the enzyme into the well as we had previously. Any resulting bubbles could shift the reads on the plate, or leave enzyme in the epi when pipetting, so we tried to centrifuge these tubes very briefly to reduce the bubbles without causing a pellet to form and need to resuspend. This caution also may have led to undermixing of the wells to prevent bubbles forming, causing the reader to potentially miss the very low concentrated enzyme if it wasn’t reacting close enough to the sensor.

HPLC

Since dicolfenac and other common micropollutants dissolved in the water do not change color as soon as they are oxidized by a laccase, it is necessary to separate and determine the products by means of spectroscopic detection using the HPLC column. We use HPLC to detect such products to verify the HPLC method.

Fig. 28: Oxidation of diclofenac by laccase.

Literature proposes 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 derivative (d).¹⁵ 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. ¹⁶ Note that the structure of product (d) shown above was established for the laccase from Trametes versicolor. ¹⁵

Diclofenac spectra confirmation

We began by confirming the peak absorbance of diclofenac at 20 μg/mL to verify that 276 nm was the correct wavelength to measure at, based on established methods to be replicated in the HPLC.¹⁵ This test would ensure that the absorbance peak wasn’t affected by the filter used and that our positive control would react with diclofenac’s established peak. The positive control of T. versicolorwas incubated with diclofenac for 10 minutes before measurement to demonstrate the laccase being deactivated. The primary sample was heat treated and filtered diclofenac sample to verify the filter used would not substantially lower the concentration and prevent accurate records. It was shown that the peaks did align as predicted with the negative control, 20 μg/mL diclofenac in pH 5 buffer, and that the filtration step did not significantly impact the measurements.

Fig. 29: Diclofenac HPLC pretests in pH 5.

Positive control: T. versicolor laccase (3570 μM) and diclofenac (20 μg/mL); HPLC Heat Filter: heat treated and filtered 20 μg/mL diclofenac sample; Negative control: diclofenac (20 μg/mL) and pH 5 buffer. All samples incubated for 10 minutes before measurement.

HPLC Diclofenac Controls

HPLC was recorded at 276nm using RP18 column running at a flow rate of 1ml/min.¹⁵ The gradient was Methanol (eluent A) and 0.1 % phosphoric acid (eluent B), starting from an initial ratio of 10 % A and 90 % B and reaching 100 % methanol within 14 min. Elution with methanol was continued for a further 6 min.
The column used was identical to the recommended protocol,¹⁵ an endcapped LiChroCART 125-4 RP18-Column with a particle size of 5 µm, pore size 100 Å, column length 125 mm, and inside diameter of 4 mm.
Using established diclofenac HPLC protocols, samples of diclofenac at different concentrations were prepared to test on the HPLC to determine the standard curve for a diclofenac peak against the reactions.¹⁵ The samples were mixed in pH 4 buffer since BaLac’s optimal pH is 4. With a sample of BaLac BL21 being tested later, we determined that the difference in reaction in our positive control laccase T. versicolor (see Fig. 19a and 19b) was minimal enough to test all samples in without significant reduction in reactivity.

Fig. 30: Diclofenac elution peak at multiple concentrations.

Diclofenac retention time was 13.1 minutes and aligned with the literature value of around 14.1 minutes .¹⁵ The large peak between 2 and 4 minutes was likely the transition between buffers methanol and phosphoric acid and just noise based on its appearance in every sample and control.

HPLC was recorded at 276nm using RP18 column running at a flow rate of 1ml/min.¹⁵ The gradient was Methanol (eluent A) and 0.1 % phosphoric acid (eluent B), starting from an initial ratio of 10 % A and 90 % B and reaching 100 % methanol within 14 min. Elution with methanol was continued for a further 6 min.
The column used was identical to the recommended protocol,¹⁵ an endcapped LiChroCART 125-4 RP18-Column with a particle size of 5 µm, pore size 100 Å, column length 125 mm, and inside diameter of 4 mm.
Using established diclofenac HPLC protocols, samples of diclofenac at different concentrations were prepared to test on the HPLC to determine the standard curve for a diclofenac peak against the reactions.¹⁵ The samples were mixed in pH 4 buffer since BaLac’s optimal pH is 4. With a sample of BaLac BL21 being tested later, we determined that the difference in reaction in our positive control laccase T. versicolor (see Fig. 19a and 19b) was minimal enough to test all samples in without significant reduction in reactivity.

HPLC Positive Controls

T. versicolor laccase was used as a standard positive reaction over the course of an hour, which appears to progress linearly until after timepoint 10, where the peak is maintained at the same area throughout the remainder of the hour. This aligns with ABTS data which shows peak saturation at around 30 minutes in Fig. 20a. The reaction appeared to occur rapidly once the T. versicolor was introduced into the sample, reducing by a little over 1/2 by t0, suggesting either that the laccase continued to oxidize after the heat treatment occurred before the laccase denatured and the reaction was halted completely, or that the laccase was very efficient in its oxidation of diclofenac.

Fig. 31: Diclofenac elution peak during reaction with T. versicolor.

Each sample held 1785 μM T. versicolor laccase and 250 μM diclofenac. Reactions were halted at assigned time with heat treatment and filtration. Diclofenac retention time was 13.1 minutes and aligned with the literature value of around 14.1 minutes ¹⁵. No obvious product peaks are visible and the area underneath the initial noise between 2 and 4 minutes does not change. Area underneath the diclofenac peaks appear to decrease at a linear rate until T10 at which time they are consistently below 100 mAU*s.

No obvious product peaks, which would be expected around 2 minutes before elution of Diclofenac, are visible. Due to time constraints, contaminated samples, and limited access to equipment, we were unable to perform replications to verify the results. However, the values are promising, reflective of what has been observed in literature and by our previous assays.

HPLC BaLac BL21

After testing the T. versicolor samples, produced BaLac was incubated with diclofenac in an identical method as seen in the positive control. Due to time constraints, no replications of the BaLac samples were able to be produced to run on the HPLC. Concentration of BaLac was accurately determined by Bradford assay to only be 3.5 μM within 200 μL, enough for 4 samples. We predicted the reaction would take much longer to occur due to the limited concentration and as a result, we decided to incubate over the course of an hour to give the enzymes the most optimal conditions.
Surprisingly, the reaction appeared to progress at nearly the same rate or slightly slower than the positive control, displaying an area of about 900 mAU*s under the peak compared to the nearly 600 mAU*S initial T. versicolor positive control. The reaction progressed at a similar rate, though due to limited sample and time constraints, replications and shorter time intervals were impossible to perform.

Fig. 32: Diclofenac elution peak during reaction with BaLac.

Each sample held 3.5 μM BaLac BL21 laccase and 250 μM diclofenac and reactions were halted at assigned time with heat treatment and filtration. Diclofenac retention time was 13.1 minutes and aligned with the literature value of around 14.1 minutes. ¹⁵ No obvious product peaks are visible and the area underneath the initial noise between 2 and 4 minutes does not change. Area underneath the diclofenac peaks appear to decrease at a linear rate until T10 at which time they are consistently below 100 mAU*s. BaLac t30 does not have a peak at 13.1, but instead at 13.4 with an area that corresponds with t60.

With such a low concentration to begin with, this sample reacting in such a clear way is suggestive that the produced laccase is not only effective, but possibly even more effective at oxidizing diclofenac than seen in the ABTS assay. It is possible that due to the errors with obtaining a protein concentration through raw absorbance calculations that the previously measured sample that was recorded at 30 μM was actually much lower, which would align with the E. coli team’s protein purification results tracing the loss of protein through each stage of the process. If this were the case, because this sample was the first to be accurately measured by Bradford assay, it is possible the yield was always so low and simply not recorded as such, suggesting the protein was much more productive than previously imagined. It is possible that the protein shows higher affinity to diclofenac than ABTS, which could also explain the results. This would need to be further explored by future work.

Sources of Error

It is important to mention due to the quality of the column that was used, measurement was difficult and required a lot of troubleshooting. Some controls showed no results, which required rerunning samples and making new stocks or reactions of the control samples that we were able to remake.
The phosphoric acid and methanol buffer gradient seemed to display a large amount of noise in our well used column (kindly donated by the Chemistry Department), which made spotting peaks in the beginning of the readouts very difficult. The machine was also a manual loading older model, meaning that accuracy in inserting samples and the duration of the injection was key to aid in precise measurements, and while accuracy was intended and caution was taken in every step, because this was the first use of the machine by the Assay team members, errors could have occurred in technique.
Additional sources of error may stem from the preparation of the samples. The injection needle was a reusable glass needle which needed to be cleaned thoroughly between uses to avoid cross contamination. Samples were all mixed individually rather than in a single epi and then split between several for incubation to ensure equal distribution of both diclofenac and enzyme. This was initially done because of the importance of stopping the reaction for t0 immediately, however with such low concentrations as those provided in the produced sample, thorough mixing is essential and should be regarded in future assays to be sure all samples contain the reaction equally.

Summary

The most promising construct was BaLac produced in E. Coli BL21 in terms of ABTS activity and HPLC results. While we recommend many replications be performed to confirm our preliminary findings and yield must be increased to make this option viable for real world application, these results suggest an alternative option to expensive technologies that are not viable at smaller water treatment facilities at this time.

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