Cycle 1: 3A4, 2D6, CAMC, reductase

Design 1.1: 3A4, 2D6, CAMC, reductase

CYPs are ultimate tools for transforming toxic substances of all kinds. These substances are released into the environment by the ton every year, entering water bodies and threatening an intact environment and thus the basis of all our lives. In order to make use of these enzymes and thus protect water, a universal human right and the basis of life, as well as the environment, the photoautotrophic organism Chlamydomas reinhardtii was employed for the expression of these universal enzymes. This photoautotrophic aquatic organism C. reinhardtii is a unicellular green alga that exhibits extremely rapid growth with a doubling time of up to 5-8 hours (The Chlamydomonas Sourcebook Second Edition Volume 1 Chapter 8.3.A page 248). C. reinhardtii is ideally suited to harness these ultimate tools, and thereby rid water that forms the basis of life in our environment, regardless of boundaries, of chemicals that are newly developed and synthesized day after day. For the project, the cell wall-less strain UVM4 is used, which is particularly well suited for transformation and high expression of transgenes.1 The basic idea of the project was to express CYPs, so-called monooxygenases that require reducing equivalents, in the chloroplast of the alga, not only to take advantage of the special properties of the enzymes, but also to increase their efficiency. After an intensive literature search, three CYPs were chosen that have been best studied from the almost infinite spectrum. These include the human cytochrome P450 enzymes from the liver 3A4 and 2D6. With a broad substrate spectrum, they metabolize drugs of all kinds that enter all our wastewater and are not filtered by conventional wastewater treatment plants. Another well-studied CYP is bacterial CAMC (camphor 5-monooxygenase), or CYP101. It originates from the organism Pseudomonas putida and converts halogenated hydrocarbons, which include, for example, chemically synthesized pesticides that enter our waters through past commercial use in agriculture. Tons of these pesticides are applied to fields every year, but their effects only become apparent decades later. Thus, catastrophic consequences are created, whose apparent solution is the application of new chemicals, creating a vicious cycle from which our world cannot break out. For this, the system of CYP enzymes, whose pool is evolving and forming every day, a silent reaction of the environment to counteract the decades of human pollution, in combination with the photoautotrophic green alga C. reinhardtii could lead to an escape route out of this vicious circle. To test the possibility of expression in general, the enzymes will initially not be targeted into any specific compartment. The natural localization is the membrane of the ER, so that it is reasonable to assume that the enzymes are natively incorporated into a membrane, so that an artificial membrane anchor may be omitted. In order to provide the huge and complicated enzymes with sufficient reduction equivalents, the human NADPH cytochrome P450 reductase should also be expressed. The sequences of all enzymes were determined via UniProt. The codon usage was adapted to the organism used and introns of the gene encoding for the small subunit of RuBisCO were inserted to increase expression levels. Specific overhangs were created to assemble the parts in the correct order, and to generate recognitions sites for TypIIS restriction enzymes, and the sequence was checked for unwanted recongtions sites. Occasionally, the sequence was adjusted in favor of synthesis performance due to GC-rich regions typical of the organism. The sequences were then synthesized by IDT or Twist. Modular cloning based on the Golden Gate method was used to create the gene constructs. The individual sequences of the synthesized parts must be assembled to generate a standardized level 0 part. To create a transcription unit for such a construct, already established and tested parts for the organism C.reinhardtii are used.

Level 0 constructs

For strong and constitutive transcription, the L0AβSAPi promoter (A1-B2) was used, followed by the coding sequence (CDS) with the generated sequences of the enzymes (B3-B4). The expression of the enzymes will be detected later via immunoblotting. The literature search revealed that specific antibodies are already available for CYP3A4, but due to the intensive cost of an antibody, and the unknown efficiency of the antibody, the sensitive L03xHA tag from the Kaiser Collection (B5) was chosen as the tag. The efficient and proven L0tRPL23 terminator from the Spaniol Collection was chosen as the terminator, which contains an intron that increases efficiency. The vector used is the pAGM1287, which allows selection of positive transformants via lac-Z based blue-white selection, and carries an ampicilin resistance cassette. This reaction was used to create the basic parts of the enzymes, in a second assembly these parts can be coupled with different promoters, tags, transit peptides and terminators. In a second and third reaction, it would also be possible to couple the transcription units of multiple enzymes, and thus transform and express multiple transcription units in C. reinhardtii in a single transformation step, generating multigene constructs.^2,3

Level 2 constructs

Since CYPs are very large and complex enzymes, the general expression should be tested first, since targeting in chloroplasts, and thus transport across two membranes, can complicate expression. The parts mentioned above were also used for this purpose, the L0-AβSAPi promoter, the L03xHA tag and the L0tRPL23 terminator served as promoters. The CDS consisted of the level 0 parts of the previously created constructs. A membrane anchor was omitted as described. Level 2 vectors, which were to be transformed into the target organism later, served as vectors. These already contain resistance to an antibiotic to which C. reinhardtii is sensitive. For each of the enzymes, a different resistance and thus vectors were chosen, as this allows for selection in a later supertransformation with multiple constructs. For 3A4, the vector L2-aadA-CRed (spectinomycin) was chosen; for 2D6, the vector L2-AphVIII-CRed (paromomycin) was chosen; for the reductase and for CAMC, the vector L2-AphVII-CRed (hygromycin) was chosen. The human reductase cannot be combined with the bacterial enzyme. The plasmids, all from the Niemeyer Collection, contain a modified bacterial operon that is responsible for canthaxanthin biosynthesis, thus ensuring a more cost-efficient red-white selection, after transformation in E.coli. ^2,3 (Figure 1)

Build 1.1

Level 0 constructs

The designed parts for one level 0 part for each enzyme and the above established parts were digested and assembled in a single reaction. The method used does not create scars that could affect function. Subsequently, the enzyme was transformed into E. Coli and identification took place via blue-white selection. The plasmids were then isolated and checked via a test digest in comparison to an in silico digest, sample showing the expected band pattern were sequenced via Sanger. These were provided by the department. The level 0 parts thus represent the basic building block for further assembly into level 2 constructs, so that different variants of constructs can be created.

Level 2 constructs

In order to check the general expression and a possible insertion into the membrane, the same established parts were used for the transcription units of the level 2 constructs, the above-mentioned ones with different resistances served as vectors. Thus, in case of successful expression of a CYP after transformation into the model organism, another transformation with another construct can follow. Positive transformants can thus be selected by the new resistance. Assembling all parts, the plasmids were then transformed into E.coli for amplification and selected via red-white selection. Therefore, the previously created level 0 Parts and the above established Parts were digested and assembled in a single reaction. Plasmids were isolated and tested by digestion as well as sequencing.

Test 1.1

For testing the expression, transformants were inoculated in liquid culture and tested by SDS-PAGE and immunoblot with the antibody against the HA Tag. The recipient strain UVM4, served as a negative control, and PKL5 served as a positive control. This strain was provided by the department and carries an HA-tagged version of the ribosomal 50S protein L5 in the chloroplast. Some transformants showed expression for 3A4, the reductase, and CAMC. In particular, the enzyme 3A4 showed comparatively strong expression in some transformants. In these first experiments, it was already successfully demonstrated that the system consisting of host external CYPs, which offer an unimaginable potential for bioremediation, and the photoautotrophic green alga C. reinhardtii is not only feasible but was already implemented by us in the first experiment. The positive transformants were again tested by rescreening for expression to verify the results and compare expression levels. Three of the four enzymes showed expression, which is an incredible success.

Freeze thaw

After proving the expression of CYP enzymes in C. reinhardtii to be possible we aimed to test whether the enzymes incorporate into the membrane of the ER, as already suspected. The strains with the comparatively strongest expression of 3A4 (E2, E13, E26) were tested. By rapidly thawing and freezing the lysed cells with liquid nitrogen, the membranes were fragmented. After centrifugation of the samples, the heavy fragments containing the membranes precipitated, while proteins not incorporated in the membrane remain in the supernatant. UVM4 served as a negative control. Samples containing the whole cell lysate were loaded onto an SDS-PAGE, as well as samples containing the pellet and supernatant. The protein cytochrome f, which should be present in the sample of the pellet, and CGE1, which is soluble, serve here as controls for the method ⁵.5 Both proteins should be detected in the samples of the whole lysate. After immunoblotting, the expressed enzyme can be detected by the HA antibody. Specific antibodies were provided by the department for the controls and the HA tag.

Learn 1.1

The enzymes CYP3A4, CAMC and the reductase were successfully expressed in Chlamy, which means that the basic idea of the project has already been implemented with extreme success, and thus represents the basis for almost infinite possibilities. The self-generated parts of the enzymes are correct, working, and the design of the level 2 constructs leads to successful expression. The enzyme 2D6 could not be expressed. Since the HA tag used is particularly sensitive but may sterically affect the protein due to its size, the next step is to use a smaller tag that has less effect on the expression of the enzymes in order to detect expression for the last of the five enzymes. The results of freeze thaw show that CYP 3A4 is also incorporated into the membrane without an anchor, presumably that of the ER, the natural localization. Consequently, there is no need to revise the gene design to add a membrane anchor. Since the reductase was also successfully expressed, the enzymes can be coupled with the reductase, in the form of tandem constructs. Since more reduction equivalents are available in this way, any stress that might arise for the cells could be reduced, so that perhaps the enzyme 2D6 would also be expressed, as Chlamydomonas is known to silence genetic constructs remarkably fast which affect the cell negative.

Cycle 1.2: Redesign of the Level 2 Parts

Design 1.2

In order to increase the number of positive transformants and to obtain expression of CYP2D6, new level 2 constructs will be generated. Since the opportunity arose to test a 3A4 antibody from Abcam, transformants could also be screened whose enzymes are not tagged, which should increase the expression levels and activity since they are in a more native state. The transcription unit composition for new level 2 constructs is identical to that in design 1, except that the part L03xHA is replaced by “pL0-CTAG-AGAG-3-CrFlag + Stop-B5” or “pL0-CTAG-Multi-Stop-B5” from Lemaire Lab. (Figure 2 and 3)

Build 1.2

The assembly of all the above parts was done in one reaction. The plasmids were then transformed into E.coli for amplification and selected via red-white selection. The constructs were isolated and verified via test digest, with prior in silico digest, and sequencing via Sanger. The constructs are then transformed into UVM4.

Test 1.2

Flag-Tag constructs

To verify the expression of Flag-tagged transformants, SDS-PAGE and an immunoblot were performed. Detections of expression were made using a specific antibody of the Flag tag. UVM4 served as negative control, and a strain expressing VIPP1 (vesicle-inducing protein in plastids 1), being Flag-tagged, was provided by the department as positive control. For each created construct, expression could be observed, so that even the last of the CYPs we chose shows expression, once again proving the success of the project idea.

mStop constructs

To check the expression of the transformants without tag, first SDS-PAGE and immunoblotting were performed with the transgenic line E26 (CYP3A4) to test the antibody provided by Abcam. E26 was used as positive control, UVM4 as negative control and the antibody itself was loaded on SDS-PAGE to test the functionality of the secondary antibody. Since a signal could be detected exclusively for the pure antibody, another SDS-PAGE and Western blot was performed using E26, UVM4, and a positive control for 3A4 from yeast provided by the university food chemistry department. The positive control from yeast were detected.

Learn 1.2

The overall number of positive transformants was not increased by the tag, but since mild expression of the 2D6 enzyme was detected, the change in tag represents a success. Based on the test results, it can be concluded that the antibody provided by Abcam does not work for C. reinhardtii, so the constructs without tag could not be pursued for the time being, however, research and possible tests were already underway to screen the transformants on the activity of the enzyme, for example, the property of the enzymes to show absorption at 450 nm.

Cycle 1.3: Tandem constructs

Design 1.3

From the point of view of efficiency and less stress on the cell, the reductase should be expressed in addition to the enzymes. Since in a multigene construct, the first transcriptional unit is increased expressed and the following one decreased, new constructs were create in which the first transcriptional unit forms the reductase and the following one the CYP (3A4, 2D6). There is some evidence that the order of genes within the multigene constructs affect the expression levels, therefore we always placed our reductase in front and the CYP enzymes at the end of our constructs. Thus, more reduction equivalents will be available and eliminate a possible bottle neck for transformation efficiency, expression level and activity to significantly increase and continuously optimize the success of the project. Level 1 constructs were first formed to represent the individual transcription units. From these level 1 constructs, level 2 constructs with multiple transcription units can then be formed in a further assembly step.

Level 1 constructs

To later guarantee the possibility of combining multiple enzymes and the reductase into level 2 plasmid, the correct level 0 parts will be combined at the level 1 with different dummies. Since the L0-AβSAPi-A1-B2 promoter and the L0tRPL23 terminator used from the Spaniol Collection proved to be effective throughout the project, these parts are also used to create level 1 modules. Likewise, the L03xHA tag was used to later test expression with a specific antibody. Considering the interference of the tag for the enzyme, the constructs were also created without a tag (stop-B5 from Leminare Lab). For the level 1 constructs, specific vectors were selected in order to assemble the parts correctly after digestion in the level 2 reaction. For each possible position of an enzyme, dummies are inserted in the level 1 construct. Since CAMC also has a specific reductase, a dummy was also included here to give the option of adding it to the multigene construct later. Similarly, another place for another CYP was created to add more CYPs to the already created collection after successful expression of the tandem constructs. Through the vectors, selection by antibiotic resistance cassette and blue-white selection is possible after transformation into E.Coli.

Level 2 constructs

Finally, multigene constructs can be created via level 1 constructs, which initially contain the transcription unit of the reductase and a human CYP as proof of concept. This was to test whether the combination of such highly complex enzymes can be expressed in the organism and whether the expression can be optimized using the reductase. For this purpose, pAGM4673 from the Weber Collection was used to select for kanamycin after transformation in E. coli. A resistance to select for Chlaymdomonas reinhardtii after later transformation was also included in the design. (Figure 4)

Build 1.3

Level 1 constructs

Assembly of all the above parts for level 1 was performed in one reaction. Plasmids were transformed into E.coli for amplification and selected via blue-white selection. Plasmids were isolated and tested via digest, with previous in silico digest, verified by Sanger-Sequencing.

Level 2 constructs

The assembly of all verified level 1 parts and the above destination vector into level 2 constructs, was performed in one reaction, the plasmids were then transformed into E. coli for amplification and selected via red-white selection. The plasmids were then isolated and tested via digestion, with prior in silico digestion, and were sequenced via Sanger. The constructs are then transformed into UVM4.

Test 1.3

Expression was tested by SDS-PAGE and Immunoblot with HA antibody provided by the department; UVM4 and PKL5 served as controls. Expression of both transcription units was successful, but the expected increased transformation efficiency did not occur.

Learn 1.3

Even though the primary expectation for the construct was not met, the evidence for the possibility of expressing two such highly complex enzymes in C. reinhardtii highlights the potential of the photoautotrophic green alga and validates the choice of target organism. Via the tandem constructs, it cannot be traced whether the corresponding CYP represents the bottleneck for the lack of the hoped-for effect, or the expressed reductase. This can be verified by transformation into strains positive for expression with the appropriate counter construct. After a discussion with the expert Hugues Renault, it was also suggested that expressing the reductase damages the endoplasmic reticulum, which of course can clearly be reflected in lower transformation efficiency. To test this, the conformation of the ER can be visualized to test the conjecture. This could be tested via constructs, by fluorescent proteins targeted into the ER, and transformed into strain expressing our construct K, as well as UVM4. Thus, the conformation of the ER of strains K12 and K2 could be checked in vivo to verify the effect of the reductase on the ER.

Cycle 1.4: Expression in the chloroplast

Design 1.4

Since the general expression of the enzymes was successful, the idea was now to target the enzymes in the chloroplasts to provide increased reduction equivalents and thus make the enzymes more efficient. A big turning point within the project orientation is the conversation with the CYP expert Hugues Renault. Not only the idea of the reductase had to be reviewed, but also the basic idea of the project was looked at very critically. CYPs are extremely large and complex enzymes. When the enzymes are targeted in chloroplasts, many steps occur that are highly unlikely to proceed correctly. These huge enzymes, whose natural localization is the membrane of the ER, must enter the chloroplast via two membranes, fold correctly, and then be incorporated into the thylakoid membrane. Despite the rather low chance of obtaining positive results for the basic idea of the project, new level 2 constructs were designed. Already at this point, based on the expert discussion, the redesign of the project was discussed and researched.

Construct design

The promoter pPsaD, which has been shown to be efficient in C. reinhardtii for proteins targeted to chloroplasts and the ctppPsaD transitpeptide was used for the gene design. Since no difference was seen between Flag tag and HA tag to expression and transformation efficiency, the tested HA tag was used again. The level 0 parts of the enzymes 3A4, 2D6 and CAMC form the CDS. Since it was already suspected that the enzymes were incorporated into the membrane, an anchor was not used. (Figure 5)

Build 1.4

The assembly of all parts was done in one reaction, the plasmids were then transformed into E. coli for amplification and selected via red-white selection. Plasmids were isolated and verified via test digestion, with prior in silico digestion. All equipment used and necessary substances were provided by the department. The constructs were subsequently transformed into UVM4.

Test 1.4

Expression was verified by SDS-PAGE and Immunoblot, and the antibodies used were provided by the department. None of the transformants showed expression.

Learn 1.4

One possibility for the fact that no expression could be detected via Immunoblotting is the possibility of processing down of the HA tag, which was observed when previously used within the laboratory. Considering that also the interviewed expert considered the idea as extremely difficile, but general expression of the CYP enzymes in C. reinhardtii is possible, the targeting CYPs in the chloroplast initial idea was discarded.

Cycle 2: 9Q3, 81A10

Design 2.1

Due to the fact that CYPs have an extremely high diversity, being present in almost every organism and developing incredibly fast, it is possible that a CYP exists, or evolves, for almost every hazardous substance that enters the environment through humans. Since we had already proven that CYPs can be expressed in Chlamydomonas reinhardtii and had thus already reached a milestone within this competition, we decided to further develop the aspect of bioremediation not only in theory. Approximately 2,000,000,000 billion tons of chemicals are newly synthesized every year. A large part is applied on fields in the form of insecticides and herbicides from where it is washed out into our environment and thereby, gets unfiltered into our lakes, rivers and even our groundwater. The damage is recorded after years, but cannot be estimated, let alone controlled. In order to further develop the aspect of bioremediation, the project was extended in the form of new enzymes. After an intensive literature search, we decided to extend the spectrum of CYPs by two enzymes that, in contrast to 3A4 and 2D6, have a very specific substrate spectrum. CYP 9Q3 is derived from Apis cerana cerana (Oriental honeybee), while CYP 81A10V7 is derived from Lolium rigidum (sweet grass). Both are expressed in eukaryotes, the latter in a plant, which in addition has already been successfully expressed in rice. Consequently, expression in our green alga seems to be only a matter of time.^6,7,8

Level 0 constructs

The previously described parts were used, as these demonstrated successful expression of the previously tested enzymes. For the part of the enzyme, the sequence of both enzymes was aligned to the codon usage for Chlamydomonas, introns were inserted, and GC-rich regions were adjusted for better gene synthesis, and appropriate overhangs and recognition sequences were appended for the restriction enzymes used. The sequence of the CYP enzymes was also searched for unwanted recognition sequences.

Level 2 constructs

Again, the parts described above were used, the vector L2-aadA-CRed was used for both constructs. Since the idea of multigene constructs cannot be pursued further in this project, different resistances in the form of other vectors are not needed. (Figure 6 and 7)

Build 2.1

To generate level 0 parts the procedure was as in section design 1. All parts were assembled in one reaction and the appropriate enzymes, transformed into E. coli, selected via blue-white selection, the construct was isolated and verified with a test digest, and sequencing via Sanger. However, a small sequence of the CDS could not be sequenced in this way as the constructs were too long. The level 2 constructs were created from the reviewed level 0 parts and the above departmental parts, assembled in a reaction, transformed into E. coli, the positive transformants were selected via red-white selection, and the constructs were isolated, and tested via digest. Sequencing of both parts was performed with primers binding to the terminator and promoter of the constructs. The level 2 constructs were transformed in UVM4.

Test 2.1

Expression was verified by SDS-PAGE followed by immunoblotting, and the used antibodies were provided by the department.

Learn 2.1

The enzyme 9Q3 was also successfully expressed in Chlamydomonas reinhardtii, once again confirming the system we have developed to remove chemicals from water, whose pollution is not a local but a global and profound problem for our planet and all living beings. These results provide further foundations for research into the inexhaustible possibilities of enzymes, which in the future may play a far greater role in bioremediation than we already imagine. Initially, it appeared surprising that one of the human CYPs could be expressed without any problems, but a plant CYP showed no expression. The sequencing results were then looked at again. Since the sequence was correct, the error had to be within the blind sequence, so new primers were created to bind to the inserted RBCS2 intron.

Cycle 3: Activity assays

Design 3.1

In order for the successful expression to be already a large part of the project, we dedicated ourselves to testing the activity in parallel to testing other constructs in order to get closer to a theoretical implementation of using C. reinhartii within a nanocapsule for bioremediation within water bodies and to further test and improve our system. The enzyme itself represents one possibility for testing activity. Due to its ability to metabolize toxic substances, which the recipient strain used is not capable of, growth tests are possible with a suitable substrate to which UVM4 is sensitive. Since the enzyme 3A4 showed expression from the beginning, and strong expression in some positive transformants, the lines E2 and E26 were used. After research, the antibiotic erythromycin, to which C. reinhardtii (paper) is sensitive, was chosen as a substrate for 3A4.

Growth tests

After transferring lines E2 and E26 to culture medium spiked with a concentration of erythromycin to which UVM4 is sensitive, cell count data [Cells/ml] can be collected over time to compare growth between transgenic lines and UVM4. With activity consistent with the expectation of substrate conversion to a less toxic form, the cell number of the transgenic lines should increase earlier from time t=0 and stagnate more rapidly as they reach stationary phase sooner. The data can be graphed, analyzed, and tested for significance.

Build 3.1

The appropriate substrate has been purchased. The cell counter for collecting the data is regularly maintained within the department.

Test 3.1

Data were collected as cell counts per ml at 24 h intervals over a 192 h period. Both selected strains, as well as UVM4 (negative control), were given in liquid culture with different concentrations of erythromycin. The cell number of UVM4 increased at a later time point than the transgenic line E26. Compared to UVM4, line A2 showed decreased growth. Liquid cultures containing pure culture medium in which all strains showed growth were used as controls. The absolute data can be displayed and evaluated graphically.

Learn 3.1

Due to conflicting data from E26 and E2 comparative to UVM4, this assay was discarded as a possibility for activity testing. Since the constructs transformed in Chlamydomonas integrate randomly into the genome and thus can lead to knock out of essential genes, it cannot be determined whether the increased growth, or the increased sensitivity can be attributed to the transgenic enzyme. Another possibility to measure the activity via growth is via analyzing the medium, forthe substrate and possible degradation product.

Design 3.2

Survivability assays

By adding the transgenic lines and UVM4 in medium mixedwith erythromycin, activity could be tested as the antibiotic should be degrade allowing growth of not resistant strains in the medium afterwards. This growth can be checked by cell count measurement.

Build 3.2

The tests were performed with culture medium and erythromycin of different concentrations. All selected strains were transferred into it and incubated, then cells were separated from the medium by centrifugation and UVM4 was added with some fresh medium.

Test 3.2

Again, time course data were collected, cell number per ml were measured at intervals of 24 h over a time span 192 h. The absolute data can be plotted and analyzed graphically. UVM4 showed growth in all media after a long incubation period.

Learn 3.2

The results of the assays already indicated the increased selection pressure to which the wild type is subjected and the reaction in the form of mutations leading to resistance. Again, increased growth of E26 was observed upon incubation with the transgenic lines.

Design 3.3

Drop tests

Another possibility is to collect absolute data by testing whether growth is possible or not. For this purpose, plates with nutrient media and different concentrations of erythromycin were poured onto which the cells were dropped in a defined volume with decreasing cell number. UVM4 served here as a negative control, E26 that did not react sensitively on the plates served as a positive control. This also provides an opportunity to screen positive transformants, including the constructs created without a tag.

Build 3.3

Plates of different concentrations were cast with the substrate and nutrient medium, on which UVM4 was plated out. Thus, the appropriate concentration to which UVM4 is sensitive could be selected.

Test 3.3

UVM4 was dropped on the plates as a negative control and E26 as a positive control with decreasing cell number. Similarly, transformants already screened positive for 3A4 were run to test growth. Similarly, for the generated transgenic lines without tag in the construct were proceeded as possible screening. UVM4, as well as lines that initially reacted sensitively, showed growth after a short time through single colonies.

Learn 3.3

Due to the increasing number of resistances against erythromycin due to a mutation in the ribosome, neither the activity can be detected via the drop test, nor can the created constructs be screened without tag. Another possibility of the activity test results from the conversion of the substrate, which can be checked via HPLC.

Design 3.4

Another way to screen transformants for functionality of CYPs to absorb at a wavelength of 450 nm after interaction with carbon monoxide. Carbon monoxide is a respiratory toxin that poses a major hazard to humans. ⁹ From the point of view of safety for team members, we decided against conducting experiments related to this hazardous substance.¹⁰ However, this approach allowed us to develop a method by which transformants can be screened for expression, providing a means to screen transformants producing untagged CYP enzymes. Heme coenzymes absorb at a wavelength of 420 nm. Since CYPs as monooxygenases possess a heme domain and thus also absorb at a wavelength of 420 nm, a screening procedure for CYPs could be designed. In order to measure absorption at 240 nm via the heme domain, the enzyme must be native and not denatured. To isolate the proteins natively, a Blue Native-PAGE sample preparation protocol can be followed. Absorbance at 420 nm can be detected using a departmental instrument. A statistical analysis of the collected data has to be performed later to test whether the additional content of CYPs in the transformants differs significantly from the natural content of molecules with heme domains in C. reinhardtii.

Build 3.4

To test the idea, liquid cultures of transformant strains E26, E2 and, as a control, strain UVM4 were prepared. These were treated similarly to a Blue Native-PAGE sample preparation. The absorbance at 420 nm can be determined using the department's NanoDrop.

Test 3.4

The absorbance of the prepared samples was measured at a wavelength of 420 nm. To normalize the measured values to the total protein content of the samples, measurements were also taken at a wavelength of 280 nm. A ratio was calculated from absorption at 420 to 280 nm, which normalizes the absorption due to heme in regard to the total protein concentration. The normalized values can be analyzed graphically and tested for significance to verify this method.

Learn 3.4

The statistical analysis shows that screening for CYP expressing transformants is possible using the method described above. Thus, not only a protocol has been created to screen transformants with untagged enzyme, but also another method for future iGEM teams to explore the inexhaustible potential of CYPs, which does not include handling high dangerous gases.

Cycle 4: Activity, HPLC

Design 4.1

Based on previous literature research, another option for verification of enzyme activity is a classical analytical experiment, where the conversion of a substrate is monitored. If the selected substrate absorbs at a defined wavelength, the change of this substrate can also be checked via high pressure liquid chromatography.⁴ A side reaction of CYP3A4 is the hydroxylation of the synthetic hormone estradiol. ¹¹ Positive transformants E13 and E26 and recipient strain were cultured in TAP medium mixed with 5 µM estradiol. After 24 hours, cells were removed by centrifugation and estradiol was purified by liquid-liquid-phase-extraction and measured by HPLC. For this, the method as such with the selected substrate estradiol in combination with the used culture medium has to be tested first, as well as suitable concentrations. A possible protocol was provided by the department, the detection limit of the substrate still had to be determined.

Build 4.1

Ethyl acetate was chosen as the solvent for the organic components, and after evaporation the samples were dissolved in acetonitrile. Thus, the method of applied liquid-liquid extraction had to be tested as well, in which the sample is concentrated 10-fold. The C18 column used is rinsed with increasing amounts of hydrophobic medium (acetonitrile) for each measurement. Research showed that estradiol absorbs at a well at a wavelength of 210 nm.

Test 4.1

After the first test showed that the substrate could be detected by the chosen method, the detection limit was tested with variable concentrations from 100 μM to 1 μΜ. The method as such, as well as the liquid-liquid extraction was successful. The change in substrate can be viewed graphically via the absorption at 210 nm and the retention time. A clear peak was obtained after a retention time of 7.9 min.

Learn 4.1

Since the method in combination with the substrate showed a clear peak at 210 nm after a retention time of 7.9 min, experiments to actually test the activity could be performed. Under the aspect of hydroxylation of estradiol by CYP3A4, it is reasonable to assume that after conversion at least one peak will be detectable at an earlier retention time than 7.9 min, with the chosen method being rinsed from aqueous to hydrophobic.

Design 4.2

In the second step, a concentration had to be discussed that would be measurably converted by our CYP-Chlamy system, given activity. For this purpose, concentrations in the range of 5 μΜ to 50 μM (concentrations without consideration of the subsequent 10-fold annealing concentration) were to be tested. For this purpose, liquid cultures were incubated at a defined cell number for 48 h and 72 h at exposure and 25 °C. As controls, in addition to E13 and E26, UVM4 was cultivated in TAP medium mixed with Estradiol and pure TAP medium was to be incubated over the same time with Estradiol. Already when planning the experiment to determine the concentration, the question of the factor of time arose. After the first measurements with 5 μM and 10 μM at incubation for 72 h, the concentration should be decreased considerably, so experiments were also performed with an estradiol concentration of 0.5 μM, 3 μΜ and 5 μM, and for comparison the samples were incubated for 24 h. In order to also identify possible signals due to incubation with C. reinhardtii, additional samples should be incubated with UVM4 but without substrate.

Build 4.2

Samples were handled similar to before as described in Building 4.1.

Test 4.2

Testing was performed for the above concentrations, incubation times with the appropriate controls. The experimental conditions were also varied with respect to the vessel (24-well plate or flask) and volume (2 ml or 6 ml). Cultures were grown to a defined cell number of 6,000,000 cells per ml, then substrate was added. The change of the substrate can be viewed graphically via the absoprtion at 210 nm. Peaks were observed after a retention time of 5.2 min, 7.9 min and 8.2 min. Peaks were observed in each of the samples with UVM4, E26 and E2, regardless of concentration and incubation time. The highest absorbance was observed at 8.2 min, then 7.9 min, and by far the lowest at 5.2 min. The measurement of the sample without substrate also showed a peak after 5.2 min.

Learn 4.2

Based on the control without substrate, it could be seen that the peak after 5.2 min is caused not by Estradiol but is most likely a component of the TAP medium, as the peak were observed in samples of TAP medium with Estradiol and samples without Estradiol. A possible degradation product was observed after a later retention time than that of the substrate, which does not correspond to the expectation, but clearly indicates activity of the green alga. The used model organism itself already shows transformation of the substrate, by measuring several replicates it should now be proven that this incredible ability of Chlamy can be optimized by our CYPs. Based on the data, an optimal concentration of 5 μM and incubation time of 24 h could be evaluated. Since the transformants used express a human CYP, the question arose whether adjusting the temperature, which was 25 °C in the previous experiments, could be adapted to the optimum of the enzyme and thus be raised to the human-body temperature of 37°C. To eliminate the possible source of error of substrate addition, in the next setup the culture should be adjusted to a defined cell number, centrifuged and then resuspended in TAP medium in which 5 μM estradiol had already been dissolved.

Design 4.3

The conversion of the substrate was observed for all lines used. In order to statistically evaluate the results and identify possible random deviations of a sample, 3 duplicates were prepared for both controls (UVM4 and TAP), as well as E13 and E26. The experiments were to be performed at 25 °C and at 37 °C.

Build 4.3

Samples were handled similar to before as described in Building 4.1.

Test 4.3

Testing was performed for the above concentrations, incubation times with the appropriate controls. The experimental conditions were also varied with respect to the vessel (24-well plate or flask) and volume (2 ml or 6 ml). Cultures were grown to a defined cell number, then after centrifugation the medium with substrate already dissolved was added. The change of the substrate can be observed graphically via the absorption at 210 nm at a retention time of 7.9 minutes. Peaks were observed after a retention time of 5.2 min,7.7 min, 7.9 min and 8.2 min, peaks was observed for each of the samples with UVM4, E26 and E13, regardless of concentration and incubation time. The highest absorbance was observed after 8.2 min, then 7.9 min and by far the lowest after 5.2 min.

Learn 4.3

The experiments at 37 °C showed increased noise within our measurements which could be due to the stress induced to Chlamydomonas by this higher temperature but also our not optimal experimental setup, as we were unable to illuminate our cultures during growth and relied on an incubator instead of a heat bath. Therefore no additional activity due to 3A4 could be measured in our samples as activity of Chlamydomonas was much prominenter and varying between individual samples. The well tested experimental setup at 25 °C delivered results with lower variance, where a higher remaining peak for Estradiol was observed in the UVM4 control than in both positive transformants hinting towards additional activity due to CYP3A4. After calculating the area of substrates and products a significantly stronger degradation of Estradiol could be observed. Still these results should be verified in future in completely independent further experiments. Also there is still much room for optimization of the method and it should be kept in mind that there are many potential causes that we misinterpret the magnitude of the degradation due to CYP3A4 activity. More definitive data could not be obtained by incubation at 37 °C. No significant difference in the amount of substrate remaining or in the possible amount of product could be detected, but this does not speak against enzyme activity. 3A4 has a very broad substrate spectrum.Estradiol is only a secondary substrate of 3A4, which is why a detection of the conversion is difficult to implement. From the point of view of substrate specificity, activity measurement with 3A4 is probably generally difficult. For further measurements, after successful expression, the CYPs 81A10V7 and 9Q3, which we have chosen, are optimally suited, as they act much more specifically against substrates. Also Dr. Hugues Renault warned that our C-terminal tags could reduce the activity severely, which is why we would like to repeat the activity tests with untagged proteins in the future. Since the substrate is hydroxylated by the reaction with 3A4, there is also the possibility that the converted substrate remains in the cells to a greater extent due to reduced permeability hiding some of the activity. Based on the results, however, it can be clearly concluded that the choice of our model organism to address the problem of the increasing amount of chemicals in our environment, which threaten ecosystems and thus the entire planet, could hardly have been better.