iGEM 2023 — Team rptu-kaiserslautern

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## WEEK #1

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/lab.jpg)

This week we were able to generate our first correct level 0 part of CYP3A4, confirmed via gel electrophoresis and sequencing. (lab-team)\*

![...](https://static.igem.wiki/teams/4806/wiki/website/note/woche-1-1.jpg)

## WEEK #2

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/lab.jpg)

Nothing special, just basic lab work. (lab-team)\*

## WEEK #3

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/lab.jpg)

This week we were able to generate our next two correct level 0 parts of CYP2D6 and the POR (human reductase), confirmed via gel electrophoresis and sequencing. Additionally, we introduced our first construct into *Chlamydomonas* via glass bead transformation, sadly this part had a point mutation. But in the same week we were able to generate our first correct level 2 part, CYP3A4 with HA-tag. (lab-team)\*

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## WEEK #4

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/lab.jpg)

We generated a new level 0 part of CYPCamC and two level 2 parts of CYP2D6 and the POR, both with HA-tag. (lab-team)\*

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## WEEK #5

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/lab.jpg)

We performed our first SDS-PAGE and westernblot to find positive transformants. Given that this specific construct contained the pointmutation, the absence of positive transformants was not as disappointing. This week we also generated our first level 1 parts of CYP3A4, CYP2D6, CYPCamC and the POR respectively with HA-tag and mStop. Additionally, we generated our first level 2 part of CYPCamC with HA-tag. (lab-team)\*

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/hp.jpg)

We met up with our local radio station here in Kaiserslautern and gave a brief interview on our project. (Désirée Ross, Tobias Krauß)
 Additionally, we had a little cooperation on Instagram with the iGEM team of Thrace where we said what iGEM is to us. (Christel Sakhel, Tobias Kraus, Désirée Ross)
 Furthermore, we had the privilege of meeting Dr.-Ing. Michael Schäfer at our wastewater treatment plant in Kaiserslautern. We not only presented our project but also engaged in discussions about the potential implementation of our system. Impressively, he provided us with an in-depth tour of the treatment plant, concluding a valuable and informative experience. (Johanna Haas-Fickinger, Désirée Ross, Luca Langenberg)

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## WEEK #6

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/lab.jpg)

We introduced our first correct constructs into *Chlamydomonas* of CYP3A4, CYP2D6 and the POR all HA-tagged. (lab-team)\*

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/hp.jpg)

We participated in a local cleanup called “Lautrer Kehrwoche” to clean up our city. (whole team)

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## WEEK #7

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/lab.jpg)

This week we generated our first multigene construct of CYP3A4 with the POR and CYP2D6 with the POR. Additionally, we introduced CYPCamC with HA-tag into *Chlamydomonas*. (lab-team)\*

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/hp.jpg)

Throughout the entire week, we had two students from France in our lab. We discussed our project with them and involved them in all the work steps we did. They even had the opportunity to perform some work themselves. (Johanna Haas-Fickinger, Luca Langenberg)

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/sponsor.jpg)

Microsynth is sponsoring us! (Luca Langenberg)
Heidolph is sponsoring us! (Luca Langenberg)

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## WEEK #8

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This week we created a lot of new level 2 constructs of CYP3A4, CYP2D6 and the POR respectively with FLAG-tag and mStop. These constructs were introduced into *Chlamydomonas* in the same week. (lab-team)\*

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/hp.jpg)

We held our first presentation in front of a school class, where we also engaged in interactive experiments involving mitosis and fluorescent proteins with them. (Désirée Ross, Tobias Krauß)
 Furthermore, we had the opportunity for a second meeting with Dr.-Ing. Michael Schäfer, this time in the company of a civil engineer. (Désirée Ross, Johanna Haas-Fickinger, Luca Langenberg)

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/sponsor.jpg)

Macherey-Nagel is sponsoring us! (Tobias Krauß)
Our university and the “TU Freundeskreis” are supporting us! (Maximilian Messmer)

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## WEEK #9

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/lab.jpg)

We performed an SDS-PAGE and a westernblot of CYP3A4, CYPCamC and the POR all tagged with HA. We were able to find our first positive transformant for every single construct. This week we performed a second SDS-PAGE and westernblot of CYP3A4, CYP2D6, CYPCamC and the POR tagged with HA and were able to find further transformants of CYP3A4, CYPCamC and the POR. (lab-team)\*

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/sponsor.jpg)

Promega is sponsoring us! (Christel Sakhel)
Greiner Bio One is sponsoring us! (Luca Langenberg)

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## WEEK #10

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We generated new level 2 constructs of CYP3A4, CYP2D6, CYPCamC and the POR all targeted to the chloroplast. Additionally, we started with our first activity assay for 3A4 using the antibiotic erythromycin. In this experiment, we incubated solutions with different concentrations of erythromycin with our two best expressing 3A4 strains and a wildtype for control. Sadly we were not able to detect activity of our enzymes. (lab-team)\*

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/hp.jpg)

During this week we did our first survey at an Edeka supermarket, where we approached strangers to gather their opinions on food labels, with the focus on “Ohne Gentechnik” ( without genetic engineering). (Désirée Ross, Luwam Gebrezgi, Christel Sakhel, Selina Pusch)

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/sponsor.jpg)

DunnLab is sponsoring us! (Johanna Haas-Fickinger)

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## WEEK #11

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/lab.jpg)

This week we performed another SDS-PAGE and Western blot, this time of our FLAG-tagged constructs of CYP2D6 and the POR and were able to detect positive transformants for both constructs. (lab-team)\*

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/hp.jpg)

We published an article in which we elaborated our project and idea in a german journal for biology called “BioSpektrum”. (Désirée Ross)
 Additionally, we sent some plasmids and parts to the iGEM team of Ionis. (Luca Langenberg)
Furthermore, we had the privilege of meeting Dr. Hugues Renault, an expert in CYPs. During our discussion, we shared details about our project, and Dr. Renault provided interesting insights from his research experience. His input not only enriched our understanding but also inspired us with valuable ideas for our next steps. Additionally, we attended one of his lectures where he elaborated the pivotal role of CYPs in the evolution of plants. (Maximilian Messmer, Luca Langenberg)

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## WEEK #12

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/lab.jpg)

We introduced our chloroplast targeted constructs of CYP3A4, CYP2D6, CYPCamC and the POR into *Chlamydomonas*. (lab-team)\*

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/sponsor.jpg)

DWK Life Science is sponsoring us! (Luca Langenberg)

## WEEK #13

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/lab.jpg)

We performed a Droptest and a Survivability Assay to measure the activity of our enzymes. Sadly, we were not able to. We also generated a new level 2 part of CYPCamC targeted to the mitochondria. (lab-team)\*

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/hp.jpg)

We hosted a MINT-Workshop at the “Gartenschau” in Kaiserslautern. It is dedicated to young kids interested in science. During our workshop, we engaged with them in discussions about DNA and did some fun experiments together. (Johanna Haas-Fickinger, Luca Langenberg)
 We had a meeting with a *Chlamydomonas reinhardtii* expert, discussing the implementation of our system. He introduced us to a new concept - an artificial system, a nano capsule, separating the environment and our algae. (Dominic Kotysch, Maximilian Messmer)

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## WEEK #14

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/lab.jpg)

Nothing special happened. Just basic lab work! (lab-team)\*

## WEEK #15

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/lab.jpg)

This week we wanted to test whether our POR is destroying our endoplasmic reticulum. For that we transformed reductase expressing lines and wildtype with roGFP in the ER, but we were not able to detect any wildtype expression. Without an existing reference we were not able to confirm our initial thoughts. (lab-team)\*

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/hp.jpg)

We had an interview with a sociologist, discussing concerns related to genetic engineering. (Selina Pusch)

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/sponsor.jpg)

Abcam is sponsoring us! (Maximilian Messmer)

## WEEK #16

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We took part in an info market at our university, directed at possible new students. We had a little booth where we endeavored to provide students with insights into the field of biology and iGEM, as well as details about our project. (Luwam Gebrezgi, Selina Pusch, Maximilian Messmer, Luca Langenberg)

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## WEEK #17

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/hp.jpg)

We held our second presentation in front of multiple school classes in Frankfurt. The topic of our discussion revolved around genetic engineering, sparking an engaging and insightful conversation with the students. (Johanna Haas-Fickinger, Luwam Gebrezgi)

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/sponsor.jpg)

Carl Roth is sponsoring us! (Désirée Ross)

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## WEEK #18

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/lab.jpg)

We tried out a new experimental approach known as freeze thaw, aimed at assessing the accurate integration of our CYP enzymes into the membrane. Unfortunately, the detection was hindered using an incorrect antibody. We are determined to retry this experiment. (lab-team)\*

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/hp.jpg)

We had a meeting with a politician of the german parliament who is the representative of our region the “Pfalz”. During our discussion, we talked about food labeling and the future of genetic engineering to fight future problems. (Tobias Krauß)
Additionally, we met with a journalist from a local newspaper to talk about an upcoming article of ours discussing genetic engineering, lab work, our project and the perception of genetic engineering of the public. (Désirée Ross, Dominic Kotysch)
Furthermore, our university released a press article that provided information about our project and the biology program of study. (Luca Langenberg)

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## WEEK #19

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We rescreened all our positive transformants to get better images of our blots. (lab-team)\*

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/hp.jpg)

We had a meeting with some people from the Lovelace Project, an initiative aiming to promote women in science. Our discussion centered around ways in which we could contribute to their mission and become actively engaged in their efforts. (Désirée Ross, Christel Sakhel)
Additionally, the Rheinpfalz article was published this week.
Also, Carl Roth got in touch with us, so we had our first meeting. We talked about an upcoming newspaper they want to release and their intention to feature an article by us. We also talked about an upcoming collaborative Instagram project. (Luca Langenberg)

## WEEK #20

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/lab.jpg)

This week we generated new level 2 parts of 3A4 and the reductase with mNeonGreen and introduced them in the same week into Chlamydomonas. Further our two new CYPs 9Q3 and 81A10V7 arrived from synthesis and we generated the level 0 parts. Continuing we generated the level 2 parts respectively with HA tag, mStop and mNeonGreen and introduced these into Chlamydomonas. (lab-team)\*

## WEEK #21

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/hp.jpg)

We collaborated with the iGEM team from Stuttgart, to create a survey to verify the lacking knowledge of the public regarding genetic engineering. (whole team)
 Additionally, we began the planning of our project promotion video. (whole-team)

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## WEEK #22

![](https://static.igem.wiki/teams/4806/wiki/website/ml/bnt.jpg)

We tried out a new method of screening our transformants. Given that the names of our CYP enzymes are derived from their characteristic peak at 450 nm, we prepared our samples in a native way, similar to a Blue native PAGE. The peak was measured photometric. Although this was our first attempt without replicates, it showed promising results. (lab-team)\*

## WEEK #23

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/lab.jpg)

This week we performed another SDS-PAGE and Western blot, this time of our HA tagged constructs of CY9Q3 and CYP81A10V7. This screening wasn’t that good with just 4 positive transformants of CYP9Q3 and none of CYP81A10V7, reflecting the strange sequencing results. Therefore, we will build new level 2 MoClo constructs. (lab-team)\*

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/hp.jpg)

We again collaborated with the iGEM team from Stuttgart to write abstracts in different difficulties, as a base to write a dictionary from us to future iGEM teams to help them communicate in their educational work. (Désirée Ross, Christel Sakhel)
 Additionally, we had our first expert interview recording for educational purposes. (Luca Langenberg)

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/sponsor.jpg)

Serva is sponsoring us! (Luca Langenberg)
 Isolab is sponsoring us ! (Maximilian Messmer)

![...](https://static.igem.wiki/teams/4806/wiki/website/jl/9q3-positiv.jpg)

## WEEK #24

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/lab.jpg)

We again screened with our new method, but this time with replicates, resulting in the same outcome. (lab-team)\*

![...](https://static.igem.wiki/teams/4806/wiki/website/resu/fig-17.jpg)

## WEEK #25

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/lab.jpg)

We built new level 2 constructs of CYP9Q3 and CYP81A10V7 with HA tag and mStop. Further, we again screened with our new method. This time we screened all our positive CYP3A4 HA tagged constructs. Simultaneously we did a SDS-PAGE and western blot with the same samples to maybe get a correlation of peak differences and the level of expression. Additionally, we designed primers for our level 2 MoClo constructs of CYP3A4, CYP9Q3 and CYP81A10V7 with mStop to remove the linker positioned before the mStop. This decision followed our research results, which indicated that a more native state of the enzymes might increase their activity. (lab-team)\*

## WEEK #26

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/hp.jpg)

We finished the work on our project promotion video. (Johanna Haas-Fickinger, Tobias Krauß)
 Further, we participated in a two-day event called “MI(N)Tmachwelt”, dedicated to bringing science closer to children. On the first day we hosted a workshop focused on fluorescent proteins and did an experiment on purification with the students. On the following day, we set up a little booth where we showcased various water samples under microscopes and plated bacterial samples from different water sources on agar, engaging the young members of our society in scientific exploration. (whole team)
Additionally, a local TV station, the SWR visited us and filmed a television segment about our project. From this segment, a radio segment and a post on the SWR’s website were also published. (whole team)

![...](https://static.igem.wiki/teams/4806/wiki/website/note/woche-26-1.jpg)

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## WEEK #27

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/lab.jpg)

We performed a PCR to remove the GS linker in between our CYP part and the mStop to get a more native protein. (lab team)\*

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/hp.jpg)

We attended the VAAM conference and gave a presentation, establishing connections with companies and other iGEM teams. (Maximilian Messmer)
 Additionally, the SWR posted about us on their Instagram account. Furthermore, the post from last week was reposted by Germanys’s largest new broadcaster, the Tagesschau.
The SWR3 radio station has also reached out to us for an interview. (Luca Langenberg)

![...](https://static.igem.wiki/teams/4806/wiki/website/note/woche-27-1.jpg)

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## WEEK #28

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HPLC Estradiol measurements. Additionally, we plated CYP81A10V7 with the removed GS linker directly on insecticides for selection, which was not successful, maybe due to a few mutations in the DNA sequence of our CYP. (lab-team)\*

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/hp.jpg)

We had another interview with Antenne Kaiserslautern to provide an update on our project. (Désirée Ross, Tobias Krauß)
 Furthermore, we conducted a school presentation at ASG and engaged in discussions with the students about water pollution. (Désirée Ross, Tobias Krauß)

![...](https://static.igem.wiki/teams/4806/wiki/website/note/woche-28-1.jpg)

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## WEEK #29

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/lab.jpg)

HPLC Estradiol measurements. Additionally, we once more performed the freeze thaw assay, this time verifying the incorporation of our enzyme into the membrane. (lab-team)\*

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/hp.jpg)

We conducted an interview with the spokesperson of the Farmers' and Winemakers' Association, during which we discussed topics such as pesticide usage, regulations in Germany, and genetic engineering in agriculture. We also had a similar conversation with a local farmer, Gerhardt Schmidt. (Désirée Ross)
Additionally, we provided mentoring for the Lovelace organization, where we explained a classroom experiment to them. (Christel Sakhel)
Moreover, we organized the iGEM Day, during which we invited students into our laboratory and carried out experiments typical for our project. (Désirée Ross, Christel Sakhel)

![...](https://static.igem.wiki/teams/4806/wiki/website/note/woche-29-1.jpg)

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## WEEK #30

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/lab.jpg)

HPLC Estradiol measurements (lab-team)\*

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/hp.jpg)

We conducted experiments with students alongside Lovelace and discussed our project. (Désirée Ross, Christel Sakhel)

![...](https://static.igem.wiki/teams/4806/wiki/website/note/woche-30-1.jpg)

## WEEK #31

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/lab.jpg)

HPLC Estradiol measurements. Additionally, we again screened for more CYP9Q3 and for our first CYP81A10V7 transformants, which was not successful. (lab-team)\*

![](https://static.igem.wiki/teams/4806/wiki/website/notebook/hp.jpg)

We delivered a final school presentation at ASG, specifically to the MSS12 students, aiming to educate them about the benefits of genetic engineering in agriculture, followed by a discussion on the topic. (Désirée Ross, Tobias Krauß)

![...](https://static.igem.wiki/teams/4806/wiki/website/note/woche-31-1.jpg)

Annotation:
In brackets, the individuals responsible are mentioned.
\*The lab team: Maximilian Messmer, Johanna Haas-Fickinger, Luca Langenberg

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* [Cycle 1: 3A4, 2D6, CAMC, Reductase](#OurMotivation1)
* [Cycle 2: 9Q3, 81A10V7](#OurMotivation2)
* [Cycle 3: Activity Assay](#OurMotivation3)
* [Cycle 4: Activity HPLC](#OurMotivation4)

# 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)

![Placeholder image](https://static.igem.wiki/teams/4806/wiki/website/engi/ha.jpg)

Figure 1: Level 2 Construct of CYP3A4 3x Ha tagged.

## 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)

![Placeholder image](https://static.igem.wiki/teams/4806/wiki/website/engi/flag.jpg)

Figure 2: Level 2 Construct of CYP3A4 3x Flag tagged.

![Placeholder image](https://static.igem.wiki/teams/4806/wiki/website/engi/mstop.jpg)

Figure 3: Level 2 Construct of CYP3A4 mStop.

## 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)

![Placeholder image](https://static.igem.wiki/teams/4806/wiki/website/engi/mgc.jpg)

Figure 4: Level 2 multigene construct of reducatse and CYP3A4.

## 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)

![Placeholder image](https://static.igem.wiki/teams/4806/wiki/website/engi/ctp.jpg)

Figure 5: Level 2 Construct of 3A4 3x Ha tagged, targeted to the chloroplast.

## 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)

![Placeholder image](https://static.igem.wiki/teams/4806/wiki/website/jl/81.jpg)

Figure 6: Level 2 construct CYP81A10V7 3xHA tagged.

![Placeholder image](https://static.igem.wiki/teams/4806/wiki/website/jl/9q3.jpg)

Figure 7: Level 2 construct CYP9Q3 3xHA tagged.

## 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.

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 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.

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.

## References

1. Verwendung der einzelligen Grünalge Chlamydomonas reinhardtii als Expressionssystem für Synthese und Export rekombinanter Proteine; Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) der Naturwissenschaftlichen Fakultät III - Biologie und Vorklinische Medizin - der Universität Regensburg vorgelegt von Alke Eichler-Stahlberg aus Regensburg Mai 2005
2. SD (2018) Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii;Pierre Crozet, Francisco J. Navarro, Felix Willmund, Payam Mehrshahi, Kamil Bakowski, Kyle J. Lauersen, Maria-Esther Pérez-Pérez, Pascaline Auroy, Aleix Gorchs Rovira, Susana Sauret-Gueto, Justus Niemeyer, Benjamin Spaniol, Jasmine Theis, Raphael Trösch, Lisa-Desiree Westrich, Konstantinos Vavitsas, Thomas Baier, Wolfgang Hübner, Felix de Carpentier, Mathieu Cassarini, Antoine Danon, Julien Henri, Christophe H. Marchand, Christophe H. Marchand, Marcello de Mia, Kevin Sarkissian, David C. Baulcombe, Gilles Peltier, José-Luis Crespo, Olaf Kruse, Poul-Erik Jensen, Michael Schroda\*, Alison G. Smith\*, Stéphane D. Lemaire\*; ACS Synth Biol;2018 Sep 21;7(9):2074-2086; doi: 10.1021/acssynbio.8b00251. Epub 2018 Sep 5.
3. Niemeyer, J., Schroda, M. New destination vectors facilitate Modular Cloning for Chlamydomonas. Curr Genet 68, 531–536 (2022). https://doi.org/10.1007/s00294-022-01239-x
4. Sheludko, Y. V., Gerasymenko, I. M., and Warzecha, H. (2018) Transient Expression of Human Cytochrome P450s 2D6 and 3A4 in Nicotiana benthamiana Provides a Possibility for Rapid Substrate Testing and Production of Novel Compounds. Biotechnol J 13, e1700696)
5. Elena Kreis, Katharina König, Melissa Misir, Justus Niemeyer, Frederik Sommer, Michael Schroda, TurboID reveals the proxiomes of Chlamydomonas proteins involved in thylakoid biogenesis and stress response, Plant Physiology, 2023;, kiad335, https://doi.org/10.1093/plphys/kiad335
6. Wang X, Shuai J, Kong Y, Li Z, Li W, Cheng J. Mechanism of the distinct toxicity level of imidacloprid and thiacloprid against honey bees: An in silico study based on cytochrome P450 9Q3. J Mol Graph Model. 2022 Nov;116:108257. doi: 10.1016/j.jmgm.2022.108257. Epub 2022 Jul 4. PMID: 35816906.
7. Mao W, Schuler MA, Berenbaum MR. CYP9Q-mediated detoxification of acaricides in the honey bee (Apis mellifera). Proc Natl Acad Sci U S A. 2011 Aug 2;108(31):12657-62. doi: 10.1073/pnas.1109535108. Epub 2011 Jul 20. PMID: 21775671; PMCID: PMC3150950.
8. Han H, Yu Q, Beffa R, González S, Maiwald F, Wang J, Powles SB. Cytochrome P450 CYP81A10v7 in Lolium rigidum confers metabolic resistance to herbicides across at least five modes of action. Plant J. 2021 Jan;105(1):79-92. doi: 10.1111/tpj.15040. Epub 2020 Nov 27. PMID: 33098711.
9. Guengerich FP, Martin MV, Sohl CD, Cheng Q. Measurement of cytochrome P450 and NADPH-cytochrome P450 reductase. Nat Protoc. 2009;4(9):1245-51. doi: 10.1038/nprot.2009.121. Epub 2009 Aug 6. PMID: 19661994; PMCID: PMC3843963.
10. Hanley ME, Patel PH. Carbon Monoxide Toxicity. [Updated 2023 Jan 23]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK430740/
11. Yu AM, Fukamachi K, Krausz KW, Cheung C, Gonzalez FJ. Potential role for human cytochrome P450 3A4 in estradiol homeostasis. Endocrinology. 2005 Jul;146(7):2911-9. doi: 10.1210/en.2004-1248. Epub 2005 Apr 7. PMID: 15817670.

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# Here you will find all protocols we used during time in the lab!

* [SDS-Page](https://static.igem.wiki/teams/4806/wiki/website/experiments/sds-page-rptu-kl-2023.pdf)
* [Bacterial DMSO-Stock](https://static.igem.wiki/teams/4806/wiki/website/experiments/bacterial-dmso-stocks-rptu-kl-2023.pdf)
* [Westernblot ECL detection](https://static.igem.wiki/teams/4806/wiki/website/experiments/ecl-detection-rptu-kl-2023.pdf)
* [LB- and TAP/HMP-media Agar-Plates](https://static.igem.wiki/teams/4806/wiki/website/experiments/lb-and-tap-plates-rptu-kl-2023.pdf)
* [HMP-media](https://static.igem.wiki/teams/4806/wiki/website/experiments/lb-media-rptu-kl-2023.pdf)
* [Ligation and Transformation in *E. coli and C. reinhardtii*](https://static.igem.wiki/teams/4806/wiki/website/experiments/moclo-and-trafo-protocol-rptu-kl-2023.pdf)
* [*C. reinhardtii* cellharvest and chlorophyll-determination](https://static.igem.wiki/teams/4806/wiki/website/experiments/protocol-cell-harvest-and-cell-lysis-for-chlorophyll-determination-rptu-kl-2023.pdf)
* [Induction and Inoculation of *E. Coli*](https://static.igem.wiki/teams/4806/wiki/website/experiments/protocol-induction-rptu-kl-2023.pdf)
* [Thermocycler settings MoClo ligation](https://static.igem.wiki/teams/4806/wiki/website/experiments/protocol-moclo-settings-thermocycler-rptu-kl-2023.pdf)
* [TAP media](https://static.igem.wiki/teams/4806/wiki/website/experiments/tap-medium-rptu-kl-2023.pdf)
* [Terrific-Broth media](https://static.igem.wiki/teams/4806/wiki/website/experiments/tb-medium-rptu-kl-2023.pdf)
* [Westernblotting](https://static.igem.wiki/teams/4806/wiki/website/experiments/western-blot-rptu-kl-2023.pdf)
* [Grwoth change assay CYP3A4](https://static.igem.wiki/teams/4806/wiki/website/experiments/growth-assay-cyp3a4-erythromycin-new.pdf)
* [Drop Test](https://static.igem.wiki/teams/4806/wiki/website/experiments/drop-test-for-activity-screening-and-positive-selection.pdf)
* [Survivability assay](https://static.igem.wiki/teams/4806/wiki/website/experiments/survivability-assay.pdf)
* [Screening for Heme-content](https://static.igem.wiki/teams/4806/wiki/website/experiments/heme-content-screening-in-cyp-enzyme-transformed-chlamydomonas-reinhardthii.pdf)

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* [Our MoClo Collection](#OurMoCloCollection)
* [Cytosolic Expression](#CytosolicExpression)
* [Chloroplast-targeted enzymes](#ExpressionInTheChloroplast)
* [The Reductase](#TheReductase)
* [Freeze Thaw](#FreezeThaw)
* [Activity Assays](#ActivityAssays)
* [Producing CYPs without Tag](#ExpressionWithoutTag)
* [Successes and failures](#SuccessesAndFailures)
* [Future Plans](#FuturePlans)
* [Considerations for replicating the experiments](#ConsiderationsForReplicatingTheExperiments)

In our project, we aimed to create a library of genes encoding for cytochrome P450 enzymes (CYPs), for production in *Chlamydomonas reinhardtii*. We successfully expressed five P450 genes of different origin and tried to exemplarily detect the activity of one of them. Successfully expressed genes include those encoding human NADPH cytochrome P450 oxidoreductase (POR), two human P450 enzymes (3A4 and 2D6), CYP enzyme 101 (CYPCamC) from *Pseudomonas putida*, and 9Q3 from the oriental honeybee *Apis cerana cerana*. A gene encoding the CYP enzyme CYP81A10V7 from the sweet grass *Lolium rigidum* was also designed and tested.
We investigated different approaches to produce these enzymes in *Chlamydomonas reinhardtii* and to measure their activity. Use the navigation bar on the left side to navigate through our results to get to whatever part you are interested in.

# Our MoClo Collection

During the experimental iGEM phase we generated six level 0 parts (being basic parts), nine level 1 parts (intermediate parts/transcriptional units) and 25 level 2 parts (functional multigene constructs) all of which are standardized and compatible with the Modular Cloning System (MoClo) so that they can be integrated seamlessly. Further, these parts are codon optimized and contain *RBCS2*-introns in their coding sequence to ensure optimal expression in *Chlamydomonas*. Overall, we contribute 39 distinct parts to the iGEM community. All our level 2 constructs shown in Figure 1 were transformed into *Chlamydomonas reinhardtii* and analyzed for expression.

![Placeholder image](https://static.igem.wiki/teams/4806/wiki/website/resu/fig-1.jpg)

Fig.1 Level 2 MoClo Constructs
(a) A visual explanation of the MoClo system with Level 0, 1, and 2 parts (Weber *et al*., 2011). (b) These level 2 constructs were designed according to the standards of the Modular Cloning System (Phytobrick) and are codon optimized for *Chlamydomonas reinhardtii*. We employed resistance cassettes (Hygromycin (HR), Paromomycin (HR) and Spectinomycin (HR)) for selection of positive transformants. The numbers 1-11 represent specific MoClo fusion sites. To create multigene constructs, we used Dummies (D61 and D63) and end linkers (El37 and El39) to connect our parts with the destination vector. For a more detailed part description check out our parts registry, especially our [best basic part](http://parts.igem.org/Part%3ABBa_K4806000).

# Cytosolic Expression

P450 enzymes are produced in the cytoplasm and integrate into the ER membrane. Therefore, our first approach was to produce the different P450 enzymes in the cytoplasm, hoping that they would integrate into the ER membrane of our microalgal host just as they do in their source organism. To accomplish this, we introduced constructs A-I (Fig. 1) into the *Chlamydomonas* strain UVM4, which is a strain suitable for high transgene expression and is easily transformable. These constructs were designed for producing cytosolic proteins harboring C-terminal 3xHA or 3xFlag tags for detection via immunoblotting.

![Placeholder image](https://static.igem.wiki/teams/4806/wiki/website/resu/fig-2.jpg)

Fig.2 Expression of CYPs in the cytosol
(1a-7a) Level 2 MoClo constructs for the production of enzymes CYP3A4, 9Q3, 2D6, the POR and CYPCamC containing either the FLAG or HA-tag were designed. (1b-7b) The UVM4 strain was transformed with the construct in (a). Up to 30 antibiotic-resistant transformants (depending on the construct) were cultivated in TAP-medium and samples taken after 3 days. Whole-cell proteins were extracted and analyzed by SDS-PAGE and immunoblotting using an anti-HA antibody. In the resultant blot, the black arrow marks our enzymes and the white arrow marks a cross reaction of antibodies. Signals were obtained for CYP3A4 (~57 kDa), CYP 9Q3 (~59 kDa), CYP2D6 (~ 56 kDa), the POR (~ 77 kDa) and CYPCamC (~ 47 kDa). The UVM4 recipient strain was used as negative control and a strain expressing the HA-tagged ribosomal chloroplastic 50S protein L5 (RPL5) or FLAG-tagged VIPP1 as positive control.

Notably, we managed to produce five out of six enzymes (Fig.2), with highest expression levels obtained for CYP3A4 and the reductase (POR).

# Chloroplast-targeted enzymes

Another idea was to target the proteins encoded on constructs T-W (Fig.1) to the chloroplast to use electrons from the light reactions to photosynthesis to fuel our enzymes. Constructs for CYP3A4, CYP2D6, CYPCamC and the POR employed an N-terminal transit peptide to target the proteins to the chloroplast and a C-terminal 3xHA tag for detection via immunoblotting.

![Placeholder image](https://static.igem.wiki/teams/4806/wiki/website/resu/fig-3.jpg)

Fig.3 Expression of CYPs in the chloroplast
(1a-4a) Level 2 MoClo constructs for the production of CYP3A4, 2D6, POR, and CYPCa,C with the CTPPSAD-transit peptide. (1b-4b) The UVM4 strain was transformed with the construct shown in (a). Up to 30 antibiotic-resistant transformants (depending on the construct) were cultivated in TAP-medium and samples taken after 3 days. Whole-cell proteins were extracted and analyzed by SDS-PAGE and immunoblotting using an anti-HA antibody. The white arrow marks cross-reactions of the antibody. No specific signals were detected for CYP2D6 (~ 56 kDa), CYP3A4 (~ 57 kDa), POR (~ 77 kDa) and CYPCamC (~ 47 kDa). The UVM4 recipient strain was used as negative control and a strain expressing HA-tagged ribosomal chloroplastic 50S protein L5 (RPL5) as positive control.

Unfortunately, we were not able to detect signals for any of our enzymes targeted to the chloroplast. This could be attributed to the unnatural environment for the enzymes leading to their degradation or to inefficient chloroplast import. Alternatively, the 3xHA-tag might be cleaved off in the chloroplast, which our lab has frequently observed for other chloroplast-targeted proteins. Since cytosolic expression was successful, we focused on the cytosolic enzymes in further experiments.

# The reductase

To optimize the supply of reducing equivalents to our CYPs, we sought to co-express them with a reductase. Our initial approach involved designing a multi-gene construct (X in Fig. 1) for producing CYP3A4 together with a POR.

![Placeholder image](https://static.igem.wiki/teams/4806/wiki/website/resu/fig-4.jpg)

Fig.4 Multigene construct for the co-expression of CYP3A4 with POR
 (a) Level 2 MoClo multigene construct for the co-expression of CYP3A4 and POR, both containing 3xHA tags. (b) The UVM4 strain was transformed with the construct shown in (a). 30 hygromycin-resistant transformants were cultivated in TAP-medium and samples were taken after 3 days. Whole-cell proteins were extracted and analyzed by SDS-PAGE and immunoblotting using an anti-HA antibody. The white arrow marks a cross-reaction of the antibody. CYP3A4 (~ 57 kDa) and POR (~ 77 kDa) could both be detected. The UVM4 recipient strain was used as negative control and a strain producing HA-tagged ribosomal chloroplastic 50S protein L5 (RPL5) as positive control.

We successfully detected cytosolic co-expression of CYP3A4 and POR from a multi-gene construct via immunoblotting (Fig.4). However, the expression level of POR was rather low.
To address this problem, we pursued an alternative approach known as a “supertransformation”. Here, previously transformed positive CYP3A4 strains were again transformed with the reductase gene (POR). Advantages of this approach were the ability to pre-select strains with known CYP3A4 expression levels and a higher probability to find a strain which shows high expression for both genes, since genes on tandem constructs (multi-gene constructs) are expressed independently of each other. The enzymes were once again designed with a C-terminal 3xHA-tag for detection.

![Placeholder image](https://static.igem.wiki/teams/4806/wiki/website/resu/fig-5.jpg)

Fig.5 Supertransformation of CYP3A4 and POR constructs
(a) (a) Level 2 MoClo constructs for the production of CYP3A4 and POR, both with a C-terminal 3xHA tag. (b) A CYP3A4-producing strain was transformed with the POR construct in (a). Up to 30 hygromycin-resistant transformants were cultivated in TAP-medium and samples were taken after 3 days. Whole-cell proteins were extracted and analyzed by SDS-PAGE and immunoblotting using an anti-HA antibody. The white arrow marks a cross-reaction of the antibody. Production of CYP3A4 (~ 57 kDa) was observed, but POR (~ 77 kDa) could not be detected. The UVM4 recipient strain was used as negative control and a strain producing HA-tagged ribosomal chloroplastic 50S protein L5 (RPL5) as positive control.

As shown in Fig.5, this approach proved to be unsuccessful as our transformants showed lower levels of CYP3A4 than the original CP3A4-producing recipient strain. Possibly, the construct for CYP3A4-expression was silenced. In an interview with Dr. Hugues Renault from the University of Strasbourg, an expert on P450 in plants, we learned that overexpression of the reductase can lead to a disruption of the ER. To investigate if the production of POR is harmful for the ER in *Chlamydomonas*, we changed our focus on measuring activity of our CYPs without the support by a human P450 reductase.

# Freeze Thaw

To be functional, CYPs must be correctly embedded within the membrane (Šrejber *et al*., 2018). To verify if our CYPs produced in *Chlamydomonas* are membrane incorporated, a freeze-thaw assay was performed, where rapid freezing and thawing leads to fragmentation of the cells. During this process, large membranes vesicles are formed which can be separated from soluble fractions by centrifugation. Soluble and membrane fractions were analyzed by immunoblotting for our strains producing 3xHA-tagged CYP3A4.

![Placeholder image](https://static.igem.wiki/teams/4806/wiki/website/ml/freeze-thaw.jpg)

Fig.6 Freeze Thaw Assay
(a) Level 2 MoClo construct for the production of 3xHA-tagged CYP3A4. (b) The UVM4 strain was transformed with the construct shown in (a). An anti-HA antibody was used to detect CYP3A4 by immunoblotting (A). For controls, antibodies against cytochrome b6f (Cyt f) and CGE1 were used, which serve as markers for membrane and soluble proteins, respectively (B).
30 spectinomycin-resistant transformants were cultivated in TAP-medium and samples were taken after 3 days. For reference, whole-cell proteins were extracted and analyzed by SDS-PAGE and immunoblotting. Cells were separated into soluble (S) and membrane (P) fractions and analyzed by SDS-PAGE and immunoblotting. CYP3A4 (~ 57 kDa) was detected in the membrane pellet and not in the supernatant (S), confirming its membrane association. CYP3A4 positive strains E2, E13 and E26 were analyzed. The UVM4 strain was used as a negative control.

We were able to detect CYP3A4 in the membrane fraction (Fig.6), indicating correct membrane targeting that is essential for its activity.

# Activity Assays

Since CYP3A4 was produced at highest levels in *Chlamydomonas*, we used CYP3A4-producing transformants to conduct first activity tests. CYP3A4 has many known substrates, one of which is erythromycin. If CYP3A4 was functional in *Chlamydomonas*, we expected our transgenic lines to exhibit better growth in the presence of erythromycin compared to the wild type, as *Chlamydomonas* is sensitive to erythromycin. We incubated our transgenic algae in TAP-medium containing 10 mg/L of erythromycin.

![Placeholder image](https://static.igem.wiki/teams/4806/wiki/website/resu/fig-8.jpg)

Fig.7 Growth Assay
(a) Level 2 MoClo construct for the production of 3xHA-tagged CYP3A4. (b) CYP3A4-producing transformants E2 and E26 were incubated for up to 192 h in TAP medium containing the indicated concentrations of erythromycin (Ery). Cells were counted once a day. The UVM4 strain was used as negative control.

The growth curves shown in Figure 7 suggest that one CYP3A4-producing strain (E2) is more sensitive to erythromycin and one is less sensitive (E26). However, this does not necessarily indicate enzyme activity, as the E26 line also exhibits faster growth in the absence of erythromycin.
 In parallel, we investigated a potential action of CYP3A4 on erythromycin via a spot assay. Here, *Chlamydomonas* cells were spotted onto plates with and without erythromycin. The erythromycin concentration was chosen to be high enough that the UVM4 recipient strains cannot grow. Therefore, only lines with active CYP3A4 should grow on plates containing erythromycin.

![Placeholder image](https://static.igem.wiki/teams/4806/wiki/website/resu/fig-10.jpg)

Fig.8 Spot Test
 (a) Level 2 MoClo construct for the production of 3xHA-tagged CYP3A4.
Four different concentrations of CYP3A4-producing transformants were spotted onto TAP plates lacking (b) or containing 8 mg/L erythromycin (c). The UVM4 strain was used as negative control.

Figure 8 shows that several transformants showed high resistance against erythromycin. Since this did not correlate with CYP3A4 expression, we concluded that spontaneous mutations in ribosomal proteins were responsible. Hence, erythromycin is no suitable substrate to test for P450 activity.
A visually less intuitive but probably more reliable activity assay involves the detection of erythromycin degradation by HPLC. For this, we first needed to detect erythromycin via our HPLC protocol.

![Placeholder image](https://static.igem.wiki/teams/4806/wiki/website/resu/ery.png)

Fig.9 HPLC signal measuring Erythromycin

Attempt at measuring Erythromycin with our HPLC protocol and column. 10 mg Erythromycin were dissolved in Methanol and the absorption was measured at 210 nm.

Unfortunately, we were not able to detect a significant peak for erythromycin with our method and column. For this reason, we had to choose a new substrate. The synthetic estrogen estradiol, among other uses, is found in birth control pillls and makes its way into the environment, where it continues to have hormonal effects, which is a strong incentive to degrade it.

![Placeholder image](https://static.igem.wiki/teams/4806/wiki/website/resu/hplc-estradiol.jpg)

Fig.10 HPLC signals of the activity of CYP3A4 against Estradiol

Cultures of the recipient strain UVM4, as well as the two CYP3A4 expressing transformants E13 and E26, were grown to a defined cell number of 6\*10^6 cells/ml. Then, TAP culture medium was exchanged via centrifugation and subsequent resuspension in TAP medium containing 5 µM estradiol. Cultures were then incubated for 24 h at 25°C. Shown is the absorption of the culture supernatant of the three stains as well as a TAP medium control containing estradiol in dependency on their retention time within the HPLC. Estradiol shows a peak at a retention time of about 7.9 minutes. Another prominent peak was observed at about 8.2 minutes. This peak negatively correlates with the amount of estradiol and is therefore suspected to be a degradation product of estradiol .
(a)Measurements of different estradiol concentrations for finding the right amount of substrate. (b) E26 with and without estradiol to identify estradiol related peaks. (c) HPLC-signals of TAP medium, and medium from UVM4, E13, and E26 cultures containing 5 µM estradiol. (d) Enlarged section of (c) without TAP medium signal.
 (e-g) Evaluated data from (c). (e) Absolute estradiol content (area of the estradiol peak at 7.9 min). (f) Total estradiol content (sum of both areas of estradiol (7.9 min) and potential product (8.2 min)). (g) Relative estradiol content (amount of estradiol in relation to the sum of both areas (7.9 min + 8.2 min)).

Before starting activity tests using estradiol as a substrate to investigate the activity of CYP3A4, pre-tests were performed to elaborate an optimal protocol for measuring estradiol containing samples via HPLC. As a first step we monitored whether estradiol could be measured by HPLC and at which concentrations. Indeed, a peak could be detected at 210 nm after 7.9 minutes (retention time) (Fig. 10(a)). Based on this experiment, we chose an estradiol concentration of 50 µM for the measurement, which relates to 5 µM estradiol concentration during cultivation. Next, we identified peaks which specifically occur due to estradiol degradation, that can therefore not be seen in the control without estradiol (Fig. 10 (b)). Thereby two peaks occurred at about 7.7 min and 8.2 min additionally to the estradiol peak (7.9 min). When incubating the culture supernatant of our CYP3A4 producing transformants with 5 µM estradiol for 24 hours at 25°C, we discovered that our recipient strain UVM4 already shows high activity against our test substrate estradiol. This shows that Chlamydomonas itself is already an excellent platform for bioremediation. But this also makes it harder to distinguish between activity that is due to Chlamydomonas and CYP3A4 related activity. Nevertheless, both transformants, E13 and E26, exhibit even higher degradation of estradiol, which was shown to be significant compared to UVM4 (Fig. 10 (g)). This proves our concept of the functional production of CYP enzymes in Chlamydomonas using CYP3A4 as an example.

These results provide the entry point for further optimization regarding culture and assay conditions, other types of P450 enzymes, P450 enzymes lacking tags, or co-expression of reductases.

# Producing CYPs without Tag

We reasoned that the C-terminal tags we used might interfere with P450 activity. Therefore, we designed constructs N-R (Fig. 1). However, due to fixed sequences in our constructs producing fusion sites for the MoClo system, a sequence encoding for a Gly/Ser linker was left between the CYP coding sequence and the stop codon. To remove that sequence, the coding sequences for CYP3A4, 9Q3, and 81A10V7 were amplified by PCR.

![Placeholder image](https://static.igem.wiki/teams/4806/wiki/website/resu/fig-13.jpg)

Fig.11 PCR to remove GS linker
Agarose gel to verify PCR products for CYP3A4, 9Q3, and 81A10V7 for the assembly of level 2 constructs with stop codons (mStop) directly downstream of the coding sequence. The primers were specifically designed to remove the GS-linker.

Figure 11 shows successful amplification of the coding sequences for CYP3A4, 9Q3, and 81A10V7 lacking the linker-encoding sequence. Removal of the linker sequence was verified by Sanger sequencing. Unfortunately, these constructs could not be further investigated due to time reasons.
 *Chlamydomonas* is sensitive to the drugs chlorsulfuron and atrazine. Both are known substrates of CYP81A10V7. We therefore transformed *Chlamydomonas* with a construct for CYP81A10V7 production and looked for resistant colonies.

![Placeholder image](https://static.igem.wiki/teams/4806/wiki/website/resu/fig-15.jpg)

Fig.12 Activity against Chlorsulfuron and Atrazine
(a) Level 2 MoClo construct for the production of CYP81A10V7 lacking any tag. (b) UVM4 cells were plated on HMP plates with and without atrazine. Moreover, UVM4 cells were transformed with the construct shown in (a) and plated on an HMP plate containing atrazine. (c) UVM4 and UVM4 transformed with the construct shown in (a) were plated on TAP plates containing chlorsulfuron.

No atrazine or chlorsulfuron resistant transformants were obtained. Sequencing revealed that, following the removal of the GS linker, mutations had occurred in the middle of the DNA sequence, which would explain the enzyme’s lack of functionality. Also, we did not succeed to find positive transformants by immunoblotting for the HA-tagged version of CYP81A10V7.
The screening of transformants generated with constructs producing P450 without tags required CYP-specific antibodies. For this, we used a commercial antibody against CYP3A4 and tested it on our positive transformants producing HA-tagged CYP3A4.

![Placeholder image](https://static.igem.wiki/teams/4806/wiki/website/resu/fig-16.jpg)

Fig.13 CYP3A4 antibody test
(a) Level 2 MoClo constructs for the production 3xHA-tagged CYP3A4.
Whole-cell proteins of transformants E2, E13, and E26 producing 3xHA-tagged CYP3A4 were extracted and analyzed by SDS-PAGE and immunoblotting using a commercial CYP3A4 antibody at different dilutions: (b) 1:1000, (c) 1:5000, (d) 1:10000, (e) 1:20000. The black arrow marks CYP3A4 and the white arrow marks a cross-reaction of the antibody. UVM4 served as negative control. (f) To confirm the expression of HA-tagged CYP3A4, the blot was detected with an anti-HA antibody.

A After testing several antibody concentrations, we can conclude that the commercial CYP3A4 antibody is not capable of detecting CYP3A4 produced in *Chlamydomonas*.
Finally, we attempted to screen transformants generated with mStop constructs spectroscopically. CYPs show a peak in absorption at 420 nm due to their heme domain. If transgenic CYPs would be produced in our transformants, this absorption should increase and changes should therefore be detectable. Initially, our transformants producing HA-tagged CYP3A4 were analyzed to see if absorption differences were detectable.

![Placeholder image](https://static.igem.wiki/teams/4806/wiki/website/ml/bnt.jpg)

Fig.14 Absorption measurements
Native whole-cell protein samples of transgenic lines E7 and E26 producing 3xHA-tagged CYP3A4. Absorption was measured using a Nano Drop 2000. The ratio of absorption at 418 nm (Heme) to the total protein content at 280 nm is depicted. UVM4 served as negative control. n = 10.

By normalizing the absorption at 418 nm by the protein content (absorption at 280 nm), an elevated ratio could be observed for CYP3A4-producing lines compared to the UVM4 control (Fig. 14). Even though there is still room for improvement of this method, it indicates that this method might be employed for the screening of CYP450-producing transformants. According to Dr. Hugues Renault, tags could severely interfere with the activity of CYP enzymes. Hence, this method would allow for identifying transformants that express untagged versions of CYPs. Nevertheless, we need to cross-validate these results with another method, as we tested it only for one specific CYP enzyme (3A4). Due to time constraints, we were not able to screen transformants producing untagged CYP variants with this method.

# Successes and failures

* Successes: We were able to produce five out of six of our P450s and reductases in the cytosol of *Chlamydomonas reinhardtii*. Among them were two CYP enzymes of human origin, which is a significant achievement in *Chlamydomonas* research, independent of our project goal.
  Using the high flexibility of the MoClo system, we generated several multi-gene constructs and successfully achieved co-expression of the genes encoding for CYP3A4 and a reductase (POR) in *Chlamydomonas*.
  We confirmed the targeting of CYP3A4 to membranes, which is essential for its activity.
  We tested several visual methods to detect enzyme activity, such as growth and survival assay. These should be repeated with other antibiotics and herbicides. .
  Furthermore, we have learned much about HPLC and have further developed it to analyze substrates such as estradiol. We have successfully implemented a protocol for measuring estradiol via HPLC, a known substrate for CYP3A4. We are confident that we can apply our knowledge to other potential CYP substrates, as long as they can be detected by UV-vis.
* Failures: We were unable to co-express the reductase (POR) in combination with our CYPs.
  Unfortunately we were not able to proof activity of our P450 enzymes against the chosen substrates. These were chosen because they represent threats to our environment.

# Future plans

We intend to continue working on this project and apply other substrates for activity testing, first focusing on known substrates of our CYPs, which would allow for an easier detection of products via HPLC. We will continue measuring enzyme activities that can be monitored in survival assays. Furthermore, we would like to expand the library of CYPs with new, highly specific enzymes in contrast to the enzymes of broad substrate specificity like CYP3A4. We will strive to refine our algal chassis and explore additional methods for its improvement, for example by optimizing the supply of reducing equivalents to facilitate enzyme activity. There are many possibilities for enhancing our system and we will continue our research on this topic as we see it as our duty to combat man-made destruction of our planet.

# Considerations for replicating the experiments

We uploaded all our parts on our registry with the specific DNA sequence. Further you can finde all the protocols we used on our [experiments page](https://2023.igem.wiki/rptu-kaiserslautern/experiments).

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