Notebook

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

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

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)^*

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)^*

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)^*

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)

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

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

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)^*

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)

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

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)^*

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)

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

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)^*

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

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)^*

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)

DunnLab is sponsoring us! (Johanna Haas-Fickinger)

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)^*

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)

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

DWK Life Science is sponsoring us! (Luca Langenberg)

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)^*

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)

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

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)^*

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

Abcam is sponsoring us! (Maximilian Messmer)

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)

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)

Carl Roth is sponsoring us! (Désirée Ross)

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)^*

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)

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

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)

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)^*

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)

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)^*

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)^*

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)

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

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

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)^*

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)

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)^*

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)

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)^*

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)

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)^*

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)

HPLC Estradiol measurements (lab-team)^*

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

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

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)