Contribution

Previous iGEM teams have worked with CYPs including iGEM14_TU_Darmstadt, iGEM15_HSNU-TAIPEI and iGEM19_Sydney_Australia. However, they have used them for medical and productive purposes, such as monitoring and diagnosing harmful liver poisons and even synthesizing substances. The iGEM team from Copenhagen in 2011, are the only ones who have used CYP proteins for bioremediation purposes, their goal was to degrade oestrogens from wastewaters.

The 2019 team from Kaiserslautern have previously worked on using Chlamydomonas to degrade micro plastics and the 2020 team from Kaiserslautern worked to address water pollution with diclofenac.

Following our predecessors, we have also created a large library of Chlamy specific Modular Cloning or MoClo parts including 6 level 0 parts, 9 level 1 parts and 25 level 2 parts for different CYPs with different chemical degradation functionalities. Our contribution to iGEM and the planet include a library of ready-to-use parts for many types of chemical pollutants. The Cytochromes we have on hand include CYP2D6, 3A4, 81A10V7, 9Q3 and CamC. We also added numerous tags in order to visualize and quantify these parts once they have been inserted.

Here is a little overview of all the parts we created!

Many of the parts we created include the level 0 part BBa_K3002017, which was created by iGEM 2019 TU Kaiserslautern, this part is specialized for Chlamy and codes for 3xHA-tag. This construct was invaluable to us, as it allowed us to easily detect if the protein was synthesized, by performing a simple Western Blot and using anti-HA-tag-antibodies. This essential part can be found in the iGEM parts registry

The experiences and the information we acquired while using these parts can be found in the iGEM registry page for parts, where they are clearly and easily accessible for future iGEM teams.

After creating these parts it was important fo us to check if the CYPs are active, so we searched for activity assays, unfortunately CYPs activity is usually measured by using carbon monoxide. Carbon monoxide is a dangerous gas that can cause nausea, dizziness and even be fatal.¹
This classic method is a spectral assay, the key idea is that the heme coenzyme can react with carbon monoxide (CO) to make a special complex, and this complex absorbs light at a particular wavelength of around 450 nm, so all we have to do is measure the wavelength, if the absorbance is around 450nm we would know our protein is correctly synthesized and active. But due to the safety concerns associated with carbon monoxide, we have opted to develop our own assay to detect production of CYP enzymes. For this assay samples were prepared similar to Native PAGE samples. Then the absorption at 420nm (heme) and the protein concentration at 280 nm are measured and compared to a wild type control that does not synthesize our CYPs.²

Our novel protocol offers enhanced safety by eliminating the need for carbon monoxide, and it is highly convenient for any laboratory since only a nanodrop is needed. The basis of our new protocol is to keep the proteins in their native folded forms instead of denaturing them first, in this way the absorption characteristics of the protein is maintained.

Our new and improved protocol is available here.

This method has been verified with 3xHA-tag protein samples in order to make sure that the method works. However the best part of our innovative method is that it also allows for a screening method of CYPs without needing a tag! This is crucial according to Dr. Hugues Renault from „Institut de biologie moléculaire des plantes“ in Strasbourg, a CYP expert we had the honor of interviewing. He discovered that any form of tag can hinder CYP activity. Which is why our protocol was so important in order to efficiently screen untagged proteins.