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Team:Copenhagen/Project
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Table of Contents
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Rescue
CyperMan - Kills fungus
We hope to be able to develop bacteria that can kill fungus. CyperMan - Saves the Fish
Cytochrome p450 CYP 1 & 2 - the source of CyperMans water cleansing power Cytochromes p450 is one of the largest gene superfamilies coding for enzymes present in the genomes in all biological kingdoms [1]. The enzymatic activities of these proteins are extremely diverse with activity in biotransformation of drugs, bioconversion of xenobiotics, biosynthesis of compounds as steroids, fatty acids, eicosanoids, fat soluble vitamins and bile acids. Furthermore, cytochrome p450’s are involved in the conversion of alkanes, terpenes and aromatic compounds as well as degradation of herbecides and insecticides [1]. We will exploit this activity of cytochromes p450 in the bioconversion of xenobiotics to neutralize damaging medical residues in waste water. Cytochromes p450 are defined as heme-thiolate proteins featuring a particular spectral signature at 450 nm, thus the name [2]. Despite the label as cytochromes, these proteins are not involved in electron transfers, but act as monooxygenases in a wide range of reactions such as epoxidation, N-dealkylation, O-dealkylation, S-oxidation and hydroxylation [3]. We will focus on the hydroxylating property of the cytochromes, which is also the defining reaction for these enzymes. The reductive activation of molecular oxygen reduces one of the oxygen atoms to a molecule of water, as the other is inserted into the substrate [2].
Fig 1
A key donor of electrons for the reduction of molecular oxygen is the NADPH dependent cytochrome p450 reductase (CPR). This protein shuttles electrons from NADPH through the FAD and FMN- coenzymes into the iron of the prosthetic heme group of cytochrome p450 [4]. A characterization of the cytochromes p450 is the main objective with this project. We will exploit the wide specificity of these cytochromes to target many different damaging agents continuously by hydroxylation, which can reduce the damaging activity of e.g. estrogen.
Cytochrome p450 CYP 79 - the source of CyperMans fungus destroying power Background information: It has been proposed in the article Dynamic Metabolons [5] that the assembly and disassembly of enzymes complexes, so-called metabolons, may differentiate plant defense responses to insect attack and fungal infection. The two CYP79 and CYP71 subfamilies are part of a metabolon complex that shifts between assembly and disassembly, according to interactions with proteins or from specific metabolic demands. The CYP79 subfamily can by itself produce a substance called 4-hydroxyphenyl-acetaldeyde oxime. This substance is generally toxic to fungi and frequently used as chemical fungicides. They function by inhibiting mitochondrial oxidases and thereby promote lipid peroxidation and the production of toxic reactive oxygen species[5]. The CYP79A1 and CYP79A2 proteins use L-Tyrosine as a substrate, which it hydroxylates twice on the amino-group in the presence of NADPH and O2. The products of these reactions are quite unstable and dehydration and decarboxylation will then produce 4-hydroxyphenyl-acetaldeyde oxime. [6] The CYP79B1 isoform is specific for tryptophan and therefore it produces indole 3-acetaldoxime instead.
The Project: Our goal is to express different CYP79’s (more specifically from the subfamilies A1, A2 and B1) in E. Coli, to determine whether they can actually be expressed as working proteins in them. Then our idea is introduce a promoter to the system, which reacts to a specific chemical signal that is sent out by fungi. We are also working with both the iGEM teams from DTU. To ensure that the protein only will be expressed in the presence of fungi, we plan to incorporate a regulating system that uses RNA-RNA interactions. A constitutive promoter will make sure that a small 9 bp piece of RNA is constantly expressed. This piece of RNA is complimentary to the ribosomal binding site of our plasmid-RNA and will therefore bind to it. This will prevent our gene of interest from being translated into protein. Another promoter that is induced by a fungal elicitor will start the production of another small 9 bp piece of RNA that is complimentary to the first 9 bp RNA when these bind there is no inhibition of the gene translation. This is a system developed by DTU-DENMARK1. Impact factors: Open source BioBricks: Our project has the aim to characterize Cytochrome p450 as a BioBrick and thus add it to the partsregistry of BioBricks, a continuously growing collection of genetic parts that can be mixed and matched to build synthetic biology devices and systems (partsregistry.org). Hence our project contributes to the expansion of the partsregistry, benefitting all researchers with interest in this particular protein, since cytochrome p450 doesn’t exist in the partsregistry. The partsregistry is founded on an open source philosophy, a philosophy we see as a great advantage for development and improvement of the number BioBricks already existing. The addition of cytochrome p450 to this catalogue will perhaps encourage other groups to look at other aspects of the many functions of p450 and thus contribute to the formation of new biological systems – further expanding the field of synthetic biology. Ecological and economical prospects: Our future aim with our project is to determine whether it would be a viable solution to use an oxime producing agent as a type of fungicide. The best scenario would be if the plants themselves was able to produce the oximes and thus protect themselves and eliminating the need for fungicides. Another future aim with our project is to improve wastewater treatments and thus contribute to a cleaner and uncontaminated drinking water for everyone. With this scientific project, we will move towards this realization, but more research is needed. Our belief is that further research on the use of cytochrome p450 will give a valid and an inexpensive product for use in wastewater treatment plants, thus benefitting the environment and our planet. References [1] F. Hannemann, A. Bichet, K. M. Ewen, R. Bernhardt: Cytochrome p450 systems – biological variations of electron transport chains. Biochemica et Biophysica Acta 1770 (2007) 330 - 344 [2] Paul R. Ortiz de Montellano. Hydrocarbon Hydroxylation by Cytochrome P450 Enzymes. Chem Rev. 2010 February 10; 110(2): 932 [3] http://www.anaesthetist.com/physiol/basics/metabol/cyp/Findex.htm [4] T. Laursen, K. Jensen,B. L. Møller: Conformational changes of the NADPH dependent cytochrome P450 reductase in the course of electron transfer to cytochromes P450. Biochemica et Biophysica Acta 1814 (2011) 132-138 [5]Moller, B. L. (2010) Plant science. Dynamic metabolons, Science 330, 1328-1329. [6]Koch, B. M., Sibbesen, O., Halkier, B. A., Svendsen, I., and Moller, B. L. (1995) The primary sequence of cytochrome P450tyr, the multifunctional N-hydroxylase catalyzing the conversion of L-tyrosine to p-hydroxyphenylacetaldehyde oxime in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench, Arch Biochem Biophys 323, 177-186.
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iGEM Medals Requirements Bronze  Team registration Complete Project Summary form Team Wiki
Present a poster and a talk at the iGEM Jamboree At least one new submitted and well-characterized standard BioBrick Part or Device.Silver
Gold For Gold Medal, either of the following must be checked
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| Comments or questions to the team? Please mail us at igemcopenhagen@gmail.com |
