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In this section you can see summaries of the research papers that Team Nevada read to help create the BAITSwitch. Additionally, you can see the beginning of our research, involving the affects of Auxin and Coronatine on Yeast Cells.
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Team Nevada
The BAITswitch
The Bioorthoginal Auxin Induceable Trigger Switch
Overview
This summer, the iGEM team of Nevada came up with a new strategy for rapid-response, protein degradation in yeast cells through our Bioorthoginal Auxin Inducible Trigger Switch (or BAITSwitch). Under biological conditions, protein degradation is a very slow process which can take anywhere from several hours to days to months! Some of these proteins are causing disease while accumulating. To combat this issue, our system is similar to that of a light switch, where a response is seen immediately after a trigger is toggled. This year we attempted to select a specifically-labeled protein for immediate degradation via the bioorthoginal auxin-inducible-degron (AID) System (Nishimura et al.2009). This Auxin system has been published and used by previous iGEM teams, but has never been combined with its parallel system, jasmonic acid, in non-plant cells. However, before we could start our journey we needed to understand some very unique characteristics about Protein Degradation.
- Understanding the Perfect Model
- Typical Protein Degradation Rates
- How Protein Degradation Works
- The BAITSwitch
How a Lightbulb Works
An easy way to explain our system is to imagine a light switch: a source of instantaneous change. A room can go from dark to light instantly by flipping the switch. Similarly, a protein can go from existing and causing havoc to being destroyed by “flipping the switch,” or adding auxin. Our research causes this change as fast as biologically possible
Understanding the Perfect Model
As seen in the graph to the right, a lightswitch is the perfect model from the fact that there is an imediate response (creation of light) after a trigger is activated (ie. flicking on the light switch). With 0 being representation of no light in a room and 1 being the room filled with light over the time span of a couple seconds.
Turning on a Light
Typical Protein Degradation Rates
One of the first things that we needed to understand was how long it usually takes a protein to degrade. As seen in the graph on the left from Junbo Liu and R. Sakari Piha, regular protein degradation can take from hours to months to eliminate the protein entirely. Hover over the graph or select the individual proteins to see their individual degradation times.
How Protein Degradation Works
After understanding that a protein's life can be fairly lengthy, we needed to learn how proteins are biologically broken down.
Ubiquitination
There are three types of enzymes involved in ubiquitination which make up the ubiquitin-proteasome system (UPS) called E1, E2, and E3. Together these three enzymes create a "three-step enzymatic cascade, resulting in the covalent transfer of ubiquitin to target proteins" (D. Kelly).
In the UPS, each enzyme serves a specific function to target the substrate protein for destruction by the 26S proteasome or alters their biochemical properties and subcellular organization in plants. Here, E1 serves as the ubiquitin activating enzyme while containing two isoforms. E2 serves as the ubiquitin-conjugating enzyme and is believed to have at least 40 different derivatives. Then, E3 serving as the ubiquitin ligase which is predicted to encode for more than a thousand potential E3 ligases. (Nitzan Shabek)
Although, most reasearch shows this system being used in several different species of plant, it also serves the same role in eukaryotic cells as well. (Nitzan Shabek)
The BAITSwitch
The Bait-Switch is composed of two plant hormone-based degron systems that work in a bioorthogonal fashion, meaning that they work independently of each other, thus eliminating the chance of adverse biological cross-talk. These two systems are analogous in component functions, and they both result in rapid and specific protein degradation. Current methods of post-translational degradation, such as RNAi, are nonspecific and can have adverse effects from off-target effects. However, previous research shows that the components of the bait-switch can be transferred from plants to yeast and other vertebrate cells. Although mammalian cells do not contain the receptors to recognize these plant hormones, they contain the same SCF degradation pathway, and this can be exploited to achieve targeted, rapid protein degradation.
Breakdown of the Auxin-Inducible-Degron (AID) system
Shown below is a breakdown of the BAITSwitch and how it is used to degrade proteins of interest.The individual components are shown below.
Our Team
Only through the joint efforts of all of these students and our wonderful advisor, Team Nevada was able to achieve all of their summer and semester goals.
Khurram Fahim
A Dual Major in Biochemistry and Neuroscience
Khurram Fahim is a senior biochemistry and neuroscience major at the University of Nevada, Reno. His future plans include attending dental school and continue pursuing research opportunities. Khurram modified and engineered new plasmids for integration into yeast. These included plasmids containing the IAA17 degron and GFP reporter. Some methods they performed are tagging of TIR1 with a Myc tag; primer design to create the insert of pTEF-GFP-IAA17; and later, work with JAZ6 in the COI1 system
Janice Bautista
A Dual Major in Biochemistry and Molecular Biology and Microbiology and Immunology
Janice Bautista was born in the Philippines and moved to Las Vegas, Nevada. She is in her fourth year at the University of Nevada, Reno majoring in Biochemistry and Molecular Biology and Microbiology and Immunology. Janice Bautista focused on the TIR1 system. She helped modified and engineered new plasmids for integration into yeast. These included plasmids containing the IAA17 degron and GFP reporter. Some methods they performed are tagging of TIR1 with a Myc tag; primer design to create the insert of pTEF-GFP-IAA17; and later, work with JAZ6 in the COI1 system.
Matthew Hawn
A Biochemistry Major
Matt Hawn is a Biochemistry Major and is in his fourth year at the University of Nevada, Reno. He is currently interested in attending medical school or working toward his PhD in Biochemistry. On the side, Matt has worked for several companies in website development. Matthew Hawn focused on the COI1 system. This included modification and engineering of appropriate plasmids to be joined to those of Khurram and Janice for the goal of a bioorthogonal system using auxin and coronatine in yeast. Matt also created many of the infographics and web design, as well as performed yeast growth assays.
Josh Beard
Biochemistry Major
Josh Beard is a biochemistry major from Las Vegas, Nevada. His interests include playing the guitar and cello! After graduating from UNR, he plans to attend graduate school in hopes of landing a career in research. Josh's main focus on this years iGEM project was to design the Auxin Inducible Degron plasmid that would ultimately be transformed into yeast. Josh Beard also worked with the COI1 system. He helped in the tagging of COI1 with HA and worked with the JAZ degron for introduction into E. coli and movement into yeast cells.
Tori Speicher
biochemistry Major
Tori Speicher was born and raised in Reno, Nevada. She is a second year Biochemistry and Molecular Biology Major at the University of Nevada, Reno. As the youngest member of the Nevada iGEM team Tori has learned a lot from her hands-on experience. Through iGEM, she has discovered her love for research and hopes to continue her education towards a PhD in biochemistry. Tori Speicher was responsible for all things parts registry related! She also performed many lab procedures such as gel electrophoresis, PCR, miniprep, etc.
Zoe Meraz
Biochemistry Major
As a first-generation college student, Zoe did not grow up talking about college but she knew she wanted to pursue science and that pursuit led her to the bounties of biochemistry. She hopes to further her studies through MD/PhD to become a coroner.Zoe focused on the synthesis of the construct of pTEF-GFP-JAZ1 and moving it from E coli into yeast. She also completed all safety form requirements. In addition, she focused on the COI1 system which included modification and engineering of appropriate plasmids to be joined to those of Khurram and Janice for the goal of a bioorthogonal system using auxin and coronatine in yeast
Nevada's Contribution to the Parts Registry
The Nevada iGEM team has submitted four new parts to the registry and will have shown that three of the parts work as expected. We have also used two parts previously submitted by the Evry 2013 team and shown that they function in yeast as a part of our BAIT switch project.Each part seen below is a part that Team Nevada contributed to the iGEM Parts Registry, along with a full description of what was added/modified. These parts were then used in various experiments to create the BAITSwitch.
Jaz1 is a degron tag utilized in the coronatine pathway. The protein of interest is tagged with Jaz1; when coronatine is added to the system, the protein and its tag are linked to the E3 ubiquitin ligase, COI1. Click the picture or the name below to be taken to the parts registry page.
RFP-Jaz1 is a degron tag utilized in the coronatine pathway. The protein of interest is tagged with RFP-Jaz1; when coronatine is added to the system, the protein and its tag are linked to the E3 ubiquitin ligase, COI1. Click the picture or the name below to be taken to the parts registry page.
Jaz6 is a degron tag utilized in the coronatine pathway. The protein of interest is tagged with Jaz6; when coronatine is added to the system, the protein and its tag are linked to the E3 ubiquitin ligase, COI1. Click the picture or the name below to be taken to the parts registry page.
COI-1 or Coronatine Insensitive-1 recruits JAZ-1 or JAZ-6 tagged substrates in the presence of Coronatine for degradation by the Proteasome.
TiRI is an ubuquitinase E3 that recognizes AID-tagged proteins and trigger their ubiquitination for further degradation. This part works with K812011 which is a AID tagged GFP. OsTirI is a protein coming from rice that have been identified as working in mammalian cells and yeast. This part takes place in a device patented by Kanemaki Masato, Kakimoto Tatsuo, Nishimura Kohei, Takisawa Haruhiko and Fukagawa Tatsuo for yeast and mammalian cells use . However the patent does not cover the use for oviparian such as frogs and chicken.
This part is a GFP coding sequence fused to an AID tag for its recognition by the ubiquitinase E3 that induces its degradation in the presence of auxin in the eukaryote cell. This is designed to be used in pSB1C3 vector. This part is a part of a device patented by Kanemaki Masato, Kakimoto Tatsuo, Nishimura Kohei, Takisawa Haruhiko and Fukagawa Tatsuo for yeast and mammalian cells use.
Our Project
Below is a breakdown of the four major components of our research involving the BAITSwitch. The project started with an understanding of previous research and was then broken down into our two systems, the Auxin and Coronatine system. We then move on to discuss our results and our future plans for the BAITSwitch. We received some parts and plasmids from the UCSF_UCB iGEM Team and the Liebman lab at the University of Nevada, Reno.
The breakdown of how Team Nevada genetically designed and engineered the Auxin Degradation system in the BAITswitch.
The breakdown of how Team Nevada genetically designed and engineered the Coronatine Degradation system in the BAITSwitch.
In this final section you can see the final results of combining the Auxin and Coronatine System to form the BAITSwitch. Furthermore, you can see the future plans that Team Nevada has for the BAITSwitch, along with its real world application.
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