Electrical circuits as models for biological circuits
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1 Electrical circuits as models for biological circuits State 1 1 State Trigger State 0 0 Time Electrical circuits (light bulb) are ideal models because a trigger (i.e. light switch) produces an immediate response (i.e. crea9on of light).
2 State 1 The Reality of Biological Circuits Immediate response In contrast to electrical circuits, biological circuits do not respond immediately 0 1 Time Biological circuits exhibit a more gradual transi9on between states. State 0 Time Gradual response What is the molecular explana9on for this phenomenon?
![The Reality of Biological Circuits DNA Protein (output) Gene/Circuit Off RNA [Protein] Gene/Circuit On Protein (output) On phase Off](/docs-images/89/98622061/images/3-0.jpg "phase Time We can turn biological circuits off by inhibi9ng gene expression, but this does not account for proteins that were already")
3 The Reality of Biological Circuits DNA Protein (output) Gene/Circuit Off RNA [Protein] Gene/Circuit On Protein (output) On phase Off phase Time We can turn biological circuits off by inhibi9ng gene expression, but this does not account for proteins that were already produced.
4 Protein stability varies widely Collagen (half- life = 117 years) GFP (half- life = 25 hours) Ubiqui9n (decreases half- life to < 15 minutes ) Natural protein destruc9on can take minutes to hours GFP, on average, takes about 25 hours
5 Auxin- Based Transcrip9onal Regula9on a. AUX Repressors Auxin- Response Gene Repression Transcrip'on Ac'vator SCF- TIR1 Complex AUX Repressor Destruc'on b. Auxin- Response Gene Ac'va'on Low auxin: AUX/IAA repressors stay bound to ARF ac9vators High auxin: AUX/IAA repressors are degraded via SCF TIR1 complex
6 The Auxin Degrada9on Pathway Repressor Protein Degrada'on via Proteasome 1. Auxin is introduced and protein a^aches to TIR1 complex. 2. TIR1 promotes ubiqui9n a^achment. 3. Repressor protein is degraded via proteasome.
7 The Corona9ne Degrada9on Pathway Repressor Protein Degrada'on via Proteasome 1. Corona9ne is introduced and protein a^aches to COI1 complex. 2. COI promotes ubiqui9n a^achment. 3. JAZ protein is degraded via proteasome.
8 Protein degrada9on occurs at varying rates. We were inspired by the auxin pathway as an elegant means of protein destruc9on. Our Mo9va9ng Ques9ons Can we create a novel pathway that u9lizes a small- molecule inducible mechanism for a modular means of targe9ng and rapidly degrading a protein? Can we move the components that are na9ve to plant cells into yeast cells?
9 Designing a Corona9ne- Inducible Protein Degrada9on System in Yeast ADH1 ptef COI1 GFP- JAZ HA Modified promoter COI1 for yeast Tagged COI1 with HA for verifica9on GFP- linked JAZ1 or JAZ6 HA
10 Recrea9ng the Auxin Inducible Protein Degrada9on System in Yeast ADH1 TIR1 ptef GFP- AID myc Modified promoter (ADH1) to be integrated into yeast Tagged TIR1 with myc myc tag for verifica9on of successful integra9on GFP- linked degron (AID)
11 Recons9tuted small- molecule degrada9on systems in yeast Destroyed Destroyed 1 2 Both 1 2 and Destroyed
12 Auxin and Corona9ne Do Not Affect Yeast Growth No Treatment 280µM Auxin No Treatment 280µM Corona9ne To determine if the small molecules have any affect on yeast growth: 284 µm of either Auxin or Corona'ne was mixed with S288C yeast cells and analyzed by automated growth curve analysis.
13 Confirming expression of E3 ligases TIR1 and COI1 COI1- HA = 69kDA TIR1-9xMyc = 78 kda We cloned tagged versions of COI1 and TIR1 and integrated them into W303 yeast cells. These cells were analyzed by Western Blot using an an9body to the tag. We were able to successfully transform myc- tagged TIR1 and HA- tagged COI1 yeast cells.
14 Tes9ng GFP- Jaz6 Degrada9on in Yeast ADH1 COI1 ptef GFP- JAZ6 HA ADH1- COI1 integrated into W303 yeast Plasmid containing ptef- GFP- JAZ6 inserted as well Ready for tests with auxin and corona9ne
15 An9cipated Effects of Auxin and Corona9ne on the COI1- Jaz- GFP Construct in Yeast No Effect Jaz6- GFP Degraded
16 Corona9ne Induced Degrada9on of GFP- Jaz6 120 DMSO Corona'ne EtOH Auxin Rela9ve GFP Expression DMSO Corona'ne 250μM Corona'ne 125μM Yeast cells containing COI1 and GFP- Jaz6 were incubated for two hours, as indicated. Rela've GFP expression compared to untreated cells is shown Addi'on of Corona'ne but not auxin lead to specific degrada'on of GFP- Jaz6 in our cells
17 Summary of Our Accomplishments Corona9ne and Auxin showed no adverse effects on yeast growth No Treatment 280µM Auxin
18 Summary of Our Accomplishments Corona9ne and Auxin showed no adverse effects on yeast growth No Treatment 280µM Auxin COI1 and TIR1 were cloned, integrated, and expressed in yeast
19 Summary of Our Accomplishments Corona9ne and Auxin showed no adverse effects on yeast growth No Treatment 280µM Auxin COI1 and TIR1 were cloned, integrated, and expressed in yeast Constructed the Jaz pathway in yeast
20 Future Direc9ons Implemen9ng RFP and GFP into a single stain with both the JAZ and auxin pathways RFP GFP Achieve the ideal electrical- like circuit Modify an exis9ng biological circuit
21 New Parts Submi^ed to the igem Registry Jaz1: BBa_K Jaz6: BBa_K COI1: BBa_K Jaz1- RFP:BBa_K
22 Exis9ng BioBricks Used in Our Project AID- GFP: BBa_K igem Evry 2012 TIR1: BBa_K igem Evry 2012
23 Nevada igem Team at the College of Agriculture, Biotechnology, and Natural Resources Field Day 550+ a^endees Field Day Theme: Feed the World. Protect the Planet. Spoke about how synthe9c biology can be used to ba^le drought and food produc9on.
24 Nevada igem Speaking at the Nevada Student Speaker Compe99on 1,100+ a^endees Spoke about synthe9c biology and ethical implica9ons of what we do as scien9sts.
25 Acknowledgements Advisors: Dr. Veronica Zepeda and Dr. Ian Wallace UCSF igem team Liebman Lab at the University of Nevada Sponsors:
26 Nevada igem 2014 Khurram Fahim Tori Speicher Ma^ Hawn Dr. Veronica Zepeda Zoe Meraz Janice Bau9sta Josh Beard
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