UMBC iGEM: The Retriever Project Sam Keating, May Li, Pranesh Navarathna
2 University of Mary ryland, Baltimore County • From Baltimore County, Maryland, USA • This is our FIRST time at the Jamboree! • Our mascot is the Chesapeake Bay Retriever • The Chesapeake Bay spans six states including Maryland
3 Project Motivation • The EPA guidelines consider copper concentrations above 1.3ppm [1] in drinking water to be toxic. • The level in the Chesapeake Bay is 8.3ppm [2] • That’s almost 6.5x the toxicity level! Potential Solution: Copper Remediating E.coli [1] -Hall, L. W., Scott, M. C. and Killen, W. D. (1998), Ecological risk assessment of copper and cadmium in surface waters of Chesapeake Bay watershed. Environmental Toxicology and Chemistry, 17: 1172 – 1189. doi:10.1002/etc.5620170626 [2] -"Basic Information about Copper in Drinking Water." Basic Information about Copper in Drinking Water . Web. 14 Sept. 2015.
4 Copper Remediating E.c .coli • Broad Goal: To engineer a robust bacterial strain capable of lowering copper amounts to a safe level without affecting the ecosystem it is in. • Summer 2015 - Proof of Concept: Engineer a strain that can lower copper concentrations in a lab setting. • Similar work done by different teams: Tokyo NoKoGen 2011, Cornell 2014, Newcastle 2009, Groningen 2009 on absorbing As, Cd, Hg.
5 Challenges of f Copper • Copper is an essential nutrient used by cells in redox reactions • Used in cytochrome c-oxidase and is necessary for energy production • Is normally held tightly by proteins inside cells • Is very toxic at small concentrations of unbound cellular copper • E. coli cells are sensitive to zeptomolar (10 -21 M) concentrations of free copper
6 Copper Metabolism in E. . coli
7 Copper Metabolism in E. . coli Native to Yeast Cu + Cu + Cu + Cu + Cysteine Group METALLOTHIONEIN Cu + Cu + • 1 cysteine group binds 1 Cu + Cu + • 2 cysteine groups bind to 1 Cu 2+ Cu 2+ Cu + Cu +
8 Ret1 Ret2 3 Avenues of Development Ret3
9 CUP1 Ret1 Part: BBa_K1811777 Codon-Optimized Ret2 CUP1 Part: BBa_K1811888 CUP1-LamB Fusion Ret3 Part: BBa_K1811666
10 Gblock Hi-Fi Assembly Cell Line Development Transformation Minipreps and Digest Copper Measurements Growth DNA Gel Experiments SDS Gel Analysis
Experimental Procedure: 11 Grow for 7 hours E. coli K-12 Inoculate Shake Flasks Overnight Culture Grow Culture on Plate Sample Every Hour for OD Measure Copper in Supernatant with Plate Reader Centrifuge SDS-PAGE
12 PRELIMINARY RESULTS
13 Ret1 Growth Experiments
14 Ret1 Copper Analysis Extracellular Copper Concentrations Copper Levels vs Time Copper Levels at End of Growth
15 Ret1 Results Analysis • The Ret1 plasmid does not hinder cell growth. Growth rates are similar for Ret1 and control. • Growth curves show that 4mM is above the threshold at which copper affects cell growth rate. • Preliminary data suggests that metallothionein may be expressed; SDS gels were inconclusive. • Minimal copper remediation relative to the control.
16 Ret2 Growth Analysis
17 Ret2 SDS-Gel Analysis 32 kD
18 • Metallothionein is not native to E. coli • Has been shown that the reducing cellular environment can degrade cysteine groups • These cysteine groups can end up forming clusters with other proteins native to E. coli
19 Ret2 Copper Analysis Extracellular Copper Concentrations Copper Levels vs Time Copper Levels at End of Growth
20 Ret2 Results Analysis • Ret2 had a slightly lower growth rate than that of the control. • Ret2 had a different protein expression profile than that of the control. • Minimal copper remediation relative to control
21 Ret3: LamB Fusion Metallothionein Gene Fusion Site: AA Position 153 Extracellular Transmembrane protein LamB: • 18 stranded antiparallel beta barrel • Maltose transporter Intracellular
22 Ret3 Growth Analysis Ret3
23 Ret3 Copper Analysis Extracellular Copper Concentrations Copper Levels vs Time Copper Levels at End of Growth Possibly due to metallothionein proteins binding copper even after cells died.
24 Ret3 Results Analysis • Growth of Ret3 was very minimal compared to that of the control. • Likely due to stress put on cells to express metallothionein with a strong constitutive promoter. • Could be affected by the increased number of LamB proteins. • Ret3 showed more copper remediation at high initial concentrations of copper.
25 Future Work @ UMBC iGEM • Explore Ret2 and Ret3 further with statistical analysis of data • Consider fine tuning cell lines with different promoters • Strong, constitutive promoter likely too demanding for cell growth • Use of a copper sensitive promoter, such as pCopA or CueR • After fine tuning, explore the addition of a gasification gene for cell harvesting, as has been done by previous iGEM teams (Groningen 2009) Entrepreneurship and Outreach: • Explore development of Retriever as a marketable product/service • Build relationships with community DIY Bio spaces and surrounding high schools to foster culture of innovation and entrepreneurship in synthetic biology
26 Acknowledgements UMBC Support: Funding Sources: • Dr. Cynthia Wagner • UMBC Student Government • Dr. Stephen Freeland Association • Professor Julie Wolf • Mrs. Felicia Felton, Assistant • Dr. William LaCourse Director of Annual Giving at UMBC. • Department of Biological Sciences • Dr. Stephen Mang • Department of Chemistry & • Dr. Hal Schreier Biochemistry • Dr. Philip Farabaugh • UMBC Honors College • Dr. Zeev Rosenzweig • College of Natural and • Dr. Elsa Garcin Mathematical Sciences at UMBC • Dr. Suzanne Ostrand-Rosenberg • Interdisciplinary Studies at UMBC • Ralph Murphy, Teaching Lab Technician
27 And Of f Course, , The Rest of f Our Team! • Alex Kusnetsov • Natithorn “Frame” Bhusri • William Larsen Angel • Mark Kerr • John Jayman • Mukta Bain • Pari Majethia • Dennis Fasciani • Tarik Hawkins • Thomas Coard
28 BACKUP SLIDES
29 Ret1 Development Miniprep #2 Miniprep #1 Control Ladder Gblock for Part BBa_K1811777 (CUP1) was synthesized by IDT NEB Hi-Fi Assembly was used to ligate Part BBa_K1811777 (CUP1) with plasmid backbone pSB1C3 Hi-Fi Assembly product was transformed into E.coli and minipreps were prepared for gel analysis (Figure) 0.5kb 0.25kb Construct Size: 247 bp Total Plasmid Size: 2.3kb Figure : Gel Analysis of Part BBa_K1811777
30 Ret3 is designed to have metallothionein bound to the outer membrane exposed to the environment. Ret3 Development This was done by fusing a membrane transport sequence called LamB 4 3 2 1 upstream of the codon optimized CUP1 coding sequence. CUP1 + LamB = 1585bp Gblock for Part: BBa_K1811666 (LamB +Codon-Optimized CUP1 NEB Hi Fi Assembly was used to ligate Part: BBa_K1811666 (LamB+Codon-Optimized CUP1) with backbone psb1c3 2 kb Hi-Fi assembly product was transformed into E. coli and minipreps were 1.6kb prepared for gel analysis
31 Ret2 Development CUP1 Gene was optimized for E.coli via IDT Gblock for Part BBa_K1811888 (Codon- Optimized CUP1) NEB Hi Fi Assembly was used to ligate Part BBa_K1811888 (Codon-Optimized CUP1) with backbone psb1c3 Hi-Fi assembly product was transformed into 0.5kb E. coli and minipreps were prepared for gel 0.25kb analysis (Figure) Figure 2: Gel analysis of Part BBa_K1811888 (Codon- Optimized CUP1), From left to right: Colonies 1-12
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