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Project Sam Keating, May Li, Pranesh Navarathna 2 University of - - PowerPoint PPT Presentation

Jul 31, 2023 390 likes •715 views

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

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UMBC iGEM: The Retriever Project

Sam Keating, May Li, Pranesh Navarathna

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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

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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.

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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.

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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

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Copper Metabolism in E. . coli

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Copper Metabolism in E. . coli

7 Cu+ Cu+ Cu+ Cu+ Cu2+ Cu+ Cu+ Cu+ Cu+ Cu+

METALLOTHIONEIN

  • 1 cysteine group binds 1 Cu+
  • 2 cysteine groups bind to 1 Cu2+

Cysteine Group Native to Yeast

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Ret1 Ret2 Ret3

3 Avenues of Development

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Ret1 Ret2 Ret3

CUP1 Codon-Optimized CUP1 CUP1-LamB Fusion

Part: BBa_K1811777 Part: BBa_K1811888 Part: BBa_K1811666 9

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Gblock Hi-Fi Assembly Transformation Minipreps and Digest DNA Gel Growth Experiments

Cell Line Development

Copper Measurements SDS Gel Analysis 10

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Experimental Procedure:

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Grow Culture on Plate Overnight Culture Inoculate Shake Flasks Grow for 7 hours Sample Every Hour for OD Measure Copper in Supernatant with Plate Reader Centrifuge SDS-PAGE

  • E. coli K-12
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PRELIMINARY RESULTS

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Ret1 Growth Experiments

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Ret1 Copper Analysis

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Copper Levels at End of Growth Copper Levels vs Time Extracellular Copper Concentrations

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Ret1 Results Analysis

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  • 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.
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Ret2 Growth Analysis

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Ret2 SDS-Gel Analysis

17 32 kD

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  • 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
  • ther proteins native to E. coli
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Ret2 Copper Analysis

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Copper Levels vs Time Extracellular Copper Concentrations Copper Levels at End of Growth

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Ret2 Results Analysis

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  • 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
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Ret3: LamB Fusion

21 Metallothionein Gene Fusion Site: AA Position 153 Transmembrane protein LamB:

  • 18 stranded antiparallel beta barrel
  • Maltose transporter

Extracellular Intracellular

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Ret3 Growth Analysis

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Ret3

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Ret3 Copper Analysis

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Copper Levels at End of Growth Extracellular Copper Concentrations Copper Levels vs Time

Possibly due to metallothionein proteins binding copper even after cells died.

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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.

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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

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Acknowledgements

UMBC Support:

  • Dr. Cynthia Wagner
  • Dr. Stephen Freeland
  • Professor Julie Wolf
  • Dr. William LaCourse
  • Dr. Stephen Mang
  • Dr. Hal Schreier
  • Dr. Philip Farabaugh
  • Dr. Zeev Rosenzweig
  • Dr. Elsa Garcin
  • Dr. Suzanne Ostrand-Rosenberg
  • Ralph Murphy, Teaching Lab Technician

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Funding Sources:

  • UMBC Student Government

Association

  • Mrs. Felicia Felton, Assistant

Director of Annual Giving at UMBC.

  • Department of Biological Sciences
  • Department of Chemistry &

Biochemistry

  • UMBC Honors College
  • College of Natural and

Mathematical Sciences at UMBC

  • Interdisciplinary Studies at UMBC
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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

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BACKUP SLIDES

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Ret1 Development

Construct Size: 247 bp Total Plasmid Size: 2.3kb 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) Figure : Gel Analysis of Part BBa_K1811777 Control Ladder Miniprep #2 Miniprep #1 0.5kb 0.25kb 29

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Ret3 Development

Ret3 is designed to have metallothionein bound to the outer membrane exposed to the environment. This was done by fusing a membrane transport sequence called LamB 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 Hi-Fi assembly product was transformed into E. coli and minipreps were prepared for gel analysis 2 kb 1.6kb 30 4 3 2 1

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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

  • E. coli and minipreps were prepared for gel

analysis (Figure)

Figure 2: Gel analysis of Part BBa_K1811888 (Codon- Optimized CUP1), From left to right: Colonies 1-12 0.5kb 0.25kb 31