Using Multi-chromatic Light McMaster University Hamilton, Ontario, - - PowerPoint PPT Presentation

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Using Multi-chromatic Light McMaster University Hamilton, Ontario, - - PowerPoint PPT Presentation

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` Automating Protein Production Using Multi-chromatic Light McMaster University Hamilton, Ontario, Canada Visit Our Wiki 2015.igem.org/Team:Hamilton_McMa ster All of the designs, constructs, and experiments presented in this poster were

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All of the designs, constructs, and experiments presented in this poster were performed by the students of the 2015 Hamilton McMaster University team. Poster and Wiki design by Kushal Panchal.

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2015.igem.org/Team:Hamilton_McMa ster

Automating Protein Production Using Multi-chromatic Light

McMaster University Hamilton, Ontario, Canada

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All of the designs, constructs, and experiments presented in this poster were performed by the students of the 2015 Hamilton McMaster University team. Poster and Wiki design by Kushal Panchal.

Visit Our Wiki

2015.igem.org/Team:Hamilton_McMa ster

Who are we?

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All of the designs, constructs, and experiments presented in this poster were performed by the students of the 2015 Hamilton McMaster University team. Poster and Wiki design by Kushal Panchal.

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2015.igem.org/Team:Hamilton_McMa ster

How do we improve or build on current biological protein expression systems?

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All of the designs, constructs, and experiments presented in this poster were performed by the students of the 2015 Hamilton McMaster University team. Poster and Wiki design by Kushal Panchal.

Visit Our Wiki

2015.igem.org/Team:Hamilton_McMa ster

Recombinant Protein Expression

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All of the designs, constructs, and experiments presented in this poster were performed by the students of the 2015 Hamilton McMaster University team. Poster and Wiki design by Kushal Panchal.

Visit Our Wiki

2015.igem.org/Team:Hamilton_McMa ster

Recombinant Protein Expression

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All of the designs, constructs, and experiments presented in this poster were performed by the students of the 2015 Hamilton McMaster University team. Poster and Wiki design by Kushal Panchal.

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2015.igem.org/Team:Hamilton_McMa ster

Gene Induction

Gene expression controlled by the addition of an inducer

How do we make gene induction easier and more efficient?

  • Requirement to make or

purchase the chemical

  • Non-reversable
  • Human intervention
  • Install expensive

automated fluidic systems Disadvantages

  • Straightforward
  • Well characterized
  • Controlled

Advantages

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All of the designs, constructs, and experiments presented in this poster were performed by the students of the 2015 Hamilton McMaster University team. Poster and Wiki design by Kushal Panchal.

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2015.igem.org/Team:Hamilton_McMa ster

Light-Inducible Systems

Phytochromes and Cyanobacteriochromes

  • Within plants and cyanobacteria like Synechocystis

PCC6803

  • Tabor et. al (2011) have demonstrated two cases

Transforms external light stimulus into a transcriptional response

Phosphotransfer Interior Exterior Phytochrome

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All of the designs, constructs, and experiments presented in this poster were performed by the students of the 2015 Hamilton McMaster University team. Poster and Wiki design by Kushal Panchal.

Visit Our Wiki

2015.igem.org/Team:Hamilton_McMa ster

Recombinant Protein Expression

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All of the designs, constructs, and experiments presented in this poster were performed by the students of the 2015 Hamilton McMaster University team. Poster and Wiki design by Kushal Panchal.

Visit Our Wiki

2015.igem.org/Team:Hamilton_McMa ster

Cell Lysis

Mechanical (French press, sonication, freeze-thaw) Chemical (detergents, alkaline lysis) Biological (lysozyme) Exist and work and serve their purpose, but:

  • Costly
  • Contaminate
  • Denature
  • Some are not applicable to all cells
  • Human intervention

How do we make cell lysis more ubiquitous and effective?

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All of the designs, constructs, and experiments presented in this poster were performed by the students of the 2015 Hamilton McMaster University team. Poster and Wiki design by Kushal Panchal.

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2015.igem.org/Team:Hamilton_McMa ster

Viral Holin and Endolysin

  • Utilized by viruses during

replication

  • Forms holes in membrane,

leading to:

  • Depolarization
  • Release of endolysin

Endolysin Peptidoglycan

Holin

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All of the designs, constructs, and experiments presented in this poster were performed by the students of the 2015 Hamilton McMaster University team. Poster and Wiki design by Kushal Panchal.

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2015.igem.org/Team:Hamilton_McMa ster

Project Design

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All of the designs, constructs, and experiments presented in this poster were performed by the students of the 2015 Hamilton McMaster University team. Poster and Wiki design by Kushal Panchal.

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2015.igem.org/Team:Hamilton_McMa ster

Plasmid Design – Chromophore

pSB1A3: AmpR Backbone BBa_I0500: pBad/araC BBa_I15008: ho1 Bba_I5009: pcya

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All of the designs, constructs, and experiments presented in this poster were performed by the students of the 2015 Hamilton McMaster University team. Poster and Wiki design by Kushal Panchal.

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2015.igem.org/Team:Hamilton_McMa ster

Plasmid Design – Red

pSB1K3: KanR Backbone Bba_I14018: p(Bla) BBa_K592018: cph8 BBa_K769001: PompC-GFP

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All of the designs, constructs, and experiments presented in this poster were performed by the students of the 2015 Hamilton McMaster University team. Poster and Wiki design by Kushal Panchal.

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2015.igem.org/Team:Hamilton_McMa ster

Plasmid Design – Green

pSB1C3: CamR Backbone Bba_I14018: p(Bla) PCR’s from pJT122: ccaS PCR’s from pJT122: ccaR PCR’s from pJT122: PcpcG2 BBa_K112000: T4Holin Bba_L112806: T4Endolysin

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All of the designs, constructs, and experiments presented in this poster were performed by the students of the 2015 Hamilton McMaster University team. Poster and Wiki design by Kushal Panchal.

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2015.igem.org/Team:Hamilton_McMa ster

Tabor et al., 2011

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All of the designs, constructs, and experiments presented in this poster were performed by the students of the 2015 Hamilton McMaster University team. Poster and Wiki design by Kushal Panchal.

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2015.igem.org/Team:Hamilton_McMa ster

Methodologies

BL21

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All of the designs, constructs, and experiments presented in this poster were performed by the students of the 2015 Hamilton McMaster University team. Poster and Wiki design by Kushal Panchal.

Visit Our Wiki

2015.igem.org/Team:Hamilton_McMa ster

Methodologies

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All of the designs, constructs, and experiments presented in this poster were performed by the students of the 2015 Hamilton McMaster University team. Poster and Wiki design by Kushal Panchal.

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2015.igem.org/Team:Hamilton_McMa ster

Results – p(bad)-ho1 (S1PC)

Ladder S1PC S1PC S1PC 1000bp 5000bp 500bp 2000bp

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All of the designs, constructs, and experiments presented in this poster were performed by the students of the 2015 Hamilton McMaster University team. Poster and Wiki design by Kushal Panchal.

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2015.igem.org/Team:Hamilton_McMa ster

Results – p(bad)-ho1

Successful transformation

  • f completed gene

section Sequence verification via MOBIX

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All of the designs, constructs, and experiments presented in this poster were performed by the students of the 2015 Hamilton McMaster University team. Poster and Wiki design by Kushal Panchal.

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2015.igem.org/Team:Hamilton_McMa ster

Results – Enzymes & Buffers

Digestions stopped working: Parallel tests to resolve

Ladder E&S E&S E&P E&P X&S X&S X&P X&P Enzymes for cut sites C.S. 3.1 C.S. 3.1 C.S. 3.1 C.S. 3.1 Buffer

CutSmart worked NEB 3.1 did not work

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All of the designs, constructs, and experiments presented in this poster were performed by the students of the 2015 Hamilton McMaster University team. Poster and Wiki design by Kushal Panchal.

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2015.igem.org/Team:Hamilton_McMa ster

Results – PCR with overhang

  • Parts required for light sensitivity
  • Not available on campus
  • 3 genes were PCR’d out of a cassette

(Addgene): Pcpcg2 (~300 bp) ccaS (~2’000 bp) ccaR (~700 bp)

  • Overhangs of E, X, S, and P, cut sites were

introduced using primer design Pending submission to iGEM as new BioBrick

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All of the designs, constructs, and experiments presented in this poster were performed by the students of the 2015 Hamilton McMaster University team. Poster and Wiki design by Kushal Panchal.

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2015.igem.org/Team:Hamilton_McMa ster

Results – Progress Report

Progress

  • Constructed majority of Chromophore plasmid
  • Learned, invested into team education
  • PCR’d parts with BioBrick compatibility

Resolved Difficulties

  • Starting late in the year ✔
  • Digestions ✔
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All of the designs, constructs, and experiments presented in this poster were performed by the students of the 2015 Hamilton McMaster University team. Poster and Wiki design by Kushal Panchal.

Visit Our Wiki

2015.igem.org/Team:Hamilton_McMa ster

Hardware

Simple by design – prototype developed

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All of the designs, constructs, and experiments presented in this poster were performed by the students of the 2015 Hamilton McMaster University team. Poster and Wiki design by Kushal Panchal.

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2015.igem.org/Team:Hamilton_McMa ster

Hardware

  • 1. Box encloses shaker within incubator
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All of the designs, constructs, and experiments presented in this poster were performed by the students of the 2015 Hamilton McMaster University team. Poster and Wiki design by Kushal Panchal.

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2015.igem.org/Team:Hamilton_McMa ster

Hardware

  • 2. Far red light (705nm) induces POI production
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All of the designs, constructs, and experiments presented in this poster were performed by the students of the 2015 Hamilton McMaster University team. Poster and Wiki design by Kushal Panchal.

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2015.igem.org/Team:Hamilton_McMa ster

Hardware

  • 3. Green light (535nm) to lyse cells
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All of the designs, constructs, and experiments presented in this poster were performed by the students of the 2015 Hamilton McMaster University team. Poster and Wiki design by Kushal Panchal.

Visit Our Wiki

2015.igem.org/Team:Hamilton_McMa ster

Human Practices – Outreach

Social Media (Facebook, Twitter) and website

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All of the designs, constructs, and experiments presented in this poster were performed by the students of the 2015 Hamilton McMaster University team. Poster and Wiki design by Kushal Panchal.

Visit Our Wiki

2015.igem.org/Team:Hamilton_McMa ster

Human Practices

Open-Access Educational Blog

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All of the designs, constructs, and experiments presented in this poster were performed by the students of the 2015 Hamilton McMaster University team. Poster and Wiki design by Kushal Panchal.

Visit Our Wiki

2015.igem.org/Team:Hamilton_McMa ster

Collaboration

  • oGEM - Met with other Ontario iGEM teams
  • Discussed projects, progress, shared ideas and tips
  • Used iGEM Wiki Generator developed by University of Toronto
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All of the designs, constructs, and experiments presented in this poster were performed by the students of the 2015 Hamilton McMaster University team. Poster and Wiki design by Kushal Panchal.

Visit Our Wiki

2015.igem.org/Team:Hamilton_McMa ster

Future Work

Project

  • Complete plasmid construction
  • Submit new BioBrick to iGEM Registry

Hardware

  • Complete prototype: narrow bandpass filters
  • Develop enclosure-box that is also an incubator

Human Practices

  • Open-Access Educational Blog
  • GEM Collaboration
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All of the designs, constructs, and experiments presented in this poster were performed by the students of the 2015 Hamilton McMaster University team. Poster and Wiki design by Kushal Panchal.

Visit Our Wiki

2015.igem.org/Team:Hamilton_McMa ster

Conclusion

  • Developed a novel application for a green/red light sensitive

expression system characterized in E. Coli

  • Our system has major applications in industry and laboratory settings
  • Commenced preliminary assembly of required plasmids
  • Developed BioBricks currently being finalized for submission to the

registry

  • Created social media outreach to engage and educate the community

about synthetic biology; corresponded with oGEM to spread synbio across Ontario

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All of the designs, constructs, and experiments presented in this poster were performed by the students of the 2015 Hamilton McMaster University team. Poster and Wiki design by Kushal Panchal.

Visit Our Wiki

2015.igem.org/Team:Hamilton_McMa ster

Acknowledgements

Supervisors and Advisors

  • Dr. Kimberley Dej, Dr. Rosa da Silva, Dr. Marie Elliot, Alison Cowie,

Katherine Cornelius Sponsors

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All of the designs, constructs, and experiments presented in this poster were performed by the students of the 2015 Hamilton McMaster University team. Poster and Wiki design by Kushal Panchal.

Visit Our Wiki

2015.igem.org/Team:Hamilton_McMa ster

Thanks for listening!

Any questions? Zone 2 - 36

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All of the designs, constructs, and experiments presented in this poster were performed by the students of the 2015 Hamilton McMaster University team. Poster and Wiki design by Kushal Panchal.

Visit Our Wiki

2015.igem.org/Team:Hamilton_McMa ster

References

  • 1. Tabor, J. J., Levskaya, A., Voigt, C. A. (2011) Multichromatic control of gene

expression in Escherichia coli. J. Mol. Biol., 405, 2, 315-324.

  • 2. Gräslund, S., Nordlund, P., Weigelt, J., Bray, J., Gileadi, O., Knapp, S.,

Oppermann, U., Arrowsmith, C., Hui, R., and Ming, J. (2008) Protein production and purification. Nat. Methods, 5, 135–146.

  • 3. Pasotti, L, Zucca, S, and Lupotto, M. (2011) Characterization of a synthetic

bacterial self-destruction device for programmed cell death and for recombinant proteins release, J. Biol. Eng., 5:8.