Perfect synthetic biology project

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Lambert Lloyd
5 years ago
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2 Perfect synthetic biology project Something beautiful! Idea Community project Present Evaluate Improve Final product

Hazardous to humans, pets and livestock. Local problem E.

3 Our problem: Algal Blooms Explosion in the growth of bluegreen algae (cyanobacteria) Production of toxins Hazardous to humans, pets and livestock. Local problem E.g. Reservoirs, lakes/lochs Global problem Clatto reservoir, Dundee

4 Microcystis aeruginosa Picture courtesy of Pauline Lang, SEPA.

5 Liver toxin: Microcystin

6 PP1 & Microcystin Microcystin Binds covalently to Protein Phosphatase 1 Blocks the active site PP1 is an essential regulatory protein Goldberg et al (1995) Nature 376, PDB code: 1FJM

7 Current methods for dealing with algal bloom toxicity are far from ideal Existing methods target the blue-green algae not the toxin We were tasked with directly targeting the toxin

8 Our idea: a biological ToxiMop PP1 PP1 PP1 PP1 PP1 PP1 PP1 (sp) PP1 PP1 PP1 PP1 PP1 PP1 PP1 PP1 PP1 PP1 PP1 PP1 PP1 PP1

9 Who s packing? E. coli vs B. subtilis

10 Transport across inner membrane PP1 (sp) PP1

11 Engineered PP1 proteins Tat-targeted TorA sp -PP1 TorA sp PP1 Sec-Targeted MalE sp -PP1 MalE sp PP1

12 TorA sp -PP1 localises to the periplasm

13 TorA sp -PP1 localises to the periplasm

14 TorA sp -PP1 localises to the periplasm

15 TorA sp -PP1 localises to the periplasm E. coli TorA sp -PP1 cells will make up our ToxiMop

16 How much PP1 wood would a a Tat woodchuck transporter chuck transport if a woodchuck if a Tat transporter could chuck could transport wood? PP1? ~200 PP1 in the periplasm ~1000 left in the cytoplasm

17 Number of PP1 in cytoplasm Number of PP1 in periplasm Predicting Transport Rates Time (s) Time (s)

18 Extracellular space Periplasm Cytoplasm

19 Testing the ability of our ToxiMop cells to clean up the toxin.

20 ToxiMop assay TorA sp -PP1 NarG-PP1 Vector Only No cells control

21 ToxiMop in action Incubate for 1 h at room temp. Centrifuge cells and take the supernatant E. coli cells

22 No Cells X ToxiMop ELISA Vector only Cytoplasmic- PP1 70% of periplasmic PP1 is bound to microcystin ToxiMop Cells 0.26 nm Microcystin

23 Are these concentrations of microcystin relevant? Alberta, Toxic drinking water 4.3 nm WHO safe-level is below 1 nm

24 Presenting to community leaders

25 Suggested addition to our project To develop a biological detector using synthetic biology.

26 Our idea: Biological toxin detector PP1 Microcystin sensor

27 E. coli osmolarity sensor: EnvZ OmpR Expression of OmpR regulated genes

28 Region to be engineered PERIPLASM PP1 CYTOPLASM Expression of OmpR regulated genes GFP expression controlled by OmpR

29 GFP reporter construct OmpC promoter RBS GFP OmpC: BBa_R0083 RBS: BBa_B0034 GFP: Bba_E0040

30 The OmpC-GFP reporter responds to environmental osmolarity in an EnvZdependent manner

31 Discussion: Algal Blooms, Clatto and Synthetic Biology We held a discussion allowing us to present our project to scientists and environmentalists.

32 Concerns raised: 1. How can we deploy our detector? 2. We have overlooked the root causes of the problem. 3. How can we use the ToxiMop without releasing GMMs?

33 Biological Detector Light Temperature Humidity ph Dissolved O 2 Webcam

34 Concerns raised: 1. We have overlooked the root causes of the problem. 2. How can we deploy our detector? 3. How can we use the ToxiMop without releasing GMMs?

35 Prototype devices Toxi-Teabag Toxi-Pump

36 The ToxiMop Works Problem We would need 70 g of cells to clean up 200 ml of microcystin contaminated buffer!

37 Potential solution

38 What have we achieved? We have made a biological mop for an environmental toxin Built the components for a biological detector A project shaped by the community

39 The Future of ToxiMop Exploring Commercialisation Okadaic Acid Other Toxins & Pollutants Expanding the ToxiMop Moptopus 2.0 Moptopus Mk. 2

40 Raw Data: Buoyancy Test

41 The Mop Campaign We re in the papers!

42 The Mop Campaign We re in the papers! Partnership with Friends of the Earth

43 The Mop Campaign We re in the papers! Partnership with Friends of the Earth Comic Book

44 The Mop Campaign We re in the papers! Partnership with Friends of the Earth Comic Book Toximop Videogame

45 The Mop Campaign We re in the papers! Partnership with Friends of the Earth Comic Book Toximop Videogame Life Science undergraduate igem practical

46 The Mop Campaign We re in the papers! Partnership with Friends of the Earth Comic Book Toximop Videogame Life Science undergraduate igem practical Stand-up comedy

51 Thank You

52 Acknowledgements Supervisors A Special thanks to: Prof Tracy Palmer Prof Frank Sargent Dr Fordyce Davidson Advisors Richard Owen Lucia Licandro Ciaran Kelly Dr Grant Buchanan Actor- Brian Cox ToxiMop comic Avril Smart (igem 2012) ToxiMop game Digital Janitors Everyone at University of Dundee Prof Tricia Cohen PP1 antibody. Prof Carol MacKintosh PP1 plasmid. Joe Fitzpatrick MSP Andrew Llanwarne George Potts and team

53 Competitive ELISA 1. Add your sample into the well.

54 Competitive ELISA 1. Add your sample into the well. 2. Add your primary antibody

55 Competitive ELISA 1. Add your sample into the well. 2. Add your primary antibody 3. Competition between microcystin at bottom of the well and in the sample for binding of the primary antibody.

56 High microcystin oncentration in the sample Wash The small number of HRP-conjugated secondary antibodys bound to the bottom of the well results in a slight colour change Add sample and primary antibody to well Add secondary antibody to well

57 Low microcystin concentration in the sample More HRP-conjugated secondary antibody at bottom of the well results in a strong colour change Wash Add sample + primary antibody to well Add secondary antibody to well

58 kda 50 Dtat strain 37

60 Don t delete.

Perfect synthetic biology project

Perfect synthetic biology project Something beautiful! Idea Community project Present Evaluate Improve Final product Our problem: Algal Blooms Explosion in the growth of bluegreen algae (cyanobacteria)