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The document describes work done by the Cambridge 2009 iGEM team to develop genetic parts for creating bacterial biosensors. They created a "Kit of Parts" including Sensitivity Tuners to allow adjustment of a biosensor's sensitivity to input, and Colour Generators to provide a visible, self-contained output for the biosensor. The team characterized 15 Sensitivity Tuners from previous work by fitting Hill functions to experimental data, determining parameters like their half-maximal induction points. This set of genetic parts is intended to facilitate future design and construction of customizable bacterial biosensors.
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- 1. Cambridge 2009 Triggering Pigment Produc/on in E. Coli Mike Davies, Shuna Gould, Siming Ma, Vivian Mullin, Megan Stanley, Alan Walbridge, Crispian Wilson Celebra2ng 800 Years of Innova2on at Cambridge University
- 2. Cambridge 2009 The Cambridge 2009 iGEM team has created a Kit of Parts that will facilitate the design and construc1on of biosensors in the future We have developed a set of Sensi1vity Tuners and a set of Colour Generators
- 3. Cambridge 2009 Bacterial Biosensors: the Detec2on of Environmental Pollutants • Bacterial biosensors ‐ an alterna/ve to chemical methods • S/ll selec/ve and sensi/ve • Inexpensive • Less labour intensive • More accessible
- 4. Cambridge 2009 Bacterial Biosensors: Problems Solu2ons S R C T E E P O U N O L N R S O E T O U R E R R R Inability to tune sensor as SensiHvity Tuners Lack of self‐contained output Colour Generators desired – – Limited by sensi/vity of PoPS converters – Reliance on reporters in – Bacterial pigments promoter – Change sensi/vity of Registry – Visible, user‐friendly output – Limited to PoPS output upstream promoter – Require addi/onal behaviour of promoter technology to read output
- 5. Cambridge 2009 Bacterial Biosensors: Easy to use S C T E O U N L N S O E O U R R R
- 6. Cambridge 2009 Bacterial Biosensors: A prototype Inducer concentra/on: 0 low high The colour readout indicates concentra/on of inducer
- 7. Cambridge 2009 Bacterial Biosensors: How to build a bacterial biosensor with these parts Chemical Pigment IN OUT COLOUR SENSOR SENSITIVITY TUNER Input GENERATOR SENSOR Input PoPS PoPS Phage SENSITIVITY TUNER T PoPS Promoter COLOUR Ac/vator Receiver ac/vator GENERATOR Pigment sensi/ve to PoPS PoPS sensi/ve producing Converter input promoter PoPS Colour device Reporter
- 8. Cambridge 2009 SensiHvity Tuners: Introduc2on Rate of Output • A Sensi/vity Tuner allows adjustment of sensi/vity to input Concentra/on • A combina/on of different Tuners in parallel allow measurements of a range of discrete input concentra/ons
- 9. Cambridge 2009 Design: an Input to Output Device PoPS in PoPS out T Phage Ac/vator ac/vator sensi/ve promoter Ac/vator PoPS in Transcrip/onal and Concentra/on PoPS out Promoter Transla/onal Characteris/cs Characteris/cs
- 10. Cambridge 2009 Previous Work: Cambridge 2007 I0500 Phage T I13507 Ac/vator I13504 pBad/AraC ac/vator mRFP sensi/ve GFP promoter “Amplifiers” ac/vators • GFP output controlled by phage P2 ogr PSP3 pag phiR73 delta promoter PF promoter I746370 I746380 I746390 promoters PO promoter I746371 I746381 I746391 • RFP output controlled by pBad PP promoter I746372 I746382 I746392 input Psid promoter I746374 I746384 I746394 PLL promoter I746375 I746385 I746395 • Characterized as an “amplifier” by ra/o of RFP to GFP
- 11. Cambridge 2009 SensiHvity Tuners: Modelling Arabinose Phage Ac/vator Ac/vator GFP GFP Conc. pBAD PoPS in Conc. Phage PoPS out Transcrip/on Conc. Transcrip/on & Promoter Promoter Transla/on & Transla/on Characteris/cs Characteris/cs Characteris/cs Characteris/cs • Model gene characteris/cs at steady where state using Law of Mass Ac/on • pBAD is repressed by repressor X* which binds to arabinose • Assume transcrip/on and transla/on are linear func/ons of PoPS • Model protein concentra/ons as dynamic, since these change slowly • Allow for protein degrada/on
- 12. Cambridge 2009 Modelling Results: Sigmoidal Behaviour • The model contains a large number of constants • A priori modelling requires arbitrary values to be chosen • Maximum reporter produc/on rate is sigmoidal with inducer concentra/on Reporter Degradation rates at multiple input Model for maximum fluorescence rate concentrations of arabinose Reporter production rate Reporter production rate time Inducer concentration
- 13. Cambridge 2009 Curve FiTng: Hill Func2on A model Sensitivity Tuner Peak rate 1 RPU Rate of GFP expression Hill coefficient Increase in rate (a) (n) Basal rate (c) Half‐maximal induc/on (k) Concentra/on of Arabinose
- 14. SensiHvity Tuners: Changing the Cambridge 2009 sensi2vity of an upstream promoter • Constructs were tested on high copy against pBAD characteris/cs • Output triggered at much lower arabinose concentra/on when Sensi/vity Tuner included pBAD -> GFP pBAD -> Construct 91 -> GFP Maximum normalised GFP Maximum normalised GFP production production Arabinose concentraion (µm) Arabinose concentraion (µm)
- 15. Cambridge 2009 SensiHvity Tuners: Characterisa2on • 15 Cambridge 2007 constructs moved down to low copy plasmid • High throughput tes/ng • 3 repeats of 3 colonies over 8 concentra/ons • OD and fluorescence P2 ogr PSP3 pag phiR73 delta measured PF promoter I746370 I746380 I746390 PO promoter I746371 I746381 I746391 • Standard Promoter included PP promoter I746372 I746382 I746392 on plate to allow for RPU Psid promoter I746374 I746384 I746394 PLL promoter I746375 I746385 I746395 measurements
- 16. Cambridge 2009 SensiHvity Tuners: SoLware • Matlab graphical interface developed to allow data to be viewed in several ways • Standard promoter data allows for RPU characterisa/on
- 17. Cambridge 2009 Curve FiTng: Hill Func2on • Non‐linear least squares method used to fit Hill func/ons to measured data • Each fit produces the parameters of the Hill func/on, enabling construct to be quan/ta/vely analysed
- 18. Cambridge 2009 SensiHvity Tuners: Parameters • A range of Sensi/vity Tuners were successfully characterised on low copy • Good range in sensi/vity: 10x range in half‐maximal induc/on • Hill coefficients of 2 – 3 when concentra/on resolu/on is sufficient • Wide range of rate increases, from 0.3RPU to 1.2RPU A model Sensitivity Tuner Peak rate 1 RPU Hill Rate of GFP expression coefficient Increase in rate (a) (n) Basal rate (c) Half‐maximal induc/on (k) Concentra/on of Arabinose
- 19. Cambridge 2009 SensiHvity Tuners: Design • A standard kit was designed using well characterised candidates • Tuners can be used with any promoter • Any device can be placed downstream of the construct P2 ogr PSP3 pag phiR73 delta PF promoter K274370 K274380 PO promoter K274371 K274381 K274391 T PP promoter K274382 K274392 ac/vator promoter Psid promoter K274374 K274384 K274394 PLL promoter K274375 K274395
- 20. Cambridge 2009 Colour Generators: Choosing pigments • Diversity: – Colour Violacein – Bacterial Origin • Design – Standard Assembly Melanin – PCR – Synthesis • PotenHal for ManipulaHon Carotenoids – Single gene systems – Mul/gene systems with colourful intermediates – Supplements to media
- 21. Cambridge 2009 Violacein: Background L-tryptophan VioD VioA • Quorum‐sensing controlled pigment from Chromobacterium violaceum VioB VioE Green VioC Violet
- 22. Cambridge 2009 Violacein: Design & Synthesis VioD VioA K274002 VCG K274002 BamHI BglII BclI VioB VioE VioC
- 23. Cambridge 2009 Violacein: Design & Synthesis K274002 VioA VioB VioC VCG VioD VioE K274002 BamHI BglII BclI GGATCC AGATCT CCTAGG TCTAGA
- 24. Cambridge 2009 Violacein: Design & Synthesis K274002 VioA VioB VioC VCG VioA VioB VioC VioD VioD VioE K274002 BamHI BglII BclI G GATCT CCTAG A K274003 GCG VioA VioB VioC VioD VioE K2742003
- 25. Cambridge 2009 Violacein: Expression & Quan2fica2on VCG K274002 GCG K2742003
- 27. Cambridge 2009 Violacein: Colour Logic A VioA, VioB, VioD, VioE Colour Output VioC B A B Output If A = constitutive, B = inducible 0 0 No colour Colour Output 1 0 GREEN Device working: 0 1 No colour 1 1 VIOLET Presence of B:
- 28. Cambridge 2009 Melanin: Background Tyrosine • Brown pigment made in many animals and bacteria via the ac/on of a tyrosinase (MelA) • Isolated melA from Rhizobium etli. MelA • Media supplemented with copper sulphate and tyrosine Dopaquinone polymerisation Brown MELANIN
- 29. Cambridge 2009 Melanin: Design K274001 Native rbs MelA BCG K274001 • Used na/ve RBS and planned to remove forbidden restric/on sites using PCR • Strong candidate for a biosensor reporter − Strong pigment produc/on − Single gene
- 32. Cambridge 2009 Carotenoids: Background pyruvate glyceraldehyde-3-phosphate Lycopene Non-mevalonate Pathway (already present in E. coli) Farnesyl pyrophosphate (Colourless precusor) CrtE CrtB CrtI β-Carotene CrtY
- 33. Cambridge 2009 Carotenoids: Standard assembly RCG K274100 OCG K274200 Farnesyl pyrophosphate (Colourless precusor) Enzymes coding CrtE sequences from CrtB Pantoea ananatis (Enterobacteria) CrtI Lycopene CrtY β-Carotene
- 34. Cambridge 2009 Carotenoids: Standard assembly RCG K274100 OCG K274200 RCT Constitutive K274110 promoter OCT K274210 Expression in E. coli strain MG1655
- 35. Cambridge 2009 Carotenoids: Expression and Quan2fica2on RCT K274110 OCT K274210
- 36. Cambridge 2009 Carotenoids: Expression and Quan2fica2on Lycopene β-Carotene Control Control 5µg carotene 474 456 β-Carotene: 1.5 µg per mL culture
- 37. Cambridge 2009 Proof of Concept: Pigment Induc2on COLOUR SENSOR SENSITIVITY TUNER GENERATOR Phage T Promoter Ac/vator Pigment ac/vator sensi/ve to sensi/ve producing input promoter device IRCT K274120 Pbad I0500 CrtE CrtB CrtI promoter IOCT K274220 I0500 CrtE CrtB CrtI CrtY
- 38. Cambridge 2009 Proof of Concept: Pigment Induc2on No arabinose Induced by 1mM arbinose Control 5 µg carotene 1mM arabinose No arabinose 456 No arabinose Induced by 1mM arbinose Control 5 µg carotene β-Carotene: 1.3 µg per mL induced culture
- 39. Cambridge 2009 BioBricks: Sensi2vity Tuners ac/vator ST promoter P2 ogr PSP3 pag phiR73 delta PF promoter ✔ ✔ ✗ K274370 K274380 PO promoter ✔ ✔ ✔ K274371 K274381 K274391 PP promoter ✗ ✔ ✔ K274382 K274392 Psid promoter ✔ ✔ ✔ K274374 K274384 K274394 PLL promoter ✔ ✗ ✔ K274375 K274395
- 40. Cambridge 2009 BioBricks: Colour Generators VCG BCG RCG K274002 K274001 K274100 GCG OCG K2742003 K274200
- 41. Cambridge 2009 BioBricks: Systems RCT K274110 R0011 CrtE CrtB CrtI OCT K274210 I0500 CrtE CrtB CrtI IRCT K274120 R0011 CrtE CrtB CrtI CrtY IOCT K274220 I0500 CrtE CrtB CrtI CrtY
- 42. Cambridge 2009 Further Work: for our Project • Show compa/bility with promoters in Registry • Expand kit of parts – Phage ac/vators and phage promoters – Pigment‐producing operons from other bacterial species
- 43. Cambridge 2009 MulHplexing InformaHon: Accessible and Informa2ve Biosensors Arsenic Mercury Lead As + Hg Ag + Pb Hg + Pb As + Hg + Pb
- 47. Cambridge 2009 Thank You… Advisors: Friends: Dr. Jim Ajioka Caitlin Cockerton Dr. Jim Haseloff Daisy Ginsberg Dr. Gos Micklem James King Dr. Tom Ellis Tuur Van Balen Dr. Duncan Rowe …and especially James Brown Summary of Achievements: Designed 23 New Biobricks Characterised 15 Biobricks already in the registry
- 48. Cambridge 2009 Thank You… Advisors: Friends: Dr. Jim Ajioka Caitlin Cockerton Dr. Jim Haseloff Daisy Ginsberg Dr. Gos Micklem James King Dr. Tom Ellis Tuur Van Balen Dr. Duncan Rowe …and especially James Brown Summary of Achievements: Designed 23 New Biobricks Characterised 15 Biobricks already in the registry
- 50. It’s Mike’s birthday today…hopefully he’s not looking at the screen! We’d like to sing him happy birthday, so join us! On 3….. 1 2 3!!!