Engineering Glow in the Dark E. coli *Header and iGEM logo not Photoshopped; drawn in bioluminescent bacteria!
Product Inverter Azure A Overview UV-A Sensor Bioluminescence Achievements Introduction Biomedical Engineering: Molecular and Cellular Biology: Genetics: Joshua Hanson Mhairi Davidson Adele Cook Cara Seggie Andrey Filipov Ye Yang Charlotte Flynn Vilija Lomeikaite Supervisors: Advisors: Dimitra Lountzi Dr Sean Colloms Dr Steph. Holt James Provan Dr Julien Reboud References:
Product Inverter Azure A Overview UV-A Sensor Bioluminescence Achievements Introduction Furri-Lux is a children’s nightlight, powered by bioluminescent bacteria. We want to use Furri-Lux as a vehicle for public engagement and education about synthetic biology, starting at a young age. Furri-Lux References:
Product Inverter Azure A Overview UV-A Sensor Bioluminescence Achievements Introduction Policy and Practices Survey School Visit Glasgow Science Centre References:
Product Inverter Azure A Overview UV-A Sensor Bioluminescence Achievements Introduction Survey We asked respondents how strongly they agreed with several points but in particular we were most interested in: • I would allow my child to have an educational toy that incorporates E. coli bacteria. • I would allow my child to have an educational toy that incorporates genetically modified bacteria. • I would allow my child to have an educational toy that incorporates bacteria. References:
Product Inverter Azure A Overview UV-A Sensor Bioluminescence Achievements Introduction Overall Opinions (n = 60) Strongly Agree Initial Opinion Final Opinion Slightly Agree Neither Agree Nor Disagree Slightly Disagree Strongly Disagree Not Sure E.coli Genetically Modified Bacteria References:
Product Inverter Azure A Overview UV-A Sensor Bioluminescence Achievements Introduction “I don’t know a lot about E. Coli and I’m sure neither do most people. However, I know that it is among the most commonly used in lab experiments and isn’t dangerous. More information about the bacteria should be available” “Still disagree with the whole "bacteria as a pet" idea. Would only consider if my kid is 1000000% explained and seen to understand that not all bacteria are harmless- quite the opposite. The fact that he has a toy pet-bacteria might create a confusing situation, leading to unforeseen events in the future.” “I don’t know enough about bacteria but I do know we are too concerned with destroying all bacteria yet we need some to be healthy so safe educational toys that explain this can only be a good thing.” “Very enlightening. Knowledge is key, people are scared of what they don’t know/understand.” References:
References: Product Design Build Design Introduction Product Print Overview UV-A Sensor Inverter Bioluminescence Azure A Achievements
References: Now for the Synthetic biology Introduction Product Overview UV-A Sensor Inverter Bioluminescence Azure A Achievements
Product Inverter Azure A Introduction UV-A Sensor Bioluminescence Achievements Overview DAYLIGHT UV-A Sensor ON Inverter ON Bioluminescence OFF NIGHT UV-A Sensor OFF Inverter OFF Bioluminescence ON References:
Product Inverter Azure A Overview Introduction Bioluminescence Achievements UV-A Sensor We made BioBricks of a UVA-light sensor system from the Cyanobacterium Synechocystis sp. PCC 6803. (Song et al. 2011) It is part of the Cyanobacteriochrome (CBCR) photoreceptor family, along with a green-light sensor - ccaS Synechocystis cultured Functionality of the green light sensor in E. coli and on plates literature detailing its optimisation was encouraging. References: Ji-Young Song, Hye Sun Cho, Jung-Il Cho, Jong-Seong Jeon, J. Clark Lagarias, and Youn-Il Parka (2011) Near-UV cyanobacteriochrome signaling system elicits negative phototaxis in the cyanobacterium Synechocystis sp. PCC 6803
Product Inverter Azure A Overview Introduction Bioluminescence Achievements UV-A Sensor Cell Cell membrane membrane UirS UirS UirS UirS P P UV-A UirR UirR UirR UirR P P UirR UirR GFP PlsiR UV-A exposure: Phosphate transfer to UirR from UirS, PlsiR promoter activated – Testing GFP expression References: Ji-Young Song, Hye Sun Cho, Jung-Il Cho, Jong-Seong Jeon, J. Clark Lagarias, and Youn-Il Parka (2011) Near-UV cyanobacteriochrome signaling system elicits negative phototaxis in the cyanobacterium Synechocystis sp. PCC 6803
Product Inverter Azure A Overview Introduction Bioluminescence Achievements UV-A Sensor Chromophore (Phycocyanobillin) uirS hoA pycA 1 K1725411 K322122 Composite: K1725445 P lsiR B0015 T uirR GFP 2 K1725427 I13500 Composite: K1725430 References: Ji-Young Song, Hye Sun Cho, Jung-Il Cho, Jong-Seong Jeon, J. Clark Lagarias, and Youn-Il Parka (2011) Near-UV cyanobacteriochrome signaling system elicits negative phototaxis in the cyanobacterium Synechocystis sp. PCC 6803
Product Azure A Overview Introduction UV-A Sensor Bioluminescence Achievements Inverter PtetR PphlF K1725001 High GFP P phlF expression GFP + TetR PhlF + 0 μ M 3 μ M K1725042 10 μ M pL-lac phlF 30 μ M Low GFP P phlF expression 100 μ M GFP IPTG K1725001 (inducer) Stanton B, Nielson A, Tamsir A, Clancy K, Peterson T, Voigt C (2013) Genomic mining of prokaryotic repressors for orthogonal logic gates References:
Product Azure A Overview Introduction UV-A Sensor Bioluminescence Achievements Inverter 1400 pL-lac driving TetR Approximate Molecules of GFP per Cell pL-lac driving PhlF pL-lac 1200 phlF 1000 Low GFP P phlF expression 800 (1000s) GFP 600 400 200 0 PtetR driving GFP PphlF driving GFP -200 Stanton B, Nielson A, Tamsir A, Clancy K, Peterson T, Voigt C (2013) Genomic mining of prokaryotic repressors for orthogonal logic gates References:
Product Inverter Azure A Overview Introduction UV-A Sensor Achievements Bioluminescence Cambridge iGEM 2010 created the LuxBrick (BBa_K325909) Complete Aliivibrio fischeri bioluminescent system Allivibrio are a in a symbiotic relationship with E. coli expressing the Cambridge LuxBrick Bobtail squid E-Glowli: 2010 Cambridge iGEM team http://2010.igem.org/Team:Cambridge References:
Product Inverter Azure A Overview Introduction UV-A Sensor Achievements Bioluminescence O 2 LuxC Lux LuxE FMNH 2 G Tetradecanal LuxD FMN Fatty Acids LuxA LuxB Luciferase Bioluminescence References:
Product Inverter Azure A Overview Introduction UV-A Sensor Achievements Bioluminescence Aliivibrio fischeri selected the original ribosome binding sites for bioluminescence over millennia of natural selection However for engineering the system for intensity in E. coli the Aliivibrio arrangement may not be optimal. Cambridge LuxBrick: luxD luxA luxE luxC luxB luxG GlasGlow: luxC luxA luxB luxG luxE luxD Plasmid 1 controlling Light generation Plasmid 2 controlling Substrate References:
Product Inverter Azure A Overview Introduction UV-A Sensor Achievements Bioluminescence TCACACANRARRG TCACACAGGAAAG B0032 Insert degenerate bases Created variations of RBS strength N = A,C,G,T R = A,G B0032 RBS degenerate ATG +20 nts Prefix Scar sequence LuxA 32 different RBS x 6 genes equals over a billion possible bioluminescence expression combinations! RBS Calculator program: https://www.denovodna.com/software References:
Product Inverter Azure A Overview Introduction UV-A Sensor Achievements Bioluminescence Plasmid 1: Under pBAD promoter Plasmid 2: Under plac promoter Lux C Lux A × 32 × 32 Lux A Lux C Lux D Lux B × 1,024 × 1,024 Lux B Lux A Lux G × 32,768 Lux C Lux D Lux E × 32,768 Two Plasmid system total Lux B Lux G Lux A Lux C Lux D Lux E × 1,073,741,824 Sequencing of a single colony Sequencing of library miniprep References:
Product Inverter Azure A Overview Introduction UV-A Sensor Achievements Bioluminescence pBAD/araC …NRARR... …NRARR... …NRARR... luxA luxB luxG Drops of decanal on lid – diffuses over plate Arabinose No Arabinose Kondo & Ishiura 1994, Decanal Vapour protocol: Circadian rhythms of cyanobacteria: monitoring the biological clocks of individual colonies by bioluminescence. References:
Product Inverter Azure A Overview Introduction UV-A Sensor Achievements Bioluminescence pBAD/araC …NRARR... …NRARR... …NRARR... luxA luxB luxG Drops of decanal on lid – diffuses over plate Arabinose No Arabinose Kondo & Ishiura 1994, Decanal Vapour protocol: Circadian rhythms of cyanobacteria: monitoring the biological clocks of individual colonies by bioluminescence. References:
Product Inverter Azure A Overview Introduction UV-A Sensor Achievements Bioluminescence pBAD/araC …NRARR... …NRARR... …NRARR... luxA luxB luxG The colony with the combination of RBS for luxABG that was the brightest was selected. Then used to select for the combination of RBS for luxCDE that was the brightest. Kondo & Ishiura 1994, Decanal Vapour protocol: Circadian rhythms of cyanobacteria: monitoring the biological clocks of individual colonies by bioluminescence. References:
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