Engineering Glow in the Dark E. coli *Header and iGEM logo not - - PowerPoint PPT Presentation

▶
engineering glow in the dark e coli
SMART_READER_LITE
LIVE PREVIEW

Engineering Glow in the Dark E. coli *Header and iGEM logo not - - PowerPoint PPT Presentation

Apr 24, 2023 354 likes •681 views

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

slide-1
SLIDE 1

Engineering Glow in the Dark E. coli

*Header and iGEM logo not Photoshopped; drawn in bioluminescent bacteria!

slide-2
SLIDE 2

References:

Overview Product UV-A Sensor Inverter Bioluminescence Azure A Achievements

Introduction

Biomedical Engineering: Adele Cook Ye Yang

Molecular and Cellular Biology: Joshua Hanson Cara Seggie Genetics: Mhairi Davidson Andrey Filipov Charlotte Flynn Vilija Lomeikaite Dimitra Lountzi James Provan

Supervisors: Dr Sean Colloms Advisors: Dr Steph. Holt Dr Julien Reboud

slide-3
SLIDE 3

References:

Overview Product UV-A Sensor Inverter Bioluminescence Azure A Achievements

Introduction

Furri-Lux

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.

slide-4
SLIDE 4

References:

Overview Product UV-A Sensor Inverter Bioluminescence Azure A Achievements

Introduction

Policy and Practices

Glasgow Science Centre School Visit Survey

slide-5
SLIDE 5

References:

Overview Product UV-A Sensor Inverter Bioluminescence Azure A Achievements

Introduction

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.

Survey

slide-6
SLIDE 6

References:

Overview Product UV-A Sensor Inverter Bioluminescence Azure A Achievements

Introduction

Slightly Agree Neither Agree Nor Disagree Slightly Disagree Strongly Disagree Not Sure Strongly Agree

E.coli Genetically Modified Bacteria

Overall Opinions (n = 60)

Initial Opinion Final Opinion

slide-7
SLIDE 7

References:

Overview Product UV-A Sensor Inverter Bioluminescence Azure A 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” “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.” “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

  • pposite.

The fact that he has a toy pet-bacteria might create a confusing situation, leading to unforeseen events in the future.”

slide-8
SLIDE 8

References:

Overview

Product

UV-A Sensor Inverter Bioluminescence Azure A Achievements Introduction

Product Design

Design Print Build

slide-9
SLIDE 9

References:

Overview

UV-A Sensor Inverter Bioluminescence Azure A Achievements Introduction Product

Now for the Synthetic biology

slide-10
SLIDE 10

References:

Overview

UV-A Sensor Inverter Bioluminescence Azure A Achievements Introduction Product

UV-A Sensor OFF Inverter OFF Bioluminescence ON UV-A Sensor ON Inverter ON Bioluminescence OFF

DAYLIGHT NIGHT

slide-11
SLIDE 11

References:

UV-A Sensor

Inverter Bioluminescence Azure A Achievements Introduction Product Overview

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 Functionality of the green light sensor in E. coli and literature detailing its optimisation was encouraging.

Synechocystis cultured

  • n plates

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

slide-12
SLIDE 12

References:

UV-A Sensor

Inverter Bioluminescence Azure A Achievements Introduction Product Overview

UV-A exposure: Phosphate transfer to UirR from UirS, PlsiR promoter activated – Testing GFP expression

UV-A

UirS UirS UirR UirR

Cell membrane

PlsiR

GFP

UirS UirS UirR UirR

Cell membrane

P P

UirR UirR

P P

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

slide-13
SLIDE 13

References:

UV-A Sensor

Inverter Bioluminescence Azure A Achievements Introduction Product Overview

uirS uirR GFP

Chromophore (Phycocyanobillin)

PlsiR

1 2

hoA pycA

K322122 K1725427 K1725411 I13500 Composite: K1725445 Composite: K1725430

B0015

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

T

slide-14
SLIDE 14

References:

Inverter

Bioluminescence Azure A Achievements Introduction Product Overview UV-A Sensor

0μM 3μM 10μM 30μM 100μM PtetR PphlF IPTG (inducer) + TetR PhlF +

GFP

PphlF High GFP expression

phlF GFP

PphlF Low GFP expression pL-lac

K1725001 K1725042 K1725001

Stanton B, Nielson A, Tamsir A, Clancy K, Peterson T, Voigt C (2013) Genomic mining of prokaryotic repressors for orthogonal logic gates

slide-15
SLIDE 15

References:

Inverter

Bioluminescence Azure A Achievements Introduction Product Overview UV-A Sensor

phlF GFP

PphlF Low GFP expression pL-lac

  • 200

200 400 600 800 1000 1200 1400 PtetR driving GFP PphlF driving GFP Approximate Molecules of GFP per Cell (1000s) pL-lac driving TetR pL-lac driving PhlF

Stanton B, Nielson A, Tamsir A, Clancy K, Peterson T, Voigt C (2013) Genomic mining of prokaryotic repressors for orthogonal logic gates

slide-16
SLIDE 16

References:

Bioluminescence

Azure A Achievements Introduction Product Overview UV-A Sensor Inverter

Cambridge iGEM 2010 created the LuxBrick (BBa_K325909) Complete Aliivibrio fischeri bioluminescent system

  • E. coli expressing the Cambridge LuxBrick

Allivibrio are a in a symbiotic relationship with Bobtail squid

E-Glowli: 2010 Cambridge iGEM team http://2010.igem.org/Team:Cambridge

slide-17
SLIDE 17

References:

Bioluminescence

Azure A Achievements Introduction Product Overview UV-A Sensor Inverter

Tetradecanal

LuxC LuxE LuxD LuxB LuxA Lux G

FMNH2 Luciferase Bioluminescence

Fatty Acids

FMN

O2

slide-18
SLIDE 18

References:

Bioluminescence

Azure A Achievements Introduction Product Overview UV-A Sensor Inverter

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: GlasGlow:

luxE luxD luxC luxG luxB luxA luxE luxD luxC luxG luxB luxA

Plasmid 1 controlling Light generation Plasmid 2 controlling Substrate

slide-19
SLIDE 19

References:

Bioluminescence

Azure A Achievements Introduction Product Overview UV-A Sensor Inverter Created variations of RBS strength

TCACACAGGAAAG TCACACANRARRG

B0032

LuxA

B0032

Insert degenerate bases N = A,C,G,T R = A,G RBS Calculator program: https://www.denovodna.com/software

32 different RBS x 6 genes equals over a billion possible bioluminescence expression combinations!

Prefix RBS degenerate sequence

Scar

ATG +20 nts

slide-20
SLIDE 20

References:

Bioluminescence

Azure A Achievements Introduction Product Overview UV-A Sensor Inverter Sequencing of library miniprep Sequencing of a single colony Lux A Lux B

×1,024

Lux A Lux B Lux G ×32,768 Lux A

×32

Lux C

×32

Lux D Lux C

×1,024

Lux D Lux C Lux E

×32,768

Plasmid 1: Under pBAD promoter Plasmid 2: Under plac promoter

Lux D Lux A Lux B Lux G Lux C

×1,073,741,824

Lux E

Two Plasmid system total

slide-21
SLIDE 21

References:

Bioluminescence

Azure A Achievements Introduction Product Overview UV-A Sensor Inverter

luxG luxB luxA

…NRARR... …NRARR... …NRARR... pBAD/araC

Arabinose No Arabinose Drops of decanal on lid – diffuses

  • ver plate

Kondo & Ishiura 1994, Decanal Vapour protocol: Circadian rhythms of cyanobacteria: monitoring the biological clocks of individual colonies by bioluminescence.

slide-22
SLIDE 22

References:

Bioluminescence

Azure A Achievements Introduction Product Overview UV-A Sensor Inverter

luxG luxB luxA

…NRARR... …NRARR... …NRARR... pBAD/araC

Arabinose No Arabinose Drops of decanal on lid – diffuses

  • ver plate

Kondo & Ishiura 1994, Decanal Vapour protocol: Circadian rhythms of cyanobacteria: monitoring the biological clocks of individual colonies by bioluminescence.

slide-23
SLIDE 23

References:

Bioluminescence

Azure A Achievements Introduction Product Overview UV-A Sensor Inverter

luxG luxB luxA

…NRARR... …NRARR... …NRARR... pBAD/araC

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.

slide-24
SLIDE 24

References:

Bioluminescence

Azure A Achievements Introduction Product Overview UV-A Sensor Inverter

luxE luxD luxC

…NRARR... …NRARR... …NRARR... pL-lac

GlasGlow Optimised Cambridge LuxBrick Transformation with degenerate RBS – colonies of different brightness

slide-25
SLIDE 25

References:

Overview

UV-A Sensor Inverter Bioluminescence Azure A Achievements Introduction Product

luxE luxD luxC luxA luxB luxG uirS uirR hoA pycA phlF Chromophore (Phycocyanobillin) PlsiR PphlF

1 2 3

K322122 K1725427 K1725000 K1725411 K1725400 Composite: K1725445

B0015

T

slide-26
SLIDE 26

References:

Azure A

Achievements Introduction Product Overview UV-A Sensor Inverter Bioluminescence

Staining DNA with Azure A

Azure A is a blue, cheap, quick, non-hazardous dye which stains DNA visible to the naked eye. We used Azure A to stain fragments for gel extraction.

Linearized RFP plasmid DNA of known concentration Ethidium bromide sensitivity: ~2ng

Azure A sensitivity: ~17ng

NCBE (2003). National Centre for Biotechnology Education | DNA50 | Staining DNA.

slide-27
SLIDE 27

References:

Achievements

Introduction Product Overview UV-A Sensor Inverter Bioluminescence Azure A

Medal Criteria

Created 110 BioBricks, and submitted 57 to the registry Favourite BioBricks:

  • PphlF – strong promoter
  • Best Basic Part - PhlF – strong orthogonal repressor
  • Part Collection – RBS library for optimised expression of lux operon
  • Recharacterisation
  • Cambridge Luxbrick operon genes turned into individual biobricks
  • Luxbrick sequenced entirely and mutations identified in sequence
  • Documented on K325909 experience page.
slide-28
SLIDE 28

References:

Achievements

Introduction Product Overview UV-A Sensor Inverter Bioluminescence Azure A

  • Expanding accessibility to Synbio with Azure A

Collaboration:

  • Cambridge Team
  • Sent them fluorescent samples driven by Pphlf to test under their
  • pensource fluorescent microscope
  • Results confirmed
  • Warwick team
  • Sent them some Azure A to test
  • Informed us that a thicker gel might not stain in the same time

as a thin gel

  • Interlab study + Extra Credit
  • Performed by Charlotte
  • Public Engagement and Education using Furri-Lux
slide-29
SLIDE 29

References:

Overview Product UV-A Sensor Inverter Bioluminescence Azure A Introduction Achievements

This iGEM team has been funded by the MSD Scottish Life Sciences Fund. As part of an ongoing contribution to Scottish life sciences, MSD Limited, a global healthcare leader, has given substantial monetary funding to the Scottish Funding Council (SFC) for distribution via the Scottish Universities Life Sciences Alliance (SULSA) to develop and deliver a high quality drug discovery research and training programme. All aspects of the programme have been geared towards attaining the highest value in terms of scientific discovery, training and impact. The opinions expressed by this iGEM team are those of the team members and do not necessarily represent those of Merck Sharp & Dohme Limited nor its affiliates.

slide-30
SLIDE 30

References:

Achievements

Introduction Product Overview UV-A Sensor Inverter Bioluminescence Azure A

Any Questions?

slide-31
SLIDE 31

References:

Overview Product UV-A Sensor Inverter Bioluminescence Azure A Introduction Achievements

  • Abbas A, Morrissey JP

, Marquez PC, Sheehan MM, Delany IR, O’Gara (2002) Characterization of Interactions between the Transcriptional Repressor PhlF and Its Binding Site at the phlA Promoter in Pseudomonas fluorescens F113

  • Delany I, Sheehan M, Fenton A, Bardin S, Aarons S and O’Gara (2000) Regulation of production of the

antifungal metabolite 2,4-diacetylphloroglucinol in Pseudomonas fluorescens F113: genetic analysis of phlF as a transcriptional repressor

  • E-Glowli: 2010 Cambridge iGEM team http://2010.igem.org/Team:Cambridge
  • 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

  • Levskaya, A., Chevalier, A.A.,

Tabor, J.J., Simpson, Z.B., Lavery, L.A., Levy, M., Davidson, E.A., Scouras, A., Ellington, A.D., Marcotte, E.M., et al. (2005).

  • NCBE (2003). National Centre for Biotechnology Education | DNA50 | Staining DNA.
  • Stanton B, Nielson A, Tamsir A, Clancy K, Peterson T,

Voigt C (2013) Genomic mining of prokaryotic repressors for orthogonal logic gates

  • Schmidl SR, Sheth RU, Wu A, Tabor JJ (2014) Refactoring and Optimization of Light Switchable

Escherichia coli Two-Component Systems

  • Yoon-Jung Moon, Seung Il Kim and

Young-Ho Chung (2012) Sensing and Responding to UV-A in Cyanobacteria