Engineering bacteria to help fight soil erosion The problem: - - PowerPoint PPT Presentation

Engineering bacteria to help fight soil erosion

31,000 hectares of arable land are degraded every day

The problem: Desertification

DRYLANDS 2010

1.3 x the area of Boston lost per day!

Lateral root growth enhances soil stability

Our solution

Engineering bacteria to move towards roots

Chemotaxis to the root

Engineering bacteria to produce auxin

Auxin production

Structure of auxin

IAA

Gene Guard

Human Practices: Informing design

Chassis Choice

Gene Guard

Human Practices: Informing design

Chassis Choice “Horizontal gene transfer happens, and at high frequencies; it is the greatest, most underestimated hazard from GMOs released into the environment” (Dr. Mae-Wan Ho)

Gene Guard

Human Practices: Informing design

Chassis Choice

Novel containment device

Gene Guard: Containing our genetic constructs

GMO Gene transfer Soil bacterium

Gene Guard Chassis Choice

• E. coli
• B. subtilis

OR

Human Practices: Informing design

We chose E. coli Chassis choice

Gene Guard Chassis Choice

• E. coli

Human Practices: Informing design

Chassis choice

Gene Guard Chassis Choice

Human Practices: Informing design

• E. coli K12 survive in soil for more than 7 weeks

Phyto-Route Auxin Xpress Gene Guard

Phyto-Route Auxin Xpress Gene Guard

Phyto-Route Auxin Xpress Gene Guard

Specifications & Design Modelling Assembly Testing & Results

Movement towards roots PA2652, malate-responsive chemoreceptor

Rewiring chemotaxis

Status

Module 1: Phyto-Route

Module 1: Phyto-Route

Optimal malate receptor expression level?

Sensitivity and saturation analysis for malate receptor

Concentration of chemoreceptors can be variable

Specifications & Design Modelling Assembly Testing & Results Status

Module 1: Phyto-Route

Phyto-Route construct

Specifications & Design Modelling Assembly Testing & Results Status

Module 1: Phyto-Route

Behaviour of bacteria upon chemoattractant saturation

Specifications & Design Modelling Assembly Testing & Results Status

20

• E. coli senses malate

Velocity (μm/s) Probability of bacterial movement at given velocity

PA2652 cells in 10 mM malate PA2652 cells in 10 mM serine Negative control (cells without construct) in 10 mM malate

Module 1: Phyto-Route

Capillary assay

Specifications & Design Modelling Assembly Testing & Results Status

Module 1: Phyto-Route

Specifications & Design Modelling Assembly Testing & Results Status

Optimum experiment duration

60 mins

% population in capillary 3D Simulation

Module 1: Phyto-Route

Chemotaxis dependence on malate concentration

Specifications & Design Modelling Assembly Testing & Results Status

• E. coli chemotaxes towards malate

Specifications & Design Modelling Assembly Testing & Results Status

Module 1: Phyto-Route

Bacteria in the root

Specifications & Design Modelling Assembly Testing & Results Status

Module 1: Phyto-Route

Bacteria chemotax towards root exudate

Status so far

Specifications & Design Modelling Assembly Testing & Results Status

Module 1: Phyto-Route

Bacteria chemotax towards root exudate Bacteria are uptaken by the root

Status so far

Phyto-Route Auxin Xpress Gene Guard

Specifications & Design Modelling Assembly Testing & Results Status

Module 2: Auxin Xpress

Release of auxin

A simple IAA producing pathway IAM pathway

Specifications & Design Modelling Assembly Testing & Results Status

Module 2: Auxin Xpress

How much bacterial auxin can we produce ?

Intracellular IAA concentration of 72 µM

Module 2: Auxin Xpress

Specifications & Design Modelling Assembly Testing & Results Status

Specifications & Design Modelling Assembly Testing & Results Status

Module 2: Auxin Xpress

What is the effect of the IAA on the root ?

The optimum is 0.1 nM

IAA concentrations (nM) Root length (cm)

Optimum IAA concentration

Simulated Arabidopsis root growth after 25 days

[IAA] = 10-6 mol/L [IAA] = 10-10 mol/L [IAA] = 10-14 mol/L

Parameters: Branching, root growth rate, gravity, twisting

Specifications & Design Modelling Assembly Testing & Results Status

Module 2: Auxin Xpress

Auxin Xpress construct

Specifications & Design Modelling Assembly Testing & Results Status

Module 2: Auxin Xpress

Salkowski assay standards Colourimetric auxin detection

Specifications & Design Modelling Assembly Testing & Results Status

Module 2: Auxin Xpress

Successful auxin production

Negative Control Auxin Xpress Negative Control Auxin Xpress

Extracellular auxin yield of 52 µM

60 50 40 30 20 10 IAA concentration (µM)

Specifications & Design Modelling Assembly Testing & Results Status

Module 2: Auxin Xpress

IAA peak confirmed by Liquid Chromatography Mass Spectrometry (LCMS) Mass 130, 176

Specifications & Design Modelling Assembly Testing & Results Status

Module 2: Auxin Xpress

DR5 Venus Construct

DR5 IAA YFP Venus

Construct kindly provided by Dr Darren Wells (University of Nottingham)

Auxin Xpress Engineered E. coli Control Wild type E. coli

Specifications & Design Modelling Assembly Testing & Results Status

Module 2: Auxin Xpress

10 20 30 40 50 60 70 80 90 100

No bacteria Auxin-producing Non-modified

Fluorescence intensity

E.coli E.coli

Can Arabidopsis detect bacterial auxin ?

Yes, it can.

Specifications & Design Modelling Assembly Testing & Results Status

Module 2: Auxin Xpress

Dendra2 photoconversion

Irreversible Photoconversion

Specifications & Design Modelling Assembly Testing & Results Status

Module 2: Auxin Xpress

Dendra2-expressing E. coli imaged in an Arabidopsis root

• E. coli remain metabolically active inside roots

Specifications & Design Modelling Assembly Testing & Results Status

Module 2: Auxin Xpress

Dendra2 photoconverted after 4 days

• E. coli remain metabolically active inside roots

Specifications & Design Modelling Assembly Testing & Results Status

Module 2: Auxin Xpress

• E. coli remain metabolically active inside roots

Root re-imaged after 24 hours

Specifications & Design Modelling Assembly Testing & Results Status

Module 2: Auxin Xpress

Mass of soil eroded (g) IAA concentration (nM)

Specifications & Design Modelling Assembly Testing & Results Status

Module 2: Auxin Xpress

d

Bacteria produce auxin

Status so far

Specifications & Design Modelling Assembly Testing & Results Status

Module 2: Auxin Xpress

d

Bacteria produce auxin Plants respond to bacterial auxin

Status so far

Specifications & Design Modelling Assembly Testing & Results Status

Module 2: Auxin Xpress

d

Bacteria produce auxin Plants respond to bacterial auxin Determined optimal auxin concentrations

Status so far

Specifications & Design Modelling Assembly Testing & Results Status

Module 2: Auxin Xpress

d

Bacteria produce auxin Plants respond to bacterial auxin Determined optimal auxin concentrations

Status so far

Dendra2: A new platform for imaging gene expression in root

Specifications & Design Modelling Assembly Testing & Results Status

Module 2: Auxin Xpress

d

Bacteria produce auxin Plants respond to bacterial auxin Determined optimal auxin concentrations

Status so far

Dendra2: A new platform for imaging gene expression in root Auxin improves soil stability

Phyto-Route Auxin Xpress Gene Guard

Specifications & Design Modelling Assembly Testing & Results Status

Module 3: Gene Guard

Preventing horizontal gene transfer

• 1. Prevent horizontal

gene transfer Holin/endolysin

• 2. Without harming
• ur GMO

Anti-holin on the genome Anti-holin sfGFP BBa_K112805 (Holin) BBa_E1010 (RFP) BBa_K515106 pSB1C3 BBa_K112806 (Endolysin)

Bba_J23103

Specifications & Design Modelling Assembly Testing & Results Status

Module 3: Gene Guard

What is the best promoter and RBS strength ratio?

A promoter strength ratio ≥ 300

52

Specifications & Design Modelling Assembly Testing & Results Status

Module 3: Gene Guard

Will it work ? Yes, it will

Specifications & Design Modelling Assembly Testing & Results Status

Module 3: Gene Guard

Stage 1

Specifications & Design Modelling Assembly Testing & Results Status

Module 3: Gene Guard

Stage 2

RBS

Specifications & Design Modelling Assembly Testing & Results Status

Module 3: Gene Guard

Stage 3

Specifications & Design Modelling Assembly Testing & Results Status

Module 3: Gene Guard

GFP & RFP GFP & RFP RFP

Horizontal Gene Transfer Experiment

Specifications & Design Modelling Assembly Testing & Results Status

Module 3: Gene Guard

GFP & RFP GFP & RFP

Horizontal Gene Transfer Experiment

Specifications & Design Modelling Assembly Testing & Results Status

Module 3: Gene Guard

Status so far

Anti-holin construct is completed

Specifications & Design Modelling Assembly Testing & Results Status

Module 3: Gene Guard

Status so far

Anti-holin construct is completed Anti-holin has been inserted into the genome

Specifications & Design Modelling Assembly Testing & Results Status

Module 3: Gene Guard

Status so far

Anti-holin construct is completed Anti-holin has been inserted into the genome

Troubleshooting toxin construct

Auxin Xpress Gene Guard Phyto-Route

Technological

Future applications

Practical

Technological

Future applications

Practical

Implementation using seed coat A world leading agri-business

How can we best implement our project?

Leaders in seed technology

Use a seed coat!

Technological

Future applications

Practical

Image provided by Dr Frans Tetteroo from Incotec

Technological

Future applications

Practical

THE BERKELEY REAFFORESTATION TRUST

Technological

Future applications

Practical

Effect of auxin on plant biomass

IAA concentration (nM) Biomass (g)

Outreach

Broadcast show Radio iGEM Artistic representation of our project by college intern Event at Natural History Museum Script writing

Experimental work Human practices Modelling

Summary

Each influenced the others to produce a compelling proof of concept

Achievements

Rewired chemotaxis Got bacteria in the root Produced auxin in our chassis Integrated anti-holin into the genome Re-characterised two fluorescent proteins 6 new working BioBricks submitted to the Registry Collaborated with WITS-CSIR team from South Africa Got Arabidopsis to express venus in response to bacterial auxin Tracked cell viability with Dendra2 inside Arabidopsis roots Designed a joint-codon optimising software Incorporated human practices and modelling into our design and implementation Created Radio iGEM IAA increases Arabidopsis biomass and soil stability

Achievements

Rewired chemotaxis Got bacteria in the root Produced auxin in our chassis Integrated anti-holin into the genome Re-characterised two fluorescent proteins 6 new working BioBricks submitted to the Registry Collaborated with WITS-CSIR team from South Africa Got Arabidopsis to express venus in response to bacterial auxin Tracked cell viability with Dendra2 inside Arabidopsis roots Designed a joint-codon optimising software Incorporated human practices and modelling into our design and implementation Created Radio iGEM IAA increases Arabidopsis biomass and soil stability

Achievements

Rewired chemotaxis Got bacteria in the root Produced auxin in our chassis Integrated anti-holin into the genome Re-characterised two fluorescent proteins 6 new working BioBricks submitted to the Registry Collaborated with WITS-CSIR team from South Africa Got Arabidopsis to express venus in response to bacterial auxin Tracked cell viability with Dendra2 inside Arabidopsis roots Designed a joint-codon optimising software Incorporated human practices and modelling into our design and implementation Created Radio iGEM IAA increases Arabidopsis biomass and soil stability

Achievements

Rewired chemotaxis Got bacteria in the root Produced auxin in our chassis Integrated anti-holin into the genome Re-characterised two fluorescent proteins 6 new working BioBricks submitted to the Registry Collaborated with WITS-CSIR team from South Africa Got Arabidopsis to express venus in response to bacterial auxin Tracked cell viability with Dendra2 inside Arabidopsis roots Designed a joint-codon optimising software Incorporated human practices and modelling into our design and implementation Created Radio iGEM IAA increases Arabidopsis biomass and soil stability

Achievements

Rewired chemotaxis Got bacteria in the root Produced auxin in our chassis Integrated anti-holin into the genome Re-characterised two fluorescent proteins 6 new working BioBricks submitted to the Registry Collaborated with WITS-CSIR team from South Africa Got Arabidopsis to express venus in response to bacterial auxin Tracked cell viability with Dendra2 inside Arabidopsis roots Designed a joint-codon optimising software Incorporated human practices and modelling into our design and implementation Created Radio iGEM IAA increases Arabidopsis biomass and soil stability

Achievements

Rewired chemotaxis Got bacteria in the root Produced auxin in our chassis Integrated anti-holin into the genome Re-characterised two fluorescent proteins 6 new working BioBricks submitted to the Registry Collaborated with WITS-CSIR team from South Africa Got Arabidopsis to express venus in response to bacterial auxin Tracked cell viability with Dendra2 inside Arabidopsis roots Designed a joint-codon optimising software Incorporated human practices and modelling into our design and implementation Created Radio iGEM IAA increases Arabidopsis biomass and soil stability

Achievements

Rewired chemotaxis Got bacteria in the root Produced auxin in our chassis Integrated anti-holin into the genome Re-characterised two fluorescent proteins 6 new working BioBricks submitted to the Registry Collaborated with WITS-CSIR team from South Africa Got Arabidopsis to express venus in response to bacterial auxin Tracked cell viability with Dendra2 inside Arabidopsis roots Designed a joint-codon optimising software Incorporated human practices and modelling into our design and implementation Created Radio iGEM IAA increases Arabidopsis biomass and soil stability

Achievements

Rewired chemotaxis Got bacteria in the root Produced auxin in our chassis Integrated anti-holin into the genome Re-characterised two fluorescent proteins 6 new working BioBricks submitted to the Registry Collaborated with WITS-CSIR team from South Africa Got Arabidopsis to express venus in response to bacterial auxin Tracked cell viability with Dendra2 inside Arabidopsis roots Designed a joint-codon optimising software Incorporated human practices and modelling into our design and implementation Created Radio iGEM IAA increases Arabidopsis biomass and soil stability

Achievements

Rewired chemotaxis Got bacteria in the root Produced auxin in our chassis Integrated anti-holin into the genome Re-characterised two fluorescent proteins 6 new working BioBricks submitted to the Registry Collaborated with WITS-CSIR team from South Africa Got Arabidopsis to express venus in response to bacterial auxin Tracked cell viability with Dendra2 inside Arabidopsis roots Designed a joint-codon optimising software Incorporated human practices and modelling into our design and implementation Created Radio iGEM IAA increases Arabidopsis biomass and soil stability

Achievements

Rewired chemotaxis Got bacteria in the root Produced auxin in our chassis Integrated anti-holin into the genome Re-characterised two fluorescent proteins 6 new working BioBricks submitted to the Registry Collaborated with WITS-CSIR team from South Africa Got Arabidopsis to express venus in response to bacterial auxin Tracked cell viability with Dendra2 inside Arabidopsis roots Designed a joint-codon optimising software Incorporated human practices and modelling into our design and implementation Created Radio iGEM IAA increases Arabidopsis biomass and soil stability

Achievements

Rewired chemotaxis Got bacteria in the root Produced auxin in our chassis Integrated anti-holin into the genome Re-characterised two fluorescent proteins 6 new working BioBricks submitted to the Registry Collaborated with WITS-CSIR team from South Africa Got Arabidopsis to express venus in response to bacterial auxin Tracked cell viability with Dendra2 inside Arabidopsis roots Designed a joint-codon optimising software Incorporated human practices and modelling into our design and implementation Created Radio iGEM IAA increases Arabidopsis biomass and soil stability

Achievements

Rewired chemotaxis Got bacteria in the root Produced auxin in our chassis Integrated anti-holin into the genome Re-characterised two fluorescent proteins 6 new working BioBricks submitted to the Registry Collaborated with WITS-CSIR team from South Africa Got Arabidopsis to express venus in response to bacterial auxin Tracked cell viability with Dendra2 inside Arabidopsis roots Designed a joint-codon optimising software Incorporated human practices and modelling into our design and implementation Created Radio iGEM IAA increases Arabidopsis biomass and soil stability

Achievements

Rewired chemotaxis Got bacteria in the root Produced auxin in our chassis Integrated anti-holin into the genome Re-characterised two fluorescent proteins 6 new working BioBricks submitted to the Registry Collaborated with WITS-CSIR team from South Africa Got Arabidopsis to express venus in response to bacterial auxin Tracked cell viability with Dendra2 inside Arabidopsis roots Designed a joint-codon optimising software Incorporated human practices and modelling into our design and implementation Created Radio iGEM IAA increases Arabidopsis biomass and soil stability

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