Engineering bacteria to help fight soil erosion
The problem: Desertification DRYLANDS 2010 31,000 hectares of arable land are degraded every day
1.3 x the area of Boston lost per day!
Our solution Lateral root growth enhances soil stability
Chemotaxis to the root Engineering bacteria to move towards roots
Auxin production IAA Structure of auxin Engineering bacteria to produce auxin
Human Practices: Informing design Gene Guard Chassis Choice
Human Practices: Informing design Gene Guard 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)
Human Practices: Informing design Novel containment device GMO Gene transfer Soil bacterium Gene Guard Chassis Choice Gene Guard: Containing our genetic constructs
Human Practices: Informing design Chassis choice Gene Guard OR Chassis Choice B. subtilis E. coli We chose E. coli
Human Practices: Informing design Chassis choice Gene Guard E. coli Chassis Choice
Human Practices: Informing design E. coli K12 survive in soil for more than 7 weeks Gene Guard Chassis Choice
Auxin Xpress Phyto-Route Gene Guard
Auxin Xpress Phyto-Route Gene Guard
Auxin Xpress Phyto-Route Gene Guard
Module 1: Phyto-Route Specifications Rewiring chemotaxis & Design PA2652, malate-responsive Movement towards roots chemoreceptor Modelling Assembly Testing & Results Status
Module 1: Phyto-Route Specifications & Design Optimal malate receptor expression level? Sensitivity and saturation analysis for malate receptor Modelling Assembly Testing & Results Status Concentration of chemoreceptors can be variable
Module 1: Phyto-Route Specifications & Design Phyto-Route construct Modelling Assembly Testing & Results Status
Module 1: Phyto-Route Specifications Behaviour of bacteria upon & Design chemoattractant saturation Probability of bacterial movement at given velocity Modelling PA2652 cells in 10 mM malate PA2652 cells in 10 mM serine Negative control (cells without construct) in 10 mM malate Assembly Testing & Results Velocity ( μ m/s) Status E. coli senses malate 20
Module 1: Phyto-Route Specifications Capillary assay & Design Modelling Assembly Testing & Results Status
Module 1: Phyto-Route Specifications & Design Optimum experiment duration 3D Simulation % population in capillary Modelling Assembly Testing & Results Status 60 mins
Module 1: Phyto-Route Specifications & Design Chemotaxis dependence on malate concentration Modelling Assembly Testing & Results Status E. coli chemotaxes towards malate
Module 1: Phyto-Route Specifications Bacteria in the root & Design Modelling Assembly Testing & Results Status
Module 1: Phyto-Route Specifications & Design Status so far Bacteria chemotax towards root exudate Modelling Assembly Testing & Results Status
Module 1: Phyto-Route Specifications & Design Status so far Bacteria chemotax towards root exudate Modelling Bacteria are uptaken by the root Assembly Testing & Results Status
Auxin Xpress Phyto-Route Gene Guard
Module 2: Auxin Xpress Specifications Release of auxin & Design A simple IAA producing IAM pathway Modelling pathway Assembly Testing & Results Status
Module 2: Auxin Xpress Specifications & Design How much bacterial auxin can we produce ? Modelling Assembly Testing & Results Status Intracellular IAA concentration of 72 µM
Module 2: Auxin Xpress Specifications & Design Modelling Assembly Testing & Results Status
Module 2: Auxin Xpress Specifications & Design What is the effect of the IAA on the root ? Modelling Root length (cm) Assembly Testing & Results IAA concentrations (nM) The optimum is 0.1 nM Status
Simulated Arabidopsis root growth after 25 days [IAA] = 10 -6 mol/L [IAA] = 10 -10 mol/L [IAA] = 10 -14 mol/L Optimum IAA concentration Parameters: Branching, root growth rate, gravity, twisting
Module 2: Auxin Xpress Specifications & Design Auxin Xpress construct Modelling Assembly Testing & Results Status
Module 2: Auxin Xpress Specifications & Design Salkowski assay standards Modelling Assembly Testing & Results Status Colourimetric auxin detection
Module 2: Auxin Xpress Specifications Successful auxin production & Design Modelling 60 IAA concentration (µM) 50 40 Assembly 30 20 10 Testing & Results 0 Negative Auxin Negative Auxin Control Xpress Control Xpress Extracellular auxin yield of 52 µM Status
Module 2: Auxin Xpress Specifications & Design Modelling Mass 130, 176 Assembly Testing & Results IAA peak confirmed by Liquid Chromatography Status Mass Spectrometry (LCMS)
Module 2: Auxin Xpress Specifications & Design DR5 Venus Construct Modelling YFP IAA Assembly DR5 Venus Testing & Results Construct kindly provided by Dr Darren Wells (University of Nottingham) Status
Auxin Xpress Control Engineered E. coli Wild type E. coli
Module 2: Auxin Xpress Specifications & Design Can Arabidopsis detect bacterial auxin ? 100 90 Modelling 80 Fluorescence intensity 70 60 50 Assembly 40 30 20 Testing & 10 Results 0 No bacteria Auxin-producing Non-modified E.coli E.coli Status Yes, it can.
Module 2: Auxin Xpress Specifications & Design Dendra2 photoconversion Modelling Irreversible Photoconversion Assembly Testing & Results Status
Module 2: Auxin Xpress Specifications & Design E. coli remain metabolically active inside roots Modelling Assembly Testing & Results Dendra2-expressing E. coli imaged in an Status Arabidopsis root
Module 2: Auxin Xpress Specifications & Design E. coli remain metabolically active inside roots Modelling Assembly Testing & Results Dendra2 photoconverted after 4 days Status
Module 2: Auxin Xpress Specifications & Design E. coli remain metabolically active inside roots Modelling Assembly Testing & Results Root re-imaged after 24 hours Status
Module 2: Auxin Xpress Specifications & Design Modelling Mass of soil eroded (g) Assembly Testing & Results Status IAA concentration (nM)
Module 2: Auxin Xpress Specifications & Design Status so far Bacteria produce auxin Modelling Assembly d Testing & Results Status
Module 2: Auxin Xpress Specifications & Design Status so far Bacteria produce auxin Modelling Plants respond to bacterial auxin Assembly d Testing & Results Status
Module 2: Auxin Xpress Specifications & Design Status so far Bacteria produce auxin Modelling Plants respond to bacterial auxin Determined optimal auxin concentrations Assembly d Testing & Results Status
Module 2: Auxin Xpress Specifications & Design Status so far Bacteria produce auxin Modelling Plants respond to bacterial auxin Determined optimal auxin concentrations Assembly d Dendra2: A new platform for imaging gene expression in root Testing & Results Status
Module 2: Auxin Xpress Specifications & Design Status so far Bacteria produce auxin Modelling Plants respond to bacterial auxin Determined optimal auxin concentrations Assembly d Dendra2: A new platform for imaging gene expression in root Testing & Results Auxin improves soil stability Status
Auxin Xpress Phyto-Route Gene Guard
Module 3: Gene Guard Specifications Preventing horizontal gene transfer & Design 1. Prevent horizontal Holin/endolysin gene transfer Modelling 2. Without harming Anti-holin on the our GMO genome Anti-holin sfGFP Assembly Bba_J23103 BBa_E1010 (RFP) Testing & Results pSB1C3 BBa_K515106 BBa_K112805 (Holin) Status BBa_K112806 (Endolysin)
Module 3: Gene Guard Specifications What is the best promoter and RBS strength ratio? & Design Modelling Assembly Testing & Results Status A promoter strength ratio ≥ 300 52
Module 3: Gene Guard Specifications & Design Will it work ? Modelling Assembly Testing & Results Status Yes, it will
Module 3: Gene Guard Specifications Stage 1 & Design Modelling Assembly Testing & Results Status
Module 3: Gene Guard Specifications & Design RBS Stage 2 Modelling Assembly Testing & Results Status
Module 3: Gene Guard Specifications & Design Stage 3 Modelling Assembly Testing & Results Status
Module 3: Gene Guard Specifications Horizontal Gene Transfer Experiment & Design Modelling Assembly Testing & Results GFP & RFP GFP & RFP RFP Status
Module 3: Gene Guard Specifications Horizontal Gene Transfer Experiment & Design Modelling Assembly Testing & Results GFP & RFP GFP & RFP Status
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