Download Part 1: Field release of free a free-living GM bacteria Oxford

Part 1: Field release of free a free-living GM bacteria
Ian Thompson
University of
Oxford
Wytham Field
Station.
1993-1994
Pseudomonas fluorescens SBW25
How did we do it? Genetic diversity of a single
pseudomond population in a single site (rDNA RFLP
ribotyping)
KpnI digests
EcoRI digests
  Over 3000 isolates
analysed over 2 growing
seasons.
  More than 2000 genotypes
detected.
  Of these only 26 genotypes
were detected more than
once.
  Only 15 commonly detected.
Chromosomal marking of P. fluorescens SBW25
Chromosome - 6.5 Mb
Bglll
Ee (4.0 Kb)
lacZY- (growth on lactose,
galactosidase)
8.4 Kb
 
 
Bglll
6 (7.2 Kb)
XylE (dioxygenase split of catechol) Km
2.4 Kb
P. fluorescens SBW 25EeZY6KX is a sugar beet phytosphere isolate
Strain released as a seed dressing, colonised the roots and leaves
of the growing crop, both sugar beet and wheat.
Monitoring GM survival in the field
Leaf prints of SBW25 distribu5on on field grown leaves Log10 cfu/g GMM dry wt. plant tissue
Phyllosphere population dynamics of P. fluorescens
SBW25EeZY6KX; field and glasshouse grown
sugar beet.
9
8
7
6
5
1993
4
1994
3
g'house
2
1
0
1
30
60
90
120
160
days after true leaf set emergence
240
GM bacteria
introduced to seed
Field release of P. fluorescens SBW25EeZY6KX:
impact of plasmid carriage on rhizosphere
colonisation
7
Count (Log cfu/g)
6
no plasmid
5
4
3
2
pQBR103,
(group 1)
1
0
0
50
100
150
200
Days from sowing
P. fluorescens SBW25EeZY6KX
P. fluorescens SBW25EeZY6KX pQBR103
330 kbp
Change in Shannon index with increasing sample size
-from sugar beet leaves at day 95
Wild type
treated
3.1
Untreated
2.9
Shannon index
2.7
Recombinant
treated
2.5
2.3
2.1
1.9
1.7
1.5
0
10
20
30
Sample size
40
50
60
Population structure/dynamics 0f
pseudomonad populations
Temporal fluctuation in the relative abundance of genotypes
•  Population size constant
Abundance
•  No extinction
•  All clonal groups persist
and proliferate when
conditions are favourable
•  Turnover between 2 and
Time
28 days
Key observations
 
 
 
 
 
 
Released GM was always detected within the
season- never across seasons.
Survived better in the glasshouse with low
competition.
Relative fitness varied with age of plant, season
and plant material sampled.
Carrying an additional genes in the form of a
plasmid impacted on detection/survival.
Acknowledgements
Mark Bailey, Andy Lilley, Paul Rainey and David
Bishop.
Part 2: Synthetic biology and
bioremediation/ clean-up
Ex-Gas works site contaminated with cyanide (Prussian blue
EFe7(CN)18)
A challenge for synthetic
biology.
Vital component of metal working. In Europe alone 2.2 millions tonnes pa
produced- it is very recalcitrant and can be very toxic. Current conventional
disposal methods are not sustainable. We are now converting the waste to
bioenergy.
We have assembled a synthetic community?
From lab to field scale
COD mg per litre
Pollutant load reduction- chemical oxygen demand
85000
80000
75000
70000
65000
60000
55000
50000
45000
40000
35000
30000
25000
20000
15000
10000
5000
0
0
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160
Time (days)
Ford Bridgend site – 80 tonne bioreactors
Synthetic biology is helping us.
2 GM lux indicator bacteria. One for
measure of metabolic response and
the other for DNA damage (Rec A).
As toxicity treatment
measure, not for the biotreatment.
Can synthetic biology help us with the
difficult challenges in remediation?
Dealing with the “treatment plateau”
50000
45000
40000
COD mg l-1
35000
30000
25000
20000
15000
10000
5000
0
Waste streams we work on.
•  Chemically mixed
industrial end-of-pipe oily
effluent (Biffa).
•  Refinery wastes provided
by BP and Exxon (including
heavy oils).
•  Ground water
contaminants such as TCE
(EA).
•  Pharmaceutical wastes
(GSK).
•  Diageo brewery effluent
(mixed humic and copper).
How can we routinely and sustainably reduce the pollution load (expressed
as COD-Carbon Oxygen Demand) to legal consent levels of <2000 mg L?
Synthetic biology approach for treating
recalcitrant
Ultrasound
Microbial community
Artificially encoded proteome
Encoding 108 de novo enyzmes
(cytochrome P450)
With Michael Hecht Metal working fluid
bioreactors
What survives?
Novel biology?
Community Function?
Enhanced Diversity?
How we are looking at the
problem.
Microbiological
Advanced Oxidation Processes
with nanoscale-Fe oxide.
Ultrasound and electrokinetics
Electron-beam
What do we need?
•  Physiology.
•  Understanding the limits of biology
and knowing when its wise to link to
physical and chemical approaches.
Some pointers:
Now
•  Avoid competition with 3.5 B years of
evolution.
•  Go for the low hanging fruit.
•  Pharmacological production in bioreactors.
•  Bioenergy produced in bioreactors.
•  Data storage on DNA.
Longer term
•  Developing tools for understanding
microbial ecology/physiology.
•  Hybrid approaches (Synbio with non-GM).