A New Chassis for Synthetic Biology: Bacteria Without a...
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A New Chassis for Synthetic Biology:
Bacteria Without a Cell Wall
L-forms
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Pros & Cons of Cell Wall
Cell membrane
DNA
ribosomes
RNA
metabolites
Bacterium
without cell wall
Cell wall
Cell membrane
Bacterium with
cell wall
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Previous work on L-forms
TEM pictures of L-forms
Gilpin, R. W., Young, F. E. & Chatterjee, A. N., 1973. Characterization of a Stable L-form of Bacillus subtilis 168. Journal of Bacteriology, 113(1), pp. 486-499.
• Discovered by Lister Institute in
1935
• Roles in diseases such as
sarcoidosis and septicemia
• Pathogens are not a good
chassis for synthetic biology
• We engineered the non-pathogen
B. subtilis to produce L-forms
• Built on pioneering work by Prof.
Jeff Errington and colleagues at
Newcastle
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Bacillus subtilis
• Gram +ve Bacteria
• Rod shape
• Non-pathogenic auxotroph
• Commonly found in soil
and human gut
• Secretion
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Aim
To develop L-forms as a chassis for the
synthetic biology community
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Synthetic Biology:
Engineering Life Cycle
Requirements
Design
Implementation
Verification
Maintenance
Refinement Requirements
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Ultimate Goals
• Develop a switch device that will selectively
turn the cell wall ON and OFF
• Demonstrate the use of L-forms for real
world applications
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Human Practice & Implications
QUESTION: Are fused cell-wall less bacteria genetically modified?
Implications of release of L-forms into the environment
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UK, EU and US Law
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Built-in Kill Switch
L-forms in soil after 1 min incubation
1sec = 1sec
L-forms in normal media NB/MSM
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Synthetic Biology:
Engineering Life Cycle
Requirements
Design
Implementation
Verification
Maintenance
Refinement Design
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Rule-based Modelling
Standard modelling (eg,
SBML)
39 species
184 reactions
Rule-based modelling
(BioNetGen)
5 molecular types
6 rules
From writer’s perspective
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Model-based Design
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Switch BioBrick: Modelling Informs Design
Mo
lecu
le n
um
ber
s M
ole
cule
nu
mb
ers
Peptidoglycan biosynthesis in the absence of xylose
Mo
lecu
le n
um
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s
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Synthetic Biology:
Engineering Life Cycle
Requirements
Design
Implementation
Verification
Maintenance
Refinement Implementation
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Switch BioBrick: Implementation
pbpB pbpb spoVD murE murE
Host chromosome
BBa_K1185000
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Synthetic Biology:
Engineering Life Cycle
Requirements
Design
Implementation
Verification
Maintenance
Refinement Verification
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Switch BioBrick:
Characterisation
0.8% (w/v)
xylose
0.5% (w/v)
xylose
No xylose
0.5% (w/v)
xylose
0.8% (w/v)
xylose
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Switch BioBrick in Action
B. subtilis rod expressing GFP B. subtilis L-form expressing GFP 1sec = 7hours
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Potential Applications
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Our Applications
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Genome Shuffling
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Genome Shuffling
BBa_K1185001 HBsu-GFP
BBa_K1185002 HBsu-RFP
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Implementing Cell Fusion
• Agarose-based single cell chemostat
chamber
• Micron-width linear tracks
• Allowing fine control of cells
movement
• Useful to study single-cell study
especially the natural heterogeneity
in growth and gene expression.
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L-forms with HBsu-GFP
tagged L-forms with Hbsu-RFP
tagged
+
Genome Shuffling
L-forms with both HBsu-
GFP and RFP tagged L-forms with both HBsu-GFP
and RFP tagged
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L-forms and plants
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L-forms Colonise Plants
Brassica pekinensis with Hbsu-GFP tagged L-
forms around the cell wall Brassica pekinensis non-innoculated negative
control
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Human Practices: Revisited
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Community Interaction
Leeds 2013 iGEM team model using BioNetGen
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Summary • A foundational advance: A new chassis for
Synthetic Biology; informed by discussion with
ethicists and the public
• We have created a genetic switch to turn the cell
wall on and off
• We demonstrated that our engineered L-forms
can be fused to shuffle their genomes
• We showed that these L-forms can inhabit plants
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Our BioBricks
BBa_K1185000: Enables B. subtilis to switch between a
cell walled rod form and cell wall removed L-form,
dependent on the presence of xylose in growth media
BBa_K1185001: Non-discriminately tags DNA, allowing
location of the DNA by glowing green under fluorescence.
BBa_K1185002: Non-discriminately tags DNA, allowing
location of the DNA by glowing red under fluorescence.
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Acknowledgments
Dr. Stach Dr. Hallinan Dr. Zuliani
Mr. Park
Dr. Smith
Mr. Gilfellon Ms. Shapiro Dr. Wu
Dr. Robertson Prof. Wipat
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Summary
• A foundational advance: A new chassis for
Synthetic Biology; informed by discussion with
ethicists and the public
• We have created a genetic switch to turn the cell
wall on and off
• We demonstrated that our engineered L-forms
can be fused to shuffle their genomes
• We showed that these L-forms can inhabit plants
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Architecture
Architecture cycle Synthethic Biology cycle