a new chassis for synthetic biology: bacteria without a...
TRANSCRIPT
A New Chassis for Synthetic Biology:
Bacteria Without a Cell Wall
L-forms
Pros & Cons of Cell Wall
Cell membrane
DNA
ribosomes
RNA
metabolites
Bacterium
without cell wall
Cell wall
Cell membrane
Bacterium with
cell wall
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
Bacillus subtilis
• Gram +ve Bacteria
• Rod shape
• Non-pathogenic auxotroph
• Commonly found in soil
and human gut
• Secretion
Aim
To develop L-forms as a chassis for the
synthetic biology community
Synthetic Biology:
Engineering Life Cycle
Requirements
Design
Implementation
Verification
Maintenance
Refinement Requirements
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
Human Practice & Implications
QUESTION: Are fused cell-wall less bacteria genetically modified?
Implications of release of L-forms into the environment
UK, EU and US Law
Built-in Kill Switch
L-forms in soil after 1 min incubation
1sec = 1sec
L-forms in normal media NB/MSM
Synthetic Biology:
Engineering Life Cycle
Requirements
Design
Implementation
Verification
Maintenance
Refinement Design
Rule-based Modelling
Standard modelling (eg,
SBML)
39 species
184 reactions
Rule-based modelling
(BioNetGen)
5 molecular types
6 rules
From writer’s perspective
Model-based Design
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
ber
s
Synthetic Biology:
Engineering Life Cycle
Requirements
Design
Implementation
Verification
Maintenance
Refinement Implementation
Switch BioBrick: Implementation
pbpB pbpb spoVD murE murE
Host chromosome
BBa_K1185000
Synthetic Biology:
Engineering Life Cycle
Requirements
Design
Implementation
Verification
Maintenance
Refinement Verification
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
Switch BioBrick in Action
B. subtilis rod expressing GFP B. subtilis L-form expressing GFP 1sec = 7hours
Potential Applications
Our Applications
Genome Shuffling
Genome Shuffling
BBa_K1185001 HBsu-GFP
BBa_K1185002 HBsu-RFP
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.
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
L-forms and plants
L-forms Colonise Plants
Brassica pekinensis with Hbsu-GFP tagged L-
forms around the cell wall Brassica pekinensis non-innoculated negative
control
Human Practices: Revisited
Community Interaction
Leeds 2013 iGEM team model using BioNetGen
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
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.
Acknowledgments
Dr. Stach Dr. Hallinan Dr. Zuliani
Mr. Park
Dr. Smith
Mr. Gilfellon Ms. Shapiro Dr. Wu
Dr. Robertson Prof. Wipat
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
Architecture
Architecture cycle Synthethic Biology cycle