Download A New Chassis for Synthetic Biology: Bacteria Without

A New Chassis for Synthetic Biology:
Bacteria Without a Cell Wall
L-forms
Pros & Cons of Cell Wall
Cell membrane
Cell wall
DNA
Cell membrane
ribosomes
RNA
metabolites
Bacterium with
cell wall
Bacterium
without cell wall
Previous work on L-forms
• 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
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.
Bacillus subtilis
Aim
To develop L-forms as a chassis for the
synthetic biology community
Synthetic Biology:
Engineering Life Cycle
Requirements
Requirements
Refinement
Design
Maintenance
Implementation
Verification
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
L-forms in normal media NB/MSM
1sec = 1sec
Synthetic Biology:
Engineering Life Cycle
Requirements
Design
Refinement
Maintenance
Design
Implementation
Verification
Rule-based Modelling
From writer’s perspective
Standard modelling (eg,
SBML)
Rule-based modelling
(BioNetGen)
39 species
5 molecular types
184 reactions
6 rules
Model-based Design
Switch BioBrick:
Molecule numbers
numbers
Molecule
Modelling Informs Design
Peptidoglycan biosynthesis in the absence of xylose
Synthetic Biology:
Engineering Life Cycle
Requirements
Implementation
Refinement
Design
Maintenance
Implementation
Verification
Switch BioBrick:
Implementation
BBa_K1185000
pbpb
pbpB
spoVD
Host chromosome
murE
murE
Synthetic Biology:
Engineering Life Cycle
Requirements
Verification
Refinement
Design
Maintenance
Implementation
Verification
Switch BioBrick:
Characterisation
0.8% (w/v)
xylose
0.8% (w/v)
xylose
0.5% (w/v)
xylose
0.5% (w/v)
xylose
No xylose
Switch BioBrick in Action
1sec = 7hours
B. subtilis rod expressing GFP
B. subtilis L-form expressing GFP
Potential Applications
Our Applications
Genome Shuffling
Genome Shuffling
BBa_K1185001 HBsu-GFP
BBa_K1185002 HBsu-RFP
Implementing Cell Fusion
Genome Shuffling
+
L-forms
L-formswith
withboth
HBsu-GFP
HBsuGFP andtagged
RFP tagged
L-forms
with with
both Hbsu-RFP
HBsu-GFP
L-forms
and RFP
tagged
tagged
L-forms and plants
L-forms Colonise Plants
Brassica pekinensis with Hbsu-GFP tagged Lforms around the cell wall
Brassica pekinensis non-innoculated negative
control
Human Practices: Revisited
Community Interaction
Leeds 2013 iGEM team model using BioNetGen
Summary
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
Prof. Wipat
Dr. Stach
Dr. Hallinan
Dr. Zuliani
Dr. Wu
Ms. Shapiro
Mr. Park
Dr. Smith Dr. Robertson
Mr. Gilfellon
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
Synthethic Biology cycle
Architecture cycle