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