Bacteria Without a Cell Wall L-forms Pros & Cons of Cell Wall - PowerPoint PPT Presentation

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 Bacterium cell wall 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 Design Maintenance Implementation Verification

Rule-based Modelling From writer’s perspective Standard modelling (eg, Rule-based modelling SBML) (BioNetGen) 39 species 5 molecular types 184 reactions 6 rules

Model-based Design

Switch BioBrick: Modelling Informs Design Peptidoglycan biosynthesis in the absence of xylose Molecule numbers Molecule numbers Molecule numbers

Synthetic Biology: Engineering Life Cycle Requirements Implementation Refinement Design Maintenance Implementation Verification

Switch BioBrick: Implementation BBa_K1185000 pbpb spoVD murE murE pbpB Host chromosome

Synthetic Biology: Engineering Life Cycle Requirements Verification Refinement Design Maintenance Implementation Verification

Switch BioBrick: Characterisation 0.8% (w/v) 0.8% (w/v) xylose xylose 0.5% (w/v) 0.5% (w/v) xylose xylose No xylose

Switch BioBrick in Action 1sec = 7hours B. subtilis L-form expressing GFP B. subtilis rod 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 with both HBsu- L-forms with HBsu-GFP L-forms with both HBsu-GFP L-forms with Hbsu-RFP GFP and RFP tagged tagged and RFP tagged tagged

L-forms and plants

L-forms Colonise Plants Brassica pekinensis with Hbsu-GFP tagged L- Brassica pekinensis non-innoculated negative forms around the cell wall 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 Dr. Stach Dr. Hallinan Prof. Wipat Dr. Zuliani Dr. Smith Dr. Robertson Ms. Shapiro Mr. Park Dr. Wu 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