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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