A New Chassis for Synthetic Biology: Bacteria Without a Cell Wall. L-forms

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Marilyn Miles
5 years ago
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1 A New Chassis for Synthetic Biology: Bacteria Without a Cell Wall L-forms

2 Pros & Cons of Cell Wall Cell wall Cell membrane Cell membrane DNA ribosomes RNA metabolites Bacterium with cell wall Bacterium without cell wall

Gilpin, R. W., Young, F. E. & Chatterjee, A. N., 1973. Characterization of a Stable L-form of Bacillus subtilis 168.

Previous work on L-forms Discovered by Lister Institute in 1935 Roles in diseases such as sarcoidosis and septicemia

3 Gilpin, R. W., Young, F. E. & Chatterjee, A. N., Characterization of a Stable L-form of Bacillus subtilis 168. Journal of Bacteriology, 113(1), pp 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

4 Bacillus subtilis

5 Aim To develop L-forms as a chassis for the synthetic biology community

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

7 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

8 Human Practice & Implications QUESTION: Are fused cell-wall less bacteria genetically modified? Implications of release of L-forms into the environment

9 UK, EU and US Law

10 Built-in Kill Switch L-forms in soil after 1 min incubation 1sec = 1sec L-forms in normal media NB/MSM

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

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

13 Model-based Design

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

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

16 Switch BioBrick: Implementation BBa_K pbpb pbpb spovd mure mure Host chromosome

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

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

19 Switch BioBrick in Action 1sec = 7hours B. subtilis rod expressing GFP B. subtilis L-form expressing GFP

20 Potential Applications

21 Our Applications

22 Genome Shuffling

23 Genome Shuffling BBa_K HBsu-GFP BBa_K HBsu-RFP

24 Implementing Cell Fusion

25 Genome Shuffling + L-forms with both HBsu-GFP GFP and tagged RFP tagged L-forms L-forms with with both Hbsu-RFP HBsu-GFP and RFP tagged tagged

26 L-forms and plants

27 L-forms Colonise Plants Brassica pekinensis with Hbsu-GFP tagged L- forms around the cell wall Brassica pekinensis non-innoculated negative control

28 Human Practices: Revisited

29 Community Interaction Leeds 2013 igem team model using BioNetGen

30 Summary

presence of xylose in growth media BBa_K1185001: Non-discriminately tags DNA, allowing location

31 Our BioBricks BBa_K : 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_K : Non-discriminately tags DNA, allowing location of the DNA by glowing green under fluorescence. BBa_K : Non-discriminately tags DNA, allowing location of the DNA by glowing red under fluorescence.

32 Acknowledgments Prof. Wipat Dr. Stach Dr. Hallinan Dr. Zuliani Dr. Smith Dr. Robertson Dr. Wu Ms. Shapiro Mr. Park 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

33 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

35 Architecture Synthethic Biology cycle Architecture cycle

igem 2009 Team Newcastle

igem 2009 Team Newcastle Introduction Environmental project Heavy metal pollution in soil Cadmium accumulation issue Image: http://www.enst.umd.edu/people/weil/researchprojects.cfm Use of engineered micro-organisms