Presentation: Project ideas (Yi and Stefan)

1. Presentation: Project ideas (Yi and Stefan) • Bacterial maze • CO2 deposition • Population oscillator

2. Bacterial maze: general idea

3. Requirements to implement the maze behaviour • Stop – wall sensing system 2. Target – wall sensing system 3. Chemorepellent system 4. Chemoattractant system

4. Implementation of the maze 0. general considerations - Need to sense non-diffusible molecule, attached to the wall: • by method proposed by Leaky et al. 1992 (Biotechnology Nov. 13(5) 738-43) • or by hydrophobic attachment - thus, a transmembrane receptor for stop and target wall sensing systems is needed - E.coli is gram negative  outer membrane makes transmembrane signalling difficult (although it does exist) - Use gram positive bacterium instead  B.subtilis is well – characterised and has lots of signalling systems - idea: use systems of B.subtilis for implementation in another gram positive bacterium to avoid cross-talk

5. Implementation of the maze Stop wall Comx ComP ComA ComAP Rep g ComQX RBS RBS Repellent ComX “Stop” response in other bacteria Repellent 1. The stop wall: ComX quorum sensing system of B.subtilis ComQ

6. Implementation of the maze AIP AgrC AgrA AgrA-P Attr g AgrD+C RBS RBS Attractant AIP “Target” response in other bacteria Attractant 2. The target wall: Agr quorum sensing system of S.aureus Target Wall P2 AgrB

7. Implementation of the maze Target and Stop Systems overview - Essentially the same system in two bacteria - peptides: AIP (S. aureus) - So a specific response can be expected - Crosstalk between Staphylococcus and Bacillus systems should be at a minimum

8. Implementation of the maze 3. The chemorepellent system - Kirsch et. al. (1993) used indole as chemorepellent for B.subtilis - indole is an intermediate of trp biosynthesis in B.subtilis: trp biosynthesis • Thus: overexpress trpC (on plasmid)  chromosomal operon will be • switched off  indole accumulates as an intermediate - Potential problem: export of indole from the cell?

9. Implementation of the maze 4. The chemoattractant system - Could be any amino acid - Propose use of glycine: - Glycine synthesis pathway: - advantage: no operon involved, just need to upregulate one gene (glyA)

10. Implementation of the maze 5. Overview of proposed systems First construct: mostly biobricked Promoter - B0034 - J64703 - B0034 - J51002 - B0015 (comA - RBS - trpC - RBS - ComQX - terminator regulated) Second construct: not yet biobricked Promoter 2 - RBS - glyA - RBS - AgrD - RBS - AgrB - terminator (AgrA regulated)

11. Implementation of the maze 5. Overview of proposed systems Third construct: mostly biobricked Promoter - B0034 - J51000 - B0034 - J51001 - B0015 (constitutive) - RBS - ComP - RBS - ComA - terminator Fourth construct: not yet biobricked Promoter - RBS - AgrC - RBS - AgrA - terminator (constitutive)

12. Implementation of the maze 6. Conclusions on the maze Interesting because: • Population behaviour • integration of quorum sensing systems from different bacteria • good if interested in modelling several interacting systems • non - diffusible initial signals – population sets up the gradient and „finds the right way“ • flexible system – if diffusion of repellent/attractant is sufficient then modify the constructs easily Remaining problems: • Find appropriate gram positive bacterium for implementation • visualisation of target or stop activation by expanding the constructs with RFP and GFP

13. 2. CO2 deposition by bacteria Spot the mistake: Would it be possible to utilize the aspartate biosynthesis pathway to make E.coli fix CO2 from the atmosphere? Project idea: - Find a way to constantly remove the aspartate from the cell maybe after further transformation?) to keep the aspartate synthesis going - Would lead to net fixation (glucose(6C) to 2*aspartate(8C)) - pycA and aspB not yet part of the registry

14. 3. Population oscillator Cellular oscillator: - Need stimulus to reset the system in all cells, otherwise the cells run out of phase - So without control the oscillations within a single cell are masked by its out-of-phase neighbours Population oscillator: - 3 distinct phenotypes that inhibit each other in the known circular manner Features: • Slow oscillations, but stable over time • evolutionary implications – model system for evolutionary instability that is observed in nature

15. Possible implementation of the population oscillator Produces AB 1 Resistant against AB 1 and 3 Susceptible to AB 2 Produces AB 2 Produces AB 3 Resistant against AB 1 and 2 Resistant against AB 2 and 3 Susceptible to AB 3 Susceptible to AB 1