Engineering a virus to select against antibiotic resistance

1. Houston, TX Engineering a virus to select against antibiotic resistance

2. Drug-Resistant Staph Germ's Toll Is Higher Than Thought Wednesday, October 17, 2007; Page A01 Tuesday, 10 October 2006 CDC concerned about drug-resistant TB Friday, March 24, 2006

3. Nearly 19,000 deaths in the U.S. in 2005 due to infections by a new virulent and rampant drug-resistant strain of Staphylococcus aureus “If the mortality estimates are correct, the number of deaths associated with the germ, methicillin-resistant Staphylococcus aureus, or MRSA, would exceed those attributed to H.I.V.-AIDS, Parkinson’s disease, emphysema or homicide each year.”

4. Mechanisms of antibiotic resistance • Efflux Pumps • Hydrolysis • Reduced Uptake • Sequestering • Enzymatic Modification The Science Creative Quarterly 2: Jan-March 2007.

5. Existing strategies to reduce microbial antibiotic resistance all have significant drawbacks StrategiesDrawbacks Use large amounts of antibiotics Merely slows the development of resistance, $$$ $$$, time consuming, unknown side effects Develop new antibiotics Select against specific mechanisms of resistance Too specific, chemical side effects Only targets resistance to specific antibiotics, hard to control Utilize mutually antagonistic antibiotics

6. Project Roadmap Circuit Design & Construction Computational Modeling Circuit Characterization

7. Project Roadmap Computational Modeling • Circuit Design & Construction • general circuit design • create constructs with reporters Circuit Characterization

8. CellS > CellR Circuit design principles: a stable method for programming phenotype-dependent fitness Pantibiotic fitness • Overarching goals: • use existing BioBricks • create a modular design • identify a robust architecture • circuit function selects regardless of resistance mechanism • self-propagating circuit

9. Nature has developed ways of detecting low [antibiotic] within cells No Tetracycline (Tc): No TetA no resistance expressed tetR tetA Low [Tc] TetA TetA TetA resistance expressed tetR tetA

10. Differentiating between sensitive (TcS) and resistant (TcR) cells affects fitness LOW [Tc] TcS bacteria Fitness increased tetR fitness TcR bacteria Smaller effect on fitness tetR fitness

11. Bacteriophage lambda can stably alter fitness Lysogeny Lysis Molecular Biology of the Cell. New York: Garland; 4th Edition, March 2002.

12. Bacteriophage lambda can stably alter fitness Robust and stable fitness alteration Lysogeny Lysis Molecular Biology of the Cell. New York: Garland; 4th Edition, March 2002.

13. Bacteriophage lambda can stably alter fitness Controllable through BioBricks Lysogeny Lysis Molecular Biology of the Cell. New York: Garland; 4th Edition, March 2002.

14. Bacteriophage lambda can stably alter fitness Self-Propagation Lysogeny Lysis Molecular Biology of the Cell. New York: Garland; 4th Edition, March 2002.

15. A circuit producing [CI] will increase phage immunity and fitness Reduced [CI] Increased [CI] l l l l l l l l lysogeny lysis Phage-encoded cI

16. A circuit producing [CI] will increase phage immunity and fitness Reduced [CI] Increased [CI] l l l l l l l l l l l l l l l l lysogeny lysis Phage-encoded cI Circuit-encoded cI

17. C C C C C C C C C C C C C C C C C C C C N N N N N N N N N N N N N N N N N N N N Our circuit design couples phage immunity to antibiotics PX PTet tetR cI PX PTet tetR cI Intracellular [tetracycline] Immunity to phage PX PTet tetR cI

18. C C C C C C C C C C C C C C C C C C C C N N N N N N N N N N N N N N N N N N N N Our circuit design couples phage immunity to antibiotics TcR bacteria PX PTet tetR cI PX PTet tetR cI TcS bacteria Intracellular [tetracycline] Immunity to phage PX PTet tetR cI

19. C C N N Reporter Tc Sensor Px PTet tetR eyfp cI generator PTet cI Our circuit consists of three interconnected pieces

20. Reporter Tc Sensor Px PTet tetR eyfp J23114 J23106 Characterization constructs include the sensor and reporter J23113 BBa_I744101 BBa_I744102 BBa_I744103

21. Reporter Tc Sensor PTet PTet tetR eyfp TetR R0040 Characterization constructs include the sensor and reporter BBa_I744104

22. C C N N The lambda cI generator construct confers immunity to phage PTet cI B0014 BBa_I744121

23. C C N N Reporter Tc Sensor PTet PTet tetR eyfp cI generator PTet cI TetR R0040 One full circuit has been constructed that uses feedback regulation B0014 BBa_I744204

24. Project roadmap Circuit Design & Construction Computational Modeling • evaluate plasmid-based circuit Circuit characterization

25. PX tetR Using the Gillespie algorithm for modeling the effect of cell type on circuit output TetA Tc TetR2 CI2 PTet cI Journal of Physical Chemistry. Vol. 81:25 p.2340-61. PNAS. Vol. 102:10 p.3581-3586.

26. Constitutive promoters produce too much TetR Px PTet Tc Sensor Immunity tetR cI Steady-state levels (copies/cell)

27. The model predicts that the the circuit can differentiate cell types PTet PTet Tc Sensor Immunity tetR cI Sensitive Molecules of cI/cell Molecules Resistant Minutes 100 Time (minutes)

28. Project Roadmap Circuit Design & Construction Computational Modeling Circuit Characterization • test circuit’s ability to differentiate between cell type

29. C C N N Reporter Tc Sensor PTet PTet tetR eyfp cI generator PTet cI EYFP output was used to characterize the full circuit Registry Part # I744204 TetR R0040 B0014

30. Control B0011 Circuit: I744204 GNB8385K=TcS Tn10 TcS+circuit GNB824=TcR TcS-circuit TcR+circuit TcR-circuit Cellular chassis for characterization Cell strains were graciously provided by G. N. Bennett Journal of Bacteriology. Vol. 177:3 p.810-814.

31. 5 7 10 5 6 10 5 5 10 TcR+circuit 5 4 10 TcR-circuit 5 3 10 5 2 10 5 1 10 0 0 20 40 60 80 100 No detectable EYFP output in TcRE. coli Fluorescence/A600 Time (min) [aTc] = 1 µM KD = 0.01nM Biochemistry. Vol. 35:23 p.7439-7446 Excitation = 514 nm, Emission = 527 nm

32. 5 7 10 5 6 10 5 5 10 TcS+circuit 5 4 10 TcS-circuit 5 3 10 5 2 10 5 1 10 0 0 20 40 60 80 100 In contrast, EYFP is produced under similar conditions in TcSE. coli Fluorescence/A600 Time (min) [aTc] = 1 µM KD = 0.01nM Biochemistry. Vol. 35:23 p.7439-7446 Excitation = 514 nm, Emission = 527 nm

33. TcR-circuit TcR+circuit TcS+circuit TcS-circuit Effects of increasing Tc on cell growth [Tc] ≤ 2 µM can be used to drive our circuit with large adverse effects on cell growth T = 240min However, the effects of Tc on EYFP (and CI) production have not yet been characterized.

34. Conclusions Circuit Design & Construction • built a variety of selection circuits Computational Modeling • created effective model and selected circuit for characterization Circuit Characterization • demonstrated circuit’s ability to differentiate between cell types

35. Future Roadmap Circuit Design & Construction • construct phage optimized circuit Computational Modeling • model circuit in phage Circuit Characterization • test circuit function within phage

36. BIOCHEMISTRY AND CELL BIOLOGY Beth Beason George Bennett Tina Chen David Kim Joff Silberg Taylor Stevenson Arielle Layman BIOENGINEERING Christie Peebles Ka-Yiu San Thomas-Segall Shapiro COMPUTATION AND APPLIED MATH Steve Cox Jay Raol CHEMICAL AND BIOMOLECULAR ENGINEERING Ken Cox Alec Walker Baylor College of Medicine Bibhash Mukhopadhyay

37. 5 7 10 5 6 10 5 5 10 5 4 10 5 3 10 5 2 10 5 1 10 0 0 0 20 20 40 40 60 60 80 80 Linking our cell detection to phage lysis Create TcR and TcS temperature sensitive lysogens and determine if circuit provides a relative fitness gain for TcS over TcR cells TcR+/-circuit TcS+/-circuit Fluorescence/A600 Time (min) Time (min)

38. Ptet Controlled Production of TetR Creates Resistance Phenotype TcS+circuit TcS Ptet tetR B0011 I744204

39. 1744204 B0011 (empty) Tetracycline Affects Growth Rates t = 30min

40. B0011 I744204 TcR TcS TcR+circuit TcS+circuit Tetracycline Affects on Growth Rates t = 240min

41. Phage Circuit Incorporation Relies Upon Recombination Plasmid lambda homologous Biobrick restriction site bleomycin resistance bleoR λ1 λ2