Synthetic Biology = design and engineering of simple biological systems

1. Synthetic Biology = design and engineering of simple biological systems that aren’t found in nature Why would we want to do this? - Want to understand natural systems. One of the best ways to understand a system is to change it or make new, related ones - To fully “understand” a system, we should be able to predict the outcome when we change the system - For molecular biology, this means: - designing new gene circuits and networks - modeling the designed systems & predicting their properties - making & testing the designs - updating our understanding from the model/test agreement

2. Engineers often look at biological systems & think that the systems are equivalent to electronic circuits e.g, fluorescent proteins light bulbs or LEDs transcription factors transistors or logic gates repressors NOT gates activators OR/AND gates polymerases (transcriptional machinery) batteries and so on... Could they be right? --> raises the possibility that biological parts (genes, proteins, etc.) could be combined using the rules established for analog/digital circuits

3. The Repressilator – an engineered genetic circuit designed to make bacteria glow in a periodic fashion = “repressor” + “oscillator” repressors (genes that turn off other genes) green fluorescent protein (glows!) Elowitz & Leibler, Nature (2000) 403:335-8

4. The Repressilator – an engineered genetic circuit designed to make bacteria glow in an oscillatory fashion Elowitz & Leibler, Nature (2000) 403:335-8

5. The repressilator in action...

6. What other kinds of circuits can be built? First, we need some more parts! Some of the other parts available include: - various sensors - light, dark, heat, cold - more switches, logic gates - more repressors, activators - parts for intracellular communication - helpful if cells could tell each what condition they’re in--> quorum sensing - parts for signaling the output of circuits - fluorescent & luminescent proteins

7. Bioluminescence – only occurs when bacteria are present at high density ==> bacteria communicate in order to establish their density Australian pinecone fish Hawaiian bobtail squid ~1011 Vibrio bacteria / ml fluid in light organ in squid mantle - squid uses for disguise (light shines downward, looks like moonlight) ~1010 Vibrio bacteria / ml fluid - fish uses to hunt for prey Nature Reviews Molecular Cell Biology3; 685-695 (2002)

8. Quorum sensing: chemical-based bacterial communication Neighboring bacteria produce HSL also - if enough bacteria around, HSL builds up, activates bioluminescence HSL diffuses in/out of cells LuxR protein (transcription factor) binds HSL, becomes active LuxI LuxI protein makes HSL (homoserine lactone) Light (bioluminescence) Bacterial cell Promoter for LuxR

9. An application of quorum sensing Programming population control into bacteria with a simple designed circuit HSL = homoserine lactone HSL makes HSL HSL- dependent activator kills cell You, Cox, Weiss, Arnold, Nature (2004)

10. & the engineered circuit works ... circuit off circuit on # of bacteria squares = experimental data lines = predictions from model

11. Importantly, the behaviour can be predicted with a simple model cell growth rate cell death rate amount of killer protein rate of cell growth amount of HSL rate of killer protein production killer protein synthesis rate killer protein degradation rate rate of HSL production HSL synthesis rate HSL degradation rate

12. Standardization of parts: MIT’s “BioBricks” project

13. Standardization of parts: MIT’s “BioBricks” project

14. A synthetic biology contest Goal: Construct a bacteria that acts as a finite state machine Finite state machine: - essentially a machine that detects an input and changes its internal state accordingly, choosing among several (finite) possible states --> the theoretical basis for computers Schools: 1st year: MIT, UT, Princeton, Boston University, Cornell 2nd year: 12 schools (the above + UK, Germany, more...) 3rd year: 37 schools, including Japan/Latin America/Korea/India/more Europe expected 4th year: >100

15. UT’s project – build a bacterial edge detector Projector Original image shine image onto cells Cells luminesce along the light/dark boundaries petri dish coated with bacteria Adapted from Zack Simpson

16. How does edge detection work in principle? A computer might visit each pixel in turn, and check to see if it is bordered by both black & white pixels. If yes, highlight the pixel. Is this pixel part of an edge? No Yes No

17. light-dependent gene expression Anselm Levskaya, Chris Voigt (UCSF) Jeff Tabor, Aaron Chevalier, et al.

18. Bacterial photography Aaron Chevalier, Jeff Tabor, Laura Lavery, et al.

19. Mask Cph1/EnvZ

20. Fiduciary Mark Fiduciary Mark Linear Gradient Transfer function: Incident light -> Color developed

21. The first bacterially-based portrait... Great moments in contemporary images: The virgin Maria on a tortilla... Andy Ellington in a petri dish.... Levskaya et al., Nature (2005) 438:441-2

22. Dark Light HSL HSL HSL HSL HSL HSL HSL HSL HSL HSL HSL HSL HSL HSL HSL HSL HSL Now for the edge detector...

23. The edge detector circuit in more detail Tabor et al., MS in prep

24. It works! Projected Mask Photo strain Edge detector strain Tabor et al., MS in prep

25. Projected Mask In silico In vivo Tabor et al., MS in prep

26. Escherichia darwinia Bacterial photography Anselm Levskaya (UCSF), Aaron Chevalier, Jeff Tabor, Zack Simpson, Laura Lavery, Matt Levy, Eric Davidson, Alex Scouras, Andy Ellington, Chris Voigt (UCSF)

27. More information for the curious-minded: Biobricks - Registry of standard biological parts http://parts.mit.edu/ iGEM - the international Genetically Engineered Machine competition http://parts.mit.edu/wiki/index.php/Main_Page From their web site: iGEM addresses the question: Can simple biological systems be built from standard, interchangeable parts and operated in living cells? Or is biology simply too complicated to be engineered in this way? Beyond trying to answer the question above, our broader goals include: - To enable the systematic engineering of biology - To promote the open and transparent development of tools for engineering biology - And to help construct a society that can productively apply biological technology” Recommended review articles: Endy, Foundations for engineering biology. Nature (2005) 438:449-53 Sprinzak & Elowitz, Reconstruction of genetic circuits. Nature (2005) 438:443-8 Hasty, McMillen & Collins, Engineered gene circuits. Nature (2002) 420:224-30