1. Synthetic Gene Circuits Small, Middle-Sized and Huge Molecules Playing Together Within a Cell

2. Outline:  WHY?  Background  Some things that cells can make from genes.  How genes make these things.  How gene activity is controlled: gene circuits.  Regulatory and ‘Epigenetic’ activity activity.  SYNTHETIC GENE CIRCUITS

3. What can genes make? (1)  Cells contain organelles that enable them to synthesize chemicals and structures from instructions in genes.  All of these organelles can reproduce themselves – and make other chemicals and structures – when the organelles follow the instructions in their genes.  Genes without cells don’t work; cells without genes do not work. They work together. Which came first – the chicken or the egg?

4. What can genes make? (2)  Genes can make any protein, following the genetic code (3 nucleotides emplace one amino acid corresponding to one codon). A gene is a one-dimensional array of nucleotides; a protein is a one-dimensional array of amino acids.  Using proteins as catalysts* genes can prescribe the manufacture of all other natural molecules – and some artificial ones as well. A catalyst is a molecule essential to a chemical reaction but neither created nor destroyed by the reaction.

5. What can genes make? (3)  The kinds of molecules that genes make is less interesting than the functions these molecules provide.  Concern here will be with these functions:  gene products (transcription factors) that directly regulate the generating gene or another gene (intrinsic regulation).  gene products that indirectly regulate a gene (extrinsic regulation).  gene products that lead to measurable changes in a cell (reporters).

6. How genes make chemicals  At least a two-step process:  Transcription – transcribe the gene’s DNA into a template RNA (amplification)  Translation – translate information encoded into the RNA into protein (more amplification)  The protein may be the end product or very often it may influence other reactions that make other chemical forms.

7. The train-on-the-track transcription and translation model Rate = Number of tracks x Number of trains x Velocity of trains / Track length

8. The train-on-the-track model: implications  Transcription and translation velocities tend to be fixed.  Length is determined by the gene. Thus …  (Molar) synthesis rate for transcription is controlled by “initiation rate” on 1 or 2 tracks  Molar synthesis rate for translation is determined by the number of mRNA “tracks”  mRNA tracks is determined by balance between synthesis and degradation: Synthesis rate = (decay constant) [mRNA] (first-order decay reaction)

9. Sooooooo …. The initiation rate for transcription* is of very great importance in determining which genes are on and which gene products are generated * The attachment and hence (in steady state) the detachment rate for RNA polymerase (RNAP)

10. What is the RNAP “train starter”?  Transcription factors.  Inducers  Repressors  These are protein molecules, made by genes, that bind to a gene at an operator site, in or near a promoter region, upstream of where transcrip- tion takes place. They often exist in two forms inactive (or quiescent) and active. Usually a small molecule induces the change: Inactive factor  small molecule  active factor

11. Transcription Factors It is important to remember that transcription factors are proteins, come from genes (like all proteins), and may influence either their predecessor gene or –often– other genes. Summary of the structure of the Engrailed homeodomain bound to DNA, as revealed by X-ray crystallography. Cylinders represent the three  -helices of the homeodomain, ribbons represent the sugar phosphate backbone of the DNA and bars symbolize the base pairs. The recognition helix (3) is shown in red.

12. Transcription factors and the molecules that activate them are crucial to determining which genes are on.

13. Transcription of the WT1 Gene Negative feedback: WT1 protein inhibits expression of its own gene and also that of PAX-2 an activator of th WT1 promoter.

14. Myogenesis Upstream regulators force differentiation to mesodermal precursor cells that then express bHLH proteins that stimulate transcription of their own genes. They also activate genes that make MEF2, which further accelerates transcription of genes for bHLH proteins. MEF2 and bHLH proteins both stimulate other muscle-specific genes. Positive feedback!

15. A caveat:  It is biological (and logical) fact that all molecular species generated in a cell degrade. For any intracellular species:

16. Unnatural Experiments  Plasmids – circles of ‘constructed’ DNA that float in bacterial cytoplasm.  Green fluorescent protein. A reporter that represents the integral of a cell’s protein synthesis rate from mRNA.

17. The ‘repressilator’ “A synthetic oscillatory network of transcriptional regulators”, Elowitz, M., Leibler, S., Nature 403 335-338 (20 January 2000)

18. Three repressors  LacI is a repressor protein made from the lacI gene, the lactose inhibitor gene of E. coli.  TetR is a repressor protein made from the tetR gene.  CI is a repressor protein made from the cI gene of phage. Each one of these, with its cognate promoter, will stop production of whatever gene is ‘downstream’ from the promoter.

19. Plasmid Construction

20. The system looks like a negative feedback loop. Does it have predictable stability properties?

21. Repressilator Steady States

22. Repressilator Simulation Results

23. Repressilator Experimental Results

24. Why?  Part of a dual strategy for identifying gene circuits:  Understand devices and low-level, device-device interactions. Elowitz is one way to attack this problem. It answers some questions and raises more.  Then recognize ‘functional motifs’, identify them, “subtract” them from a circuit diagram, and identify the macroscopic circuit design. (Alon*) *Shai S. Shen-Orr, Ron Milo, Shmoolik Mangan & Uri Alon Network motifs in the transcriptional regulation network of Escherichia coli, Nature Genetics, Published online: 22 April, 2002

25. Motifs? – Or in the eye of the believer?

26. The engineering analysis of Gene Circuits is just beginning.