2. Synthetic Mammalian Transgene Negative Autoregulation Harpreet Chawla April 2, 2015 Vinay Shimoga, Jacob White, Yi Li, Eduardo Sontag & Leonidas Bleris

3. Outline  Introduction  Transgene  Transcriptional Network  Auto Regulation  Synthetic biology  Network Motifs  Cellular Noise & Its Types  Two Reporter Experiment  Circuit Integration  Results  Analysis of Noise  References

4. Connections Transcriptional n/w Auto regulation TF’s Genes Network Motifs Synthetic Circuits Cellular Noise

5. Gene/Transgene  A Gene is "a locatable region of genomic sequence”, corresponding to a unit of inheritance.  A Transgene is gene/genetic material which has been transferred from one organism to another (naturally or by genetic engineering)

6. Transcriptional Network  The rate of production of each protein  Consist of interactions between Transcription factors (TFs) & Genes.  TF’s bind to the promoter region of the gene & affects the transcription rate. TFs can act as activators or as repressors.  TFs are themselves encoded by genes, which are regulated by yet another TFs, and so on. This set of interactions forms a Transcription Network.

7. Gene Transcription Regulation

8. TF as Activator/Repressor

9. Autoregulation  Self Loop  Autoregulation - Genes regulated by its own product.  Autoregulation - Process that works to adjust system's response to stimuli.  Can be positive or negative.

10. NAR/PAR

11. Synthetic Biology : Engineering life to examine it  Synthetic biological circuits are designed to perform logical functions mimicking those observed in electronic circuits.  Like electronic circuits, they can take a number of different inputs and deliver a particular kind of output.  Are used to control and manipulate living cells. These circuits serve as a method to modify cellular functions, create cellular responses to environmental conditions, or influence cellular development.

13. E Coli transcriptional Network

15. Network Motifs  Isomorphic Subgraphs  Patterns that occur in the real network significantly more often than in randomized networks.  Recurrent and Statistically significant sub graphs or patterns  NAR is a common network motif in transcriptional networks.

16. What this paper is about  Synthetic Circuits stably integrated into Human Kidney cells (Mammalian) to study effect of Noise on genes & cells (Transgene) using NAR (Negative Autoregulation).  Network Motif – Negative feedback loop.  Study the effects of negative feedback regulation on cells and genes (extrinsic/intrinsic noise).  Study effects of negative feedback on total noise.

17.  Cells continuously change their internal state to adapt to environmental changes, look for nutrients or coordinate with other cells.  Do always cells in identical conditions produce the same response (output) to an external stimulus (input)? No two cells are identical, even in a clonal population under the same conditions (pH, T, nutrients etc.) Cellular Noise

19. Two Types : (a) the Intrinsic noise - originating from fluctuations internal to the module (b) the Extrinsic noise - originating from external fluctuations that impinge on the module.  Intrinsic noise refers to variation in identically-regulated quantities within a single cell: for example, the intra-cell variation in expression levels of two identically-controlled genes  Extrinsic noise refers to variation in identically-regulated quantities between different cells: for example, the cell-to-cell variation in expression of a given gene. Types Of Noise

20. The gene encoding a fluorescent protein (GFP) is placed under the control of a promoter, allowing gene expression to be monitored. Experimental Measurement

21. Previous Work : Two Reporter Experiment  Two color experiments  Two identical copies of a gene under precisely the same conditions.  Two equivalent repressible fluorescent reporter genes inserted in the E. coli chromosome controlled by the same promoter, and on opposite sites and equidistant from the replication origin.

22. Previous Work : Two Reporter Experiment Promoters unrepressed due to addition of IPTG high transcription (high number of mRNA molecules) low intrinsic noise Promoters repressed by wild type repressor (lacI) gene low transcription(low number of mRNA molecules) high intrinsic noise  Limitation - Two identically regulated reporters

23. Integration of the circuit

24. Results

25. Analysis of Noise  The total noise observed arises through the combination of global (extrinsic) fluctuations together with the fluctuations in that protein’s local regulation (intrinsic).  The intrinsic noise and extrinsic noise squared, sums to the CV-squared of the fluorescent reporter.

26. Y the constitutive reporter (dsRed) X the regulated reporter - controlled by an inducer IPTG (zsGreen1) α is the coefficient that is 1 for two constitutive promoters with identical reporter statistics but varies depending on the regulation of X.  Result - Negative feedback results in significant total noise reduction by reducing extrinsic noise while marginally increasing intrinsic noise. Analysis of Noise

27. References  Abdullah Hamadeh & Domitilla Del Vecchio, Mitigation of resource competition in synthetic genetic circuits through feedback regulation.  Alon U, Network motifs: theory and experimental approaches.  Rosenfeld N, Elowitz MB and Alon U, Negative auto regulation speeds the response times of transcription networks.  Timothy K Lu, Ahmad S Khalil & James J Collins, Next generation synthetic gene circuits.  Katherine A. Riccione, Robert P. Smith, Anna J. Lee and Lingchong You, A Synthetic Biology approach to understanding cellular information.  Abhyudai Singh1 and Joao Hespanha1, Noise suppression in auto regulatory gene networks.  Hui Zhang, Yueling Chen and Yong Chen, Noise Propagation in gene regulation networks involving interlinked positive and negative feedback loops.

28. Questions

29. THANK YOU