Synthetic Mammalian Transgene Negative Autoregulation Vinay Shimoga Jacob

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

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

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

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

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

Gene Transcription Regulation

TF as Activator/Repressor

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

NAR/PAR

Synthetic Biology : Engineering life to examine it q Synthetic biological circuits are designed to perform logical functions mimicking those observed in electronic circuits. q Like electronic circuits, they can take a number of different inputs and deliver a particular kind of output. q 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.

E Coli transcriptional Network

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

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

Cellular Noise q Cells continuously change their internal state to adapt to environmental changes, look for nutrients or coordinate with other cells. q 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 (p. H, T, nutrients etc. )

Cellular Noise

Types Of Noise 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. q 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 q 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.

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

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

Previous Work : Two Reporter Experiment • Promoters repressed by wild type repressor (lac. I) gene • low transcription(low number of m. RNA molecules) • high intrinsic noise • Promoters unrepressed due to addition of IPTG • high transcription (high number of m. RNA molecules) • low intrinsic noise q Limitation - Two identically regulated reporters

Integration of the circuit

Results

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

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

References q Abdullah Hamadeh & Domitilla Del Vecchio, Mitigation of resource competition in synthetic genetic circuits through feedback regulation. q Alon U, Network motifs: theory and experimental approaches. q Rosenfeld N, Elowitz MB and Alon U, Negative auto regulation speeds the response times of transcription networks. q Timothy K Lu, Ahmad S Khalil & James J Collins, Next generation synthetic gene circuits. q Katherine A. Riccione, Robert P. Smith, Anna J. Lee and Lingchong You, A Synthetic Biology approach to understanding cellular information. q Abhyudai Singh 1 and Joao Hespanha 1, Noise suppression in auto regulatory gene networks. q Hui Zhang, Yueling Chen and Yong Chen, Noise Propagation in gene regulation networks involving interlinked positive and negative feedback loops.

Questions

THANK YOU