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The Value of Tools in Biology Smolke Lab talk 11-1-06 Framework • Thesis: our ability to understand and manipulate biology is limited by the quality and scope of our tools – cellular understanding - what determines the cell's behavior? – cellular manipulation - how can we control the cell's behavior? Quantizing Biology • cellular behavior is determined by physical properties and their variation in time: – Structures – Locations – Energies – Numbers • Cellular processes often manipulate these quantities in tandem Natural Systems • For example, transcriptional processes separate mechanisms for controlling protein (Number) vs (Structure): – Structure then determines the protein’s Location and Energies, and thereby its function Independence of Tools • If we could manipulate cellular quantities independently, then more states would be reachable. – Analogy: like building a house with (nails, a hammer, and a saw) vs with a (nailshammer-saw) • We can reappropriate natural systems for our own purposes, but their independent use is limited. – Example: PCR borrows from the transcriptional network. Some sequences of DNA are difficult to amplify. • Complete independence is not always possible – Example: the necessary connection between protein Structure and Energy, which limits functions. A closer look at Number • Control over protein number is affected by cellular noise sources – Extrinsic noise: variation in environmental conditions. (temperature, nutrients, signals) – Intrinsic noise: follows from the stochastic nature of protein formation • Laboratory experiments often focus on reducing extrinsic noise – Repeated trials reduces measurement variance A Simple model • Protein produced at an average rate of λ proteins/sec – No RNA, no protein decay – Instrinsic noise is the single cell probability distribution – Extrinsic noise is the sum of many cellular distributions Adding the effects of Translation • Translation efficiency is a major source of noise – variance of many small steps is less than that of fewer large steps – Translation amplifies transcriptional variation in addition to adding noise Ozbodak PMID: 11967532 Qualities of protein Number • Mean and the Variance are both important for cellular behavior • Example: robustness – Mean influences most probable action • Cellular robustness through error control averaging – Variance influences probability of alternative actions • population robustness through diversity Independent control of protein Number • Goal: control over the mean and variance of cellular protein – Mean controlled by protein production rates – Variance controlled by feedback on rates • negative feedback on protein production reduces variance – More protein lower rate less production less protein – Less protein higher rate more production more protein Protein Auto-regulation • Transcriptional feedback: production of a repressor that inhibits transcription • Becskei PMID: 10850721 • Translational feedback: production of a protein that decreases RNA stability – More efficient at reducing relative variance – Higher metabolic cost • Swain PMID: 15544806 A Physical Feedback Mechanism • Translational regulation via modulation of RNA decay rate – RNA degraded though endogeneous endo/exonuclease pathways in E. Coli – 5’ and 3’ hairpins increase the stability of RNA RNA modulation • Removal of protective hairpins decreases stability of RNA transcript less protein produced – Yeast Rnt1p cleaves RNA hairpins with high sequence specificity • Express Rnt1p from the protected RNA transcript, closing the feedback loop – Possibility of an orthogonal, modular feedback system RNA hairpin substrate specificity • Rnt1p recognizes sequence dependent domains • E. Coli RNaseIII also cleaves dsRNA with some sequence dependence • Goal: high Rnt1p activity, low E. Coli RNaseIII activity – Orthogonal system Lamontagne PMID: 14581474 System Modularity • Independence of functional parts: – 5’ and 3’ protective hairpin sequences determine lifetime control of protein number mean – Rnt1p hairpin sequence determines level of feedback control of protein number variance – Hairpin libraries tuning of variance and mean Correlated Expression of YFGOI • Polycistronic coding regions have correlated expression levels – Express any other protein on the same transcript – Use GFPuv for testing purposes – Additional correlation if using same RBS Controls • Open loop system: Rnt1p on separate plasmid no feedback 1. Test for Rnt1p substrate cleavage and RNA destabilization after the expression of Rnt1p 2. Test for no destabilization with non-active Rnt1p hairpins 3. Test for no destabilization without Rnt1p hairpins 4. Test for no destabilization without protectice 5’ and 3’ hairpins – With additional combinations for individual 5’ vs 3’ testing if necessary Applications of controlled variance • Any decision can be modelled as maximizing over some Utility function • Cells make decisions to express or not express a specific protein with a certain probability – Rewarded if choice is correct – Penalized if choice is incorrect • Engineering systems have their own Utility functions Low Number protein expression • Proteins toxic in large numbers • Low number expression is difficult, due to relatively high variance at small N • Variance control through feedback provides higher net population fitness Signal Rectification • Electronic Digital circuits scale well due to voltage rectification after every computation • In contrast, in electronic Analog circuits, errors can propogate and amplify uncontrollably • Chemical rectification may be a useful method for reducing error propogation between separate circuit elements – Allowing for larger, more complicated synthetic circuits and computations Measurement Probe • Remember that every measurement is actually the result of many individual measurements of individual cells – Reducing intrinsic cellular noise increases the accuracy of measurements Conclusions • Tools for Independent manipulation of cellular quantities are intrinsically useful • Negative Feedback as a method for control of number variance • Modular Rnt1p system for orthogonal control of protein variance in E. Coli • Circuit designs using low variance systems Future plans • • • • • Cloning Cloning Cloning Cloning More cloning…