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The Programming of a Cell

The Programming of a Cell. By L Varin and N Kharma Biology and Computer Engineering Departments Concordia University. Motivation. Cells have advantages over silicon: They have/can have built-in interfaces, to sense and produce many biological substances

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The Programming of a Cell

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  1. The Programming of a Cell By L Varin and N Kharma Biology and Computer Engineering Departments Concordia University

  2. Motivation • Cells have advantages over silicon: • They have/can have built-in interfaces, to sense and produce many biological substances • They are easy to mass produce, store and distribute • They are generally more robust than man-made systems • They are optimizable via (real) evolution The Programming of a Cell

  3. Motivation – precisely • The aim is to produce a cell that implements a configurable Boolean logic function in 2 var’s • Ultimately, we would like to use intercellular signalling to compile larger circuits using many smaller ones A mechanical AND gate The Programming of a Cell

  4. Outline • Motivation & Outline • Biological Background • Problem Statement • Alternative Methods • Biological Realization • Practical Significance The Programming of a Cell

  5. Biological Background:Flow of genetic information DNA RNA Protein We can easily manipulate DNA Gene expression Transcription Translation The Programming of a Cell

  6. Biological Background:Gene expression (promoters) +1 RNA Pol -10 Box TATAA -35 Box TTGTCA RNA Core promoter = Binding site for RNA Polymerase In this configuration transcription is ON The Programming of a Cell

  7. Biological Background:Gene expression (promoters) +1 X R -10 box -35 box operator R = Repressor In this configuration RNA Polymerase cannot bind transcription is OFF The Programming of a Cell

  8. Biological Background:Gene expression (synthetic gene) Modular structure • Construct a promoter • Insert an operator • Select a coding sequence (output) Output -10 box -35 box operator The Programming of a Cell

  9. Biological Background:Biological regulatory network • The lactose operon of E. coli lacI repressor X R -35 O -10 Transcription is OFF R Active repressor The Programming of a Cell

  10. Biological Background:Biological regulatory network • The lactose operon of E. coli lacI repressor RNA Pol -35 O -10 X Transcription is ON R Inactive repressor = inducer (lactose) The Programming of a Cell

  11. Biological Background:Artificial regulatory network • Select an output gene • Select a promoter • Select an operator-repressor system • Assemble the parts together The Programming of a Cell

  12. Biological Background:Artificial regulatory network - lactose X OFF lacI l C1 repressor Repressed by lac repressor Lac promoter ON l promoter Green Fluorescent protein Repressed by C1 repressor The Programming of a Cell

  13. Biological Background:Artificial regulatory network + lactose R X l C1 repressor Repressed by lac repressor Lac promoter C1 X C1 l promoter Green Fluorescent protein Repressed by C1 repressor OFF The Programming of a Cell

  14. Problem Statement ++ • Synthesize a cell that can be configured to implement any one of 16 different Boolean functions in 2 variables • Such a project will involve 4 phases: 1 Designing a regulatory network 2 Constructing a configurable cell 3 Configuring the cell 4 Using the cell The Programming of a Cell

  15. Methodology 1: Using Repressilators A B Output R1 Anti-sense DNA R2 The Programming of a Cell

  16. A B’ A B A’ B A’ B’ Output Output Output Output R5 R1 R7 R3 Anti-sense DNA Anti-sense DNA Anti-sense DNA Anti-sense DNA R2 R8 R4 R6 Methodology 1: Full Picture The Programming of a Cell

  17. Methodology 2: Using Excision • A Boolean function in 2 variables has 16 possible truth tables • They all involve 1-4 (3) different terms of 2 variables A B Output 00 ? 01 ? 11 ? 10 ? The Programming of a Cell

  18. Methodology 2: Chosen Path • Design A regulatory network implementing the 4 terms and allowing for subsequent excision of any term • Construct The regulatory network by embedding 4 gene networks corresponding to the 4 terms in a real organism (e.g. e.coli) • Configure The cell by excising those gene networks corresponding to the unwanted terms • Use The configured cell by adding the inducers (variables) it is designed to respond to, and monitoring the output The Programming of a Cell

  19. Biological Realization • 2 variables A and B • A = lactose • B = arabinose • 1 promoter • 4 repressors • 1 ouput gene (Green Fluorescent Protein) • 4 terms (A B), (A B), (A B), (A B) The Programming of a Cell

  20. Biological Realization Output (GFP) (A B) = Lac operon operator (bound by LacI repressor) = Arabinose operon operator (bound by AraR repressor) In the absence of A and B X LacI AraR Output (GFP) The Programming of a Cell

  21. Biological Realization (A B) LacI AraR X Output (GFP) In the presence of A and B ( lactose and arabinose) LacI AraR X X Output (GFP) The Programming of a Cell

  22. Biological Realization Output (GFP) (A B) = l Pr operator (bound by C1 repressor) = Arabinose operon operator (bound by AraR repressor) X LacI C1 In the absence of A and in presence of B AraR X Output (GFP) The Programming of a Cell

  23. Biological Realization Output (GFP) (A B) = lactose operon operator (bound by lacI repressor) = l Prm operator (bound by CRO repressor) X AraR CRO In the presence of A and in absence of B LacI X Output (GFP) The Programming of a Cell

  24. Biological Realization Output (GFP) (A B) = l Pr operator (bound by C1 repressor) X AraR CRO = l Prm operator (bound by CRO repressor) X LacI C1 In the absence of A and in absence of B Output (GFP) The Programming of a Cell

  25. Biological Realization Output (GFP) (A B) (A B) (A B) (A B) Output (GFP) Output (GFP) Output (GFP) The Programming of a Cell

  26. Practical Significance Limited ConfigurableDecision Logic Inputs tied to a particular application: lactose, arabinose etc. Outputs tied to a particular application: GFP etc. The Programming of a Cell

  27. Practical Significance Application-specific outputs Application-specific inputs Input Interface Output Interface Extended Decision Logic Standardized Signals The Programming of a Cell

  28. Summary • A specific Boolean logic function in 5 variables has been recently realized in living cells, but never a configurable bio-logic device  theoretic value • We believe we have found a simple means of realizing a configurable 2-input Boolean function in an e.coli cell simple methodology • Both the logic functionality and the practical value of the work can be considerably enhanced with the use of intercellular signaling  broader vision • First experiments (for the Method 2) will start in January 2008  and we’ll update you! The Programming of a Cell

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