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

Artificial Life GroupThe Programming of a Cell2 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

Artificial Life GroupThe Programming of a Cell3 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

Artificial Life GroupThe Programming of a Cell4 Outline Motivation & Outline Biological Background Problem Statement Alternative Methods Biological Realization Practical Significance

Artificial Life GroupThe Programming of a Cell5 Biological Background: Flow of genetic information DNARNAProtein We can easily manipulate DNA TranscriptionTranslation Gene expression

Artificial Life GroupThe Programming of a Cell6 Biological Background: Gene expression (promoters) Box TATAA -35 Box TTGTCA RNA Core promoter = Binding site for RNA Polymerase In this configuration transcription is ON RNA Pol

Artificial Life GroupThe Programming of a Cell box -35 box Biological Background: Gene expression (promoters) operator R R = Repressor In this configuration RNA Polymerase cannot bind transcription is OFF X

Artificial Life GroupThe Programming of a Cell8 Construct a promoter Insert an operator Select a coding sequence (output) Biological Background: Gene expression (synthetic gene) -10 box -35 box operator Modular structure Output

Artificial Life GroupThe Programming of a Cell9 Biological Background: Biological regulatory network The lactose operon of E. coli R lacI repressor R -35 O -10 Transcription is OFF Active repressor X

Artificial Life GroupThe Programming of a Cell10 Biological Background: Biological regulatory network The lactose operon of E. coli R lacI repressor -35 O -10 Transcription is ON Inactive repressor = inducer (lactose) RNA Pol X

Artificial Life GroupThe Programming of a Cell11 Biological Background: Artificial regulatory network Select an output gene Select a promoter Select an operator-repressor system Assemble the parts together

Artificial Life GroupThe Programming of a Cell12 Biological Background: Artificial regulatory network Green Fluorescent protein C1 repressor promoter Lac promoter Repressed by lac repressor Repressed by C1 repressor lacI ON OFF - lactose X

Artificial Life GroupThe Programming of a Cell13 Green Fluorescent protein C1 repressor promoter Lac promoter Repressed by lac repressor Repressed by C1 repressor OFF R + lactose X X C1 Biological Background: Artificial regulatory network

Artificial Life GroupThe Programming of a Cell14 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

Artificial Life GroupThe Programming of a Cell15 Methodology 1: Using Repressilators A B Output R1 R2 Anti-sense DNA

Artificial Life GroupThe Programming of a Cell16 Methodology 1: Full Picture A B’ Output R3 R4 Anti- sense DNA A B Output R1 R2 Anti- sense DNA A’ B’ Output R5 R6 Anti- sense DNA A’ B Output R7 R8 Anti- sense DNA

Artificial Life GroupThe Programming of a Cell17 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 ?

Artificial Life GroupThe Programming of a Cell18 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

Artificial Life GroupThe Programming of a Cell19 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) Biological Realization

Artificial Life GroupThe Programming of a Cell20 Biological Realization (A B) Output (GFP) = Lac operon operator (bound by LacI repressor) = Arabinose operon operator (bound by AraR repressor) In the absence of A and B Output (GFP) LacIAraR X

Artificial Life GroupThe Programming of a Cell21 Biological Realization (A B) Output (GFP) In the presence of A and B ( lactose and arabinose) Output (GFP) LacIAraR LacIAraR X XX

Artificial Life GroupThe Programming of a Cell22 Biological Realization (A B) Output (GFP) = Pr operator (bound by C1 repressor) In the absence of A and in presence of B Output (GFP) X C1 LacI = Arabinose operon operator (bound by AraR repressor) AraR X

Artificial Life GroupThe Programming of a Cell23 Biological Realization (A B) Output (GFP) = lactose operon operator (bound by lacI repressor) In the presence of A and in absence of B Output (GFP) X CRO = Prm operator (bound by CRO repressor) LacI X AraR

Artificial Life GroupThe Programming of a Cell24 Biological Realization (A B) Output (GFP) = Pr operator (bound by C1 repressor) In the absence of A and in absence of B Output (GFP) X CRO = Prm operator (bound by CRO repressor) AraR C1 LacI X

Artificial Life GroupThe Programming of a Cell25 Biological Realization (A B) Output (GFP)

Artificial Life GroupThe Programming of a Cell26 Practical Significance Limited Configurable Decision Logic Inputs tied to a particular application: lactose, arabinose etc. Outputs tied to a particular application: GFP etc.

Artificial Life GroupThe Programming of a Cell27 Practical Significance Extended Decision Logic Input Interface Output Interface Application- specific inputs Application- specific outputs Standardized Signals

Artificial Life GroupThe Programming of a Cell28 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!