From 2006.igem.org

Revision as of 08:05, 17 October 2005

Abstract. We report here the design and implementation in vivo of a gene circuit that can count up to 4. In essence, it uses two toggle switches, each storing 1 bit, to keep track of the 4 states. The design of the counter is highly modular, with the hope that it can be included as a unit in larger circuits, and also combined with further counter instances to keep track of a much larger number of states, up to (2^(n+1)) with n units. To facilitate further developments and integration to other projects, the counter is available in form of BioBricks. Among many exciting applications, the availability of a counter enables the execution of sequential instructions, and paves the way for the execution of artifical programs inside living cells.

Introduction

The past few years have seen the emergence of the field of synthetic biology, in which functional units are designed and built into cells to generate a particular behaviour, and ultimately to better understand Life's mechanisms. Previous efforts include the creation of gene circuits that generate oscillating behaviour (Elowitz00), toggle switch functionality (Atkinson03), artificial cell-cell communication (Bulter04) or pattern-forming behaviour (Basu2005). The present document describes the design and realization of a gene circuit that counts to 4.

Design of the Counter

The counter is a genetic circuit that has 1 input and 4 outputs. It uses the input signal to switch from one of the four output to the next. When the input signal is high, either output 1 or 3 is active, when it is low, output 2 or 4 is active. Thus, output 1 and 3 alternatively keep track of high input signal, while output 2 and 4 alternatively keep track of low input signals.

As depicted above, the counter is made of two parts, serially linked:

• the "Input" module, which splits the input into two opposite signals.
• the "NOR" module, which uses these two signals to sequencially switch through the outputs 1, 2, 3 and 4.

Note that all interfaces have flows described in Polymerase Per Second (PoPS), is explained in details on the [http://partsregistry.org/cgi/htdocs/AbstractionHierarchy/index.cgi abstraction hierarchy] of the MIT Registry of Parts. For instance, the input can be of any nature as long as an adequate promoter is available (e.g. heat-shock using a sigma32 promoter, IPTG using a LacI promoter, AHL using quorum sensing promoters...)

Input Module

The input module splits the input into two opposite signals. It is best described through its system boundaries. One of the outputs should be high and the other low when S is high and vice versa when S is low:

To achieve such behaviour, we use the λ-system, with IPTG as inductor. It is relatively easy to handle/debug, and does not restrict the module from being extended to work with other types of inputs. More importantly, it is already available as a BioBrick (Registry package 7.05) in its unidirectional flavour (In nature, the λ-system is bidirectional, with Pr on one DNA strand and Prm on the other, overlapping). The following picture shows the gene circuit of the input module in details:

cI is a dimer and regulates the activity of the two promoter regions, Pr and Prm, on the λ-system. Pr is constitutively active and is repressed when cI binds to the two operator regions it overlaps with (OR1, OR2). Conversly, Prm has low basal activity, and is activated by cI.

Since the two promoters are regulated by the same protein-operator interactions, repression and activation should be symmetrical (as very desirable proprety, see results from simulation below). For more details, please consult the page Input-module.

NOR Module

Simulation

Implementation

Results and Discussion

Applications and Perspecitves