Computational Methods for Strain Design - Synthetic Biology - page 136

where the T3 RNAP is linked to the T7 promoter and the GFP is linked to the T3 promoter:
[P Ltet0-1 -ribreg-T7RNAP]; [P T7 -ribreg-T3RNAP]; [P T3 -ribreg-GFP]. 15
The DIC counter uses recombinase that can invert DNA between the respective and
oppositely oriented recombination target (FRT) sites, for example the recombinases Cre and
Flp e with the FRT loxP and flp e respectively, to construct a counter module. 15 This counter
constructs a single invertase memory module (SIMM) that is placed in sequence with each
other, each with different FRT sites, and the promoter for each consecutive module is
oriented in the opposite direction within the previous module. The first module, whose
promoter is oriented in the forward direction, becomes activated by a pulse, resulting in the
expression of the recombinase and the inversion of the module. When the module becomes
inverted, the inverted promoter is reoriented to the forward direction, and is able to activate
the expression of the next module in the sequence when a second pulse is experienced. The
components of the SIMM consists of an inverted promoter (P inv ), a recombinase gene ( rec ),
an ssrA tag (this causes rapid protein degradation to prepare the system for the next pulse),
a terminator (Term), and two recombination target sites for rec placed at both ends of the
module. The sequence of these components between the recombination target sites is: P inv -
rec -ssrA-Term. The number of SIMM used determines the number of pulses the system is
able to count. For instance, two SIMMs, one with the Cre recombinase and the other with
the Flp e recombinase, with the overall sequence of P BAD -SIMM ( flp e )-SIMM ( cre )-gfp, and
the inverted promoter in each SIMM is P BAD , the system is able to count three pulses of
arabinose before a green fluorescent signal is detected. In this example, the first pulse of
arabinose flips the first SIMM ( flp e ), so that the inverted promoter (P BAD ) is able to activate
the next SIMM and so on in a cascade, resulting in the production of GFP. 15

Cellular Signaling System
The edge detection program employs a signal processing system to synthetically engineer
the cell to detect the edges or changes in the environment. 13 This edge detection program
was demonstrated using a light image sensing program to produce a signal and produce
pigment in response to light by Escherichia coli . The Boolean logic program for detecting the
edge between light and dark is: [IF NOT light

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- produce signal] and[IF signal AND NOT

produce pigment]. 13 Thus, for cells at the boundary between light and dark,
pigment is produced. This program was implemented as a genetic circuit by employing a
chimeric light-sensitive protein, Cph8, a cell

(NOT light)

-

cell communication signal 3-oxohexanoyl-

homoserine lactone (AHL), and -galactosidase. The first component is the light detection

module which consists of a NOT gate that inhibits the expression of the signal protein in
the presence of light. In the presence of light, the gene luxI , which produces AHL (IF signal),
and the gene cI , which encodes for a transcriptional repressor from phage

β

, are repressed.
AHL complexed with the constitutively expressed transcriptional factor luxR in the absence
of cI (NOT(NOT light)) allows for the expression from the P lux- λ promoter, which in turn,
leads to the expression of the

λ

-galactosidase gene lacZ in cells located at the boundary of
light and dark, resulting in the cleavage of a substrate in the media to produce the black
pigment. This system was also simulated computationally by using a sigmoidal function
( f light ) for the [NOT light] gate and a Shea-Ackers formalism ( f logic ) for the [IF signal AND
NOT(NOT light)] gate.

β

A more complex signaling system is the predator prey ecosystem that employs two

different strains to control the populations of the two organisms. 20 To demonstrate this
system, two different E. coli strains were engineered to communicate with each other
through quorum sensing to control gene expression and survival of the two strains via
engineered bidirectional gene circuits. Two quorum sensing molecules were employed:
N-3-oxododecanoyl homoserine lactone, 3OC12HSL; and 3-oxohexanoyl-homoserine
lactone, 3OC6HSL. 20 In the predator E. coli strain, the suicide gene, ccdB , is constitutively