1. Environmental signal integration by a modular AND gate
J Christopher Anderson, Christopher A Voigt and Adam P Arkin
Molecular Systems Biology (2007) 3:133

2. AND gate
INPUT
OUTPUT A B
A AND B 1

3. Amber stop codon
Nonsense mutation = point mutation in DNA sequence resulting in premature stop codon or nonsense codon in transcribed mRNA -> truncated/non-functional protein product.
Supressor = tRNA that can “read” nonsense codon and cause appropriate amino acid to be incorporated into peptide sequence.

4. Two promoters:
Input 1 = salacylic acid sensitive promoter Psal
Input 2 = Arabinonse sensitive promoter PBAD
Input 1 results in transcription of the amber supressor gene supD encoding the appropriate supressor tRNA
Input 2 results in transcripton of the RNA polymerase T7ptag containing the amber stop codon; however the functional molecule cannot be translated without the amber supressor encoded downstream of the Psal promoter.
To have a functional T7 polymerase, which can produce PoPS output at the PT7 promoter, you need both inputs. The output in the first case was GFP.

5. Functionality
A – Integration of the two promoters by the AND gate. The fluorescence was measured at 64 combinations of inducer in a fluorimter.
B – The fluorescence was measured in individual cells using a flow cytometer to determine the population level behaviour.

6. Mathematical model Individual transfer functions for input promoters.
The transfer functions were measured by fusing each promoter to gfp, measuring the fluorescence as a function of the concentration of small molecule inducer (salicylate or arabinose). The transfer functions were used to parameterize the AND gate model.
The left panel shows the activation of Psal in response to salicylate. This promoter is leaky even in the complete absence of inducer.
The right panel shows the transfer functions for the PBAD parent rbs (black triangle), F11 (red square), and B9 (blue circle) clones. The first construct that was made showed output irrespective of the concentration of arabinose as input because the parent rbs was strong. The authors made a saturation mutagenesis library and selected the rbs that made the mods and-gate like behaviour for characterisation.
Then the fit to the data is shown.

7. Modularity
The AND gate is designed to be modular, where the inputs and outputs can be changed by swapping promoters and directing mutagenesis to the RBS. Using this device any two environmental signals can be connected to the AND gate as long as they can lead to transcriptional output. Similarly the T7 promoter can be used to drive a cellular response of to act as the input to another downstream circuit.
To demonstrate this modularity, two constructs containing different inputs and outputs were designed, constructed and analysed.
Inputs swapped for two natural promoeter (Quorum sensing – input = quorum sensing molecule; Mg2+ responsive and is repressed in presence of Mg2+); output is GFP
Output is swapped from GFP to invasin gene, which enables the bacteria to invade mammalian cells (inputs are Sal and Mg2+ .)

8. Concluding remarks
Independent promoters detecting different aspects of the environment (O2, pH, cell density, lactate, glucose) could be integrated to sense a specific environment.