Transcriptional NOT-gate: RNA polymerase (RNAP) flux as signal.
cl-family repressors.
RNAP: 2 subunits: (1) catalytic core, (2) binding unit called specificity factor (sigma factor).
Some like sigma70 for housekeeping.
Sigma factors bind to DNA sequences in promoter regions. Can sequester with anti-sigma factors.
Extracytoplasmic function sigma factors (ECF): orthogonal to host processes, high dynamic range, low toxicity.
He built NOT-gates with ECF20 and ECF16. Inducible.
“Initial development of plasmid based NOT gates”: We began with a set of 20 repressor:promoter pairs. Construct NOT gates and cloned onto p15a plasmids.
“From these experiments, we identified a set of 12 repressors and promoters that exhibited large dynamic ranges and near-perfect orthogonality. For simplicity, these repressors were named JR#, with their original names and sources are provided Table 2.3-2 (JR1 is cI repressor from phage lambda). NOT-gates were then constructed based on these repressors.” p22
Schematic of NOT-gate JR1-3 as an example. IPTG and aTc as inducers?
JR1 data sheet:
And as plasmid:
“Genome-encoded NOR-gates use two copies of the same repressor gene, each of which is independently connected to an input promoter.”
Development of genome based cell-cell communication devices: Each channel was based on a sender device that produces the chemical signal and a receiver device that responds to it. 4 signals that don’t cross-react with each other: