Programming Escherichia coli to function as a digital display

Abstract

Synthetic genetic circuits offer the potential to wield computational control over biology, but their complexity is limited by the accuracy of mathematical models. Here, we present advances that enable the complete encoding of an electronic chip in the DNA carried by Escherichia coli (E. coli). The chip is a binary‐coded digit (BCD) to 7‐segment decoder, associated with clocks and calculators, to turn on segments to visualize 0–9. Design automation is used to build seven strains, each of which contains a circuit with up to 12 repressors and two activators (totaling 63 regulators and 76,000 bp DNA). The inputs to each circuit represent the digit to be displayed (encoded in binary by four molecules), and output is the segment state, reported as fluorescence. Implementation requires an advanced gate model that captures dynamics, promoter interference, and a measure of total power usage (RNAP flux). This project is an exemplar of design automation pushing engineering beyond that achievable “by hand”, essential for realizing the potential of biology.

P recision design of stable genetic circuits carried in highly‐insulated E. coli genomic landing pads

“…Indeed, this design has several problems that are exacerbated when carried on the genome. The first is that the upstream promoter is inhibited by the binding of repressor to the downstream promoter ("roadblocking"), which is more problematic at lower copy number (Nielsen et al, 2016;Shin et al, 2020). In addition, maximum repressor expression is less than the sum of the input promoter activities.…”

“…Each circuit was designed to produce the same 3-input logic operation, encoded using Verilog, but different UCFs were used to map the circuit to a DNA sequence. The first was designed using the Eco1C2G2T2 UCF for the p15a plasmid in E. coli DH10b and was published previously (Nielsen et al, 2016;Shin et al, 2020). The second was designed using the Eco2C1G3T1 for the E. coli MG1655 genome.…”

“…Genetic circuits were first split and cloned into two plasmids, plYJP066 (KanR) and plYJP070 (CmR), that target Landing Pads #1 and #2, respectively, using Type II assembly as previously described (Nielsen et al, 2016;Shin et al, 2020). The order of transcription units within a circuit was assigned by Cello 2.0 based on EUGENE rules.…”

“…The plasmid-encoded 0xF1 circuit was designed using the UCF for the p15a plasmid (Eco1C2G2T2) and was constructed in E. coli DH10b (Nielsen et al, 2016;Shin et al, 2020). The E. coli MG1655 strain harboring the genome-encoded 0xF1 circuit was streaked on LB agar (2%) plates with Kan (50 lg/ml) and Cm (35 lg/ml) antibiotics and grown overnight.…”

A standardized broad host range inverter package for genetic circuitry design in Gram-negative bacteria

“…This in turn can be used as the input for another node of the circuit and so on 2,3 . Further abstraction of biological circuits as wholes of Boolean logic gates enables a superior level of complexity, as shown by a suite of examples involving rewiring of stress responses, detection of environmental contaminants, implementation of cellular calculators and others [4][5][6][7] . Yet, as the demand for increasingly complex circuits grows 5,8,9 , so does the interest in automation of their design and execution.…”