We report the design, synthesis, and assembly of the 1.08-mega-base pair Mycoplasma mycoides JCVI-syn1.0 genome starting from digitized genome sequence information and its transplantation into a M. capricolum recipient cell to create new M. mycoides cells that are controlled only by the synthetic chromosome. The only DNA in the cells is the designed synthetic DNA sequence, including "watermark" sequences and other designed gene deletions and polymorphisms, and mutations acquired during the building process. The new cells have expected phenotypic properties and are capable of continuous self-replication.

Cellular processes are carried out by many interacting genes and their study and optimization requires multiple levers by which they can be independently controlled. The most common method is via a genetically-encoded sensor that responds to a small molecule (an "inducible system"). However, these sensors are often suboptimal, exhibiting high background expression and low dynamic range. Further, using multiple sensors in one cell is limited by cross-talk and the taxing of cellular resources. Here, we have developed a directed evolution strategy to simultaneously select for less background, high dynamic range, increased sensitivity, and low crosstalk. Libraries of the regulatory protein and output promoter are built based on random and rationally-guided mutations. This is applied to generate a set of 12 high-performance sensors, which exhibit >100-fold induction with low background and crossreactivity. These are combined to build a single "sensor array" and inserted into the genomes of E. coli MG1655 (wild-type), DH10B (cloning), and BL21 (protein expression). These "Marionette" strains allow for the independent control of gene expression using 2,4-diacetylphophloroglucinol (DAPG), cuminic acid (Cuma), 3-oxohexanoyl-homoserine lactone (OC6), vanillic acid (Van), isopropyl -D-1thiogalactopyranoside (IPTG), anhydrotetracycline (aTc), L-arabinose (Ara), choline chloride (Cho), naringenin (Nar), 3,4-dihydroxybenzoic acid (DHBA), sodium salicylate (Sal), and 3hydroxytetradecanoyl-homoserine lactone (OHC14). Advances in biology are often tied to new methods that use external stimuli to control the levels of gene expression 1-3. Pioneered in the early 1980s, so-called inducible systems were developed that allow genes to be turned on by adding a small molecule inducer to the growth media 4. These consist of a protein transcription factor (e.g., LacI) whose binding to a DNA operator in a promoter is controlled by the inducer (e.g., IPTG). Initially co-opted from natural regulatory networks, over the years many versions were designed to improve performance. In the 1990s, additional systems were developed that responded to other inducers, notably arabinose and aTc, which became common tools in the field. In 1997, Lutz and Bujard published a seminal paper that combined three (IPTG, arabinose, aTc) that could be easily interchanged on a two-plasmid system 5. Its organizational simplicity, compatibility, and quantified response functions were revolutionary. Beyond providing a new tool to biologists to control multiple genes with independent "strings," it facilitated researchers with quantitative backgrounds to enter biology 6-7. Armed with the new ability to control two genes with precision, physicists and engineers built the first synthetic genetic circuits, performed single molecule experiments inside cells, deconstructed the origins of noise in gene expression, determined how enzyme balancing impacts metabolic flux, elucidated rules underlying the assembly of molecular machines, and built synthetic symbiotic microbial commu...

The interaction specificities of extracytoplasmic function (ECF) sigma (σ) factors with promoters and their negative regulators (anti-σs) were mapped to identify non-crossreacting parts. These orthogonal sets represent a synthetic biology toolbox of genetic switches.

Synthetic genetic sensors and circuits enable programmable control over the timing and conditions of gene expression. They are being increasingly incorporated into the control of complex, multigene pathways and cellular functions. Here, we propose a design strategy to genetically separate the sensing/circuitry functions from the pathway to be controlled. This separation is achieved by having the output of the circuit drive the expression of a polymerase, which then activates the pathway from polymerase-specific promoters. The sensors, circuits and polymerase are encoded together on a ‘controller’ plasmid. Variants of T7 RNA polymerase that reduce toxicity were constructed and used as scaffolds for the construction of four orthogonal polymerases identified via part mining that bind to unique promoter sequences. This set is highly orthogonal and induces cognate promoters by 8- to 75-fold more than off-target promoters. These orthogonal polymerases enable four independent channels linking the outputs of circuits to the control of different cellular functions. As a demonstration, we constructed a controller plasmid that integrates two inducible systems, implements an AND logic operation and toggles between metabolic pathways that change Escherichia coli green (deoxychromoviridans) and red (lycopene). The advantages of this organization are that (i) the regulation of the pathway can be changed simply by introducing a different controller plasmid, (ii) transcription is orthogonal to host machinery and (iii) the pathway genes are not transcribed in the absence of a controller and are thus more easily carried without invoking evolutionary pressure.

Synthetic genetic systems share resources with the host, including machinery for transcription and translation. Phage RNA polymerases (RNAPs) decouple transcription from the host and generate high expression. However, they can exhibit toxicity and lack accessory proteins (σ factors and activators) that enable switching between different promoters and modulation of activity. Here, we show that T7 RNAP (883 amino acids) can be divided into four fragments that have to be co-expressed to function. The DNA-binding loop is encoded in a C-terminal 285-aa ‘σ fragment’, and fragments with different specificity can direct the remaining 601-aa ‘core fragment’ to different promoters. Using these parts, we have built a resource allocator that sets the core fragment concentration, which is then shared by multiple σ fragments. Adjusting the concentration of the core fragment sets the maximum transcriptional capacity available to a synthetic system. Further, positive and negative regulation is implemented using a 67-aa N-terminal ‘α fragment’ and a null (inactivated) σ fragment, respectively. The α fragment can be fused to recombinant proteins to make promoters responsive to their levels. These parts provide a toolbox to allocate transcriptional resources via different schemes, which we demonstrate by building a system which adjusts promoter activity to compensate for the difference in copy number of two plasmids.

The internal environment of growing cells is variable and dynamic, making it difficult to introduce reliable parts, such as promoters, for genetic engineering. Here, we applied control-theoretic ideas to design promoters that maintained constant levels of expression at any copy number. Theory predicts that independence to copy number can be achieved by using an incoherent feedforward loop (iFFL) if the negative regulation is perfectly non-cooperative. We engineered iFFLs into Escherichia coli promoters using transcription-activator-like effectors (TALEs). These promoters had near-identical expression in different genome locations and plasmids, even when their copy number was perturbed by genomic mutations or changes in growth medium composition. We applied the stabilized promoters to show that a three-gene metabolic pathway to produce deoxychromoviridans could retain function without re-tuning when the stabilized-promoter-driven genes were moved from a plasmid into the genome.

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Bacterial regulators of transcriptional elongation are versatile units for building custom genetic switches, as they control the expression of both coding and noncoding RNAs, act on multigene operons and can be predictably tethered into higher-order regulatory functions (a property called composability). Yet the less versatile bacterial regulators of translational initiation are substantially easier to engineer. To bypass this tradeoff, we have developed an adaptor that converts regulators of translational initiation into regulators of transcriptional elongation in Escherichia coli. We applied this adaptor to the construction of several transcriptional attenuators and activators, including a small molecule-triggered attenuator and a group of five mutually orthogonal riboregulators that we assembled into NOR gates of two, three or four RNA inputs. Continued application of our adaptor should produce large collections of transcriptional regulators whose inherent composability can facilitate the predictable engineering of complex synthetic circuits.