Engineering enzymes, pathways, and microbes through the use of an automated organism engineering foundry

Ginkgo Bioworks is an organism engineering company that applies high-throughput approaches to engineer enzymes and pathways in a variety of hosts. The process of engineering microbes is conducted within our custom-built foundries, which leverage proprietary enzyme sourcing and DNA design software, high volume DNA synthesis, next-generation sequencing, metabolomics, High Resolution Accurate Mass LC-MS, proteomics, and automated bioprocess development to rapidly develop and screen prototype enzymes and strains. Foundry processes are heavily automated and are tracked within Ginkgo’s custom software, to permit simultaneous handling of thousands of samples and strains across a single experiment. In most cases, the resulting engineered strains are then cultured to produce chemicals and ingredients of interest via fermentation. In this talk, we will describe how the foundry model can be applied to optimize the production of multiple high value and distinct chemicals. Specifically, we will provide examples of how the foundry can be utilized to engineer not only single enzymes, but pathways as well within the context of distinct hosts. These examples will illustrate how foundry-scale approaches can be harnessed to overcome challenges inherent to biological engineering across a variety of enzyme classes

Systematic Transfer of Prokaryotic Sensors and Circuits to Mammalian Cells

Prokaryotic regulatory proteins respond to diverse signals and represent a rich resource for building synthetic sensors and circuits. The TetR family contains >10[superscript 5] members that use a simple mechanism to respond to stimuli and bind distinct DNA operators. We present a platform that enables the transfer of these regulators to mammalian cells, which is demonstrated using human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells. The repressors are modified to include nuclear localization signals (NLS) and responsive promoters are built by incorporating multiple operators. Activators are also constructed by modifying the protein to include a VP16 domain. Together, this approach yields 15 new regulators that demonstrate 19- to 551-fold induction and retain both the low levels of crosstalk in DNA binding specificity observed between the parent regulators in Escherichia coli, as well as their dynamic range of activity. By taking advantage of the DAPG small molecule sensing mediated by the PhlF repressor, we introduce a new inducible system with 50-fold induction and a threshold of 0.9 μM DAPG, which is comparable to the classic Dox-induced TetR system. A set of NOT gates is constructed from the new repressors and their response function quantified. Finally, the Dox- and DAPG- inducible systems and two new activators are used to build a synthetic enhancer (fuzzy AND gate), requiring the coordination of 5 transcription factors organized into two layers. This work introduces a generic approach for the development of mammalian genetic sensors and circuits to populate a toolbox that can be applied to diverse applications from biomanufacturing to living therapeutics.United States. Defense Advanced Research Projects Agency (DARPA-BAA-11-23)National Institutes of Health (U.S.) (P50GM098792)Life Technologies, Inc. (Research Contract A114510)United States. Office of Naval Research. Multidisciplinary University Research Initiative (N00014-13-1-0074)National Institute of General Medical Sciences (U.S.) (Award R01 GM095765

Genomic mining of prokaryotic repressors for orthogonal logic gates

Genetic circuits perform computational operations based on interactions between freely diffusing molecules within a cell. When transcription factors are combined to build a circuit, unintended interactions can disrupt its function. Here, we apply 'part mining' to build a library of 73 TetR-family repressors gleaned from prokaryotic genomes. The operators of a subset were determined using an in vitro method, and this information was used to build synthetic promoters. The promoters and repressors were screened for cross-reactions. Of these, 16 were identified that both strongly repress their cognate promoter (5- to 207-fold) and exhibit minimal interactions with other promoters. Each repressor-promoter pair was converted to a NOT gate and characterized. Used as a set of 16 NOT/NOR gates, there are >10[superscript 54] circuits that could be built by changing the pattern of input and output promoters. This represents a large set of compatible gates that can be used to construct user-defined circuits.United States. Air Force Office of Scientific Research (Award FA9550-11-C-0028)American Society for Engineering Education. National Defense Science and Engineering Graduate Fellowship (32 CFR 168a)United States. Defense Advanced Research Projects Agency. Chronical of Lineage Indicative of Origins (N66001-12-C-4016)United States. Office of Naval Research (N00014-13-1-0074)National Institutes of Health (U.S.) (GM095765)National Science Foundation (U.S.). Synthetic Biology Engineering Research Center (SA5284-11210

Ribozyme-based insulator parts buffer synthetic circuits from genetic context

Synthetic genetic programs are built from circuits that integrate sensors and implement temporal control of gene expression. Transcriptional circuits are layered by using promoters to carry the signal between circuits. In other words, the output promoter of one circuit serves as the input promoter to the next. Thus, connecting circuits requires physically connecting a promoter to the next circuit. We show that the sequence at the junction between the input promoter and circuit can affect the input-output response (transfer function) of the circuit. A library of putative sequences that might reduce (or buffer) such context effects, which we refer to as 'insulator parts', is screened in Escherichia coli. We find that ribozymes that cleave the 5′ untranslated region (5′-UTR) of the mRNA are effective insulators. They generate quantitatively identical transfer functions, irrespective of the identity of the input promoter. When these insulators are used to join synthetic gene circuits, the behavior of layered circuits can be predicted using a mathematical model. The inclusion of insulators will be critical in reliably permuting circuits to build different programs.Life Technologies, Inc.United States. Defense Advanced Research Projects Agency (DARPA CLIO N66001-12-C-4018)United States. Office of Naval Research (N00014-10-1-0245)National Science Foundation (U.S.) (CCF-0943385)National Institutes of Health (U.S.) (AI067699)National Science Foundation (U.S.). Synthetic Biology Engineering Research Center (SynBERC, SA5284-11210

Allelic Exchange of Pheromones and Their Receptors Reprograms Sexual Identity in Cryptococcus neoformans

Cell type specification is a fundamental process that all cells must carry out to ensure appropriate behaviors in response to environmental stimuli. In fungi, cell identity is critical for defining “sexes” known as mating types and is controlled by components of mating type (MAT) loci. MAT–encoded genes function to define sexes via two distinct paradigms: 1) by controlling transcription of components common to both sexes, or 2) by expressing specially encoded factors (pheromones and their receptors) that differ between mating types. The human fungal pathogen Cryptococcus neoformans has two mating types (a and α) that are specified by an extremely unusual MAT locus. The complex architecture of this locus makes it impossible to predict which paradigm governs mating type. To identify the mechanism by which the C. neoformans sexes are determined, we created strains in which the pheromone and pheromone receptor from one mating type (a) replaced the pheromone and pheromone receptor of the other (α). We discovered that these “αa” cells effectively adopt a new mating type (that of a cells); they sense and respond to α factor, they elicit a mating response from α cells, and they fuse with α cells. In addition, αa cells lose the α cell type-specific response to pheromone and do not form germ tubes, instead remaining spherical like a cells. Finally, we discovered that exogenous expression of the diploid/dikaryon-specific transcription factor Sxi2a could then promote complete sexual development in crosses between α and αa strains. These data reveal that cell identity in C. neoformans is controlled fully by three kinds of MAT–encoded proteins: pheromones, pheromone receptors, and homeodomain proteins. Our findings establish the mechanisms for maintenance of distinct cell types and subsequent developmental behaviors in this unusual human fungal pathogen

Genetic Programs Constructed from Layered Logic Gates in Single Cells

Genetic programs function to integrate environmental sensors, implement signal processing algorithms and control expression dynamics[1]. These programs consist of integrated genetic circuits that individually implement operations ranging from digital logic to dynamic circuits[2, 3, 4, 5, 6], and they have been used in various cellular engineering applications, including the implementation of process control in metabolic networks and the coordination of spatial differentiation in artificial tissues. A key limitation is that the circuits are based on biochemical interactions occurring in the confined volume of the cell, so the size of programs has been limited to a few circuits[1, 7]. Here we apply part mining and directed evolution to build a set of transcriptional AND gates in Escherichia coli. Each AND gate integrates two promoter inputs and controls one promoter output. This allows the gates to be layered by having the output promoter of an upstream circuit serve as the input promoter for a downstream circuit. Each gate consists of a transcription factor that requires a second chaperone protein to activate the output promoter. Multiple activator–chaperone pairs are identified from type III secretion pathways in different strains of bacteria. Directed evolution is applied to increase the dynamic range and orthogonality of the circuits. These gates are connected in different permutations to form programs, the largest of which is a 4-input AND gate that consists of 3 circuits that integrate 4 inducible systems, thus requiring 11 regulatory proteins. Measuring the performance of individual gates is sufficient to capture the behaviour of the complete program. Errors in the output due to delays (faults), a common problem for layered circuits, are not observed. This work demonstrates the successful layering of orthogonal logic gates, a design strategy that could enable the construction of large, integrated circuits in single cells.United States. Defense Advanced Research Projects Agency (Chronicle of Lineage Indicative of Origins N66001-12-C-4018)United States. Office of Naval Research (N00014-10-1-0245)National Science Foundation (U.S.) (CCF-0943385)National Institutes of Health (U.S.) (AI067699)National Science Foundation (U.S.) (Synthetic Biology Engineering Research Center SA5284-11210

Sexual Development in Cryptococcus neoformans Requires CLP1, a Target of the Homeodomain Transcription Factors Sxi1α and Sxi2a▿

Sexual development in the human fungal pathogen Cryptococcus neoformans is a multistep process that results in the formation of spores, the likely infectious particles. A critical step in this developmental process is the transition from bud-form growth to filamentous growth. This transition is controlled by the homeodomain transcription factors Sxi1α and Sxi2a, whose targets are largely unknown. Here we describe the discovery of a gene, CLP1, that is regulated by Sxi1α and Sxi2a and is essential for sexual development. In vitro binding studies also show that the CLP1 promoter is bound directly by Sxi1α and Sxi2a. The deletion of CLP1 leads to a block in sexual development after cell fusion but before filament formation, and cells without CLP1 are unable to grow vegetatively after cell fusion. Our findings lead to a model in which CLP1 is a downstream target of the Sxi proteins that functions to promote growth after mating and to establish the filamentous state, a critical step in the production of spores

α x α^a crosses proceed through sexual development only in the presence of Sxi2a.

<p>(A) Sexual development assays were carried out on V8 for 72 hours, and the peripheries of crosses are shown under 200× magnification. Panels: 1) <b>a</b> x α, 2) <b>a</b> x α<i>^mfαΔ</i>, 3) <b>a</b> x α<i>^{mfαΔ, STE3a}</i>, 4) <b>a</b> x α<b>^a</b>, 5) α x α, 6) α x α<i>^mfαΔ</i>, 7) α x α<i>^{mfαΔ, STE3}</i>^{<b><i>a</i></b>}, 8) α x α<b>^a</b>, 9) <b>a </b><i>sxi2</i><b><i>a</i></b><i>Δ</i> x α, 10) α <i>sxi1αΔ</i> + <i>SXI2</i><b><i>a</i></b> x α<b>^a</b>. Complete sexual development is observed only in panels 1 and 10. (B) Dikaryotic filaments are produced in the α <i>sxi1αΔ</i> + <i>SXI2</i><b><i>a</i></b> x α<b>^a</b> cross. Calcofluor stained filaments appear blue, and Sytox Green strained nuclei appear green. Both an <b>a</b> x α cross (left) and the α <i>sxi1αΔ</i> + <i>SXI2</i><b><i>a</i></b> x α<b>^a</b> cross (right) produce dikaryotic filaments (400× magnification). (C) Basidia and spores are produced in the α <i>sxi1αΔ</i> + <i>SXI2</i><b><i>a</i></b> x α<b>^a</b> cross. High resolution microscopy reveals the formation of basidia and spores from an <b>a</b> x α cross (left), and from the α <i>sxi1αΔ</i> + <i>SXI2</i><b><i>a</i></b> x α<b>^a</b> cross (right) (1000× magnification). (D) Addition of the <i>SXI2</i><b><i>a</i></b> gene to α<b>^a</b>/α diploids results in sexual development. Diploids were incubated on V8 for 72 hours, and test spot peripheries are shown under 200× magnification as follows: wild type <b>a</b>/α diploid (panel 1), α<b>^a</b>/α diploid (panel 2), and α<b>^a</b>/α + <i>SXI2</i><b><i>a</i></b> (panel 3).</p

Strains.

<p>Strains.</p