SynBioFromLeukipposInstitute

Engineering synthetic gene regulatory circuits proceeds through iterative cycles of design, building, and testing. Initial circuit designs must rely on often-incomplete models of regulation established by fields of reductive inquiry-biochemistry and molecular and systems biology. As differences in designed and experimentally observed circuit behavior are inevitably encountered, investigated, and resolved, each turn of the engineering cycle can force a resynthesis in understanding of natural network function. Here, we outline research that uses the process of gene circuit engineering to advance biological discovery. Synthetic gene circuit engineering research has not only refined our understanding of cellular regulation but furnished biologists with a toolkit that can be directed at natural systems to exact precision manipulation of network structure. As we discuss, using circuit engineering to predictively reorganize, rewire, and reconstruct cellular regulation serves as the ultimate means of testing and understanding how cellular phenotype emerges from systems-level network function.

Synthetic biology is a rapidly growing multidisciplinary branch of science which aims to mimic complex biological systems by creating similar forms. Constructing an artificial system requires optimization at the gene and protein levels to allow the formation of entire biological pathways. Advances in cell-free synthetic biology have helped in discovering new genes, proteins, and pathways bypassing the complexity of the complex pathway interactions in living cells. Furthermore, this method is cost- and time-effective with access to the cellular protein factory without the membrane boundaries. The freedom of design, full automation, and mimicking of in vivo systems reveal advantages of synthetic biology that can improve the molecular understanding of processes, relevant for life science applications. In parallel, in vitro approaches have enhanced our understanding of the living system. This review highlights the recent evolution of cell-free gene design, proteins, and cells integrated with microfluidic platforms as a promising technology, which has allowed for the transformation of the concept of bioprocesses. Although several challenges remain, the manipulation of biological synthetic machinery in microfluidic devices as suitable 'homes' for in vitro protein synthesis has been proposed as a pioneering approach for the development of new platforms, relevant in biomedical and diagnostic contexts towards even the sensing and monitoring of environmental issues.

Synthetic biology is a novel branch of biological sciences. The creation of a synthetic microorganisms with minimal genome compatible with life by Craig Venter had provoked the prior hot philosophical discussions about the nature of life. The recent advancements in synthetic biology brings new philosophical analysis about hybrid entities “synthetic organisms” and “living machines” and fundamental difference between biological machines and the living world. This paper is a critical analysis of the philosophical perspective on the design of synthetic organisms, minimal genome and artificial life. It also presents a critical view on knowledge-making practices in synthetic biology on Richard Feynman’s statement: “What I cannot create, I do not understand.”

BACKGROUND:
When using the microbial cell factories for green manufacturing, several important issues need to be addressed such as how to maintain the stability of biocatalysts used in the bioprocess and how to improve the synthetic efficiency of the biological system. One strategy widely used during natural evolution is the creation of organelles which can be used for regional control. This kind of compartmentalization strategy has inspired the design of artificial organelle-like nanodevice for synthetic biology and "green chemistry".
RESULTS:
Mimicking the natural concept of functional compartments, here we show that the engineered thermostable ketohydroxyglutarate aldolase from Thermotoga maritima could be developed as a general platform for nanoreactor design via supramolecular self-assembly. An industrial biocatalyst-(+)-γ-lactamase was selected as a model catalyst and successful encapsulated in the nanoreactor with high copies. These nanomaterials could easily be synthesized by Escherichia coli by heterologous expression and subsequently self-assembles into the target organelle-like nanoreactors both in vivo and in vitro. By probing their structural characteristics via transmission electronic microscopy and their catalytic activity under diverse conditions, we proved that these nanoreactors could confer a significant benefit to the cargo proteins. The encapsulated protein exhibits significantly improved stability under conditions such as in the presence of organic solvent or proteases, and shows better substrate tolerance than free enzyme.
CONCLUSIONS:
Our biodesign strategy provides new methods to develop new catalytically active protein-nanoreactors and could easily be applied into other biocatalysts. These artificial organelles could have widely application in sustainable catalysis, synthetic biology and could significantly improve the performance of microbial cell factories.

The CRISPR Journal

Introduction: The discovery and domestication of biomolecules that respond to light has taken a light of its own, providing new molecular tools with incredible spatio-temporal resolution to manipulate cellular behavior.

Areas covered: The authors herein analyze the current optogenetic tools in light of their current, and potential, uses in cancer drug discovery, biosafety and cancer biology.

Expert opinion: The pipeline from drug discovery to the clinic is plagued with drawbacks, where most drugs fail in either efficacy or safety. These issues require the redesign of the pipeline and the development of more controllable/personalized therapies. Light is, aside from inexpensive, almost harmless if used appropriately, can be directed to single cells or organs with controllable penetration, and comes in a variety of wavelengths. Light-responsive systems can activate, inhibit or compensate cell signaling pathways or specific cellular events, allowing the specific control of the genome and epigenome, and modulate cell fate and transformation. These synthetic molecular tools have the potential to revolutionize drug discovery and cancer research.

Bacteria and bacteriophages arm themselves with various defensive and counterdefensive mechanisms to protect their own genome and degrade the other’s. CRISPR (clustered regularly interspaced short palindromic repeat)–Cas (CRISPR-associated) is an adaptive bacterial defense mechanism that recognizes short stretches of invading phage genome and destroys it by nuclease attack. Unexpectedly, we discovered that the CRISPR-Cas system might also accelerate phage evolution. When Escherichia coli bacteria containing CRISPR-Cas9 were infected with phage T4, its cytosine hydroxymethylated and glucosylated genome was cleaved poorly by Cas9 nuclease, but the continuing CRISPR-Cas9 pressure led to rapid evolution of mutants that accumulated even by the time a single plaque was formed. The mutation frequencies are, remarkably, approximately six orders of magnitude higher than the spontaneous mutation frequency in the absence of CRISPR pressure. Our findings lead to the hypothesis that the CRISPR-Cas might be a double-edged sword, providing survival advantages to both bacteria and phages, leading to their coevolution and abundance on Earth.

Synthetic biology: Reframing cell therapy for cancer

Genome editing with CRISPR/Cas9 is a promising new approach for correcting or mitigating disease-causing mutations. Duchenne muscular dystrophy (DMD) is associated with lethal degeneration of cardiac and skeletal muscle caused by more than 3000 different mutations in the X-linked dystrophin gene (DMD). Most of these mutations are clustered in “hotspots.” There is a fortuitous correspondence between the eukaryotic splice acceptor and splice donor sequences and the protospacer adjacent motif sequences that govern prokaryotic CRISPR/Cas9 target gene recognition and cleavage. Taking advantage of this correspondence, we screened for optimal guide RNAs capable of introducing insertion/deletion (indel) mutations by nonhomologous end joining that abolish conserved RNA splice sites in 12 exons that potentially allow skipping of the most common mutant or out-of-frame DMD exons within or nearby mutational hotspots. We refer to the correction of DMD mutations by exon skipping as myoediting. In proof-of-concept studies, we performed myoediting in representative induced pluripotent stem cells from multiple patients with large deletions, point mutations, or duplications within the DMD gene and efficiently restored dystrophin protein expression in derivative cardiomyocytes. In three-dimensional engineered heart muscle (EHM), myoediting of DMD mutations restored dystrophin expression and the corresponding mechanical force of contraction. Correcting only a subset of cardiomyocytes (30 to 50%) was sufficient to rescue the mutant EHM phenotype to near-normal control levels. We conclude that abolishing conserved RNA splicing acceptor/donor sites and directing the splicing machinery to skip mutant or out-of-frame exons through myoediting allow correction of the cardiac abnormalities associated with DMD by eliminating the underlying genetic basis of the disease.

Transcriptional biosensors have various applications in metabolic engineering, including dynamic pathway control and high-throughput screening of combinatorial strain libraries. Previously, various biosensors have been created from naturally occurring transcription factors (TFs), largely relying on native sequences with- out the possibility to modularly optimize their response curve. The lack of design and engineering techniques thus greatly hinders the development of custom biosensors. In view of the intended application this is detrimental. In contrast, a bottom-up approach to design tailor-made biosensors was pursued here. Novel biosensors were created that respond to N-acetylneuraminic acid (Neu5Ac), an important sugar moiety with various biological functions, by employing native and engineered promoters that interact with the TF NanR. This bottom-up approach, whereby various tuned modules, e.g., the ribosome binding site (RBS) controlling NanR translation can be combined, enabled the reliable engineering of various response curve characteristics. The latter was validated by testing these biosensors in combination with various Neu5Ac-producing pathways, which allowed to produce up to 1.4 ± 0.4 g/L extracellular Neu5Ac. In this way, the repertoire of biosensors was expanded with seven novel functional Neu5Ac-responsive biosensors.

The ultimate goal of synthetic biology is to build customized cells or organisms to meet specific industrial or medical needs. The most important part of the customized cell is a synthetic genome. Advanced genomic writing technologies are required to build such an artificial genome. Recently, the partially-completed synthetic yeast genome project represents a milestone in this field. In this mini review, we briefly introduce the techniques for de novo genome synthesis and genome editing. Furthermore, we summarize recent research progresses and highlight several applications in the synthetic genome field. Finally, we discuss current challenges and future prospects.

Synthetic cells, artificial cell-like particles, capable of autonomously synthesizing RNA and proteins based on a DNA template, are emerging platforms for studying cellular functions and for revealing the origins-of-life. Here, it is shown for the first time that artificial lipid-based vesicles, containing the molecular machinery necessary for transcription and translation, can be used to synthesize anticancer proteins inside tumors. The synthetic cells are engineered as stand-alone systems, sourcing nutrients from their biological microenvironment to trigger protein synthesis. When pre-loaded with template DNA, amino acids and energy-supplying molecules, up to 2 × 107 copies of green fluorescent protein are synthesized in each synthetic cell. A variety of proteins, having molecular weights reaching 66 kDa and with diagnostic and therapeutic activities, are synthesized inside the particles. Incubating synthetic cells, encoded to secrete Pseudomonas exotoxin A (PE) with 4T1 breast cancer cells in culture, resulted in killing of most of the malignant cells. In mice bearing 4T1 tumors, histological evaluation of the tumor tissue after a local injection of PE-producing particles indicates robust apoptosis. Synthetic cells are new platforms for synthesizing therapeutic proteins on-demand in diseased tissues.

Society’s strong dependence on fossil fuels and petroleum-based products leads not only to a rapid decline of natural oil reserves but contributes massively to global warming and environmental damage. This consequently urges society to look into more sustainable alternatives. Microorganisms present such sustainable alternative if converted into so-called microbial cell factories. Instead of crude oil, cell factories use renewable resources or waste products as source material. The challenge is, however, that microbial production needs to be economically feasible to compete with the classical chemical production. The development of a microbial cell factory typically takes up to 8 years of research and costs over $50 million. The production and selection of heterologous pathway proteins are major bottlenecks encountered in the construction of a cell factory. Thus, new approaches for the optimization of recombinant protein production and screening techniques with high capacity for the identification of the best performing enzymes are continually developed. This thesis aims to equip researchers with a fundamental knowledge about protein biosynthesis necessary for the understanding of protein production bottlenecks. Moreover, the thesis guides through the possible causes of low protein yields and presents available approaches for optimization of the protein and the host. The main work presented in this thesis provides and applies a new synthetic biology approach for the optimization and selection of recombinant proteins. A major bottleneck during production is translation initiation. By creating sequence libraries of the translation initiation region, protein production can be improved substantially in Gram-negative and Gram-positive bacteria. The design of versatile and tuneable translational coupling devices and their fusion to antibiotic selection markers enables subsequent selection of high-expressing constructs. The approach is a simple and inexpensive alternative to advanced screening techniques. In addition, a second synthetic biology approach provides the means for fast and efficient plasmid backbone swapping and is a versatile tool for the design and construction of optimal protein production constructs.