scholarly journals Synthetic and systems biology principles in the design of programmable oncolytic virus immunotherapies for glioblastoma

2021 ◽  
Vol 50 (2) ◽  
pp. E10
Author(s):  
Dileep D. Monie ◽  
Archis R. Bhandarkar ◽  
Ian F. Parney ◽  
Cristina Correia ◽  
Jann N. Sarkaria ◽  
...  
Keyword(s):  
Systems Biology ◽  
Synthetic Biology ◽  
Laboratory Data ◽  
Immunological Response ◽  
Analytical Framework ◽  
Oncolytic Viruses ◽  
Genetic Circuit ◽  
Genetic Circuits ◽  
Synthetic Dna ◽  
Network Analyses

Oncolytic viruses (OVs) are a class of immunotherapeutic agents with promising preclinical results for the treatment of glioblastoma (GBM) but have shown limited success in recent clinical trials. Advanced bioengineering principles from disciplines such as synthetic and systems biology are needed to overcome the current challenges faced in developing effective OV-based immunotherapies for GBMs, including off-target effects and poor clinical responses. Synthetic biology is an emerging field that focuses on the development of synthetic DNA constructs that encode networks of genes and proteins (synthetic genetic circuits) to perform novel functions, whereas systems biology is an analytical framework that enables the study of complex interactions between host pathways and these synthetic genetic circuits. In this review, the authors summarize synthetic and systems biology concepts for developing programmable, logic-based OVs to treat GBMs. Programmable OVs can increase selectivity for tumor cells and enhance the local immunological response using synthetic genetic circuits. The authors discuss key principles for developing programmable OV-based immunotherapies, including how to 1) select an appropriate chassis, a vector that carries a synthetic genetic circuit, and 2) design a synthetic genetic circuit that can be programmed to sense key signals in the GBM microenvironment and trigger release of a therapeutic payload. To illustrate these principles, some original laboratory data are included, highlighting the need for systems biology studies, as well as some preliminary network analyses in preparation for synthetic biology applications. Examples from the literature of state-of-the-art synthetic genetic circuits that can be packaged into leading candidate OV chassis are also surveyed and discussed.

scholarly journals CRIMoClo plasmids for modular assembly and orthogonal chromosomal integration of synthetic circuits in Escherichia coli

2019 ◽  
Vol 13 (1) ◽  
Author(s):  
Stefano Vecchione ◽  
Georg Fritz
Keyword(s):  
Escherichia Coli ◽  
Synthetic Biology ◽  
Integrated Circuits ◽  
High Efficiency ◽  
Genetic Circuit ◽  
Dna Assembly ◽  
Chromosomal Integration ◽  
Golden Gate ◽  
Genetic Circuits ◽  
E Coli

Abstract Background Synthetic biology heavily depends on rapid and simple techniques for DNA engineering, such as Ligase Cycling Reaction (LCR), Gibson assembly and Golden Gate assembly, all of which allow for fast, multi-fragment DNA assembly. A major enhancement of Golden Gate assembly is represented by the Modular Cloning (MoClo) system that allows for simple library propagation and combinatorial construction of genetic circuits from reusable parts. Yet, one limitation of the MoClo system is that all circuits are assembled in low- and medium copy plasmids, while a rapid route to chromosomal integration is lacking. To overcome this bottleneck, here we took advantage of the conditional-replication, integration, and modular (CRIM) plasmids, which can be integrated in single copies into the chromosome of Escherichia coli and related bacteria by site-specific recombination at different phage attachment (att) sites. Results By combining the modularity of the MoClo system with the CRIM plasmids features we created a set of 32 novel CRIMoClo plasmids and benchmarked their suitability for synthetic biology applications. Using CRIMoClo plasmids we assembled and integrated a given genetic circuit into four selected phage attachment sites. Analyzing the behavior of these circuits we found essentially identical expression levels, indicating orthogonality of the loci. Using CRIMoClo plasmids and four different reporter systems, we illustrated a framework that allows for a fast and reliable sequential integration at the four selected att sites. Taking advantage of four resistance cassettes the procedure did not require recombination events between each round of integration. Finally, we assembled and genomically integrated synthetic ECF σ factor/anti-σ switches with high efficiency, showing that the growth defects observed for circuits encoded on medium-copy plasmids were alleviated. Conclusions The CRIMoClo system enables the generation of genetic circuits from reusable, MoClo-compatible parts and their integration into 4 orthogonal att sites into the genome of E. coli. Utilizing four different resistance modules the CRIMoClo system allows for easy, fast, and reliable multiple integrations. Moreover, utilizing CRIMoClo plasmids and MoClo reusable parts, we efficiently integrated and alleviated the toxicity of plasmid-borne circuits. Finally, since CRIMoClo framework allows for high flexibility, it is possible to utilize plasmid-borne and chromosomally integrated circuits simultaneously. This increases our ability to permute multiple genetic modules and allows for an easier design of complex synthetic metabolic pathways in E. coli.

Multimaterial bioprinting—minus the printer: Synthetic bacterial patterning with UV-responsive genetic circuits

2020 ◽  
pp. 147807712096337
Author(s):  
Gizem Gumuskaya
Keyword(s):  
Industrial Revolution ◽  
Vibrio Fischeri ◽  
Raw Material ◽  
Genetic Circuit ◽  
Elastic Tissue ◽  
Genetic Circuits ◽  
Bacterial Colony ◽  
Synthetic Dna ◽  
Macro Scale ◽  
Bacterial Lawn

In this paper, we argue that synthetic biology can help us employ living systems’ unique capacity for self-construction and biomaterial production toward developing novel architectural fabrication paradigms, in which both the raw material production and its refinement into a target structure can be merged into a single computational process embedded in the living structure itself. To demonstrate, here we introduce bioPheme, a novel biofabrication method for engineering bacteria to build biomaterial(s) of designer’s choice into arbitrary 2D geometries specified via transient UV tracing. To this end, we present the design, construction, and testing of the enabling synthetic DNA circuit, which, once inserted into a bacterial colony, allows the bacteria to execute spatial computation by interacting with one another based on the if-then rules encoded in this circuit. At the heart of this genetic circuit is a pair of UV sensor – actuator, and a pair of cell-to-cell signal transmitter – receptor modules, created with genes extracted from the virus λ Phage and marine bacterium Vibrio fischeri, respectively. These modules are wired together to help designers engineer bacteria to build macro-scale structures with seamlessly integrated biomaterials, thereby bridge the molecular and architectural scales. In this way, a bacterial lawn can be programmed to produce different objects with complementary biomaterial compositions, such as a biomineralized superstructure and an elastic tissue filling in-between. In summary, this paper focuses on how scientists’ increasing ability to harness the innate computational capacity of living cells can help designers create self-constructing structures for architectural biofabrication. Through the discussions in this paper, we aim to initiate a shift in today’s biodesign practices toward a greater appreciation and adoption of bottom-up governance of living structures. We are confident that such a paradigm shift will allow for more efficient and sustainable biofabrication systems in the 4th industrial revolution and beyond.

scholarly journals Rosa26 docking sites for investigating genetic circuit silencing in stem cells

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Michael Fitzgerald ◽  
Mark Livingston ◽  
Chelsea Gibbs ◽  
Tara L Deans
Keyword(s):  
Stem Cells ◽  
Synthetic Biology ◽  
Cell Lines ◽  
Pluripotent Stem Cells ◽  
Genetic Circuit ◽  
Genetic Circuits ◽  
Immortalized Cell Lines ◽  
Site Specific Integration ◽  
Docking Sites ◽  
Immortalized Cell

Abstract Approaches in mammalian synthetic biology have transformed how cells can be programmed to have reliable and predictable behavior, however, the majority of mammalian synthetic biology has been accomplished using immortalized cell lines that are easy to grow and easy to transfect. Genetic circuits that integrate into the genome of these immortalized cell lines remain functional for many generations, often for the lifetime of the cells, yet when genetic circuits are integrated into the genome of stem cells gene silencing is observed within a few generations. To investigate the reactivation of silenced genetic circuits in stem cells, the Rosa26 locus of mouse pluripotent stem cells was modified to contain docking sites for site-specific integration of genetic circuits. We show that the silencing of genetic circuits can be reversed with the addition of sodium butyrate, a histone deacetylase inhibitor. These findings demonstrate an approach to reactivate the function of genetic circuits in pluripotent stem cells to ensure robust function over many generations. Altogether, this work introduces an approach to overcome the silencing of genetic circuits in pluripotent stem cells that may enable the use of genetic circuits in pluripotent stem cells for long-term function.

scholarly journals Rosa26 docking sites for investigating genetic circuit silencing in stem cells

2019 ◽  
Author(s):  
Michael Fitzgerald ◽  
Mark Livingston ◽  
Chelsea Gibbs ◽  
Tara L. Deans
Keyword(s):  
Stem Cells ◽  
Synthetic Biology ◽  
Cell Lines ◽  
Pluripotent Stem Cells ◽  
Genetic Circuit ◽  
Genetic Circuits ◽  
Immortalized Cell Lines ◽  
Site Specific Integration ◽  
Docking Sites ◽  
Immortalized Cell

ABSTRACTApproaches in mammalian synthetic biology have transformed how cells can be programmed to have reliable and predictable behaviour, however, the majority of mammalian synthetic biology has been accomplished using immortalized cell lines that are easy to grow and easy to transfect. Genetic circuits that integrate into the genome of these immortalized cell lines remain functional for many generations, often for the lifetime of the cells, yet when genetic circuits are integrated into the genome of stem cells gene silencing is observed within a few generations. To investigate the reactivation of silenced genetic circuits in stem cells, the Rosa26 locus of mouse pluripotent stem cells was modified to contain docking sites for site-specific integration of genetic circuits. We show that the silencing of genetic circuits can be reversed with the addition of sodium butyrate, a histone deacetylase inhibitor. These findings demonstrate an approach to reactivate the function of genetic circuits in pluripotent stem cells to ensure robust function over many generations. Altogether, this work introduces an approach to overcome the silencing of genetic circuits in pluripotent stem cells that may enable the use of genetic circuits in pluripotent stem cells for long-term function.

scholarly journals TXO: Transcription-Only genetic circuits as a novel cell-free approach for Synthetic Biology

2019 ◽  
Author(s):  
Felipe A. Millacura ◽  
Mengxi Li ◽  
Marcos Valenzuela-Ortega ◽  
Christopher E. French
Keyword(s):  
Synthetic Biology ◽  
Real World ◽  
Genetically Modified Organisms ◽  
Rna Aptamers ◽  
Genetic Circuit ◽  
Environmental Concerns ◽  
Genetic Circuits ◽  
Transcriptional Units ◽  
Biosensor Applications ◽  
Real World Problems

AbstractWhile synthetic biology represents a promising approach to solve real-world problems, the use of genetically modified organisms is a cause of legal and environmental concerns. Cell-free systems have emerged as a possible solution but much work is needed to optimize their functionality and simplify their usage for Synthetic Biology. Here we present TXO, transcription-only genetic circuits, independent of translation or post-translation maturation. RNA aptamers are used as reaction output allowing the generation of fast, reliable and simple-to-design transcriptional units. TXO cell-free reactions and their possible applications are a promising new tool for fast and simple bench-to-market genetic circuit and biosensor applications.

scholarly journals SBOL-OWL: An ontological approach for formal and semantic representation of synthetic genetic circuits

2018 ◽  
Author(s):  
Goksel Misirli ◽  
Renee Taylor ◽  
Angel Goni-Moreno ◽  
James Alastair McLaughlin ◽  
Chris Myers ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Semantic Representation ◽  
Genetic Circuit ◽  
Free Text ◽  
Genetic Circuits ◽  
Biological Ontologies ◽  
Text Document ◽  
Ontological Approach ◽  
New Perspective ◽  
Synthetic Biology Open Language

Standard representation of data is key for the reproducibility of designs in synthetic biology. The Synthetic Biology Open Language (SBOL) has already emerged as a data standard to represent genetic circuit designs, and it is based on capturing data using graphs. The language provides the syntax using a free text document which is accessible to humans only. Here, we provide SBOL-OWL, an ontology for a machine understandable definition of SBOL. This ontology acts as a semantic layer for genetic circuit designs. As a result, computational tools can understand the meaning of design entities in addition to parsing structured SBOL data. SBOL-OWL not only describes how genetic circuits can be constructed computationally, it also facilitates the use of several existing Semantic Web tooling for synthetic biology. Here, we demonstrate some of these features, for example, to validate designs and check for inconsistencies. Through the use of SBOL-OWL, queries are simplified and become more intuitive. Moreover, existing reasoners can be used to infer information about genetic circuit designs that can't be directly retrieved using existing querying mechanisms. This ontological representation of the SBOL standard provides a new perspective to the verification, representation and querying of information about synthetic genetic circuits and is important to incorporate complex design information via the integration of biological ontologies.

scholarly journals Properties of alternative microbial hosts used in synthetic biology: towards the design of a modular chassis

2016 ◽  
Vol 60 (4) ◽  
pp. 303-313 ◽  
Author(s):  
Juhyun Kim ◽  
Manuel Salvador ◽  
Elizabeth Saunders ◽  
Jaime González ◽  
Claudio Avignone-Rossa ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Genetic Information ◽  
Essential Element ◽  
Model Organisms ◽  
Biotechnological Applications ◽  
Cell Models ◽  
Genetic Circuits ◽  
Metabolic Diversity ◽  
Translational Machinery ◽  
Metabolic Fluxes

The chassis is the cellular host used as a recipient of engineered biological systems in synthetic biology. They are required to propagate the genetic information and to express the genes encoded in it. Despite being an essential element for the appropriate function of genetic circuits, the chassis is rarely considered in their design phase. Consequently, the circuits are transferred to model organisms commonly used in the laboratory, such as Escherichia coli, that may be suboptimal for a required function. In this review, we discuss some of the properties desirable in a versatile chassis and summarize some examples of alternative hosts for synthetic biology amenable for engineering. These properties include a suitable life style, a robust cell wall, good knowledge of its regulatory network as well as of the interplay of the host components with the exogenous circuits, and the possibility of developing whole-cell models and tuneable metabolic fluxes that could allow a better distribution of cellular resources (metabolites, ATP, nucleotides, amino acids, transcriptional and translational machinery). We highlight Pseudomonas putida, widely used in many different biotechnological applications as a prominent organism for synthetic biology due to its metabolic diversity, robustness and ease of manipulation.

scholarly journals A standard-enabled workflow for synthetic biology

2017 ◽  
Vol 45 (3) ◽  
pp. 793-803 ◽  
Author(s):  
Chris J. Myers ◽  
Jacob Beal ◽  
Thomas E. Gorochowski ◽  
Hiroyuki Kuwahara ◽  
Curtis Madsen ◽  
...  
Keyword(s):  
Systems Biology ◽  
Synthetic Biology ◽  
Markup Language ◽  
Design Tools ◽  
Data Standards ◽  
Data Repositories ◽  
Simulation Tools ◽  
Systems Biology Markup Language ◽  
Visualization Tools ◽  
Synthetic Biology Open Language

A synthetic biology workflow is composed of data repositories that provide information about genetic parts, sequence-level design tools to compose these parts into circuits, visualization tools to depict these designs, genetic design tools to select parts to create systems, and modeling and simulation tools to evaluate alternative design choices. Data standards enable the ready exchange of information within such a workflow, allowing repositories and tools to be connected from a diversity of sources. The present paper describes one such workflow that utilizes, among others, the Synthetic Biology Open Language (SBOL) to describe genetic designs, the Systems Biology Markup Language to model these designs, and SBOL Visual to visualize these designs. We describe how a standard-enabled workflow can be used to produce types of design information, including multiple repositories and software tools exchanging information using a variety of data standards. Recently, the ACS Synthetic Biology journal has recommended the use of SBOL in their publications.

scholarly journals Development of oriC-Based Plasmids for Mesoplasma florum

2017 ◽  
Vol 83 (7) ◽  
Author(s):  
Dominick Matteau ◽  
Marie-Eve Pepin ◽  
Vincent Baby ◽  
Samuel Gauthier ◽  
Mélissa Arango Giraldo ◽  
...  
Keyword(s):  
Systems Biology ◽  
Synthetic Biology ◽  
Genetic Engineering ◽  
Genome Engineering ◽  
Antibiotic Resistance Genes ◽  
Viable Cell ◽  
Pathogenic Potential ◽  
Strong Basis ◽  
Content Type ◽  
Basic Biology

ABSTRACT The near-minimal bacterium Mesoplasma florum constitutes an attractive model for systems biology and for the development of a simplified cell chassis in synthetic biology. However, the lack of genetic engineering tools for this microorganism has limited our capacity to understand its basic biology and modify its genome. To address this issue, we have evaluated the susceptibility of M. florum to common antibiotics and developed the first generation of artificial plasmids able to replicate in this bacterium. Selected regions of the predicted M. florum chromosomal origin of replication (oriC) were used to create different plasmid versions that were tested for their transformation frequency and stability. Using polyethylene glycol-mediated transformation, we observed that plasmids harboring both rpmH-dnaA and dnaA-dnaN intergenic regions, interspaced or not with a copy of the dnaA gene, resulted in a frequency of ∼4.1 × 10−6 transformants per viable cell and were stably maintained throughout multiple generations. In contrast, plasmids containing only one M. florum oriC intergenic region or the heterologous oriC region of Mycoplasma capricolum, Mycoplasma mycoides, or Spiroplasma citri failed to produce any detectable transformants. We also developed alternative transformation procedures based on electroporation and conjugation from Escherichia coli, reaching frequencies up to 7.87 × 10−6 and 8.44 × 10−7 transformants per viable cell, respectively. Finally, we demonstrated the functionality of antibiotic resistance genes active against tetracycline, puromycin, and spectinomycin/streptomycin in M. florum. Taken together, these valuable genetic tools will facilitate efforts toward building an M. florum-based near-minimal cellular chassis for synthetic biology. IMPORTANCE Mesoplasma florum constitutes an attractive model for systems biology and for the development of a simplified cell chassis in synthetic biology. M. florum is closely related to the mycoides cluster of mycoplasmas, which has become a model for whole-genome cloning, genome transplantation, and genome minimization. However, M. florum shows higher growth rates than other Mollicutes, has no known pathogenic potential, and possesses a significantly smaller genome that positions this species among some of the simplest free-living organisms. So far, the lack of genetic engineering tools has limited our capacity to understand the basic biology of M. florum in order to modify its genome. To address this issue, we have evaluated the susceptibility of M. florum to common antibiotics and developed the first artificial plasmids and transformation methods for this bacterium. This represents a strong basis for ongoing genome engineering efforts using this near-minimal microorganism.

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