Era Of Synthetic Biology Research Articles

Novel pressure sensors and bioreporters in the synthetic biology era

Miguel A. Matilla was supported by the Spanish Ministry of Economy and Competitiveness Research Program, Juan de la Cierva (JCI-2012–11815).

Synthetic biology for microbial heavy metal biosensors.

Using recombinant DNA technology, various whole-cell biosensors have been developed for detection of environmental pollutants, including heavy metal ions. Whole-cell biosensors have several advantages: easy and inexpensive cultivation, multiple assays, and no requirement of any special techniques for analysis. In the era of synthetic biology, cutting-edge DNA sequencing and gene synthesis technologies have accelerated the development of cell-based biosensors. Here, we summarize current technological advances in whole-cell heavy metal biosensors, including the synthetic biological components (bioparts), sensing and reporter modules, genetic circuits, and chassis cells. We discuss several opportunities for improvement of synthetic cell-based biosensors. First, new functional modules must be discovered in genome databases, and this knowledge must be used to upgrade specific bioparts through molecular engineering. Second, modules must be assembled into functional biosystems in chassis cells. Third, heterogeneity of individual cells in the microbial population must be eliminated. In the perspectives, the development of whole-cell biosensors is also discussed in the aspects of cultivation methods and synthetic cells.

생명산업 시대의 신학적 생명 : CRISPR 기술을 중심으로

‘Synthetic Biology’ is a genome editing practice that endeavors to produce new life forms. The CRISPR-cas9 is a new technique that amount to a set of Genetic Scissors, the most advanced technology in the Synthetic biology fields, outpaces other genetic technologies because it has the accuracy and economical potential. The advent of the synthetic biology era has raised a wide range of questions, including ‘what is life’, and what relationship is there between ‘life’ associated with the ‘economic approach’ and life that is already happening. The moral challenge of synthetic biology is not a matter that should be discussed only in science and technology or social sciences, but it is a problem to be discussed in our Christian theology which is concerned with ‘life’ and eternal life from a religious perspective. Therefore, this paper explains in detail the CRISPR technology, which greatly improved synthetic biology as a whole, and then discusses the social, economic, and cognitive changes that synthetic biology has undergone and the theological questions that can be raised through synthetic biology.BR Furthermore, I would like to focus on Georgio Agamben’s life politics and life-ruling thought as an answer to the question of the definition of life. Agamben has divided human life largely into Zoe and Bios, and has focused on social and political life beyond simple biological life. The human endeavor to gain control over life has been ongoing since the beginning of human history, he says. Agamben further asserts that the notion of sovereign power over life has sometimes shifted from killing life to sustaining life. The modern politics of sovereign power operates in a way to promote the survival of people within the system, not just to threaten and kill them. In the end, mankind is not living on his/her own, but living a life that is totally dependent on the sovereign power. Therefore, considering CRISPR technology as an extension of this life rule, the technology of synthetic biology is not an entirely positive development. Human progress in the scientific world tells us that we are governed by capital and sovereign power. Therefore, this paper tries to reconsider this CRISPR technique from the theological point of view, and to reveal the violence of life politics hidden behind the optimistic view of science.

Phage Therapy in the Era of Synthetic Biology.

For more than a century, bacteriophage (or phage) research has enabled some of the most important discoveries in biological sciences and has equipped scientists with many of the molecular biology tools that have advanced our understanding of replication, maintenance, and expression of genetic material. Phages have also been recognized and exploited as natural antimicrobial agents and nanovectors for gene therapy, but their potential as therapeutics has not been fully exploited in Western medicine because of challenges such as narrow host range, bacterial resistance, and unique pharmacokinetics. However, increasing concern related to the emergence of bacteria resistant to multiple antibiotics has heightened interest in phage therapy and the development of strategies to overcome hurdles associated with bacteriophage therapeutics. Recent progress in sequencing technologies, DNA manipulation, and synthetic biology allowed scientists to refactor the entire bacterial genome of Mycoplasma mycoides, thereby creating the first synthetic cell. These new strategies for engineering genomes may have the potential to accelerate the construction of designer phage genomes with superior therapeutic potential. Here, we discuss the use of phage as therapeutics, as well as how synthetic biology can create bacteriophage with desirable attributes.

CRISPR-Cas-Assisted Multiplexing (CAM): Simple Same-Day Multi-Locus Engineering in Yeast.

Demands on the industrial and academic yeast strain engineer have increased significantly in the era of synthetic biology. Installing complex biosynthetic pathways and combining point mutations are tedious and time-consuming using traditional methods. With multiplex engineering tools, these tasks can be completed in a single step, typically achieving up to sixfold compression in strain engineering timelines. To capitalize on this potential, a variety of yeast CRISPR-Cas methods have been developed, differing largely in how the guide RNA (gRNA) reagents that direct the Cas9 nuclease are delivered. However, in nearly all reported protocols, the time savings of multiplexing is offset by multiple days of cloning to prepare the required reagents. Here, we discuss the advantages and opportunities of CRISPR-Cas-assisted multiplexing (CAM), a same-day, cloning-free method for multi-locus engineering in yeast. J. Cell. Physiol. 231: 2563-2569, 2016. © 2016 Wiley Periodicals, Inc.

Natural Product Biosynthesis in Escherichia coli: Mentha Monoterpenoids.

The era of synthetic biology heralds in a new, more "green" approach to fine chemical and pharmaceutical drug production. It takes the knowledge of natural metabolic pathways and builds new routes to chemicals, enables nonnatural chemical production, and/or allows the rapid production of chemicals in alternative, highly performing organisms. This route is particularly useful in the production of monoterpenoids in microorganisms, which are naturally sourced from plant essential oils. Successful pathways are constructed by taking into consideration factors such as gene selection, regulatory elements, host selection and optimization, and metabolic considerations of the host organism. Seamless pathway construction techniques enable a "plug-and-play" switching of genes and regulatory parts to optimize the metabolic functioning in vivo. Ultimately, synthetic biology approaches to microbial monoterpenoid production may revolutionize "natural" compound formation.

Abstract IA10: CARs for leukemia and beyond?

Abstract Relapsed hematologic malignancies pose a substantial unmet therapeutic challenge despite aggressive therapies. Engineered T cells have shown promise for a variety of malignancies and chronic infections. In this session we will review the current status of trials testing chimeric antigen receptor (CAR)-modified T cells for acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML) and for myeloma. As of April 2014, twenty-five children and 5 adults with relapsed or refractory ALL were treated with anti-CD19:4-1BB:CD3zeta CAR T cells (CTL019). Complete remissions (CR) were achieved in 27/30 patients (90%). CTL019 cells proliferated up to 1000-fold and were detectable in blood, bone marrow, and cerebrospinal fluid of responding patients. Sustained remissions were achieved in 15/22 evaluable patients with median follow-up of 7 months (2-24 months), with event-free survival of 67% (95% confidence interval [CI], 51% to 88%) and overall survival of 78% (95% CI, 65% to 95%) measured at 6 months. CTL019-mediated B cell aplasia persisted for up to 2 years and is ongoing. Cytokine release syndrome (CRS) was observed in all patients. Severe CRS, seen in 27% of patients, was associated with high disease burden and was effectively treated with the IL-6 receptor antibody tocilizumab. More than 50 patients with advanced refractory chronic lymphocytic leukemia have been treated with CTL019. Two of the first three patients treated remain in complete remission nearly 4 years after infusion. An ongoing randomized phase II trial testing a high and a low dose of CTL019 has not shown a difference in the response rate. The lack of an obvious dose response rate is consistent with the high level proliferation of CAR T cells in vivo and suggests that once a threshold dose is reached, further dose escalation does not increase efficacy. Persistent B cell aplasia is observed in responding patients (more than 3 years in CLL patients and more than 2 years in pediatric ALL patients). No patient with persistent B cell aplasia has relapsed with CD19+ tumor. A major issue in the field is whether it is necessary to have ongoing immunosurveillance by CAR T cells or whether they induce “sterile remissions” where every last tumor cell is eradicated. CAR T cells can target very low levels of targets expressed at the cell surface. Because some myeloma cells express low levels of CD19, and because B cells may be a precursor of malignant myeloma cells, an ongoing pilot trial is testing whether CTL019 cells might recognize and eliminate malignant cells in patients with advanced plasma cell myeloma. In preclinical studies we have found that CD33 and CD123 CAR T cells have potent therapeutic effects in mice bearing xenografts with AML. At issue is whether off tumor toxicity on normal cells will be tolerable with CD33 and CD123 specific CARs. In summary, CAR-modified T cell therapy directed against CD19 is highly effective as salvage therapy for children and adults with relapsed/refractory ALL and CLL. Moreover, long-term remissions may be possible in some patients. Our findings support the advancement of CAR-modified T cell therapy into multicenter phase 2 clinical trials. 1. M. Kalos, C. H. June, Adoptive T cell transfer for cancer immunotherapy in the era of synthetic biology. Immunity 39, 49 (2013). 2. S. A. Grupp et al., Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. New England Journal of Medicine 368, 1509 (2013). 3. D. L. Porter, B. L. Levine, M. Kalos, A. Bagg, C. H. June, Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia New England Journal of Medicine 365, 725 (2011). 4. M. Kalos et al., T cells expressing chimeric receptors establish memory and potent antitumor effects in patients with advanced leukemia. Science Translational Medicine 3, 95ra73 (2011). 5. S. Maude et al., Sustained Remissions with Chimeric Antigen Receptor T Cells for Leukemia. New England Journal of Medicine, in press (2014). Citation Format: Carl H. June, David Porter, Stephan Grupp, Bruce L. Levine. CARs for leukemia and beyond? [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr IA10.

A Delicate Nanoscale Motor Made by Nature-The Bacterial Flagellar Motor.

The bacterial flagellar motor (BFM) is a molecular complex ca. 45 nm in diameter that rotates the propeller that makes nearly all bacteria swim. The motor self‐assembles out of ca. 20 different proteins and can not only rotate at up to 50 000 rpm, but can also switch rotational direction in milliseconds and navigate its environment to maneuver, on average, towards regions of greater benefit. The BFM is a pinnacle of evolution that informs and inspires the design of novel nanotechnology in the new era of synthetic biology.

Method of preparing an equimolar DNA mixture for one-step DNA assembly of over 50 fragments.

In the era of synthetic biology, techniques for rapidly constructing a designer long DNA from short DNA fragments are desired. To realize this, we attempted to establish a method for one-step DNA assembly of unprecedentedly large numbers of fragments. The basic technology is the Ordered Gene Assembly in Bacillus subtilis (OGAB) method, which uses the plasmid transformation system of B. subtilis. Since this method doesn’t require circular ligation products but needs tandem repeat ligation products, the degree of deviation in the molar concentration of the material DNAs is the only determinant that affects the efficiency of DNA assembly. The strict standardization of the size of plasmids that clone the DNA block and the measurement of the block in the state of intact plasmid improve the reliability of this step, with the coefficient of variation of the molar concentrations becoming 7%. By coupling this method with the OGAB method, one-step assembly of more than 50 DNA fragments becomes feasible.

Abstract PL07-04: Engineered T cell transfer for cancer immunotherapy in the era of synthetic biology: Challenges and opportunities.

Abstract Adoptive T cell transfer for cancer and chronic infection is an emerging field that shows promise in recent trials. Synthetic-biology-based engineering of T lymphocytes to express high-affinity antigen receptors can overcome immune tolerance, which has been a major limitation of previous immunotherapy-based strategies. Advances in cell engineering and culture approaches to enable efficient gene transfer and ex vivo cell expansion have facilitated broader evaluation of this technology, moving adoptive transfer from a "boutique"’ application to the cusp of a mainstream technology. Recent observations from ongoing clinical trials in patients with leukemia and carcinomas will be discussed. The major scientific challenge currently facing the field is identification of cancer specific targets to avoid on-target, off-tissue toxicity. As the field of adoptive transfer technology matures, the major engineering challenge is the development of automated cell culture systems, so that the approach can extend beyond specialized academic centers and become widely available. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PL07-04. Citation Format: Carl H. June. Engineered T cell transfer for cancer immunotherapy in the era of synthetic biology: Challenges and opportunities. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr PL07-04.

Opportunities for bioprocess monitoring using FRET biosensors

Bioprocess monitoring is used to track the progress of a cell culture and ensure that the product quality is maintained. Current schemes for monitoring metabolism rely on offline measurements of samples of the extracellular medium. However, in the era of synthetic biology, it is now possible to design and implement biosensors that consist of biological macromolecules and are able to report on the intracellular environment of cells. The use of fluorescent reporter signals allows non-invasive, non-destructive and online monitoring of the culture, which reduces the delay between measurement and any necessary intervention. The present mini-review focuses on protein-based biosensors that utilize FRET as the signal transduction mechanism. The mechanism of FRET, which utilizes the ratio of emission intensity at two wavelengths, has an inherent advantage of being ratiometric, meaning that small differences in the experimental set-up or biosensor expression level can be normalized away. This allows for more reliable quantitative estimation of the concentration of the target molecule. Existing FRET biosensors that are of potential interest to bioprocess monitoring include those developed for primary metabolites, redox potential, pH and product formation. For target molecules where a biosensor has not yet been developed, some candidate binding domains can be identified from the existing biological databases. However, the remaining challenge is to make the process of developing a FRET biosensor faster and more efficient.

Single-cell analysis - Methods and protocols

This is certainly a timely volume in the Methods in molecular biology series: we already entered the synthetic biology era and thus we need to be aware of the new methodological advances able to fulfill the new and necessary needs for biologists, biotechnologists and nano-biotechnologists. Notably, among these, the possibility to perform single cell analysis allow researchers to capture single cell responses....

Synthetic Biology: Applying Engineering to Life Sciences to Develop Rationally Designed Biological Parts, Devices, and Systems

Synthetic Biology: Applying Engineering to Life Sciences to Develop Rationally Designed Biological Parts, Devices, and Systems

Consensus is needed at a global level concerning dual‐use research

Consensus is needed at a global level concerning dual‐use research

News items

News items

The ten grand challenges of synthetic life.

The construction of artificial life is one of the main scientific challenges of the Synthetic Biology era. Advances in DNA synthesis and a better understanding of regulatory processes make the goal of constructing the first artificial cell a realistic possibility. This would be both a fundamental scientific milestone and a starting point of a vast range of applications, from biofuel production to drug design. However, several major issues might hamper the objective of achieving an artificial cell. From the bottom-up to the selection-based strategies, this work encompasses the ten grand challenges synthetic biologists will have to be aware of in order to cope with the task of creating life in the lab.

Synthesis of Empty Bacterial Microcompartments, Directed Organelle Protein Incorporation, and Evidence of Filament-Associated Organelle Movement

Compartmentalization is an important process, since it allows the segregation of metabolic activities and, in the era of synthetic biology, represents an important tool by which defined microenvironments can be created for specific metabolic functions. Indeed, some bacteria make specialized proteinaceous metabolic compartments called bacterial microcompartments (BMCs) or metabolosomes. Here we demonstrate that the shell of the metabolosome (representing an empty BMC) can be produced within E. coli cells by the coordinated expression of genes encoding structural proteins. A plethora of diverse structures can be generated by changing the expression profile of these genes, including the formation of large axial filaments that interfere with septation. Fusing GFP to PduC, PduD, or PduV, none of which are shell proteins, allows regiospecific targeting of the reporter group to the empty BMC. Live cell imaging provides unexpected evidence of filament-associated BMC movement within the cell in the presence of PduV.

The path to next generation biofuels: successes and challenges in the era of synthetic biology

Volatility of oil prices along with major concerns about climate change, oil supply security and depleting reserves have sparked renewed interest in the production of fuels from renewable resources. Recent advances in synthetic biology provide new tools for metabolic engineers to direct their strategies and construct optimal biocatalysts for the sustainable production of biofuels. Metabolic engineering and synthetic biology efforts entailing the engineering of native and de novo pathways for conversion of biomass constituents to short-chain alcohols and advanced biofuels are herewith reviewed. In the foreseeable future, formal integration of functional genomics and systems biology with synthetic biology and metabolic engineering will undoubtedly support the discovery, characterization, and engineering of new metabolic routes and more efficient microbial systems for the production of biofuels.

The dawn of synthetic genomics.

The first month of 2008 was unusually quiet in terms of microbial genome sequencing. Still, even the relatively short list of newly released genomes includes several interesting environmental microorganisms, such as the anoxygenic phototroph Chloroflexus aurantiacus, the toxic bloom-forming cyanobacterium Microcystis aeruginosa, and the methylotroph Methylobacterium extorquens (Table 1). However, arguably the biggest news was the announcement by J. Craig Venter and colleagues that they ‘have synthesized a 582 970 bp Mycoplasma genitalium genome’ (Gibson et al., 2008). The authors used chemically synthesized oligonucleotides ∼50 nucleotides in length to assemble ‘cassettes’ 5–7 kb in length, then to join them by in vitro recombination to produce intermediate assemblies, gradually increasing in size. Finally, four 144 kb pieces were cloned in Escherichia coli as bacterial artificial chromosomes, transferred into yeast and assembled into a full-length genome. The genome of the resulting strain, named M. genitalium JCVI-1.0, was virtually identical to the genome of the original strain M. genitalium G37. It was not immediately clear whether this technically very challenging and truly monumental work had any purpose beyond just serving as a proof of principle. However, J. C. Venter and colleagues have a record of overcoming enormous technical challenges and launching entirely new areas of biotechnology. It might be simply too early right now to ask them to explain the future of this work. In any case, the era of synthetic biology has officially begun and who knows what kind of molecules people will be synthesizing 10 or 20 years from now.