A deterministic genotyping workflow reduces waste of transgenic individuals by two-thirds.

We present a deterministic workflow for genotyping single and double transgenic individuals directly upon nascence that prevents overproduction and reduces wasted animals by two-thirds. In our vector concepts, transgenes are accompanied by two of four clearly distinguishable transformation markers that are embedded in interweaved, but incompatible Lox site pairs. Following Cre-mediated recombination, the genotypes of single and double transgenic individuals were successfully identified by specific marker combinations in 461 scorings.

An inexpensive anaerobic chamber for the genetic manipulation of strictly anaerobic bacteria.

Strictly anaerobic bacteria are important to both human health and industrial usage. These bacteria are sensitive to oxygen, therefore, it is preferable to manipulate these microbes in an anaerobic chamber. However, commercial anaerobic chambers (CACs) are expensive, making them less accessible to scientists with a limited budget, especially to those in developing countries. The high price of commercial chambers has hindered, at least partially, the progress of research on anaerobes in developing countries. In the research presented here, we developed an inexpensive and reliable anaerobic chamber and successfully achieved routine maintenance of eleven strictly anaerobic bacterial strains. Furthermore, genetic manipulation examples have been set for both Clostridioidesdifficile 630 and Clostridiumbeijerinckii NCIMB 8052 strains to validate that the chamber could applied to advanced genetic engineering of strictly anaerobes. C. difficile and C. beijerinckii were both genetically manipulated in this chamber, showing it's utility for the genetic engineering of anaerobes. Most importantly, the anaerobic chamber was 76% - 88% less expensive than a CACs and has similar functionality with regards to the cultivation and manipulation of strictly anaerobic bacteria. The anaerobic chamber described in this study will promote the research of anaerobes in developing counties and scientists who have limited research budgets.

The Daunting Economics of Therapeutic Genome Editing.

There is no shortage of enthusiasm for the clinical potential of CRISPR-based genome editing: many life-changing cures appear to be just around the corner. However, as mature genetic therapies reach the market, it seems that million-dollar price tags are the new normal. Several factors contribute to the extreme pricing of next-generation medicines, including the need to recoup development costs, the undeniable value of these powerful therapies, and the inherent technical challenges of manufacture and delivery. CRISPR technology has been hailed as a great leveler and a democratizing force in biomedicine. But for this principle to hold true in clinical contexts, therapeutic genome editing must avoid several pitfalls that could substantially limit access to its transformative potential, especially in the developing world.

Towards Automated Manufacturing for Cell Therapies.

PURPOSE OF REVIEW: Many cell therapy products are beginning to reach the commercial finish line and a rapidly escalating pipeline of products are in clinical development. The need to develop manufacturing capability that will support a successful commercial business model has become a top priority as many cell therapy developers look to secure long-term visions to enable both funding and treatment success. RECENT FINDINGS: Manufacturing automation is both highly compelling and very challenging at the same time as a key tactic to address quality, cost of goods, scale, and sustainability that are fundamental drivers for commercially viable manufacturing. This paper presents an overview and strategic drivers for application of automation to cell therapy manufacturing. It also explores unique automation considerations for patient-specific cell therapy (PSCT) where each full-scale lot is for one patient vs off-the-shelf cell therapy (OTSCT) where a full-scale lot will treat many patients, and finally some practical considerations for implementing automation.

Efficient vector systems for economical and rapid epitope-tagging and overexpression in Candida albicans.

Candida albicans is an opportunistic pathogenic fungus which causes superficial and systemic infections in immunocompromised patients. It is important to characterize the roles of genes involved in its pathogenesis, virulence, and drug resistance. Several genetic manipulation toolkits have been developed for gene function research in C. albicans. Here, we describe efficient vector systems that allow economical and rapid C-terminal and N-terminal epitope-tagging, inducible and constitutive promoter replacements, and ectopic gene overexpression in C. albicans. These systems use modularized genetic elements (conventional and non-conventional selection markers, epitope tags and promoters) and universal primers. These advantages should greatly reduce laboratory work and costs of strain construction for C. albicans.

Biosynthesis of butyric acid by Clostridium tyrobutyricum.

Butyric acid (C3H7COOH) is an important chemical that is widely used in foodstuffs along with in the chemical and pharmaceutical industries. The bioproduction of butyric acid through large-scale fermentation has the potential to be more economical and efficient than petrochemical synthesis. In this paper, the metabolic pathways involved in the production of butyric acid from Clostridium tyrobutyricum using hexose and pentose as substrates are investigated, and approaches to enhance butyric acid production through genetic modification are discussed. Finally, bioreactor modifications (including fibrous bed bioreactor, inner disk-shaped matrix bioreactor, fibrous matrix packed in porous levitated sphere carriers), low-cost feedstocks, and special treatments (including continuous fermentation with cell recycling, extractive fermentation with solvent, using different artificial electron carriers) intended to improve the feasibility of commercial butyric acid bioproduction are summarized.

Fusion expression of the PGLa-AM1 with native structure and evaluation of its anti-Helicobacter pylori activity.

Helicobacter pylori (H. pylori) shows increasingly enhanced resistance to various antibiotics, and its eradication has become a major problem in medicine. The antimicrobial peptide PGLa-AM1 is a short peptide with 22 amino acids and exhibits strong antibacterial activity. In this study, we investigated whether it has anti-H. pylori activity for the further development of anti-H. pylori drugs to replace existing antibiotics. However, the natural antimicrobial peptide PGLa-AM1 shows a low yield and is difficult to separate, limiting its application. A good strategy to solve this problem is to express the antimicrobial peptide PGLa-AM1 using gene engineering at a high level and low cost. For getting PGLa-AM1 with native structure, in this study, a specific protease cleavage site of tobacco etch virus (TEV) was designed before the PGLa-AM1 peptide. For convenience to purify and identify high-efficiency expression PGLa-AM1, the PGLa-AM1 gene was fused with the polyhedrin gene of Bombyx mori (B. mori), and a 6 × His tag was designed to insert before the amino terminus of the fusion protein. The fusion antibacterial peptide PGLa-AM1 (FAMP) gene codon was optimized, and the gene was synthesized and cloned into the Escherichia coli (E. coli) pET-30a (+) expression vector. The results showed that the FAMP was successfully expressed in E. coli. Its molecular weight was approximately 34 kDa, and its expression level was approximately 30 mg/L. After the FAMP was purified, it was further digested with TEV protease. The acquired recombinant antimicrobial peptide PGLa-AM1 exerted strong anti-H. pylori activity and therapeutic effect in vitro and in vivo.

Synthetic Biology in the Driving Seat of the Bioeconomy.

Synthetic biology is revolutionising the biotech industry and is increasingly applied in previously unthought-of markets. Here, we discuss the importance of this industry to the bioeconomy and two of its key factors: the synthetic biology approach to research and development (R&D), and the unique nature of the carefully designed, stakeholder-inclusive, community-directed evolution of the field.

Economic co-production of poly(malic acid) and pullulan from Jerusalem artichoke tuber by Aureobasidium pullulans HA-4D.

BACKGROUND: poly(L-malic acid) (PMA) is a water-soluble polyester with many attractive properties in medicine and food industries, but the high cost of PMA fermentation has restricted its further application for large-scale production. To overcome this problem, PMA production from Jerusalem artichoke tubers was successfully performed. Additionally, a valuable exopolysaccharide, pullulan, was co-produced with PMA by Aureobasidum pullulans HA-4D. RESULTS: The Jerusalem artichoke medium for PMA and pullulan co-production contained only 100 g/L hydrolysate sugar, 30 g/L CaCO3 and 1 g/L NaNO3. Compared with the glucose medium, the Jerusalem artichoke medium resulted in a higher PMA concentration (114.4 g/L) and a lower pullulan concentration (14.3 g/L) in a 5 L bioreactor. Meanwhile, the activity of pyruvate carboxylase and malate dehydrogenas was significantly increased, while the activity of α-phosphoglucose mutase, UDP-glucose pyrophosphorylase and glucosyltransferase was not affected. To assay the economic-feasibility, large-scale production in a 1 t fermentor was performed, yielding 117.5 g/L PMA and 15.2 g/L pullulan. CONCLUSIONS: In this study, an economical co-production system for PMA and pullulan from Jerusalem artichoke was developed. The medium for PMA and pullulan co-production was significantly simplified when Jerusalem artichoke tubers were used. With the simplified medium, PMA production was obviously stimulated, which would be associated with the improved activity of pyruvate carboxylase and malate dehydrogenas.

A Rapid, Cost-Effective Method to Prepare Recombinant Adeno-Associated Virus for Efficient Gene Transfer to the Developing Mouse Inner Ear.

There is keen interest to define gene therapies aimed at restoration of auditory and vestibular function in the diseased or damaged mammalian inner ear. A persistent limitation of regenerative medical strategies that seek to correct or modify gene expression in the sensory epithelia of the inner ear involves efficacious delivery of a therapeutic genetic construct. Our approach is to define methodologies that enable fetal gene transfer to the developing mammalian inner ear in an effort to correct defective gene expression during formation of the sensory epithelia or during early postnatal life. Conceptually, the goal is to atraumatically introduce the genetic construct into the otocyst-staged mouse inner ear and transfect otic progenitors that give rise to sensory hair cells and supporting cells. Our long-term goal is to define therapeutic interventions for congenital deafness and balance disorders with the expectation that the approach may also be exploited for therapeutic intervention postnatally.In the inaugural volume of this series, we introduced electroporation-mediated gene transfer to the developing mouse inner ear that encompassed our mouse survival surgery and transuterine microinjection protocols (Brigande et al., Methods Mol Biol 493:125-139, 2009). In this chapter, we first briefly update our use of sodium pentobarbital anesthesia, our preferred anesthetic for mouse ventral laparotomy, in light of its rapidly escalating cost. Next, we define a rapid, cost-effective method to produce recombinant adeno-associated virus (rAAV) for efficient gene transfer to the developing mouse inner ear. Our immediate goal is to provide a genetic toolkit that will permit the definition and validation of gene therapies in mouse models of human deafness and balance disorders.

Enhanced Generation of Integration-free iPSCs from Human Adult Peripheral Blood Mononuclear Cells with an Optimal Combination of Episomal Vectors.

We previously reported the generation of integration-free induced pluripotent stem cells from adult peripheral blood (PB) with an improved episomal vector (EV) system, which uses the spleen focus-forming virus U3 promoter and an extra factor BCL-XL (B). Here we show an ∼100-fold increase in efficiency by optimizing the vector combination. The two most critical factors are: (1) equimolar expression of OCT4 (O) and SOX2 (S), by using a 2A linker; (2) a higher and gradual increase in the MYC (M) to KLF4 (K) ratio during the course of reprogramming, by using two individual vectors to express M and K instead of one. The combination of EV plasmids (OS + M + K + B) is comparable with Sendai virus in reprogramming efficiency but at a fraction of the cost. The generated iPSCs are indistinguishable from those from our previous approach in pluripotency and phenotype. This improvement lays the foundation for broad applications of episomal vectors in PB reprogramming.

Screening Strategies for TALEN-Mediated Gene Disruption.

Targeted gene disruption has rapidly become the tool of choice for the analysis of gene and protein function in routinely cultured mammalian cells. Three main technologies capable of irreversibly disrupting gene-expression exist: zinc-finger nucleases, transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas9 system. The desired outcome of the use of any of these technologies is targeted insertions and/or deletions (indels) that result in either a nonsense frame shift or splicing error that disrupts protein expression. Many excellent do-it-yourself systems for TALEN construct assembly are now available at low or no cost to academic researchers. However, for new users, screening for successful gene disruption is still a hurdle. Here, we describe efficient and cost-effective strategies for the generation of gene-disrupted cell lines. Although the focus of this chapter is on the use of TALENs, these strategies can be applied to the use of all three technologies.

The emergence of commodity-scale genetic manipulation.

Since the 1970s technological advancements in the fields of synthetic biology and metabolic engineering have led to a dramatic reduction in both time and cost required for generating genomic mutations in a variety of organisms. The union of genomic editing machinery, DNA inkjet printers, and bioinformatics algorithms allows engineers to design a library of thousands of unique oligos as well as build and test these designs on a ∼2 months time-scale and at a cost of roughly ∼0.3 cents per base pair. The implications of these capabilities for a variety of fields are far-reaching, with potential impacts in defense, agricultural, human health, and environmental research. The explosion of synthetic biology applications over the past two decades have led many to draw parallels between biological engineering and the computer sciences. In this review, we highlight some important parallels between these fields and emphasize the importance of engineering design strategies.

The Bacterial Origins of the CRISPR Genome-Editing Revolution.

Like most of the tools that enable modern life science research, the recent genome-editing revolution has its biological roots in the world of bacteria and archaea. Clustered, regularly interspaced, short palindromic repeats (CRISPR) loci are found in the genomes of many bacteria and most archaea, and underlie an adaptive immune system that protects the host cell against invasive nucleic acids such as viral genomes. In recent years, engineered versions of these systems have enabled efficient DNA targeting in living cells from dozens of species (including humans and other eukaryotes), and the exploitation of the resulting endogenous DNA repair pathways has provided a route to fast, easy, and affordable genome editing. In only three years after RNA-guided DNA cleavage was first harnessed, the ability to edit genomes via simple, user-defined RNA sequences has already revolutionized nearly all areas of biological science. CRISPR-based technologies are now poised to similarly revolutionize many facets of clinical medicine, and even promise to advance the long-term goal of directly editing genomic sequences of patients with inherited disease. In this review, we describe the biological and mechanistic basis for these remarkable immune systems, and how their engineered derivatives are revolutionizing basic and clinical research.