Construction of Small Genes with Repetitive Elements Using Oligo Extension and Golden Gate Assembly.

Gene synthesis efficiency has greatly improved in recent years but is limited when it comes to repetitive sequences and results in synthesis failure or delays by DNA synthesis vendors. Here, we describe a method for the assembly of small synthetic genes with repetitive elements: First, a gene of interest is split in silico into small synthons of up to 80 base pairs flanked by Golden Gate-compatible overhangs. Then synthons are made by oligo extension and finally assembled into a synthetic gene by Golden Gate assembly.

Gene synthesis design: a pythonic approach.

Researchers often need to synthesize genes of interest in this era of synthetic biology. Gene synthesis by PCR assembly of multiple DNA fragments is a quick and economical method that is widely applied. Up to now, there have been a few software solutions for designing fragments in gene synthesis. However, some of these software solutions use programming languages that are not popular now, other software products are commercial or require users to visit servers. In this study, we propose a Python program to design DNA fragments for gene synthesis. The algorithm is designed to meet the experimental needs. Also, the source code with detailed annotation is freely available for all users. Furthermore, the feasibility of the algorithm and the program is validated by experiments. Our program can be useful for the design of gene synthesis in the labs and help the study of gene structure and function.

How advancements in molecular biology impact education and training.

Molecular biology, broadly defined as the investigation of complex biomolecules in the laboratory, is a rapidly advancing field and as such the technologies available to investigators are constantly evolving. This constant advancement has obvious advantages because it allows students and researchers to perform more complex experiments in shorter periods of time. One challenge with such a rapidly advancing field is that techniques that had been vital for students to learn how to perform are now not essential for a laboratory scientist. For example, while cloning a gene in the past could have led to a publication and form the bulk of a PhD thesis project, technology has now made this process only a step toward one of these larger goals and can, in many cases, be performed by a company or core facility. As teachers and mentors, it is imperative that we understand that the technologies we teach in the lab and classroom must also evolve to match these advancements. In this perspective, we discuss how the rapid advances in gene synthesis technologies are affecting curriculum and how our classrooms should evolve to ensure our lessons prepare students for the world in which they will do science.

Modern Approaches to de novo Synthesis of Extended DNA Fragments: Assembly of a Wide Repertoire of Sequences.

The standardization of DNA fragment assembly methods for many tasks of synthetic biology is crucial. This is necessary for synthesizing a wider repertoire of sequences, as well as for further automation and miniaturization of such reactions. In this work, we proposed conditions for the assembly of DNA fragments from chemically synthesized oligonucleotides and we identified the errors occurring in the sequence under these conditions. Additionally, we proposed conditions for further combining synthetic fragments into larger DNA fragments. We showed that the optimized conditions are suitable for the assembly of a wide range of sequences.

Porcine Mx proteins inhibit pseudorabies virus replication through interfering with early gene synthesis.

Pseudorabies virus (PRV) is an enveloped, linear double-stranded DNA herpesvirus that resulted in huge financial losses to the swine industry. In addition to vaccination, the development of antiviral molecules is also a beneficial supplement to the control of Pseudorabies (PR). Although our previous studies have shown that porcine Mx protein (poMx1/2) significantly inhibited the proliferation of RNA virus, it was unknown whether poMx1/2 could inhibit porcine DNA virus, such as PRV. In this study, it was investigated the inhibitory effect of porcine Mx1/2 protein on PRV multiplication. The results showed that both poMx1 and poMx2 had anti-PRV activities, which required GTPase ability and stable oligomerization. Interestingly, the two GTPase deficient mutants (G52Q and T148A) of poMx2 also had the antiviral ability against PRV, which was consistent with previous reports, indicating that these mutants recognized and blocked the viral targets. Mechanistically, the antiviral restriction of poMx1/2 came from their inhibition of the early gene synthesis of PRV. Our results for the first time shed light on the antiviral activities of two poMx proteins against DNA virus. The data from this study provide further insights to develop new strategies for preventing and controlling the diseases caused by PRV.

PCR-Based Assembly of Gene Sequences by Thermodynamically Balanced Inside-Out (TBIO) Gene Synthesis.

The ability to enzymatically assemble DNA oligonucleotides into longer DNA duplexes in a process known as gene synthesis has wide-ranging applications in the fields of genetic engineering and synthetic biology. Thermodynamically balanced inside-out (TBIO) gene synthesis is one of several PCR-based primer extension gene synthesis protocols that have been developed. In TBIO gene synthesis, overlapping primers with equivalent melting temperatures (Tms) are designed so that the 5' half of the DNA is encoded by sense primers and the 3' half of the DNA molecule is encoded by antisense primers. Primer extension is initiated at the center of the DNA and continues bidirectionally to progressively elongate the DNA molecule. Here we provide the protocols necessary for performing TBIO gene synthesis to generate a DNA molecule of interest.

Learning from Gobind Khorana: "The difficult we do today, the impossible tomorrow".

Prof. Har Gobind Khorana was one of the greatest scientists of the twentieth century. Drawing on his strong roots in organic chemistry, he had a remarkable ability to select and focus his intellect on successfully addressing some of the most important challenges in modern biology in a career spanning nearly 6 decades. His pioneering contributions in gene synthesis and protein structure-function studies, and more broadly in what he termed "chemical biology," continue to have a major impact on modern biomedical science.

Simple Webserver-Facilitated Method to Design and Synthesize Artificial miRNA Gene and Its Application in Engineering Viral Resistance.

Plant viruses impose serious threats on crop production. Artificial miRNAs can mediate specific and effective gene silencing in plants and are widely used in plant gene function studies and to engineer plant viral resistance. To facilitate the design of artificial miRNA genes, we developed a webserver, AMIRdesigner, which can be used to design oligos for artificial miRNA synthesis using wild-type and permutated MIR171 and MIR164 backbones. The artificial miRNA genes designed by AMIRdesigner can be easily assembled into miRNA clusters for multiple target sites. To validate the server functionality, we designed four artificial miRNA genes targeting four conserved regions in the potato leafroll virus genome using AMIRdesigner. These genes were synthesized with the server-designed oligos and further assembled into a quadruple miRNA cluster, which was cloned into an overexpression vector and transformed into potato plants. Small RNA Northern blot and virus inoculation analyses showed that a high level of artificial miRNA expression and good viral resistance were achieved in some of the transgenic lines. These results demonstrate the utility of our webserver AMIRdesigner for engineering crop viral resistance.

Triple gene expressions in yeast, Escherichia coli, and mammalian cells by transferring DNA fragments amplified from a mother yeast expression plasmid.

Escherichia coli, Saccharomyces cerevisiae, and mammalian culture cells are standard host organisms for genetic engineering and research, thus various plasmid vectors have been developed. However, the vectors are designed only for a single host owing to their host-specific genetic elements such as promoters and selection markers. In this study, we developed a yeast expression plasmid that enables the expression of the same gene in E. coli and mammalian cells via the transfer of PCR products amplified from the plasmid as a template. The yeast plasmid YHp26352 was constructed to contain the following regions sequentially: yeast TDH3 promoter (TDH3p), red fluorescent protein (eEmRFP), SV40 terminator (SVpA), E. coli origin (ori), ampicillin resistant gene (AmpR), mammalian cytomegalovirus promoter (CMVp), E. coli srlA promoter (srlAp), and yeast selection marker URA3, which expressed eEmRFP in yeast. To express eEmRFP in mammalian cells, an end-promoter DNA fragment encompassing the eEmRFP-SVpA-ori-AmpR-CMVp region was amplified by PCR and directly used for transfection to mammalian culture cells, resulting in gene expression in mammalian cells through non-homologous end joining. Homologous recombination-mediated circularization was carried out for E. coli cloning and expression by attaching a short overlapping sequence to the 5'-end of a PCR primer, which was used to amplify the eEmRFP-SVpA-ori-AmpR-CMVp-srlAp fragment, after which E. coli transformation was performed. Proof-of-concept experiments were performed by expressing GFP-fused human synaptobrevin VAMP1, and wild-type and codon-changed CLuc luciferase genes in yeast, E. coli, and HEK293 cells. This is the first all-in-one plasmid applicable for expression in three host organisms.

An Integrated Algorithm for Designing Oligodeoxynucleotides for Gene Synthesis.

The design and construction of large synthetic genes can be a slow, difficult, and confusing process, especially in the key step of oligodeoxynucleotide design. Herein we present an integrated algorithm to design oligonucleotide sets for gene synthesis by both ligase chain reaction and polymerase chain reaction. It offers much flexibility with no constraints on the gene to be synthesized. Firstly, it divides the long-input DNA sequence by a greedy algorithm based on the length of the oligodeoxynucleotide overlap region. Secondly, it tunes the length of the overlap region iteratively in an attempt to minimize the melting temperature variance of overlap. Thirdly, dynamic programming algorithm is used to achieve the uniform melting temperature of the oligodeoxynucleotide overlaps. Finally, the oligodeoxynucleotides with homologous melting temperature necessary for ligase chain reaction-based or two-step assembly PCR-based synthesis of the desired gene are outputted.

Generation of a Library of Carbohydrate-Active Enzymes for Plant Biomass Deconstruction.

In nature, the deconstruction of plant carbohydrates is carried out by carbohydrate-active enzymes (CAZymes). A high-throughput (HTP) strategy was used to isolate and clone 1476 genes obtained from a diverse library of recombinant CAZymes covering a variety of sequence-based families, enzyme classes, and source organisms. All genes were successfully isolated by either PCR (61%) or gene synthesis (GS) (39%) and were subsequently cloned into Escherichia coli expression vectors. Most proteins (79%) were obtained at a good yield during recombinant expression. A significantly lower number (p < 0.01) of proteins from eukaryotic (57.7%) and archaeal (53.3%) origin were soluble compared to bacteria (79.7%). Genes obtained by GS gave a significantly lower number (p = 0.04) of soluble proteins while the green fluorescent protein tag improved protein solubility (p = 0.05). Finally, a relationship between the amino acid composition and protein solubility was observed. Thus, a lower percentage of non-polar and higher percentage of negatively charged amino acids in a protein may be a good predictor for higher protein solubility in E. coli. The HTP approach presented here is a powerful tool for producing recombinant CAZymes that can be used for future studies of plant cell wall degradation. Successful production and expression of soluble recombinant proteins at a high rate opens new possibilities for the high-throughput production of targets from limitless sources.

Oligo Pools as an Affordable Source of Synthetic DNA for Cost-Effective Library Construction in Protein- and Metabolic Pathway Engineering.

The construction of custom libraries is critical for rational protein engineering and directed evolution. Array-synthesized oligo pools of thousands of user-defined sequences (up to ∼350 bases in length) have emerged as a low-cost commercially available source of DNA. These pools cost ≤10 % (depending on error rate and length) of other commercial sources of custom DNA, and this significant cost difference can determine whether an enzyme engineering project can be realized on a given research budget. However, while being cheap, oligo pools do suffer from a low concentration of individual oligos and relatively high error rates. Several powerful techniques that specifically make use of oligo pools have been developed and proven valuable or even essential for next-generation protein and pathway engineering strategies, such as sequence-function mapping, enzyme minimization, or de-novo design. Here we consolidate the knowledge on these techniques and their applications to facilitate the use of oligo pools within the protein engineering community.

[Current status and prospect of DNA synthesis in industrial biotechnology].

DNA synthesis is one of the most basic, widely-used tools in life science as well as a key enabling technology in synthetic biology. The rapid development of industrial biotechnology promoted by synthetic biology is creating an insatiable demand for large-scale DNA synthesis from more convenient, economical and safe sources. Industrial DNA synthesis platforms have remarkable advantages in terms of throughput, cost and speed. The research and development processes of industrial biotechnology benefit from these advantages, achieving a higher efficiency and lower cost. However, challenges in DNA manufacturing process remain, such as the use of large amounts of organic reagents, waste of resources and so on. With the continuous and rapid increase of DNA synthesis scale, the hazard of toxic chemicals, cost burden and environmental burden are becoming prominent. Based on our practical work on DNA synthesis, we discuss the demand and strategies for large-scale DNA synthesis in industrial biotechnology as well as the issues and potential solutions for sustainable development.

[Application of Array-Based Oligonucleotides for Synthesis of Genetic Designs].

The application of array-based oligonucleotides in biological studies is described. These oligonucleotides are mainly used to design large libraries of various nucleotide sequences, which are applied to study protein-nucleic acid interactions, splicing, transcription, translation, and other regulatory processes in mammalian, yeast, and bacterial systems. The application of gene libraries generated by array-based nucleotides along with advanced methods of the combination of DNA duplexes will make it possible to obtain complex genetic designs for synthetic biology.

Mutation Maker, An Open Source Oligo Design Platform for Protein Engineering.

Protein engineering is the discipline of developing useful proteins for applications in research, therapeutic, and industrial processes by modification of naturally occurring proteins or by invention of de novo proteins. Modern protein engineering relies on the ability to rapidly generate and screen diverse libraries of mutant proteins. However, design of mutant libraries is typically hampered by scale and complexity, necessitating development of advanced automation and optimization tools that can improve efficiency and accuracy. At present, automated library design tools are functionally limited or not freely available. To address these issues, we developed Mutation Maker, an open source mutagenic oligo design software for large-scale protein engineering experiments. Mutation Maker is not only specifically tailored to multisite random and directed mutagenesis protocols, but also pioneers bespoke mutagenic oligo design for de novo gene synthesis workflows. Enabled by a novel bundle of orchestrated heuristics, optimization, constraint-satisfaction and backtracking algorithms, Mutation Maker offers a versatile toolbox for gene diversification design at industrial scale. Supported by in silico simulations and compelling experimental validation data, Mutation Maker oligos produce diverse gene libraries at high success rates irrespective of genes or vectors used. Finally, Mutation Maker was created as an extensible platform on the notion that directed evolution techniques will continue to evolve and revolutionize current and future-oriented applications.

Development and evaluation of recombinant GRA8 protein for the serodiagnosis of Toxoplasma gondii infection in goats.

BACKGROUND: The development of sensitive and specific methods for detecting Toxoplasma gondii infection is critical for preventing and controlling toxoplasmosis in humans and other animals. Recently, various recombinant proteins have been used in serological tests for diagnosing toxoplasmosis. The production of these antigens is associated with live tachyzoites obtained from cell cultures or laboratory animals for genomic extraction to amplify target genes. Synthetic genes have gained a key role in recombinant protein production. For the first time, we demonstrated the production of the recombinant protein of the T. gondii dense granular antigen 8 (TgGRA8) gene based on commercial gene synthesis. Recombinant TgGRA8 plasmids were successfully expressed in an Escherichia coli system. The recombinant protein was affinity-purified and characterized via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Furthermore, the diagnostic potential of the recombinant protein was assessed using 306 field serum samples from goats via indirect enzyme-linked immunosorbent assay (iELISA) and the latex agglutination test (LAT). RESULTS: Western blotting using known positive serum samples from goats identified a single antigen at the expected molecular weight of TgGRA8 (27 kDa). iELISA illustrated that 15.40% of goat samples were positive for T. gondii-specific IgG antibodies. In addition, TgGRA8 provided high sensitivity and specificity, with significant concordance (91.83) and kappa values (0.69) compared with the results obtained using LAT. CONCLUSION: Our findings highlight the production of a recombinant protein from a synthetic TgGRA8 gene and the ability to detect T. gondii infection in field samples. The sensitivity and specificity of TgGRA8 demonstrated that this protein could be a good serological marker for detecting specific IgG in goat sera.

An improved gene synthesis method with asymmetric directions of oligonucleotides designed using a simulation program.

Artificial gene synthesis based on oligonucleotide augmentation is known as overlap extension PCR which generates a variety of intermediate synthetic products. The orientation and concentration of oligomers can be adjusted to reduce the synthesis of intermediates and optimize the full-length process of DNA synthesis, using a simulation program for serial oligomer extension. The efficiency of the serial oligomer extension process is predicted to be greatest when oligomers are in a 'forward-reverse-reverse-reverse' direction. Oligomers with such designed directions demonstrated generation of the desired product in the shortest time (number of cycles) by repeated annealing and elongation. This method, named Asymmetric Extension supported by a Simulator for Oligonucleotide Extension (AESOE), has shown efficiency and effectiveness with potentials for future improvements and optimal usage in DNA synthesis.

Efficient and Low-Cost Error Removal in DNA Synthesis by a High-Durability MutS.

Enzyme-based error correction is a key step in de novo DNA synthesis, yet the inherent instability of error-correction enzymes such as MutS has hindered the throughput and efficiency of DNA synthesis workflows. Here we introduce a process called Improved MICC (iMICC), in which all error-correction steps of oligos and fragments within a complete gene-synthesis cycle are completed in a simple, efficient, and low-cost manner via a MutS protein engineered for high durability. By establishing a disulfide bond of L157C-G233C, full-activity shelf life of E. coli MutS (eMutS) was prolonged from 7 to 49 days and was further extended to 63 days via cellulose-bound 4 °C storage. In synthesis of 10 Cas9 homologues in-solution and 10 xylose reductase (XR) homologues on-chip, iMICC reduced error frequency to 0.64/Kb and 0.41/Kb, respectively, with 72.1% and 86.4% of assembled fragments being error-free. By elevating base accuracy by 37.6-fold while avoiding repetitive preparation of fresh enzymes, iMICC is more efficient and robust than the wild-type eMutS, and it is 6.6-fold more accurate and 26.7-fold cheaper than CorrectASE. These advantages promise its broad applications in industrial DNA synthesis.

Strengthening Security for Gene Synthesis: Recommendations for Governance.

Since the inception of gene synthesis technologies, there have been concerns about possible misuse. Using gene synthesis, pathogens-particularly small viruses-may be assembled "from scratch" in the laboratory, evading the regulatory regimes many nations have in place to control unauthorized access to dangerous pathogens. Progress has been made to reduce these risks. In 2010, the US Department of Health and Human Services (HHS) published guidance for commercial gene synthesis providers that included sequence screening of the orders and customer screening. The industry-led International Gene Synthesis Consortium (IGSC) was formed in 2009 to share sequence and customer screening methods, and it now includes the major international gene synthesis providers among its members. Since the 2010 HHS Guidance was released, however, there have been changes in gene synthesis technologies and market conditions that have reduced the efficacy of these biosecurity protections, leading to questions about whether the 2010 HHS Guidance should be updated, what changes could make it more effective, and what other international governance efforts could be undertaken to reduce the risks of misuse of gene synthesis products. This article describes these conditions and recommends actions that governments should take to reduce these risks and engage other nations involved in gene synthesis research.

Programmed assembly of long DNA synthons: design, mechanism, and online monitoring.

Synthesis of custom de novo DNA sequences is highly demanded by fast-growing field of synthetic biology. Usually DNA sequences with length more than 1 kb are assembled from smaller synthetic DNA fragments (synthons) obtained by PCR assembly. The ability to synthesize longer synthons sufficiently reduces efforts and time for DNA synthesis. We developed a novel rational oligonucleotide design and programmed approach for the assembly of synthetic DNA synthons up to 1550 bp. The developed procedure was thoroughly investigated by synthesis of cholesterol oxidase gene from Streptomyces lavendulae (1544 bp). Our approach is based on combined design, oligonucleotide concentration gradient, and specialized assembly program that directs assembly reaction to full-length gene in a stepwise manner. The process includes conventional thermodynamically balanced assembly, thermodynamically balanced inside-out elongation, and further amplification. The ability of DNA polymerase to perform programmed assembly is highly influenced by the presence of 5' → 3'-exonuclease activity. Oligonucleotide probing of PCR assembly products allowed us to shed light on the nature of high molecular weight spurious by-products and to understand the mechanism of their formation. For the first time, we applied light scattering techniques for tracking of oligonucleotide annealing, analysis of gene assembly products, and even for real-time monitoring of gene assembly process.