Rational de novo gene synthesis by rapid polymerase chain assembly (PCA) and expression of endothelial protein-C and thrombin receptor genes

The assembly of synthetic oligonucleotides into genes and genomes is an important methodology. Several methodologies for such synthesis have been developed, but they have two drawbacks: (1) the processes are slow and (2) the error frequencies are high (typically 1-3 errors/kb of DNA). Thermal damage is a major contributor to biosynthetic errors. In this paper, we elucidate the advantages of rapid gene synthesis by polymerase chain assembly (PCA) when used in combination with smart error control strategies. We used a high-speed thermocycler (PCRJet) to effectively minimize thermal damage and to perform rapid assembly of synthetic oligonucleotides to construct two different genes: endothelial protein C receptor (EPCR) and endothelial cell thrombin receptor, thrombomodulin (TM). First, the intact EPCR gene (EPCR-1, 612 bp) and a mutant EPCR-2 (576 bp) that lacked 4 N-linked glycosylation sites were constructed from 35 and 33 oligonucleotides, respectively. Next, for direct error comparison, another longer gene, the 1548 bp TM gene was constructed from 87 oligonucleotides by both rapid and conventional PCA. The fidelity and accuracy of the synthetic genes generated in this manner were confirmed by sequencing. The combined steps of PCA and DNA amplification are completed in about 10 and 22 min for EPCR-1, 2 and TM genes, respectively with comparable low errors in the DNA sequence. Furthermore, we subcloned synthetic TM, EPCR-1, EPCR-2 and native EPCR-1 (amplified from cDNA) into a Pichia pastoris expression vector to evaluate the expression ability, and to compare them with the native gene. Here, we illustrate that the synthetic genes, assembled by rapid PCA, successfully directed the expression of functional proteins. And, importantly, the synthetic and the native genes expressed proteins with the same efficiency.     

A Method for Multiplex Gene Synthesis Employing Error Correction Based on Expression

Our ability to engineer organisms with new biosynthetic pathways and genetic circuits is limited by the availability of protein characterization data and the cost of synthetic DNA. With new tools for reading and writing DNA, there are opportunities for scalable assays that more efficiently and cost effectively mine for biochemical protein characteristics. To that end, we have developed the Multiplex Library Synthesis and Expression Correction (MuLSEC) method for rapid assembly, error correction, and expression characterization of many genes as a pooled library. This methodology enables gene synthesis from microarray-synthesized oligonucleotide pools with a one-pot technique, eliminating the need for robotic liquid handling. Post assembly, the gene library is subjected to an ampicillin based quality control selection, which serves as both an error correction step and a selection for proteins that are properly expressed and folded in E. coli. Next generation sequencing of post selection DNA enables quantitative analysis of gene expression characteristics. We demonstrate the feasibility of this approach by building and testing over 90 genes for empirical evidence of soluble expression. This technique reduces the problem of part characterization to multiplex oligonucleotide synthesis and deep sequencing, two technologies under extensive development with projected cost reduction.     

Microfluidic PicoArray synthesis of oligodeoxynucleotides and simultaneous assembling of multiple DNA sequences

Large DNA constructs of arbitrary sequences can currently be assembled with relative ease by joining short synthetic oligodeoxynucleotides (oligonucleotides). The ability to mass produce these synthetic genes readily will have a significant impact on research in biology and medicine. Presently, high-throughput gene synthesis is unlikely, due to the limits of oligonucleotide synthesis. We describe a microfluidic PicoArray method for the simultaneous synthesis and purification of oligonucleotides that are designed for multiplex gene synthesis. Given the demand for highly pure oligonucleotides in gene synthesis processes, we used a model to improve key reaction steps in DNA synthesis. The oligonucleotides obtained were successfully used in ligation under thermal cycling conditions to generate DNA constructs of several hundreds of base pairs. Protein expression using the gene thus synthesized was demonstrated. We used a DNA assembly strategy, i.e. ligation followed by fusion PCR, and achieved effective assembling of up to 10 kb DNA constructs. These results illustrate the potential of microfluidics-based ultra-fast oligonucleotide parallel synthesis as an enabling tool for modern synthetic biology applications, such as the construction of genome-scale molecular clones and cell-free large scale protein expression.     

Error correction of microchip synthesized genes using Surveyor nuclease

The development of economical and high-throughput gene synthesis technology has been hampered by the high occurrence of errors in the synthesized products, which requires expensive labor and time to correct. Here, we describe an error correction reaction (ECR), which employs Surveyor, a mismatch-specific DNA endonuclease, to remove errors from synthetic genes. In ECR reactions, errors are revealed as mismatches by re-annealing of the synthetic gene products. Mismatches are recognized and excised by a combination of mismatch-specific endonuclease and 3'→5' exonuclease activities in the reaction mixture. Finally, overlap extension polymerase chain reaction (OE-PCR) re-assembles the resulting fragments into intact genes. The process can be iterated for increased fidelity. With two iterations, we were able to reduce errors in synthetic genes by >16-fold, yielding a final error rate of ～1 in 8700 bp.     

Improved PCR-based gene synthesis method and its application to the Citrobacter freundii phytase gene codon modification

Gene synthesis technologies provide a powerful tool for increasing protein expression through codon optimization and gene modification. Here we describe an improved PCR-based gene synthesis technology, which is accurate, simple and cheap. The improved PCR-based gene synthesis (IPS) method consists of two steps. The first one is the synthesis of 300-400 bp fragments by PCR reaction with Pfu DNA polymerase from 60-mer and 30-mer oligonucleotides with a 15 bp overlap. The second one is assembling of fragments from the first step into the full-length gene by PCR reaction. Using this approach, we have successfully synthesized a modified phytase gene with 1256 bp in length with optimal codons for expression in Pichia pastoris. P. pastoris strain that expressed the modified phytase gene (phyA-mod) showed a 50% increase in phytase activity level. In addition, we propose an inexpensive method for error correction, based on overlap-extension PCR (OE-PCR).     

Chemical gene synthesis: strategies, softwares, error corrections, and applications

Chemical synthesis of DNA sequences provides a powerful tool for modifying genes and for studying gene structure, expression and function. Modified genes and consequently protein/enzymes can bridge genomics and proteomics research or facilitate commercial applications of gene and protein technologies. In this review, we will summarize various strategies, designing softwares and error correction methods for chemical gene synthesis, particularly for the synthesis and assembly of long DNA molecules based on polymerase cycling assembly. Also, we will briefly discuss some of the major applications of chemical synthesis of DNA sequences in basic research and applied areas.     

Herpes simplex virus gene expression in neurons: viral DNA synthesis is a critical regulatory event in the branch point between the lytic and latent pathways.

Herpes simplex virus establishes a latent infection in peripheral neurons. We examined viral gene expression in rat peripheral neurons in vitro and determined that viral gene expression is attenuated and delayed in these neurons compared with that in Vero cells. In addition, using pharmacologic and genetic blocks to viral DNA synthesis, we found that viral alpha and beta gene expression was upregulated by viral DNA synthesis. Although maximal gene expression in neurons requires viral DNA synthetic activity, activation of viral gene expression was seen even in the presence of herpes simplex virus DNA polymerase inhibitors, but not in the absence of the origin-binding protein. Initiation of viral DNA synthesis is apparently a key regulatory event in the balance between the lytic and latent pathways in peripheral neurons.     

Protein expression from synthetic genes: selection of clones using GFP

Construction of synthetic genes is today the most elegant way to optimize the heterologous expression of a recombinant protein. However, the selection of positive clones that incorporate the correct synthetic DNA fragments is a bottleneck as current methods of gene synthesis introduce 3.5 nucleotide deletions per kb. Furthermore, even when all predictable optimizations for protein production have been introduced into the synthetic gene, production of the protein is often disappointing: protein is produced in too low amounts or end up in inclusion bodies. We propose a strategy to overcome these two problems simultaneously by cloning the synthetic gene upstream of a reporter gene. This permits the selection of clones devoid of frame-shift mutations. In addition, beside nucleotide deletion, an average of three non-neutral mutations per kb are introduced during gene synthesis. Using a reporter protein downstream of the synthetic gene, allows the selection of clones with random mutations improving the expression or the folding of the protein of interest. The problem of errors found in synthetic genes is then turned into an advantage since it provides polymorphism useful for molecular evolution. The use of synthetic genes appears as an alternative to the error-prone PCR strategy to generate the variations necessary in protein engineering experiments.     

Protein-mediated error correction for de novo DNA synthesis

The availability of inexpensive, on demand synthetic DNA has enabled numerous powerful applications in biotechnology, in turn driving considerable present interest in the de novo synthesis of increasingly longer DNA constructs. The synthesis of DNA from oligonucleotides into products even as large as small viral genomes has been accomplished. Despite such achievements, the costs and time required to generate such long constructs has, to date, precluded gene-length (and longer) DNA synthesis from being an everyday research tool in the same manner as PCR and DNA sequencing. A critical barrier to low-cost, high-throughput de novo DNA synthesis is the frequency at which errors pervade the final product. Here, we employ a DNA mismatch-binding protein, MutS (from Thermus aquaticus) to remove failure products from synthetic genes. This method reduced errors by >15-fold relative to conventional gene synthesis techniques, yielding DNA with one error per 10000 base pairs. The approach is general, scalable and can be iterated multiple times for greater fidelity. Reductions in both costs and time required are demonstrated for the synthesis of a 2.5 kb gene.     

DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis

The availability of sequences of entire genomes has dramatically increased the number of protein targets, many of which will need to be overexpressed in cells other than the original source of DNA. Gene synthesis often provides a fast and economically efficient approach. The synthetic gene can be optimized for expression and constructed for easy mutational manipulation without regard to the parent genome. Yet design and construction of synthetic genes, especially those coding for large proteins, can be a slow, difficult and confusing process. We have written a computer program that automates the design of oligonucleotides for gene synthesis. Our program requires simple input information, i.e. amino acid sequence of the target protein and melting temperature (needed for the gene assembly) of synthetic oligonucleotides. The program outputs a series of oligonucleotide sequences with codons optimized for expression in an organism of choice. Those oligonucleotides are characterized by highly homogeneous melting temperatures and a minimized tendency for hairpin formation. With the help of this program and a two-step PCR method, we have successfully constructed numerous synthetic genes, ranging from 139 to 1042 bp. The approach presented here simplifies the production of proteins from a wide variety of organisms for genomics-based studies.     

Streamlined cell-free protein synthesis from sequence information

In this study, we describe a cell-free protein synthesis consolidated with polymerase chain reaction (PCR)-based synthetic gene assembly that allows for streamlined translation of genetic information. In silico-designed fragments of target genes were PCR-assembled and directly expressed in a cell-free synthesis system to generate functional proteins. This method bypasses the procedures required in conventional cell-based gene expression methods, integrates gene synthesis and cell-free protein synthesis, shortens the time to protein production, and allows for facile regulation of gene expression by manipulating the oligomer sequences used for gene synthesis. The strategy proposed herein expands the flexibility and throughput of the protein synthesis process, a fundamental component in the construction of synthetic biological systems.     

Use of synthetic DNA in expression of foreign proteins

Synthetic DNAs and oligonucleotides, which can be prepared conveniently by combining chemical synthesis and enzymatic methods, have been used extensively in recombinant DNA research. Examples include total gene synthesis, probes for the isolation of specific genes from cDNA or genomic libraries, linkers containing specific restriction sites for cloning, primers for DNA and RNA sequencing, and primers for the construction of specific mutations (either deletion, insertion or point mutations) by oligonucleotide-directed site-specific mutagenesis.This article reviews recent advances in the chemical and enzymatic synthesis of oligo- and polynucleotides and the application of synthetic DNA to the expression of foreign proteins. The synthesis of genes, including structural genes and regulatory genes are reviewed. Oligonucleotide-directed site-specific mutagenesis and use of synthetic DNA to optimize foreign protein expression are also discussed.     

Cell-free protein synthesis: the state of the art

Cell-free protein synthesis harnesses the synthetic power of biology, programming the ribosomal translational machinery of the cell to create macromolecular products. Like PCR, which uses cellular replication machinery to create a DNA amplifier, cell-free protein synthesis is emerging as a transformative technology with broad applications in protein engineering, biopharmaceutical development, and post-genomic research. By breaking free from the constraints of cell-based systems, it takes the next step towards synthetic biology. Recent advances in reconstituted cell-free protein synthesis (Protein synthesis Using Recombinant Elements expression systems) are creating new opportunities to tailor the reactions for specialized applications including in vitro protein evolution, printing protein microarrays, isotopic labeling, and incorporating nonnatural amino acids.     

Redundant control of adenovirus late gene expression by early region 4.

A series of human adenovirus type 5 derivatives carrying deletion mutations in early region 4 (E4) were constructed and characterized with respect to viral late protein synthesis, viral cytoplasmic late message accumulation, viral DNA accumulation, and plaquing ability. Viral late protein synthesis was essentially normal in cells infected by mutants expected to produce either the E4 open reading frame (ORF) 3 product or the E4 ORF 6 product. In cells infected by mutants lacking both ORF 3 and ORF 6, late protein synthesis was dramatically reduced. The basis for this reduction appears to be a concomitant reduction in cytoplasmic late message levels. Our results suggest that the products of ORFs 3 and 6 are redundant, since they are individually able to satisfy the requirement for E4 in late gene expression. Two of the mutants examined were defective for viral late protein synthesis but showed no measurable defect in viral DNA accumulation. The defect in late gene expression is not, therefore, a reflection of a primary defect in viral DNA synthesis. Finally, mutants expected to express ORF 3 or ORF 6 formed plaques with normal or only modestly reduced efficiency, whereas mutants expected to express neither ORF formed plaques with an efficiency less than 10(-6) that of wild-type virus. Thus, plaque-forming ability reflected late protein synthetic ability, suggesting that among these mutants late protein synthetic proficiency is the principle determinant of plaquing efficiency.     

Error correction in gene synthesis technology

Accurate, economical and high-throughput gene and genome synthesis is essential to the development of synthetic biology and biotechnology. New large-scale gene synthesis methods harnessing the power of DNA microchips have recently been demonstrated. Yet, the technology is still compromised by a high occurrence of errors in the synthesized products. These errors still require substantial effort to correct. To solve this bottleneck, novel approaches based on new chemistry, enzymology or next generation sequencing have emerged. This review discusses these new trends and promising strategies of error filtration, correction and prevention in de novo gene and genome synthesis. Continued innovation in error correction technologies will enable affordable and large-scale gene and genome synthesis in the near future.     

Advances in molecular cloning

“Molecular cloning” meaning creation of recombinant DNA molecules has impelled advancement throughout life sciences. DNA manipulation has become easy due to powerful tools showing exponential growth in applications and sophistication of recombinant DNA technology. Cloning genes has become simple what led to an explosion in the understanding of gene function by seamlessly stitching together multiple DNA fragments or by the use of swappable gene cassettes, maximizing swiftness and litheness. A novel archetype might materialize in the near future with synthetic biology techniques that will facilitate quicker assembly and iteration of DNA clones, accelerating the progress of gene therapy vectors, recombinant protein production processes and new vaccines by in vitro chemical synthesis of any in silico-specified DNA construct. The advent of innovative cloning techniques has opened the door to more refined applications such as identification and mapping of epigenetic modifications and high-throughput assembly of combinatorial libraries. In this review, we will examine the major breakthroughs in cloning techniques and their applications in various areas of biological research that have evolved mainly due to easy construction of novel expression systems.     

Toward genetic transformation of mitochondria in mammalian cells using a recoded drug-resistant selection marker

Due to technical difficulties, the genetic transformation of mitochondria in mammalian cells is still a challenge. In this report, we described our attempts to transform mammalian mitochondria with an engineered mitochondrial genome based on selection using a drug resistance gene. Because the standard drug-resistant neomycin phosphotransferase confers resistance to high concentrations of G418 when targeted to the mitochondria, we generated a recoded neomycin resistance gene that uses the mammalian mitochondrial genetic code to direct the synthesis of this protein in the mitochondria, but not in the nucleus (mitochondrial version). We also generated a universal version of the recoded neomycin resistance gene that allows synthesis of the drug-resistant proteins both in the mitochondria and nucleus. When we transfected these recoded neomycin resistance genes that were incorporated into the mouse mitochondrial genome clones into mouse tissue culture cells by electroporation, no DNA constructs were delivered into the mitochondria. We found that the universal version of the recoded neomycin resistance gene was expressed in the nucleus and thus conferred drug resistance to G418 selection, while the synthetic mitochondrial version of the gene produced no background drug-resistant cells from nuclear transformation. These recoded synthetic drug-resistant genes could be a useful tool for selecting mitochondrial genetic transformants as a precise technology for mitochondrial transformation is developed.     

Error removal in microchip-synthesized DNA using immobilized MutS

The development of economical de novo gene synthesis methods using microchip-synthesized oligonucleotides has been limited by their high error rates. In this study, a low-cost, effective and improved-throughput (up to 32 oligos per run) error-removal method using an immobilized cellulose column containing the mismatch binding protein MutS was produced to generate high-quality DNA from oligos, particularly microchip-synthesized oligonucleotides. Error-containing DNA in the initial material was specifically retained on the MutS-immobilized cellulose column (MICC), and error-depleted DNA in the eluate was collected for downstream gene assembly. Significantly, this method improved a population of synthetic enhanced green fluorescent protein (720 bp) clones from 0.93% to 83.22%, corresponding to a decrease in the error frequency of synthetic gene from 11.44/kb to 0.46/kb. In addition, a parallel multiplex MICC error-removal strategy was also evaluated in assembling 11 genes encoding ∼21 kb of DNA from 893 oligos. The error frequency was reduced by 21.59-fold (from 14.25/kb to 0.66/kb), resulting in a 24.48-fold increase in the percentage of error-free assembled fragments (from 3.23% to 79.07%). Furthermore, the standard MICC error-removal process could be completed within 1.5 h at a cost as low as $0.374 per MICC.