Construction Of Synthetic Genes Research Articles

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.

Engineering combinatorial and dynamic decoders using synthetic immediate-early genes

Many cell- and tissue-level functions are coordinated by intracellular signaling pathways that trigger the expression of context-specific target genes. Yet the input–output relationships that link pathways to the genes they activate are incompletely understood. Mapping the pathway-decoding logic of natural target genes could also provide a basis for engineering novel signal-decoding circuits. Here we report the construction of synthetic immediate-early genes (SynIEGs), target genes of Erk signaling that implement complex, user-defined regulation and can be monitored by using live-cell biosensors to track their transcription and translation. We demonstrate the power of this approach by confirming Erk duration-sensing by FOS, elucidating how the BTG2 gene is differentially regulated by external stimuli, and designing a synthetic immediate-early gene that selectively responds to the combination of growth factor and DNA damage stimuli. SynIEGs pave the way toward engineering molecular circuits that decode signaling dynamics and combinations across a broad range of cellular contexts.

Efficient assembly of very short oligonucleotides using T4 DNA Ligase

BackgroundIn principle, a pre-constructed library of all possible short oligonucleotides could be used to construct many distinct gene sequences. In order to assess the feasibility of such an approach, we characterized T4 DNA Ligase activity on short oligonucleotide substrates and defined conditions suitable for assembly of a plurality of oligonucleotides.FindingsLigation by T4 DNA Ligase was found to be dependent on the formation of a double stranded DNA duplex of at least five base pairs surrounding the site of ligation. However, ligations could be performed effectively with overhangs smaller than five base pairs and oligonucleotides as small as octamers, in the presence of a second, complementary oligonucleotide. We demonstrate the feasibility of simultaneous oligonucleotide phosphorylation and ligation and, as a proof of principle for DNA synthesis through the assembly of short oligonucleotides, we performed a hierarchical ligation procedure whereby octamers were combined to construct a target 128-bp segment of the beta-actin gene.ConclusionsOligonucleotides as short as 8 nucleotides can be efficiently assembled using T4 DNA Ligase. Thus, the construction of synthetic genes, without the need for custom oligonucleotide synthesis, appears feasible.

Tandem multimer expression of angiotensin I‐converting enzyme inhibitory peptide in Escherichia coli

It is common for small tandem peptide multimer genes to be indirectly inserted into expression vectors and fused with a protein tag. In this study, a multimer of the tandem angiotensin I-converting enzyme inhibitory peptide (ACE-IP) gene was directly transferred to a commercially available vector and the designed gene was expressed as a repeated peptide in Escherichia coli BL21(DE3)pLysS. The process further developed in our study was the construction of six-repeated ACE-IP synthetic genes and their direct insertion. Protein expression in inclusion bodies was confirmed by SDS-PAGE and Western blot. Acid hydrolysis of inclusion bodies produced single-unit peptides through cleavage of the aspartyl-prolyl bonds. This cleaved recombinant peptide (rACE-IP) was purified using immuno-affinity chromatography followed by reversed phase-HPLC. 105-115 mg of the lyophilized recombinant peptide was obtained from 1 L E. coli culture. In vitro biological activity of rACE-IP was indistinguishable from that of the natural peptide produced by hydrolysis in artificial gastric juice or by acidic hydrolysis. The rACE-IP prepared by recombinant DNA technology and solid-phase synthesis methods showed a similar IC(50). This strategy could be used for the expression of important peptides, which have N-terminal proline (P) and C-terminal aspartic acid residues (D) for commercial applications, e.g. functional foods and drinks.

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.

Optimal encoding rules for synthetic genes: the need for a community effort

Mol Syst Biol. 3: 134 A paradigm shift is underway within the methodology of heterologous protein expression. Specifically, researchers are moving away from conventional techniques of cloning genes from cDNA libraries and moving toward the rational design and de novo synthesis of entire protein‐coding sequences from pre‐annealed oligonucleotides (Libertini and Di Donato, 1992; Gustafsson et al , 2004). It was the invention of polymerase chain reaction (PCR) that allowed efficient construction of synthetic genes. Since then, the steadily increasing accuracy and decreasing cost of oligonucleotide synthesis (now as low as $0.10 per base; Carlson, 2003; Carr et al , 2004; Kong et al , 2007, see Figure 1) has created a research environment in which gene synthesis offers three main advantages over molecular cloning: cost efficiency, scope and flexibility of redesign (Libertini and Di Donato, 1992). As a result, the emerging field of synthetic biology is highly motivated to improve this approach, as it seeks to expand the sophistication of human‐engineered genetic architectures, leading ultimately to the synthesis of entire genomes (Yount et al , 2000; Smith et al , 2003). Figure 1. The cost, per base, of commercial oligonucleotide assembly from 1999 to 2006. The price of gene synthesis has decreased almost 30‐fold in the past 7 years (data for years 1999–2003 are taken from Carlson, 2003). Data for years 2004–2006 reflect the lowest price found in advertisements placed within Science magazine). Current research into synthetic gene construction has focused largely on improving PCR‐based methods. Areas under active investigation include the following: increasing the accuracy of gene products by reducing errors in oligonucleotide construction and PCR synthesis/amplification (Ciccarelli et al , 1991; Young and Dong, 2004), reducing the relatively high cost of post‐synthesis sequencing (Young and Dong, 2004), increasing the length of genes …

Recombinant production of bacterial toxins and their derivatives in the methylotrophic yeast Pichia pastoris

The methylotrophic yeast Pichia pastoris is a popular heterologous expression host for the recombinant production of a variety of prokaryotic and eukaryotic proteins. The rapid emergence of P. pastoris as a robust heterologous expression host was facilitated by the ease with which it can be manipulated and propagated, which is comparable to that of Escherichia coli and Saccharomyces cerevisiae. P. pastoris offers further advantages such as the tightly-regulated alcohol oxidase promoter that is particularly suitable for heterologous expression of foreign genes. While recombinant production of bacterial toxins and their derivatives is highly desirable, attempts at their heterologous expression using the traditional E. coli expression system can be problematic due to the formation of inclusion bodies that often severely limit the final yields of biologically active products. However, recent literature now suggests that P. pastoris may be an attractive alternative host for the heterologous production of bacterial toxins, such as those from the genera Bacillus, Clostridium, and Corynebacterium, as well as their more complex derivatives. Here, we review the recombinant production of bacterial toxins and their derivatives in P. pastoris with special emphasis on their potential clinical applications. Considering that de novo design and construction of synthetic toxin genes have often been necessary to achieve optimal heterologous expression in P. pastoris, we also present general guidelines to this end based on our experience with the P. pastoris expression of the Bacillus thuringiensis Cyt2Aa1 toxin.

Expression of the genomic form of the bovine viral diarrhea virus E2 ORF in a bovine herpesvirus-1 vector.

Bovine viral diarrhea virus (BVDV) is a ubiquitous pathogen of cattle with a world-wide distribution. Recently, the possibility of using recombinant virus vectors to immunize cattle against selected BVDV genes has gained widespread interest. Among the virus vectors tested, bovine herpesvirus-1 (BHV1) provides many unique advantages. However, results of recent studies have raised the possibility that the codon usage pattern required for optimal expression in a BHV1-infected cell may be incompatible with the codon usage pattern of BVDV. If true, use of BHV1 to express BVDV proteins would require construction of synthetic BVDV genes that have been modified to resemble the codon pattern of BHV1. To explore this possibility, we constructed a BHV1 recombinant containing the genomic form of the BVDV (NADL) E2 ORF and compared expression of the E2 protein with that of the endogenous BHV1 gD protein. We observed that E2 was expressed at a significant rate compared to that of the gD protein. We conclude that codon usage problems are unlikely to constitute a serious problem for expression of BVDV proteins in BHV1 vectors.

Construction of Synthetic Genes for Analogs of Spider Silk Spidroin 1 and Their Expression in Tobacco Plants

To obtain transgenic tobacco plants expressing recombinant analogs of spider dragline silk spidroin 1, artificial 1f5 and 1f 9 coding for spidroin 1 analogs were 3"-fused in-frame with the reporter lichenase gene. The Tr2" weak constitutive promoter of Agrobacterium tumefaciens T-DNA and the strong constitutive promoter of the cauliflower mosaic virus 35S RNA gene were used as regulatory elements. The expression cassettes were used to transform agrobacteria and then introduced in tobacco leaf disks. On evidence of Southern hybridization, transgenic plants each carried a single copy of a hybrid gene, which corresponded in size to the constructed one. Zymography and Western blotting revealed full-length hybrid proteins in leaf extracts of transgenic plants. The results testified that plants can maintain and express synthetic genes for spider silks and, consequently, may be used as a convenient producer of recombinant silk analogs.

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.

In-frame cloning of synthetic genes using PCR inserts.

Because many genes of biological interest are larger than the maximum size that current synthetic oligonucleotide synthesizers can produce (approx 110 bases), there is a need for methods that allow rapid production and expression of genes constructed from multiple synthetic DNA fragments. A number of synthetic genes have been generated using the recursive polymerase chain reaction (PCR) method, but problems concerning primer design and incorrect final gene sequence, especially with large synthetic genes, are a concern (-). The cloning method described here follows a series of steps in which multiple PCR products or synthetic duplex oligonucleotides are positionally cloned into a plasmid vector (). A synthetic gene of practically any sequence or length can be built using the in-frame cloning method. Genes are assembled such that open reading frames are maintained by linking DNA fragments through the use of six basepair blunt-end restriction sites. Each cloning step uses an anchored sticky-end restriction site and a variable blunt-end restriction site that result in specific insert orientation and high cloning efficiencies. The overall strategy of in-frame cloning allows the researcher total control over nucleotide sequence, codon usage, promoter and other regulatory elements, and the placement of unique restriction sites throughout the recombinant construct. One advantage of the in-frame cloning method described here is that it allows for flexible yet precise construction of synthetic genes using standard recombinant techniques. Another advantage is that it employs inexpensive, readily available materials.

Gene Synthesis by a LCR-Based Approach: High-Level Production of Leptin-L54 Using Synthetic Gene inEscherichia coli

Synthetic genes are very useful in genetic and protein engineering. Here we propose a general method for construction of synthetic genes. Short oligonucleotides are joined through ligase chain reaction (LCR) in high stringency conditions to make “unit fragments” which are then fused to form a full-length gene sequence by polymerase chain reaction. The procedure is simple and accurate and does not place constraints on sequence and length. In this report, a recombinant leptin gene was synthesized according to the codon preference ofEscherichia coli.Besides, a substitution of the only Met at position 54 for Leu and an addition of a Met at the N-terminus were introduced in the synthetic gene. The gene was cloned in the pQE-31 expression vector and was expressed inE. coli.A large amount of recombinant leptin containing 6× His tag was produced and purified by Ni-NTA affinity column. Finally, intact leptin-L54 was released after removing the tag by CNBr cleavage at the Met residue.

A facile method for the construction of synthetic genes

A simple protocol for rapid assembly of chemically synthesized deoxyoligonucleotides into double stranded DNA is described. Several parameters of a ligation-free method were investigated to allow efficient assembly of a large number of oligonucleotides into double stranded DNA by polymerase chain reaction. Synthesis of a 701 bp DNA was carried out in a single reaction by assembling 28 oligonucleotides designed with partial overlaps at complementary ends. An estimate of error rate was made by sequencing several independent clones of the synthesized DNA

Construction, Cloning, and Expression of Synthetic Genes Encoding Spider Dragline Silk

Synthetic genes encoding recombinant spider silk proteins have been constructed, cloned, and expressed. Protein sequences were derived from Nephila clavipes dragline silk proteins and reverse-translated to the corresponding DNA sequences. Codon selection was chosen to maximize expression levels in Escherichia coli. DNA "monomer" sequences were multimerized to encode high molecular weight synthetic spider silks using a "head-to-tail" construction strategy. Multimers were cloned into a prokaryotic expression vector and the encoded silk proteins were expressed in E. coli upon induction with IPTG. Four multimer, ranging in size from 14.7 to 41.3 kDa, were chosen for detailed analysis. These proteins were isolated by immobilized metal affinity chromatography and purified using reverse-phase HPLC. The composition and identity of the purified proteins were confirmed by amino acid composition analysis, N-terminal sequencing, laser desorption mass spectroscopy, and Western analysis using antibodies reactive to native spider dragline silk. Circular dichroism measurements indicate that the synthetic spider silks have substantial beta-sheet structure.

Construction and expression of synthetic wild-type and mutant genes encoding porcine pancreatic colipase: tryptophan fluorescence studies

Based on the known (95-residue) amino acid (aa) sequence of porcine pancreatic colipase (CLP), a cofactor of pancreatic lipase, a 297 bp gene was designed and assembled from eight synthetic, overlapping DNA fragments. Optimized for expression in bacteria, the CLP-encoding gene (CLP) was inserted into the lacZ gene fragment contained in the small expression vector, pUC8, and cloned in Escherichia coli JM109. Expression of this construct yielded a protein approx. 11 kDa in size, equivalent to CLP, with an Mr of 10,336, plus ten additional amino acids at the N-terminus. The recombinant CLP (reCLP) was solubilized from bacterial inclusion bodies and then purified and refolded. A mutant CLP gene, changing Tyr-55 to Trp, was then constructed by site-directed mutagenesis. Since porcine CLP contains no Trp, this strategy provided a protein with an internal fluorescent probe for biophysical studies. The presence of Trp in the mutant protein was confirmed using fluorescence spectroscopy. Both wild-type (wt) and mutant reCLP reacted on Western blots with an affinity-purified rabbit anti-CLP antibody, raised against native CLP. The Tyr-55 to Trp exchange did not affect the activity of reCLP. Fluorescence studies of the interaction between reCLP and the bile salt, taurodeoxycholate (TDOC), showed that Trp-55 in the hydrophobic binding site of mutant reCLP inserted into the interior of the bile salt micelle.

Construction of synthetic genes using PCR after automated DNA synthesis of their entire top and bottom strands

A new method is described for the direct construction of synthetic genes by applying a modified version of the polymerase chain reaction (PCR) to crude oligonucleotide mixtures made by automated solid phase DNA synthesis. Construction of the HIV-1 393 bp rev gene and the 655 bp nef gene by this method is illustrated. The sequences for the entire top and bottom strands of rev were each programmed into an automated DNA synthesizer. Following DNA synthesis, the two crude oligonucleotide solutions were mixed together, specific primers were added, and the target gene was amplified by a modified PCR technique. Although the longer (greater than 200 bases) strands comprise a very small percentage of the total DNA after solid phase synthesis, this method uses PCR to 'find' and amplify such strands to create the target gene. The rev gene constructed by this method was found to contain 4 sequence errors, which were subsequently corrected by site-directed mutagenesis. In order to evaluate the source of sequence errors, several nef genes were made from the top and bottom strand DNA synthesis solutions using independent PCR's. Results suggest that sequence errors arose from both DNA synthesis and PCR. The utility of this method in producing a functional gene is demonstrated by expression of rev in E.coli.

Two methods for rapid assembly and oligomerization of synthetic genes: construction of human calcitonin-encoding sequences

Two fast, simple and reliable methods for construction of synthetic genes are described. The first represents a one-vial method for assembly and cloning of monomeric genes, and the second allows a simultaneous assembly, oligomerization and cloning of synthetic genes. Both methods consist of ligation of the synthetic oligodeoxyribonucleotides (oligos) building up the gene directly into the cloning vector, followed by screening of the recombinant clones for the presence of specific gene constructs. The average length of the oligomeric genes thus constructed is directly proportional to the molar ratio between the vector DNA and the synthetic oligos used. Both methods are illustrated by the construction of a series of human calcitonin-encoding genes.

Shotgun concatenation of synthetic genes: construction of concatemeric human calcitonin genes

A new fast and reliable approach for construction of concatemeric synthetic genes is described. The method consists of shotgun polymerization of three truncated monomeric gene units using a specific linker, followed by cloning of the recombinant clones and screening them for the presence of concatemeric genes of defined length. The method is illustrated by constructing a series of tandemly repeated human calcitonin (hCT) genes.

Rapid construction of large synthetic genes: total chemical synthesis of two different versions of the bovine prochymosin gene

We have tested several different synthesis designs and assembly methodologies to develop an improved gene synthesis strategy which enables significantly longer nucleotide sequences to be easily constructed. This strategy, based in part upon our ability to synthesize high-quality extended-length oligodeoxynucleotides (over 100-mer in length), together with the use of chemical 5 '-phosphorylation, and simplified low-melting-temperature agarose gel purification methods, combines ease, speed and high overall efficiency. We show that it is now feasible to synthesize routinely even long genes (at least 1–2 kb). To demonstrate this capability we have chemically synthesized and assembled two different versions of the gene encoding the bovine enzyme prochymosin (prorennin). One gene is essentially the natural bovine prochymosin gene sequence. In the second gene the codons have been optimized with regard to the codon bias of highly expressed yeast genes. Each synthetic gene was in excess of 1100 bp, yet they were assembled from only 13 or 14 pairs of complementary oligodeoxynucleotides (oligos), the average lengths of which were 87 and 82 bp, respectively. The ‘mutation’ rate was low enough to assess that more than 75% of all such oligo pairs (160–170 total nt) were error-free.

The construction and cloning of synthetic genes coding for artificial proteins and expression studies to obtain fusion proteins

Synthetic genes coding for artificial proteins with predefined and nutritionally valuable amino acid compositions have been constructed and cloned in bacterial plasmid vector pKK233-2. The genes were constructed from three easily interchangeable 'cassettes' encoding either essential, non-essential or branched-chain amino acid residues. A potential hairpin loop structure in the mRNA around the region of the ribosome binding site was probably the reason for blockage of translation from this vector. Two selected genes, AHB (containing one copy of each cassette) and A6 (consisting of six copies concatemerized A cassette) were cloned into pUR300, a beta-Gal fusion vector and expressed as fusion proteins beta-Gal-AHB and beta-Gal-A6.