Tag: gene synthesis

Gene Synthesis and Cloning

Gene Synthesis and Cloning is an integral part of many research pipelines. There are two popular methods of genetic cloning: DNA amplification by use of subcloning and polymerase chain reaction (PCR). The former needs more time and resource consuming, while the latter spends less time and cost effectively. Traditionally, subcloning was commonly used for amplification. But recently PCR has become much more effective and popular. The popularity of amplification by subcloning may have dwindled because it requires multiple steps. The process starts with the removal of the genetic sequence of interest using specific restriction enzymes. This same restriction enzyme is then used to open up the plasmid where the genetic sequence will be inserted into. This creates “sticky ends” which allow the genetic sequence to be more easily inserted. The genetic sequences is then added into a plasmid and sealed in place with DNA ligase. The plasmid is subcequently inserted into bacteria where the genetic sequence of interest is amplified and later extracted. This is a somewhat laborious process when compared to PCR. PCR has very simple steps: extracting the genetic sequence of interest and loading it into the machine. The sequence of interest is then rapidly amplified with a high quality output. The amplified sequence can then be used to in various research topics specific to the user. Amplification of these sequences were once restricted to only allow preexisting sequences to be extracted and amplified, but recent technology has allowed a small change in this.

The interesting aspect that Synbio Technologies offers is the ability to synthesize the genetic sequence of interest, as opposed to extracting the preexisting sequence from an organism. This technology is referred to as our Syno®2.0 Platform, which allows Synbio Technologies to successfully synthesize any desired genetic sequence, specified by the user. The lengths of these sequences can vary from a small as a few hundred base pairs to up to 100kb or even more in length. The Syno®2.0 Platform provides an extremely effective and revolutionary approach to genetic research. Traditionally, the sequence of interest must be present within a genome of an organism to then be extracted and later amplified. This platform allows us to synthetically create a sequence that does not need to be preexisting within any organism. The Syno®2.0 Platform allows us to move from a text file of the user specified sequence to the physical genetic constructs, bypassing the previous stipulation of extracting the preexisting sequence. The resulting synthetic products is then analyzed using Sanger Sequencing in order to verify and guarantee one hundred percent accuracy. Once successful, Synbio Technologies offers the option to clone this sequence as many times as necessary. This is done by use of either PCR or subcloning, both of which are extremely effective mechanisms to amplify the genetic sequence. The process of gene synthesis and later gene cloning allows Synbio Technologies to supply the researcher with a sufficient amount of genetic material needed within an efficient timeframe.

Synbio Technologies also offers PCR cloning if the requested sequence of interest is already present and does not need to be synthetically engineered. First the provided sequence will be verified using Sanger Sequencing in order to account for one hundred percent accuracy. The resulting verified sequence will then be put through our flexible and reliable pipeline of gene cloning. Using patent pending, Syno®2.0 Platform and Clone®3.1 system, Synbio Technologies is able to achieve inserting any sequence of interest into any site of a vector specified by the user. This allows the user to specify which restriction enzyme to use, both for the extraction of genetic sequence, as well as the restriction enzyme used on the plasmid. These wide range of possibilities allows the researcher to design a location within a specific vector in order to amplify the sequence of interest. This specificity is something that Synbio Technologies is very proud of, allowing us to adhere to any requests that the user might have while also providing a high quality output in the process.

At Synbio Technologies we pride ourselves as being one of the leading companies in the biotechnology industry, especially when referring to gene synthesis. We offer both accurate and time effective approaches to gene synthesis and gene cloning. The genetic sequence of interest will be Sanger Sequenced and verified for both genetically synthesized sequences as well as sequences provided by the user. This allows us to verify that the sequence, as well as the resulting amplified sequences, are extremely accurate before shipping the resulting product to the user. If the DNA sequence of interest has not yet been sequenced with high quality, we will conduct sequence validation in order to verify the accuracy before amplification. In addition to the accuracy and time efficiency, Synbio Technologies offers competitive prices for both gene synthesis and cloning of your sequence of interest. With competitive prices, along with the verified accuracy and time efficiency, Synbio Technologies is ready to offer customers a unique pipeline to utilize and rely on when conducting various types of genetic research that including gene synthesis and gene cloning.

Gene Synthesis Methods and Applications

Introduction

Over the last few decades, gene synthesis and assembly technology have developed rapidly. Since the 1960s, our capability to synthesize genes has skyrocketed from less than 100bp to more than 1,000,000bp. Gene synthesis methods and applications have a profound impact in metabolic engineering, genetic network design, and vaccine design. However, existing gene synthesis methods still have their fair share of drawbacks and inconsistencies, such as low yield, high error rate, and lack of scalability both on the size of the project and the size of the gene.

Gene Synthesis Methods

Gene synthesis methods are not able to replace each other, and each occupies its own niche depending on the requirements of the project. The following is a brief overview of several common gene synthesis methods:

  • Solid-phase synthesis

    • The traditional oligonucleotide synthesis uses a small volume of solution processed in a column full of chemicals. The oligonucleotides are synthesized by attaching nucleotide residues stepwise to the end of the chain, one-by-one. The addition of each oligonucleotide consists of four steps: de-blocking, coupling, capping, and oxidation. The integrity of the sequence and the productivity of the synthesis are hindered for products longer than 200bp, and thus this method is generally limited by DNA sequence length. The primary advantage of this method is its high accuracy, compensating for its high expense and low output.

  • Chip-based DNA synthesis

    • As the name implies, Chip-based synthesis utilizes microarray chips utilizing a series of electrochemical techniques. Different kinds of oligonucleotides are able to be synthesized in different specific parts of the chips, called assembly subpools. Following this piecewise synthesis, gene fragments in subpools are amplified and then aggregated and assembled into the finished product. Chip-based DNA synthesis is cheaper than solid-phase synthesis and can yield a larger amount of the target gene, but its accuracy suffers in comparison.

  • PCR based enzyme synthesis

    • PCR-based enzyme synthesis generates gene fragments through a variety of cell systems. Using the Yeast system as an example, by using different incision enzymes and label markers, different kind of genes can be added to Yeast chromosomes. Due to the nature of gene insertion the target gene could have no limit to its length as long as the chromosomes can accommodate. This method performs well in synthesizing large gene fragments, and with the help of the cell systems the accuracy of the gene sequence is guaranteed.

Gene synthesis applications

Synthetic genes have wide implications on a variety of fields, ranging from genetic circuits to metabolic improvement to many more. As our technology advances and our understanding of genetics continues to develop, scientists could modify and design genes, The scientists have already synthesized and assembled a gene fragment of over 100kbp. When inserted into a host bacterium lacking its own genetic material, the bacterium was able to successfully produce new cells.

Gene synthesis methods can even be used to construct new metabolic systems in living cells. For example, Jay D. Keasling constructed a new metabolic system in E. coli and S. cerevisiae in the mid2000s to produce artemisinin. An important component in some anti-malarial drugs, Keasling’s construct reduced the cost of artemisinin production by tenfold, showing that gene synthesis has a vast amount of potential in medicine and countless other fields.

Gene Synthesis Codon Optimization

Gene synthesis refers to the technology of artificially synthesizing double-stranded DNA molecules in vitro through reverse transcription of mRNA of a known template gene. Intelligent synthetic gene design is critical to gene engineering and the efficient production of recombinant protein through heterologous hosts.

Unfortunately, not all genes can be successfully and effectively expressed in heterologous expression systems. The intrinsic sequence characteristics of genes including stability, codon bias, GC content, and mRNA secondary structure play unexpected roles in regulating translation. The genetic code consists of 64 different tri-nucleotide codons which correspond to only 20 amino acids. This degeneracy allows multiple synonymous codons to encode the same protein. Codon optimization , described as altering codons within the gene to improve recombinant protein expression, is an important part of efficient synthetic gene design.

The Origins of Codon Usage Bias

Codon bias arises from the observed uneven usage of codons across different organisms. In Escherichia coli and Saccharomyces cerevisiae (yeast), certain synonymous codons are optimal and preferred to match the most abundant tRNAs in the cell or bind to those tRNAs with best binding strength. The preferred codons might tend to be read by abundant tRNA molecules while low-usage codons might tend to be read by scarce tRNA. The reason why some highly expressed genes possess preferentially selected codons is still unknown. One conventional perspective is optimal codons would be translated faster than rare codons, enhancing the efficiency of translation. Another alternative assumption is that using preferred codons may increase translation accuracy.

The Functional Impact of Codon Optimization

Codon usage bias is correlated with gene expression levels. In heterologous protein expression, the gene of interest can be overexpressed. Their products can take up to 30% of the cell’s total proteins. The attempt to generate more protein by changing codon assignments led to broad use of codon-optimized mRNAs. Originally, codons within the gene were altered by replacing rare codons with synonymous counterparts, which were more preferable and more frequently used by hosts. It was found that optimized codons led to an increase in corresponding protein expression in both plants and mammalian cells. Surprisingly, expression of viral proteins has also been found to decrease after substitution of synonymous codons or adjacent codons. The many unanswered questions related to codon optimization may have profound significance in exploring novel methods of vaccine design.

Strategies of Codon Optimization

A variety of approaches and programs can design and produce various codon-optimized mRNA sequences. The quantification of codon usage as well as the completion of codon changes must be considered. Synthetic codon optimization tends to substitute rare codons with synonymous counterparts used at a higher frequency. Another variation referred to as codon harmonization alters codons within gene sequences to correlate with the codon usage bias of the host organism.

Admittedly, for protein expression, optimizing codon usage alone is not sufficient to perfect the design of synthetic genes. Many other factors can potentially interfere; for example, mRNA secondary structure can affect gene transcription. Additionally, cryptic splice sites, polyadenylation signals, and other regulatory elements ought to be avoided, as they can lead to undesirable processing of mRNA. GC content has a direct impact on the binding stability and annealing temperature of DNA sequences. Translation initiation and termination efficiency also influence protein output and solubility. Only by taking all of above factors into consideration can gene synthesis codon optimization achieve maximum value.

Gene Synthesis Applications：gene cloning technology and application

Gene synthesis was first successfully conducted in 1972 on a yeast tRNA by Har Gobind Khrona and associates.Since 1972, the technology and mechanisms of gene synthesis have flourished in order to successfully obtain the genetic sequence specific to a wide range of research topics.Gene synthesis offers many major advantages over the traditional gene cloning methods: molecular cloning and polymerase chain reaction (PCR).These two traditional methods have two major restrictions,the first is that these methods are only capable of gene amplification and cloning, as opposed to gene synthesis.The other restriction molecular cloning and polymerase chain reaction have is that the preexisting genetic sequence of interest must be obtained and then amplified.The important advantage gene synthesis offers is that the genetic code you wish to study does not need to be physically obtained in order to be amplified.The technology currently allows us to synthesize “de novo” sequences.This means that a sequence of interest,whether it is viral DNA or a cancer cell’s mutation in a specific gene, can be synthesized without the physical copy of the gene being present.Without the physical copy of the gene of interest being needed,the necessary amount of time and money is drastically reduced when compared with traditional methods of cloning, amplification,targeted mutagenesis,and gene target knockouts.Due to this advantage gene synthesis has been becoming more and more popular in various fields of research.

Due to the ease of use and cost effective mechanism, gene synthesis has been used countless amounts of times in varying fields of scientific research. Gene synthesis has most notably and recently been used to conduct viral research in order to produce safer, more effective DNA vaccines. It has also been used to better understand mechanisms for cancer cell metabolic regulation. Gene synthesis is currently being used for targeted gene therapy, and other topics of interest that seemed almost impossible 30 years ago. In addition to the research that has already been conducted, gene synthesis offers a myriad of possibilities of application ranging from gene regulation to better understanding evolution and antibiotic resistance. Gene synthesis makes it possible to build variant libraries, genes, operons, increase the function of proteins, and even test all the orthologs of a particular gene.

The advantages gene synthesis offers are seemingly endless.The main advantage that gene synthesis has to offer is the ease of synthetically manipulating or cloning a gene of interest with or without the physical copy of the gene itself.Synbio Technologies offers, through our Syno®DNA Platform, an extremely accurate, time efficient, and cost effective mechanism to analyze your sequence of interest and successfully synthesize the sequence.All sequences constructed by Synbio Technologies are verified using Sanger sequencing, and are guaranteed to be 100 percent identical to your sequence of interest.In addition to the high quality synthesized sequences, Synbio Technologies offers competitive prices on gene synthesis for a wide range of gene lengths up to 100 kb.With the competitive prices and accuracy, on top of the additional money and resources needed for PCR and molecular cloning, it is clear that gene synthesis is the more effective mechanism to utilize when conducting certain types of genetic research.

Gene Library Synthesis : genomic libraries and cDNA libraries

Gene synthesis has revolutionized genetic research over the past twenty years by allowing the convenience of DNA storage for a wide range of genome sizes. These libraries have allowed researchers to archive the genes of interested and be able to obtain each gene with relative ease at his or her leisure. Another great advantage ofgene synthetic libraries is the convenient location of the genetic foundation of the organism at your disposal. The genetic foundation can either be stored as the entire genome or more specifically the coding regions of the genome. Gene libraries are mainly broken down into two distinct categories: genomic libraries and cDNA libraries. The two have the same principles and similar output, but are created in different ways and have many different aspects.

First, the genomic libraries is representative of the organism’s entire genome, making the library quite large. The process of creating a genomic library is as follows: first the selected DNA is isolated from the cells by a specific restriction endonuclease. The resulting DNA fragments are then inserted into a selected vector using DNA ligase. Once the DNA is located within the vector, the vector is inserted into a bacteriophage to be amplified. After amplification, the cloned DNA is then isolated again and inserted into a genomic library. This process is then repeated until the entirety of the organism’s genome is isolated, amplified, isolated again, and inserted into the genomic library. Since it is the entire organism’s genome is located in these libraries, including both coding and noncoding regions, these libraries can become quite large. Genomic libraries offer many advantages, such as being able to study gene regulation, or off target effects of a particular mutation. The large amounts of data allow researchers to better understand how mutations, located outside of the coding region of a gene, affect the organism. This extra information is essential to better understand certain mutations, but the large amounts of data can sometimes be problematic. In conclusion, genomic libraries offer more information about the organism, but in turn are more laborious to create and are much larger in size when compared to cDNA libaries.

Second, cDNA library is representative of the organism’s exonic regions, meaning that only the coding regions of the genome are recorded and stored in the library. The process of creating a cDNA library is as follows: mRNA is isolated from a cell of interest and collected. Reverse transcriptase is then used to generate the double stranded cDNA corresponding to the mRNA sequence. Once this is complete, the DNA ends are cleaved to become single stranded and is inserted into a selected vector using DNA ligase. The vector is then inserted into a bacteriophage and amplified. After amplification, the cDNA is isolated and collected in the cDNA library. The cDNA library is much smaller than that of genomic libraries as it only represents the exonic portion of the organism’s genome. This small size is not always problematic, cDNA libraries offer a unique approach to study variant mutations present within coding regions of a gene. In addition to studying variant mutations located within a gene, cDNA libraries also offer a more convenient approach to study protein function, interaction and expression.

Whether you are interested in studying an organism with a very small genome or one as complex and large as a human genome, these two types of libraries are one of the best ways to store data. At Synbio Technologies we offer gene synthesis service verify the genes you are interested.A library, either constructed by you or our team of experts, will be generated for a competitive price and in an efficient timeframe. We offer virtually any library type to fit your need, ranging from scanning, to triplet codon, to modular. In addition to this Synbio Technologies also offers error-free gene sequence with a 100 percent guarantee of the generated sequence quality. This also allows us to create a synthetic gene library, generated by genes of interest without the physical copy of the gene being needed. Synbio Technologies offers many quality assessments to make sure that your gene library is exactly how you designed it to be and is as effective as possible. With the high quality library Synbio Technologies offers, and at a competitive price, your library will be synthesized and be ready to analyze with ease and convenience.

Gene Synthesis Driving Synthetic Biology Applications

DNA sequencing has been vital to the development of synthetic biology in many ways. Sequencing enables researchers to determine the DNA sequence in just about any gene, and enables the construction of vast databases that can hold entire genomes. Genome databases are an important resource for downstream synthetic biology applications such as protein expression, directed evolution, and metabolic engineering. In addition, the low cost of DNA sequencing enables more efficient quality control of large DNA constructs, a key step in gene synthesis.

Reductions in the production costs of genes and their key raw materials, oligos, are driving demand for synthetic biology products. Gene synthesis is key to many synthetic biology applications, and their availability at low cost increases the number of gene synthesis applications and customers, driving sales up.

The growing proteomics market is driving demand for more efficient protein expression in novel host systems. This in turn is driving the demand for synthetic genes that have been optimized for heterologous gene expression. Such optimized genes allow for expressing the desired protein product more efficiently, since they can be tailored to the intended host cell system. Gene synthesis will penetrate into the genetic engineering market among pharmaceutical and biotech companies developing new products. Gene synthesis provides a high level of flexibility to the customer and, as its cost decreases, its services are rapidly permeating the classical genetic engineering market to become standard tools among end users.

Synthetic biology applications driven by gene synthesis technology:

  • HGP-Write

    • The Human Genome Project – Write, formally announced on 2 June 2016, is a ten-year extension of the Human Genome Project, to synthesize the human genome. The human genome consists of three billion DNA nucleotides, which were described in the Human Genome Project – Read program, completed in 2003. With the advancement of gene synthesis technology, the time and cost of gene synthesis is approaching Moors’ Law. Many researchers expect that the ability to synthesize large portions of the human genome could lead to many scientific and medical advances.

  • Antibody Library

    • The traditional humanized antibody library refers to a group of re-expressed antibodies which have been transformed by gene cloning and DNA recombination technology based on mouse monoclonal antibody. Synbio Technologies designed and developed unique antibody humanization strategies based on advanced concepts and technologies in synthetic biology. This, combined with the integration of efficient phage display and cell surface display technology, have allowed Synbio Tech to easily provide fast and efficient humanized antibody services.

  • DNA storage

    • DNA storage (using DNA as a data carrier) technology is a relatively new discovery that may have monumental implications on the future of bioinformatics and data science. Text, images, audio, and video documents could be transformed into “A, T, C, G” format and stored in artificial synthesized DNA. Scientists from Synbio Technologies have mastered next generation gene synthesis technology which greatly reduces the manufacturing cost of DNA synthesis. Combined with our patented DNA StudioTMsoftware, DNA storage is closer than ever to spearheading the next generation of storage technology.

Gene Synthesis Related Services

Synbio Technologies’ Gene Synthesis Process

Synbio Technologies’s proprietary Syno®2.0 gene synthesis process includes computational design of short oligos, which can then be reliably synthesized, assembled, and cloned into the desired vector. An available precursor to this process is optimization of the gene sequence for improved protein expression and other purposes.

gene synthesis process

Fig. 1 Syno®2.0 gene synthesis process
A large number of oligos can be synthesized in parallel on gene chips. The Syno®3.0 next generation DNA synthesis platform offers revolutionary large-scale gene synthesis in an efficient, low cost manner that will open up new avenues for the development and industrialization of synthetic biology applications.

gene synthesis process

Fig. 2 Syno®3.0 gene synthesis process
Gene synthesis process–Codon Optimization:

Codon optimization refers to the use of preferred codons – that is, to avoid the usage of rare codons with low utilization – to simplify secondary structure of mRNA after gene transcription. It also involves the replacement of motifs that hinder efficient expression with those that promote it, as well as the adjusting of GC content and other factors in order to optimize gene expression.

Learn more about Codon Optimization:(click here to learn more:codon optimization)

  • Codon Usage Bias
  • Synbio Technologies’s NGTMCodon Optimization Software
  • Synbio Technologies’s Codon Optimization Strategy

Gene synthesis process–Oligo Synthesis:
Syno®2.0 gene synthesis technology:

  • Higher flexibility
  • Less cost effective
  • Limited throughput

Syno®3.0 gene synthesis technology:

  • Lower price (starting from $0.09/bp)
  • High throughput (synthesis of over 500,000 nucleobases and building DNA strands as long as 30,000bp on a single chip)
  • Limited flexibility

Learn more about Synbio Technologies’ Synotype platform

Gene synthesis process—Gene Assembly:

Comparison of popular DNA assembly technologies:

Gibson AssemblyYeast Assembly
DNA polymerase/Taq ligaseNeedNo need
Yeast has homologous recombination capability
Multiple fragmentOne-time assemblyOne-step assembly
Large DNA fragment/Genome< 20K base pairsUp to 1.08M base pairs

How to order


Tel
+1 732-230-3003
Email
service@synbio-tech.com
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+1 609 228 5911
Online Inquiry
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Artificial Gene Synthesis and Traditional Molecular Cloning

Synbio Technologies is a professional company dealing with gene synthesis and cloning. The company can provide artificial gene synthesis and traditional molecular services through our Syno®1.0, Syno®2.0 and Syno®3.0 gene synthesis platforms. Our Syno® platforms can conduct a variety of functions, including construction of a humanized antibody library, optimization of industrial enzymes, chromosomes/genome synthesis, development of genetic engineering vaccines and DNA information storage technology.

What is traditional molecular cloning?
Prior to the 1970s, molecular cloning had served as the foundation of technical expertise in labs worldwide for 30 years. Molecular cloning is a set of experimental methods that are used to assemble recombined DNA molecules and to direct their replication within host organisms.

Why gene synthesis?
Gene synthesis can artificially synthesize double-stranded DNA in vitro, with an assembly capacity of 50bp to 12Kb products. Gene synthesis differs from traditional molecular cloning and PCR cloning in several ways. The traditional molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombined DNA molecules and to direct their replication within host organisms. However, not every gene has high-efficiency expression in these systems, meaning that molecular cloning may not be the best option for these genes. Instead, through gene synthesis, it is possible to avoid this problem by creating a new system with high-efficiency expression of the target gene.

The advantages of artificial gene synthesis:
(1)Saves time and labor
(2)Guarantees 100% sequencing accuracy
(3)Changes all target codons simultaneously, so it only needs to be done once
(4)It is possible to create new base pairings which could greatly expand the possibility of biological form.

The unique advantages of Synbio Technologies’ gene synthesis and cloning:
(1) Syno® 2.0 gene synthesis platform: The proprietary gene synthetic platform can synthesis any gene perfectly. At present, Synbio Technologies can synthesize over 10 million base pairs per month.
(2) NGTM Codon Optimization Technology: Codon optimization can increase protein expression and promote proper protein folding.
(3) High Quality: Effectively deliver high-quality complex sequences including those with repetitive sequences, strong hairpin structures, high GC content, poly structure, etc.
(4) Fast Turnaround: A normal sequence order can be completed within 5 business days with 100% accuracy.
(5) Cost-effective: Starting from 1 cent/nt by Syno® 3.0 high throughput DNA synthesis platform.
(6) Capability: Synbio Technologies can synthesize single 200 kB DNA fragments with high fidelity.

Gene Synthesis Related Services

Synbio Tenchologies can also design sequencing with codon optimization software -NGTMCodon Optimization Technology at no cost.