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in vitro Transcription (RNA Synthesis)

RNA plays an important role in gene regulation. Although RNA overexpression in cells has made some achievements, in vitro transcription is still a common research method, which is popular due to convenient operations and wide uses. In vitro transcription is to use DNA as a template to simulate the intracellular transcription process to generate RNA in vitro cell-free system. That can control the genes of transcription, the process of transcription and the use of post transcriptional RNA.

In vitro transcription of RNA is mainly applied to labeled probes synthesis and lots of unlabeled RNA generation. In addition, the capped RNA synthesized in the transcription reaction can be used for hybridization analysis, microinjection, biochemical and genetic researches, etc. Synbio Technologies has developed nucleotides, polymerases, modified enzymes and kits for highly-efficient transcription of capped RNA, large-scale transcription and transcription reaction based on short DNA template.

Case Study

CRISPR-Cas system offers versatile genome engineering technologies. While the ongoing discoveries of new Cas nucleases and anti-CRISPR proteins far exceeds the rate of proficient implementation. Here, researchers used E. coli cell-free transcription-translation (TXTL) systems to significantly improve the speed and scalability of CRISPR characterization and validation.

TXTL can express active CRISPR mechanism from added plasmids and linear DNA. Researchers used TXTL to measure the quantitative dynamics of DNA cleavage and gene repression for single- and multi-effector CRISPR nucleases, predict gene repression strength, determine specificities of 24 anti-CRISPR proteins, and develop a fast and scalable screen method for protospacer-adjacent motifs that was successfully applied to five uncharacterized Cpf1 nucleases. This example illustrated how TXTL can facilitate the characterization and application of CRISPR technologies in different uses[1].

in-vitro-transcription

Applications of in vitro transcription

  • sgRNA synthesis for gene editing
  • RNA probe synthesis for hybridization
  • dsRNA / siRNA synthesis for RNA interference
  • RNA virus genome synthesis for virus research
  • mRNA synthesis for in vitro transcription
  • Synthesis of mRNA / cRNA for electrophysiological research
  • Synthesis of various types of RNA for noncoding RNA research

Related Services

RNA Synthesis
CRISPR sgRNA Design and Construction
DNA Plasmid Preparation
Oligo Synthesis

Reference

[1] RyanMarshall, Colin S.Maxwell, Scott P.Collins, etc. Rapid and Scalable Characterization of CRISPR Technologies Using an E. coli Cell-Free Transcription-Translation System. Molecular Cell, 2018. 69: 146-157. https://doi.org/10.1016/j.molcel.2017.12.007

The Wide Applications of Molecular Biology

Molecular biology clarifies regularities of cell and receals the essence of life.Molecular biology technologies have been widely adapted for recombinant protein production, genetic modification of organisms, gene therapy, environmental protection, etc.

Production of recombinant proteins

  • Recombinant Insulin production
  • For very long time, the insulin that was used to treat diabetic patients is solely purified from bovine or porcine pancreas. 100kg pancreas can only extract 4-5g of insulin. The development in the field of genetic engineering introduced chemically synthesized insulin cDNA in E.coli and allowed insulin production in microorganisms, yielding 100g insulin from every 2000L microorganism’s culture. The massive industrial scale production of insulin not only solves the yield problem but also drives its price down by 30% -50%.

  • Genetic engineering drugs
  • Genetic engineering is used to mass-produce interferon, artificial blood, interleukin, hepatitis B vaccine and many other drugs which played a huge role of lifting the human suffering, improving human health.Genetic engineering drugs has greatly been embraced by man to improve on his well-being.

  • Genetically modified animal
  • For certain protein drugs that require complex modifications or are needed in large supply, production in transgenic animals seems most efficient. The current strategy to achieve these objectives is to couple the DNA for the protein drug with a DNA signal directing production in the mammary gland.

  • Herbicide resistant crops
  • Herbicide resistant crops are genetically modified to tolerate broad-spectrum herbicides, which kill the surrounding flora, but leave the cultivated crop intact.

  • Genetically modified microorganism
  • Microorganisms are most commonly used in genetic engineering due to their inexpensive nature. Cheese production requires the use of a protein called chymosin which is a proteolitic enzyme usually obtained from calf stomachs. Production of chymosin in genetically engineered microorganism provides an alternative way of producing cheese that does not require the sacrifice of large amount of animals. Moreover, microorganism production of recombinant chymosin offers an easy way of increasing the production of chymosin compared to the amount that can be obtained from young calves.

  • Gene therapy
  • Gene therapy is a technique to treat genetic diseases by introducing foreign DNA which usually contains a functioning gene to correct the effects of a disease-causing mutation.

  • Environmental protection
  • The genetically engineered organisms can be used as bioindicators to readily reflecting pollution level on a habitat, community, or ecosystem. Moreover, these bioindicators are engineered to resist pollutant-leaded mortality and potentially has the capability in bioremediation of toxic chemicals.

Codon Usage Bias

The genetic information in DNA is transcribed into mRNA then translated into protein, in which codons played important roles during the process. Total 64 combinations of nucleotide triplet (codon) encode all 22 amino acids. Each amino acid corresponds to at least one codon, e.g. methionine and tryptophan. In other cases, amino acids are encoded by 2 to 6 different codons. The codons encode the same amino acid are referred as synonymous codons. The frequency of synonymous codon usage varies widely among different organisms, and these differences have important implications for the regulation of protein expression. In the process of protein synthesis, a particular species tends to use a set of specific codons, which are called optimal codons. This phenomenon is also known as codon usage bias. So the use of different species in the codon usage bias, optimizing the use of codons can increase the protein expression.

Codon usage bias and protein

At the same time, accurate polypeptide elongation could minimize the energy waste caused by translation errors. Therefore, a codon with optimal translation speed and high fidelity will result high translation efficiency and an increased protein expression. The combination of codons and translation efficiency are not only contributing to the codon usage bias of the whole genome, but also the distribution of codons with different translation efficiency in different regions. The codon usage bias in different regions can regulate genes expression at different stages, such as affecting the mRNA expression during transcription, the speed and accuracy of translation, and the folding of polypeptide.

In the heterologous protein expression system, selecting the codon combinations that control the speed of translation is essential to avoid formation of inclusion bodies due to high expression or not obtaining any protein at all. Thus a well-tuned system for controlled gene expression is the key component for protein production in industrial and scientific research.

Synbio Tech proprietary NGTMCodon Optimization software can intelligently optimize codons based on different expression systems and effectively improve the protein expression in order to meet the needs of scientific research and industrial production.

Long Gene Synthesis

Artificial gene synthesis is a critically important technique in modern synthetic biology. The ability to easily customize and create DNA sequences based off of a single gene, gene clusters, or completely from scratch enables an enormous amount of versatility and a wide range of applications for synthetic DNA.

The necessity of long gene synthesis

Researchers often investigate the genetic causes of a particular target phenotype, frequently by examining the effects of site-directed mutations. Through artificial gene synthesis, inducing mutations, stretches, elongations, or other changes to a target gene sequence becomes substantially easier than before, allowing for efficient study of these mutant phenotypes.

In recent years, the importance of being able to analyze entire genomes or large collections of genes has become more and more pronounced. The necessity of having to look at mutations in the context of an organism’s genome means that being able to synthesize huge amounts of DNA with near-perfect fidelity is becoming increasingly relevant to many current experiments.

Gene Synthesis Scheme

Current solid-phase oligonucleotide synthesis technology can yield chemically synthetic gene constructs. However, it is generally limited to sequences of roughly 200 nucleotides in length. Another approach named Gibson assembly shares a similar drawback in that it is restricted to plasmids of around 10kb or less.

The need for technology that could handle synthesis of very large DNA sequences turned to yeast as the system of choice. Yeast can provide self-connection between multiple DNA fragments easily and rapidly without the application of polymerase or ligase. Synbio Technologies’s own proprietary Syno® platform, combined with the built-in homologous recombination technology of yeast, enables rapid and accurate assembly of various long DNA fragments and genomes of up to 150 Kb.

Synbio Technologies delivers more than 2 million base pairs of DNA sequences every month, all over the world. Any DNA sequence, even those possessing difficult characteristics such as high or low GC content, hairpin structures, or highly long/complex sequences, is able to be synthesized with 100% accuracy guaranteed. Synbio Technologies can provide large sale and low cost methods to accomplish gene synthesis including assembling long DNA segments at high accuracy and yield.

Gene Synthesis Related Services

  • Codon Optimization
  • Vector Construction
  • Small Genome Synthesis
  • Pathway Synthesis

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-01

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

  • Synotype platform
  • Syno®2.0 gene synthesis
  • Syno®3.0 gene synthesis

Gene synthesis process—Gene Assembly:

Comparison of popular DNA assembly technologies:

 

 

career

Synbio Technologies is a multinational corporation with rapid growth, we have companies both in the United States and China. As the need of development of company, we want to recruit a variety of talents. We provide equal employment opportunities with no bias. For more details, you can contact:

Synbio Technologies

1 Deer Park Drive, Suite L-1, Monmouth Junction, NJ, 08852

Tel: +1 732-230-3003

Email: hr@synbio-tech.com

Position: Regional Sales Manager

Responsibilities:

  • Responsible for leading the region to achieve all assigned sales goals and revenue targets (Regions includes mainly CA, WA, NC, MD and DC area)
  • Develop and implement sales and marketing strategies and tactics for company brand, products and services; building strategic sales planning/ business plan, including specific, measurable objectives in accordance with regional and national sales goals
  • Drive strategic business expansion with large pharma/ biotech companies in the U.S and international markets; gaining new clients while maintaining, expanding and developing partnership between prospective clients
  • Execute sales and marketing campaigns through digital advertising, social media and trades shows
  • Consistent closing abilities throughout the sales cycle, managing multiple new customer accounts and critical project deadlines. Work closely with Project Management team on customer projects/orders progress and deadlines

Requirements:

  • Demonstrated track record of success with measurable and significant revenue generation
  • Minimum of B.S. in biology (biochemistry, synthetic biology, molecular biology or equivalent), M.S. or MBA preferred.
  • 1-3 years of direct selling experience to BioPharma, diagnostic, pharmaceutical or relevant biotech companies required
    Impeccable verbal and written communication skills
  • Frequent business travel (> 60%) is required throughout the territory
  • Comply with all corporate policies

Position: R & D Manager

Responsibilities:

  • Lead on R&D long-term strategic planning and innovation of new technology platforms and product research
  • Responsible for annual planning, budgeting of the company’s R & D projects, including their organization,implementation, progress reports, patent applications and industrial transformation of all research and development
  • Oversee R&D group’s team building and employees’ training; supervise and influence R & D team’s overall business performance and development, create positive working environment
  • Maintain high sensitivity and acumen about the industry trends and market new changes; initiate and develop effective plans and strategies of technology development
  • Communicate and collaborate with the company’s business units to ensure that production and project management groups receive full technical support from R&D team

Requirements:

  • Master’s degree in Molecular Biology or other related discipline
  • At Least three years’ experience in molecular biology, protein or antibody engineering, modification, and characterization
  • Oversea working experience preferred

Position: Product manager

Responsibilities:

  • Assist with the strategic direction of product/service development and to formulate annual development direction, plan and long-term competitive strategy of the product;
  • Conduct investigation and comprehensive analysis of the industry; understand the product market and customers’ requirements; provide market basis and suggestions for enterprise strategic decision;
  • Supervise the whole process of product design, development, packaging, distribution, pricing, launch, etc.;
  • Set the strategy, plan and promotion plan for the company and products; organize the team to execute, track and feedback;
  • Collect the feedback information after the launch of products; manage the product brand and cost;
  • Be responsible for product development; follow the progress and quality of products; optimize product quality and promote the value of the products;
  • Coordinate with related departments (such as sales, marketing, production, research and development) to plan, organize and implement product promotion activities, academic lectures and other marketing activities;
  • Maintain communication with customers; provide professional and efficient technical support; coordinate with customers’ feedback and keep continuous improvement;
  • Assist to complete other products related works.

Requirements:

  • Doctoral degree, major in biology or related fields, 2 years overseas working experience;
  • Good data analysis and judge capability; be able to conduct market analysis independently
  • Strong innovation and pioneering ability; be fond of designing the content of promotional materials;
  • Good management skills, communication skills, adaptability and organization coordination ability;
  • Hardworking; able to travel or work abroad.