Cusabio Virus & Bacteria Recombinants


The maintenance and manipulation of large genomes of DNA and RNA viruses had presented an obstacle to virological research. BAC vectors provided a solution to both problems as they can harbour large DNA sequences and can be efficiently modified using well-established mutagenesis techniques in Escherichia coli. Numerous herpesvirus and poxvirus DNA virus genomes were cloned into mini-F vectors.

Furthermore, several reverse genetic systems could be established for RNA viruses, such as members of Coronaviridae and Flaviviridae, based on BAC constructs. Transfection into susceptible eukaryotic cells of cloned viral DNA such as BAC allows reconstitution of recombinant viruses and bacteria. In this paper, we provide an overview of strategies that can be used for the generation of BAC vectors from viruses and also on systems that are currently available for various virus species.

In addition, we address common mutagenesis techniques that allow modification of BACs from single nucleotide substitutions to deletion of viral genes or insertion of foreign sequences. Finally, we review the reconstitution of viruses from BAC vectors and the removal of bacterial sequences from the virus genome during this process.

Cosmid-based approach

An alternative strategy often used for the generation of BACs from cell-associated viruses uses cosmid vectors to initially maintain overlapping parts of the DNA virus genome. The mini-F is subsequently inserted into one of the cosmids by ligation or homologous recombination in E. coli. Transfection of the overlapping cosmids into eukaryotic cells results in recombination between homologous sequences and reconstitution of infectious viruses.

During the process, the cosmid containing the mini-F cassette is incorporated into the virus genome, all resulting viruses harbour mini-F, and no laborious selection steps are required to obtain recombinant clones. As described above, circular virus DNA is isolated and transformed into E. coli and clones are analyzed for the integrity of the virus genomes they contain.

In vitro ligation

Recently, it has been shown that the mini-F replicon can be inserted into herpesvirus genomes by direct ligation. For this purpose, concatemeric virus DNA is isolated from herpes virus-infected cells and cleaved with a restriction enzyme that cuts only at a single locus within the virus genome. The resulting full-length viral genome is then ligated with a linearized mini-F vector containing compatible DNA ends. To prevent ligation of mini-F with cellular fragments, restriction enzymes that recognize an interrupted palindrome and allow the generation of desired directional sticky ends such as SfiI or BstXI can be used.

This strategy has been successfully applied to the generation of a BAC system for human herpesvirus 6A (HHV-6A). There are, however, several drawbacks to this method. First, the strategy requires a fully sequenced virus genome to determine potential restriction sites that can be used for the ligation procedure. Second, many virus genomes do not possess a single restriction site that is suitable for the approach.

Third, the mini-F insertion site is limited to the location of the single restriction site. Insertion into open reading frames (ORFs) or promoters of the virus genome can affect or abrogate the infectivity of BAC-derived viruses. Last but not least, the ligation and transformation procedures for large BAC vectors are very inefficient, making cloning attempts difficult.

Poxvirus Strategy

As described in Section 2.1, the cellular recombination machinery in mammalian cells can facilitate the insertion of mini-F sequences into the poxvirus genome. However, unlike herpesviruses, poxviruses do not produce a circular shape of the virus genome during replication. This poses a major obstacle to the transfer of recombinant poxvirus constructs to E. coli. To overcome the problem, infected cells are treated with isatin-β-thiosemicarbazone which promotes the accumulation of unresolved genomic concatemers.

For the generation of some poxvirus BAC clones, it was sufficient to transform E. coli with concatemeric DNA, a procedure that probably resulted in a recombination event that allowed circularization of the replicon. Alternatively, the isolated poxvirus DNA was circularized prior to E. coli transformation using the Cre/loxP or Flp/FRT recombination system.


Since the establishment of the first BAC system in 1997, BAC technology has contributed substantially to our understanding of the life cycle of large DNA and RNA viruses. Various techniques have been developed that facilitate the insertion of mini-F sequences into the virus genome. The methods enabled the generation of BAC systems for a plethora of virus species, including members of the Herpesvirales, Poxviridae, Coronaviridae, and Flaviviridae. The well-established mutagenesis techniques described herein facilitate site-specific manipulation of the virus genome in E. coli.

Various strategies can be used to introduce any desired modification, including deletions of viral sequences or insertions of foreign sequences. Reconstitution of recombinant viruses can be achieved by transfecting purified BAC DNA into susceptible mammalian cells, while in some cases additional helper viruses or expression vectors are required in this process. Finally, several techniques have been established that allow the excision of mini-F sequences from virus genomes without leaving behind an unwanted sequence.

Cusabio Others Recombinants


We Other Utopians is the first book to explore recombinant DNA/genome editing issues based on ethnographic research in the post-communist context. The book focuses on the topics of human DNA editing and genome repair at two levels. First, inspired by texts that analyze the concept of life and the body in general, it works conceptually and analytically with various approaches to designed life and incarnations from the perspective of anthropology, sociology, and science and technology studies.

Second, it presents an analysis of artificial life and biotechnological achievements in specific technologies: genome editing, other recombinant DNA and biological computing. The book explores the topic of genome editing based on ethnographic research carried out in a biochemical laboratory in the Czech Republic. The fieldwork was carried out between 2017 and 2019, mainly in a laboratory focused on DNA damage and genomic risk of complex diseases or genetic vulnerabilities such as breast cancer, infertility and aging.

Recombinant DNA is understood here as the exchange of DNA strands to produce and design new arrangements of nucleotide sequences to cure or improve human bodies and health in the future. The book examines various economies of hope, hype, expectations, the politics and poetics of false promises and better or worse predictions from the standpoint of sociology, anthropology, and science and technology studies.

Linked gene recombinants are those combinations of genes that are not found in the parents.

  • Recombinants are produced as a result of crossing over of genetic material during prophase I of meiosis.
  • If linked genes are separated by a chiasm, there will be an allele exchange between non-sister chromatids.
  • This creates new combinations of alleles that are different from the parent.

The frequency of recombinant phenotypes within a population will normally be less than that of non-recombinant phenotypes.

  • This is because crossing over is a random process and chiasmata do not form in the same places with each meiotic division.

The relative frequency of recombinant phenotypes will depend on the distance between linked genes.

  • The frequency of recombination between two linked genes will be higher when the genes are further apart on the chromosome.
  • This is because there are more possible locations where a chiasm could form between genes.

Cusabio N-terminal 10xHis-tagged Recombinant

Manufacturer/trade name: Novagen®

Quality level: 400

Storage condition: do not freeze

Sent in: environment

Storage temperature: 2-8°C

General description

Ni-NTA His•Bind resin is high-performance Ni2+-loaded agarose used for the rapid, one-step purification of proteins containing an N-terminal 10x His•Tagged recombinant sequence by metal chelation chromatography. NTA chemistry minimizes metal leaching during purification and is compatible with 10 mM β-mercaptoethanol or 1 mM tris(hydroxypropyl)phosphine (THP) for disulfide bond reduction. Ni-NTA His•Bind resin has a binding capacity of 5 to 10 mg of His•Tag fusion protein per ml of resin. Supplied as a 50% suspension; quantity/package is based on the amount of settled resin.


  • 10 ml in plastic ampoule
  • 100, 25, 500 ml in a glass bottle


Toxicity: Flammable (J)

Other notes

Due to the nature of the hazardous materials in this shipment, additional shipping charges may apply to your order. Certain sizes may be exempt from additional shipping charges for hazardous materials. Contact your local sales office for more information on these charges.

Legal information

  • HIS-BIND is a registered trademark of Merck KGaA, Darmstadt, Germany
  • NOVAGEN is a registered trademark of Merck KGaA, Darmstadt, Germany