The biopharmaceutical company, Altravax (www.altravax.com; Fargo, ND, and Sunnyvale, CA, USA), is focused on discovery and development of improved vaccines, as well as establishing new methods for vaccine research. The company primarily uses high-throughput workflows, including their proprietary Molecular Breeding™ technology. This directed, molecular evolution process employs in vitro DNA recombination to produce large libraries of recombined, chimeric DNA sequences that express potential vaccine candidates. The most promising vaccine candidates from these libraries are evaluated using high throughput ParallelaVax™ vaccine screening, a technique developed by Altravax in collaboration with Aldevron LLC (Fargo, ND, USA).
While these newer, high-throughput methods are central to their research, Dr Bob Whalen, Altravax CSO, explains, “…modern vaccine research takes full advantage of all of the commonly available molecular biology techniques, including synthetic biology methods.” In fact, because Altravax is very active in early-stage discovery research, they employ a greater mix of high throughput and traditional molecular methods than other companies involved in later stages of vaccine development.
The Altravax high-throughput workflows are initially developed via basic research done at the bench. This often includes substantial molecular cloning and cell culture that can be significantly time consuming. Thus, Altravax scientists are constantly looking to improve on experimental efficiency. A recent advance that has proved time saving was the introduction of gBlocks® Gene Fragments as a highly reliable way to replace PCR-based cloning methods in the group’s reagent synthesis applications.
Senior scientist, Dr Kristin Narayan, is investigating flu vaccines at Altravax. Like a lot of the basic research there, Dr Narayan’s work requires cloning and testing flu genes and gene variants to determine which genes and resulting peptide motifs make the best potential vaccine targets. Traditionally, this would have been accomplished by standard PCR from a viral template (a wait of weeks for physical sample availability after the onset of a pandemic), or by assembling multiple synthetic oligonucleotides using PCR.
While PCR is a robust method for cloning and gene assembly, the amplification and purification procedures are time consuming. Generating constructs by PCR can also produce unexpected mutations and artifacts, and may require additional optimization and troubleshooting to obtain the desired sequence. The ability to order a synthetic, double-stranded DNA product that is sequence-verified allows researchers to skip the PCR step altogether, and minimize the potential pitfalls associated with PCR, something that is appreciated by Dr Narayan and her colleagues.
Rapid assembly of synthetic vaccine constructs
Using gBlocks Gene Fragments to replace inserts produced by PCR for restriction-based cloning increases lab throughput. With gBlocks Gene Fragments, no physical template samples are needed, only the desired sequence information for ordering. Further gains in efficiency can be made by moving to more recently developed synthetic gene assembly techniques. gBlocks fragments are compatible with most synthetic gene assembly methods, many of which are substantially faster than restriction cloning. Constructs are also easier to design since they do not require specific DNA restriction sites in the sequences.
Dr Sam Hoot, a staff scientist at Altravax, also uses gBlocks Gene Fragments in his research on HIV antibodies. With a length of up to 3000 bp, he finds that gBlocks fragments are an ideal size for cloning antibody variable regions, which are the domains necessary for antigen recognition. Dr Hoot assembles these using the Gibson Assembly™ Method, an isothermal technique that joins multiple DNA elements with overlapping ends easily, in a single, 1-hour isothermal reaction. As shown in Figure 1, this method has worked well for Dr Hoot to quickly assemble 2 gBlocks fragments into a plasmid vector, creating a 1 kb insert. He and his colleagues anticipate that the method will be useful for larger assemblies in the future. Previous DECODED articles have covered cloning of antibody variable regions , and the Gibson Assembly Method [2, 3].
Figure 1. Assembly of gBlocks® Gene Fragments using the Gibson Assembly™ Method. 1) Two or more gBlocks Gene Fragments are designed with sequences that overlap one another and the desired insertion region of the multiple cloning site (MCS) or other plasmid location. The plasmid is linearized by restriction digest or PCR. Linearized plasmid and fragments are combined in a tube with Gibson Assembly™ Master Mix. In a simple, 1 hr reaction, 2) exonucleases digest the 5’ DNA ends; 3) the resulting complementary overhangs anneal; 4) high-fidelity polymerases fill in remaining gaps; and 5) a ligase covalently joins DNA fragments to the plasmid to generate the complete construct. Many segments of DNA can be assembled in this way to generate a desired construct .
Vaccine research involves screening many possible antigens or antibodies and identifying a small subset of the most efficacious of these. Until recently, this kind of research was relatively confined to academic research because of the limitations of the tools and methods. However, the new high-throughput methods developed by Altravax, and recently introduced synthetic biology tools, including gBlocks Gene Fragments, make commercialization possible with the promise of a healthier future for us all.
Figure 2. The research team at Altravax. Front row (l–r): Janelle Muranaka, Kristin Narayan, and Sam Hoot. Back row (l–r): Fiona Fernandes, Erika Navarro, Robert Whalen, and Sudha Gudavalli.
- IDT, A Next Generation Understanding of Immune Response, http://www.idtdna.com/pages/ decoded/decoded-2-4.
- IDT, Isothermal Assembly: Quick, Easy Gene Construction, http://www.idtdna.com/pages/ decoded/decoded-2-1.
- Gibson DG, Young L, et al. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods, 6(5):343–345.
gBlocks® Gene Fragments
gBlocks® Gene Fragments are double-stranded, sequence-verified genomic blocks from 125 to 3000 bp in length that can be shipped in 2–8 working days for affordable and easy gene construction or modification. gBlocks fragments are synthesized using the same industry-leading, high fidelity synthesis chemistries developed by IDT for Ultramer® Oligonucleotides, and are sequence verified prior to shipping. Using the isothermal assembly method, 4 gBlocks fragments can be assembled into a multi kb gene in less than 1 hour; a simple, 30 nt sequence overlap is required when designing the gene fragments.
Learn more about gBlocks Gene Fragments at www.idtdna.com/gblocks.
Author: Hans Packer, PhD, is a Scientific Writer at IDT.
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