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flashBAC incorporates the advantages of the existing baculovirus expression vector system, but also has additional
improvements to take it to the next level of expression vector technology.
Most importantly, flashBAC has been modified to remove the necessity of a plaque-purification step.
flashBAC lacks part of an essential gene and contains a bacterial artificial chromosome (BAC) at the polh locus.
The essential gene deletion prevents virus replication within insect cells but the BAC allows the viral DNA to
be maintained and propagated, as a circular genome within bacterial cells. Circular viral DNA is then isolated
from the bacterial cells and purified. This is the flashBAC DNA provided in this kit.
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Homologous recombination between flashBAC DNA and a transfer vector containing a gene of your choice restores
the function of the essential gene and simultaneously removes the BAC sequence.
The flashBAC system is back compatible with all baculovirus transfer vectors based on homologous
recombination in insect cells at the polyhedrin locus. This includes vectors such as pBacPAK8/9,
pAcUW31 and pBacPAK-His1/2/3 (BD Biosciences Clontech) but not vectors such as pFastBac™, which are
designed for site-specific transposition in E. coli using the Bac-to-Bac® system (GibCo-BRL)13.
The recombinant virus subsequently replicates to produce a genetically homogenous recombinant virus
population without the need to perform a plaque assay. After five days, virus can be added directly
to insect cells to amplify a high titre working virus stock. This one-step procedure for making recombinant
baculoviruses greatly facilitates the process of high throughput production of baculovirus expression vectors
via automated systems.
The flashBAC system also maximises protein secretion and membrane protein targeting. Baculovirus genomes
contain several auxillary genes, which are non-essential for replication, including a chitinase (chiA),
with exo- and endochitinase activity14. In an infected insect chitinase (together with cathepsin) facilitate
host cuticle breakdown and tissue liquefaction at the very late stages of infection, so releasing the virus
to infect more hosts15.
Confocal and electron microscopy observations of insect cells infected with AcMNPV
have shown that the endoplasmic reticulum (ER) is densely packed with chitinase16, completely blocking up
the secretory pathway. Deletion of chiA from flashBAC (Click here for details)
has improved the efficacy of the secretory pathway and
resulted in a greatly enhanced (up to 60-fold in some instances) yield of recombinant proteins that are
secreted or membrane targeted (in comparison with recombinant viruses that synthesise chitinase).
The flashBAC Protocol Schema
- 1. AcMNPV genomic DNA was modified to remove the AvrII site within egt (a gene which is non-essential for replication in cell culture). The starter virus also lacked a functional chitinase (chiA) gene.
- 2. A low copy number BAC replicon was inserted in place of the polyhedrin gene to create AcBAC. A gene essential for replication in insect cells, was modified to include an AvrII site within its coding region
- 3. AcBAC DNA was digested with AvrII to remove the 3’ coding region of the essential gene, religated and used to transform Escherichia coli DH10B cells via electroporation to derive AcBACD (flashBAC)
- 4. flashBAC was mixed with a transfer vector containing Your Favourite Gene (pAcYFG) and used to co-transfect insect cells to derive AcYFG without the need for a plaque assay; because all progeny virus are recombinants.
- 5. During homologous recombination between pAcYFG and flashBAC the BAC replicon is removed concomitant with the insertion of YFG
- 6. The final expression vector contains YFG at the polyhedrin locus under the control of the polyhedrin gene promoter (or other promoter used in the transfer vector). No BAC sequences remain in the expression vector.
 Download the FlashBAC User Guide PDF document
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