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flashBAC™ is a family products which use major new platform technology based on baculovirus protein expression technology. The technology enables fast and simultaneous production of multiple recombinant viruses, lending itself to use for high-throughput systems.
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RAPID - One-step procedure with no tedious selection stages. |
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FLEXIBLE - Suitable for the production of single or multiple viruses, by manual or automated methods. |
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SIMPLE - No plaque assays or other selection procedures. |
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| Technical Description |
| flashBAC™ incorporates the advantages of the existing baculovirus expression Plasmid system, but also has additional improvements to take it to the next level of expression Plasmid 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 these kits. |
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| flashBAC Products |
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flashBAC
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flashBACGOLD |
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flashBAC10 |
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flashBACULTRA |
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| Specific Product/Application Areas: |
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baculoQUANT Virus Titration |
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titrePLUS Virus Titration and Protein Expression
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pOET Transfer Plasmids
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NEW Products |
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NEW Transfer Plasmids pOET2™, pOET3™ and pOET4™ |
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NEW flashBAC™ Selection Box |
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NEW 96 Reaction flashBAC™ Kits |
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| Literature |
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Download flashBAC Technical Literature |
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Go back to Oxford Expression Technologies Products and Applications |
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| Product |
Technical Overview |
flashBAC™
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Homologous recombination between flashBAC™ DNA and a transfer plasmid 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 (Invitrogen)1.
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 activity1. 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 hosts1.
Confocal and electron microscopy observations of insect cells infected with AcMNPV have shown that the endoplasmic reticulum (ER) is densely packed with chitinase, completely blocking the secretory pathway. Deletion of chiA from flashBAC™ 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).
Note 1: Contact us for information on the references cited |
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flashBACGOLD™
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flashBACGOLD™ enhance yields for difficult to express proteins.
Baculovirus genomes contain several auxiliary genes, which are non-essential for replication in insect cell culture. Two of these are chitinase (chiA), which encodes an enzyme with exo- and endochitinase activity1 and a cathepsin-like cysteine protease (v-cath)1. In an infected insect, chitinase and cathepsin facilitate host cuticle breakdown and tissue liquefaction at the very late stages of infection, so releasing the virus to infect more hosts.
Confocal and electron microscopy observations of insect cells infected with AcMNPV have shown that chitinase is targeted to the endoplasmic reticulum (ER), where it is densely packed in a para-crystalline array, blocking and severely compromising the function and efficacy of the secretory pathway1. V-cath accumulates in the ER at early times post-infection as an inactive proenzyme (pro-v-cath) and is then activated by proteolytic cleavage upon cell death, but is sensitive to the cysteine protease inhibitor E-641.
It has optimum activity at pH 5.0-5.5, although it also shows measurable activity up to pH 7.01. Chitinase may act as a chaperone for the proper folding of pro-v-cath in the ER1. Together these enzymes compete with the recombinant protein for limiting cellular resources, putting a huge burden on the protein translocational machinery1. As a protease, v-cath will also degrade susceptible recombinant proteins, particularly in the later stages of infection when the polh promoter is most active.
The deletion of both chiA and v-cath from flashBACGOLD™ has improved the efficacy of the secretory pathway and resulted in a greatly enhanced yield of recombinant proteins that are secreted or membrane targeted (in comparison to recombinant viruses that encode chiA and v-cath). Results also show a significant reduction in degradation of protease-sensitive targets and increased production and stability of some intra-cellular proteins1.
Note 1: Contact us for information on the references cited |
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flashBAC10™
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flashBAC10™ builds upon this existing technology by the removal of the p10 gene from the flashBAC10™ genome. p10 is a 10 kDa protein, expressed concurrently with polyhedrin (polh) late in infection and is nonessential in cell culture1. Both p10 and polh promoters share a 12-nucleotide consensus sequence1 containing the transcription initiation ATAAG motif. p10 is activated a few hours before polh1 and has been demonstrated to compete with polh at a transcriptional level1. However, inhibition and deletion of the p10 promoter has been shown to result in increased polh-controlled protein production1 and polh mRNA levels1. p10 also associates with occlusion bodies (OBs)1 and is believed to mediate nuclear disintegration late in infection as its disruption has been shown to prevent OB release1. Recent work has also shown that it forms extensive cytoskeleton-associated or cytoskeletal-like structures in the nucleus and cytoplasm, potentially de-stabilizing the cells cytoskeleton1 and further depleting cellular resources.
Deletion of p10 increases polh activity providing more recombinant protein, increases nuclear and cellular stability, ensuring a longer timeframe for protein expression and removes a major competitor for limiting cellular resources.
Note 1: Contact us for information on the references cited
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flashBACULTRA™
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flashBACULTRA™ has taken this technology a step further, by the removal of three more virus genes (p10, p74 and p26) from the flashBACULTRA™ genome.
p10 is a 10 kDa protein, expressed concurrently with polyhedrin (polh) late in infection and is nonessential in cell culture. Both p10 and polh promoters share a 12-nucleotide consensus sequence containing the transcription initiation ATAAG motif. p10 is activated a few hours before polh and has been demonstrated to compete with polh at a transcriptional level. However, inhibition and deletion of the p10 promoter has been shown to result in increased polh-controlled protein production and polh mRNA levels. p10 also associates with occlusion bodies (OBs) and is believed to mediate nuclear disintegration late in infection as its disruption has been shown to prevent OB release. Recent work has also shown that it forms extensive cytoskeleton-associated or cytoskeletal-like structures in the nucleus and cytoplasm, potentially de-stabilizing the cells cytoskeleton and further depleting cellular resources.
Deletion of p10 increases polh activity providing more recombinant protein, increases nuclear and cellular stability, ensuring a longer timeframe for protein expression and removes a major competitor for limiting cellular resources.
P74 is non-essential in cell culture but is essential for oral infectivity of occlusion-derived virus (ODV) in the host where it plays a role in midgut attachment and fusion. Deletion of p74 has been shown to have no effect on virus production in vitro.
Deletion of p74 further increases the biosafety profile of recombinant baculoviruses in the environment, making them unable to traverse the insect gut wall.
P26 is an early gene that codes for a 240-amino acid polypeptide of unknown function and has the same 5′ terminus as p10. Deletion of the 3’-end of p26 and fusion to lacZ or p10 have previously been shown to have no effect on virus replication in vitro.
Deletion of p10, p74 and p26 removes an unnecessary genetic burden from the recombinant virus genome, providing a more efficient baculovirus expression vector. |
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