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flashBAC is a major new platform technology enabling the fast and simultaneous production of multiple recombinant viruses, lending itself to use for high-throughput systems.
flashBAC is now available for 5, 24 or 96 reactions. 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. Technology OverviewHomologous 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). Protocol Overview
Click here to download the flashBAC Feature Comparison table (pdf). These are our interpretation of the manufacturer's protocols. While we have taken every precaution to make a fair comparison of the kits listed, we strongly recommend that a thorough comparison should be carried out by individuals with reference to the manufacturer's protocols. The flashBAC system maximises production of secreted and membrane-targeted proteins by deletion of the chitinase gene from the virus genome. Tests with a wide range of secreted and membrane targeted proteins indicate that this chitinase-deficient virus can increase the yield of protein by up to 60-fold (in comparison with recombinant viruses that synthesise chitinase) (see ref. 1) However, chitinase deletion has no effect on the yield of recombinant proteins targeted to the cytoplasmic or nuclear compartments. Baculovirus genomes contain several auxillary genes, which are non-essential for replication in insect cell culture. One of these is chitinase (chiA), which encodes an enzyme with exo- and endochitinase activity (see ref 2). In an infected insect, chitinase (together with cathepsin) facilitates host cuticle breakdown and tissue liquefaction at the very late stages of infection, so releasing the virus to infect more hosts (see ref 3). This function is obviously not required in cell culture. 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, severely compromising the function and efficacy of the secretory pathway (refs 4,5). Deletion of chiA from flashBAC has no effect on virus replication, production of recombinant virus or the function of the polyhedrin gene promotor (controlling foreign gene expression). Reference Papers
How big is flashBAC?flashBAC is approximately 136 kb in size, What is the transfer vector supplied in the flashBAC kit?The transfer vector supplied with the kit contains a marker gene (lacz) under control of the polyhedrin gene (polh) and is supplied as a positive control only, i.e. to confirm that recombination within the insect cells has occurred and the virus is expressing. This would be done alongside your own transfer vector containing your gene of interest. What vectors are compatible with flashBAC?All vectors based on homologous recombination at the polh locus (see list) Can I use transfer vectors BlueBacHis2 and pMelBac from Invitrogen with flashBAC?Unfortunately these vectors are designed for use only with Invitrogens Bac-n-Blue system and as such are not compatible with flashBAC. Is it necessary to linearize the transfer vector before doing the homologous recombination reaction?No. Can I use T.ni cells for amplifying my virus?We recommend not using T.ni cells for virus amplification because of their propensity for the generation of defective viruses which has been observed over many yearse.g. Kumar S, Miller LK. ‘Effects of serial passage of Autographa californica nuclear polyhedrosis virus in cell culture.’ Virus Res. 1987 Jun;7(4):335-49.The protocol we prefer to use is the one contained within the flashBAC manual, using Sf9 cells for co-transfections and amplifications and Sf21 cells for plaque assays. Can I use T.ni cells for my protein production?Yes. T.ni cells can give excellent protein production results and can be used for this purpose without problems. We would recommend a comparison with Sf9 cells to evaluate optimal protein production for your specific protein. Why do I not need to do a plaque assay to separate the virus?In the initial stages a plaque assay is not required - ie there is no need to go through the stage that is necessary in systems based on the old technology of separating parental from recombinant virus by plaque picking, because all virus produced is recombinant (http://www.expressiontechnologies.com/flashBAC/technology.asp) It's not possible to know an exact moi without doing a plaque assay at the end, but often all that is required is a sample of the virus for a quick protein production for screening, in which case it is not necessary to know the titre. We routinely add 0.5 ml seed stock (untitred) to 200ml cells to produce working inoculum, it can then be titred at this stage if we need an accurate moi, but the titre is usually in the high 10^7 / 10^8 pfu/ml. Do I need to do a plaque assay before I produce protein?This depends on whether you just want a protein sample for screening purposes or if you want to produce optimal yield of protein. If your aim is simply to screen, it is not necessary to know the titre. Simply use 100 – 200ul to infect cells in a 35mm dish, or 400 – 500 ul to infect cells in a T25 flask. For larger quantities of inoculm, 0.5 ml untitred seed stock can be added to 200ml cells to produce a working inoculum. If required, this can then be titred to give an accurate m.o.i. for infection, but the titre is usually in the region of high 10^7 / 10^8.The problem with not titrating working stocks of virus is that sometimes virus amplification does not go according to plan (often due to the cell culture – see above) and the final titre can be much lower than 10^7 pfu/ml. If this happens there is unlikely to be good protein expression. Is there an easy way to titre my virus?Visit the BaculoQuantTM page of our website for our quick baculovirus titration service
For more information on commercial licences, please contact Helen Irving at This e-mail address is being protected from spam bots, you need JavaScript enabled to view it |
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