The bacmid is essentially a very large plasmid containing the baculovirus genome modified to carry a lacZ gene and an attTn7 docking site inserted in the lacZ coding region. … The most commonly used cell line for expressing recombinant proteins from baculovirus vectors is Sf9.
How do you make Bacmid?
Isolation of bacmid DNA
- Prepare a glycerol stock of each recombinant bacterial clone by mixing 0.5 ml of overnight culture with 0.5 ml of sterile 30% glycerol in a cryovial. …
- Spin down the remaining volume of cells (~3.5 ml) in a 1.5 ml microcentrifuge tube.
- Resuspend the cells in 0.3 ml of Solution I.
What is baculovirus expression system?
The Baculovirus Expression Vector System (BEVS) is used to produce recombinant proteins. … A baculovirus is an enveloped double-stranded DNA virus that belongs to the Baculoviridae family, which affects approximately 600 different insect species.
What is polyhedrin promoter?
As has been already mentioned above, the polyhedrin promoter is by far the most frequently exploited promoter for recombinant protein expression using baculovirus vectors. Initially, transfer vector pAc373 (Smith et al., 1985) was used to target the AcNPV polyhedrin gene locus for foreign gene expression.
How do you make a dh10bac competent cell?
Start a 5ml culture SOB with Kanamycin 50ug/ml and Tetracycline 10ug/ml final concentration. Incubate at 37°C overnight. The day after, start a 100ml SOB culture with Kanamycin 50ug/ml and Tetracycline 10ug/ml final concentration and inoculate with 1ml from the 5ml culture.
What is a Bacmid Wikipedia?
Noun. bacmid (plural bacmids) (cytology) A shuttle vector that can be propagated in both E.coli and insect cells.
What are Sf9 cells used for?
Sf9 cells, a clonal isolate of Spodoptera frugiperda Sf21 cells (IPLB-Sf21-AE), are commonly used in insect cell culture for recombinant protein production using baculovirus. They were originally established from ovarian tissue. They can be grown in the absence of serum, and can be cultured attached or in suspension.
What is Bac to Bac system?
The Bac-to-Bac baculovirus expression system enables the efficient production of recombinant baculovirus for expression testing in insect cells. The system relies on generation of recombinant baculovirus by site-specific transposition in E. coli rather than homologous recombination in insect cells.
What is the function of baculovirus?
Baculoviruses are insect pathogenic viruses that are used for pest insect control and as tools to produce recombinant proteins. Numerous studies show that both natural baculoviruses and genetically modified (GM) baculoviruses hold clear and substantial benefits for crop protection.
What is mammalian expression system?
Mammalian expression is the system of choice for studying the function of a particular protein in the most physiologically relevant environment, because it allows for the highest level of posttranslational processing and functional activity of the protein.
Can baculovirus infect human cells?
The final phase of infection requires the transfer of the viral genome to the nuclear compartment. We have shown that baculovirus can infect nondividing mammalian cells (Fig. 2 and 3), which implies transport of the viral genome through the nuclear membrane.
Is baculovirus a biocontrol agent?
Baculovirus are biocontrol agents of genus nucleopolyhedrovirus.
What is p10 promoter?
The ubiquitous polyhedrin gene promoter, from which most baculovirus expression vectors are developed, has an oft forgotten rival – the p10 gene promoter. … These sequences remain under the control of the polyhedrin gene promoter to obtain high levels of protein production.
What is a CMV promoter?
The CMV promoter is a commonly used promoter for the production of high level recombinant protein in mammalian cells17. However, the expression level of the transgene driven by CMV promoter decreases with extended culture times because of transcriptional silencing, which is associated with DNA methylation18 ,19.
What is polyhedral occlusion bodies?
Polyhedrins are a type of viral protein that form occlusion bodies (also called polyhedra), large structures that protect the virus particles from the outside environment for extended periods until they are ingested by susceptible insects.
Why is BAC used?
A bacterial artificial chromosome (BAC) is an engineered DNA molecule used to clone DNA sequences in bacterial cells (for example, E. coli). BACs are often used in connection with DNA sequencing. … As the bacterial cells grow and divide, they amplify the BAC DNA, which can then be isolated and used in sequencing DNA.
How do you make a human artificial chromosome?
The first is to create a small minichromosome by altering a natural human chromosome. This is accomplished by truncating the natural chromosome, followed by the introduction of unique genetic material via the Cre-Lox system of recombination. The second method involves the literal creation of a novel chromosome de novo.
What is a pET vector?
pET Bacterial Recombinant Protein Vector. Overview. The pET vector system is a powerful and widely used system for expressing recombinant proteins in E.coli. The gene of interest is cloned into the pET vector under the control of the strong bacteriophage T7 transcription and translation regulatory system.
Where do Sf9 cells come from?
The Sf9 insect cell line is a clonal isolate derived from the parental Spodoptera frugiperda cell line IPLB-Sf-21-AE, and it is a suitable host for expression of recombinant proteins from baculovirus expression systems (e.g., Invitrogen’s Bac-to-Bac® and Bac-N-Blue™ Expression Systems).
What is the meaning of Sf9?
Sensation Feeling 9 The name SF9 means “Sensation Feeling 9,” in hopes that the boys will shock the world with their talent. The Korean word for nine also sounds like the Chinese character (久) meaning length, which points to the group’s desire to last a long time.
How do you count insect cells?
Procedure for Counting Insect Cells
- Mix insect cell culture with trypan blue.
- Pipette 20 µl of the mixture solution into sample introduction port of a disposable counting chamber (CHT4).
- Insert chamber slide into Auto T4 instrument.
- Obtain cell concentration, viability and cell size diameters.
How is baculovirus used as biological control agent?
What are baculoviruses? The baculoviruses used for biocontrol are naturally occurring viruses which are able to infect insects. Once these natural pathogens are ingested by a suitable insect host they reproduce within gut cells of the insect causing it to become ill and die.
Graduated from ENSAT (national agronomic school of Toulouse) in plant sciences in 2018, I pursued a CIFRE doctorate under contract with Sun’Agri and INRAE in Avignon between 2019 and 2022. My thesis aimed to study dynamic agrivoltaic systems, in my case in arboriculture. I love to write and share science related Stuff Here on my Website. I am currently continuing at Sun’Agri as an R&D engineer.