BEVS + Protein expression (BEVS)

Question 1

What are the four steps to protein production?

Answer 1

1) cDNA synthesis.
2) Expression in the expression system.
3) Protein purification.
4) Protein characterisation.

Question 2

What is BEVS an acronym for?

Answer 2

Baculovirus Expression Vector System

Question 3

What cells are used in the baculovirus expression vector system (BEVS)?

Answer 3

Insect cells.

Insect cells grow easily in suspensions, they are easy to cultivate.

Question 4

What enzymes do you use to detach cells from their base layer?

Answer 4

1) Normally, Trypsin
2) If multiple cell layers, trypsin + collagenase
3) If they should detach as sheets, dispase

Question 5

Why should you use freezing vials when freezing at -150?

Answer 5

1. They’re being frozen in liquid nitrogen, it may leak in.
2. The tubes may blow up in ya face if they are heated quickly.

Question 6

Flip for 2 random BEVS facts

Answer 6

1. 600 different BEVS can infect insect cells.
2. Lipid envelope which is covered in a polyhedren (protein matrix).

Question 7

Explain the BEVS life cycle, where do genetic inserts come into play?

Answer 7

1. The virus infect cells via receptors on lipid envelope. (GP64 is vital for the entry into the cell)
2. The genome is translocated to the nucleus.
3. Early genes are activated, viral genome is amplified.
4. Budded viruses (BV) are produced. These are genomes encompassed in the cell’s membrane.
5. Late genes are activated, and cells apoptotize 24h after infection.
6. Polyhedren is produced (survival mode).

The inserts exchange the genes encoding the polyhedron. Activated as cells die.

Question 8

What does ‘MN’ stand for in AcMNPV?

Answer 8

Multiple nuclei.

Question 9

Name the most important protein forviral proliferation.

Answer 9

GP64.

Question 10

How do you incorporate genetic information in the BEVS genome? (there’s another more detailed question)

Answer 10

Homologous recombination. The efficiency is low (P = 0.001).

Question 11

What distinguishes FlashBac compared to general baculoviruses.

Answer 11

It infects e.coli.

Question 12

Explain the BacToBac BEVS system.

Answer 12

1. Special strand of e.coli includes a BacMid (artificial bacterial plasmid).
2. Donor plasmid is designed with two transposition domains, a promoter, and the gene of interest.
3. The donor plasmid is transformed into e.coli.
4. The corresponding transposition sites for the plasmid are located inside of the lacZ gene (which is only expressed in the BacMid, not in the e.coli genome). The contents within the transposition borders are transfected into the bacMid. With dysfunctional LacZ, there’s no beta-galactosidase, meaning that the bacteria can’t metabolize X-gal. The bacteria remains transparent, if LacZ is functional, the bacteria is blue.
5. Blue/white screening w/ LacZ / beta-galactosidase.
6. Isolate the BacMid from selected colonies.
7. Transfect insect cells with DNA.
8. Viruses propagate

Question 13

Explain the FlashBac system.

Answer 13

1. Co-transfect flashBac vector (containing the entire viriome) with transfer vector into e.coli.
2. The flashBac vector importantly includes the lef2 gene, the BAC gene and a dysfunctional ORF1629. lef2 and dysfunctional ORF1629 will allow for homologous recombination of whatever gene is in the place of the BAC gene in the transfer vector.
3. There is homologous recombination due to homology in flanking regions (lef2, ORF1629). The recombination repairs ORF1629 and inserts the gene of interest. W/o ORF1629, the virome won’t be able to propagate.
4. Transfect insect cells –> Upscale cultures.

Question 14

What do you use plaque assays for? How are they performed?

Answer 14

Finding the amount of infections in cultures.
1. Infect cells
2. Immobilize by adding agarose layer.
3. 3-6 day incubation
4. Stain living cells.
5. Find plaques of dead bacteria (small dots).

Question 15

Explain the general BEVS workflow.

Answer 15

1. Generate donor plasmid containing gene of interest.
2. Generate viruses using FlashBaq or BaqToBaq.
3. Determine the virustiter (the amount).
4. Testing and analysis.

Repeat the workflow in bigger culture.

Question 16

What’s the general principle behind multiBac?

Answer 16

Multiple plasmids can be combined to one plasmid, then inserted into a single BEVS. 22 proteins can be produced at once.