BacPAK6, Bac-N-Blue, BaculoDirect, Bac3000 (Nov
agen), MultiBac (ETH Zurich/Redbiotec AG), flash
back, flash BACGOLD, AND
Bac-to-Bac
Baculovirus
 A family of large rod-shaped viruses
 Circular double-stranded genome ranging from 80-180 kbp.
 AcMNPV
: Autographa califonica multiple nuclear polyhedrosis virus
: the best characterized and undergoes a succession of early,
late and very late gene expression during its infection cycle.
: the strong polyhedrin promoter-> the transcriptional
control.
Bac-to-Bac Baculovirus Expression System
 An efficient site-specific transposition system to generate
baculovirus for high-level expression of recombinant proteins
 Advantage
1. recombinant virus DNA isolated from selected colonies is
not mixed with parental, nonrecombinant virus
- Easy colony screening : lacZα gene (Bluo-gal or X-gal)
2. Time-saving expression to identify and purify a
recombinant virus
Bac-to-Bac Baculovirus Expression System
 Used vectors:
1. Donor plasmid vector into which the gene(s) of
interest will be cloned
2. Baculovirus shuttle vector (bacmid)
: mini-attTn7 target site, lacZ, kanamycin resistance
marker
3. Helper plasmid
: transposase, tetracycline resistance marker
pFastBac1Transfer vector, donor plasmid
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Multiple cloning site
SV40 polyadenylation signal
Tn7L
f1 origin (f1 intergenic region)
Ampicillin resistance gene
pUC origin
Tn7R
Gentamicin resistance gene
(complementary strand)
 Polyhedrin promoter (PPH)
7
8
Bacmid -baculovirus shuttle vector
<bacmid>
•kanamycin resistance marker
• LacZ-mini-attTn7
AcMNPV bacmid
(136kb)
LacZ-mini-attTn7
Kan
( a short segment containing the attachment site for
the bacterial transposon Tn7)
mini-F
• a low-copy-number miniFertility replicon
Ppolh
Tn7R
SV40 P(A)
gusA
Gen
Donor plasmid(pFastBac)
(6.7kb)
Tn7R
<donor plasmid>
• Multiple cloning site
• SV40 polyadenylation signal
• Tn7R(bacteria transposon)
• gusA
• Ppolh(promoter)
• Gentamicin resistance gene
site-specific transposition
transposed donor vector
sequence
Bacterial primer sequence
Transposon & transpostion
 Sequences of DNA that can move around to different positions within the
genome of a single cell.
 A process called transposition
 Transposase: an enzyme
 Inverted repeats(IR): a sequence of nucleotides that is the reversed
complement of another sequence further downstream.
Diagram of the Bac-to-Bac system
“Bac-to-Bac” Flow Chart
pFastBac donor plasmid
Clone gene of interest
pFastBac Recombinant
Transform into E.coli DH10Bac
E.Coli Colonies +Rec Bacmid
Grow overnight culture
Isolate Rec. Bacmid DNA
(do not freeze&thaw)
Transfection into insect cells
Rec. Baculovirus
Analyzing Recombinant Bacmid DNA
 analyze recombinant bacmid DNA using PCR
 Note: It is possible to verify successful transposition to
the bacmid by using agarose gel electrophoresis to
look for the presence of high molecular weight DNA.
This method is less reliable than performing PCR
analysis as high molecular weight DNA can be difficult
to visualize.
Transfection
Transfecting Insect Cells with Baculovirus DNA, procedure to transfect Sf9 insect cells in a
6-well format . You will need log-phase cells with >95% viability to perform a successful
transfection.
 1. Plate 5 x 105 Sf9 cells in 2 ml of Insect medium containing antibiotics. Allow cells to
attach for at least 1 hour.
 2. For each transfection sample, prepare complexes as follows:
 a. Dilute 1-2 μg of baculovirus DNA in 100 μl of Insect medium without antibiotics.
 b. Mix Cellfectin® before use, then dilute 1.5-9 μl in 100 μl of Insect medium without
antibiotics.
 c. Combine the diluted DNA with diluted Cellfectin® (total volume = 200 μl).
 Mix gently and incubate for 15-45 minutes at room temperature (solution may appear
cloudy).
 3. Remove the growth medium from the cells and wash once with Insect medium without
antibiotics. Remove the wash medium.
 4. Add 0.8 ml of Insect medium to the complexes (Step 2c), mix gently and add to the
cells. Incubate cells at 27°C for 5 hours.
 5. Remove the transfection mixture and replace with 2 ml of Insect medium containing
antibiotics. Incubate cells at 27°C for 48 hours.
 6. Harvest virus at 48-72 hours post-transfection.
Signs of Infection Phenotype Description
 Increased cell diameter , 25-50% increase in cell diameter
 Early (first 24 hours) Increased size of cell nuclei may
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appear to "fill" the cells.
Stop of cell growth compared to a cell-only control
Late (24-72 hours)Granular appearance, Signs of viral
budding; vesicular appearance of cells
Detachment Cells release from the plate or flask
Very Late (>72 hours) Cell lysis, signs of clearing in the
monolayer
Sf9 control cells
Tranfected sf9 cells
Preparing the P1Viral Stock
 1. Once the signs of late stage infection (e.g. 72 hours post-transfection)
occurred, collect the medium containing virus from each well (~2 ml) and
transfer to sterile 15 ml snap-cap tubes.Centrifuge the tubes at 500 x g for 5
minutes to remove cells and large debris.
 2. Transfer the clarified supernatant to fresh 15 ml snap-cap tubes. This is
the P1 viral stock. Store at +4ºC, protected from light.
 3. amplify the p1 viral stock and prepare p2 and p3 viral stock(low MOI)
 Note: If you wish to concentrate your viral stock to obtain a higher titer,
you may filter your viral supernatant through a 0.2 µm, low protein binding
filter after the low-speed centrifugation step, if desired.
Storage information
• Store viral stock at +4ºC, protected from light.
• If medium is serum-free ,add fetal bovine serum to a final
concentration of 2%. Serum proteins act as substrates for
proteases.
• For long-term storage, store an aliquot of the viral stock at
-80ºC for later reamplification.
• Do not store routinely used viral stocks at temperatures below
+4ºC. Repeated freeze/thaw cycles can result in a 10- to 100fold decrease in virus titer.
• Plaque purify your baculoviral construct, if desired
Expression of Recombinant Protein
 Once you have generated a baculoviral stock with a
suitable titer (e.g. 1 x 108 pfu/ml), you are ready to use
the baculoviral stock to infect insect cells and assay for
expression of your recombinant protein.
Analyzing Protein Expression
To detect expression of your protein by western blot
analysis, you may use a Monoclonal antibody to your
protein of interest.
Purifying Recombinant Protein
 You may use any method of choice to purify your
recombinant protein of interest.
 Note: If you have cloned your gene of interest in frame
with the 6xHis tag, you may purify your recombinant
protein using a metal chelating resin such as ProBond or
Ni-NTA .
<Principle of rapid generation of multigene baculoviruses>
•
Two transfer vectors, each expressing two genes
•
recombine with the baculovirus genome
maintained in E. coli as a bacmid.
•
Two genetic loci in the baculovirus genome are
used,
•
recombination is controlled by Tn7 transposition
•
the bacmid DNA is transfected into insect cells,
where it directs expression of all four genes at
high levels.
•
The improved expression characteristics of the
eukaryotic expression system promote assembly
of correctly processed and folded tetrameric
protein complexes.
<Baculovirus life cycle>
Presented by:
Dr. Fotouhi
Virologist
Insects & Insect cells
 Baculovirus infects lepidopteran insects (butterflies )
and insect cell lines
 Commonly used cell lines are sf9 & sf21 derived from
the pupal ovarian tissue of the fall army worm
spodoptera frugiperda and high five derived from the
ovarian cells of the cabbage looper
Insect Medium
 Grace’s Insect medium- unsupplemented but contains L
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glutamine
Grace’s Insect medium supplemented-contains additional TC
yeastolate & Lactalbumin hydrolysate
Trichoplusia ni Medium formulation hink (TNM-FH)
Serum Free Medium(SFM)
Aditives: FBS 10-20%, L-Glutamine, non-essential amino
acids, antibiotics(penecillin 100 unit/ml, streptomycin 100
ug/ml), antimycotic (fungine 10-50 ugml)
Requirements for proper cell culture
 Temperature- Optimal range is 27-28 C
 pH- Optimal range is 6.1 to 6.4
 Aeration-Requires passive 02 diffusion for optimal
growth & recombinant protein expression
 Osmolality- Optimum is 345-380 mOsm/kg
 FBS- Working with suspension culture it is
advisable to use (10-20% FBS) to gave protection
from cellular shear forces
Biological Hood
Types of cell culturing
 Monolayer culture
 Suspension culture
Disk/flask
Seeding density of cells
Seeding density
(cell number)
Vol. of medium
(ml)
35mm dia. Petri dish
1x 105
1.5-2
60mm dia. Petri dish
2 x 105
3-4
25 cm2 flask
1x 106
4-5
75 cm2 flask
.5-1 x 107
10
Spinner culture
1-2 x 105/ml
40-500
Methods of sub culturing adherent cells
 Three methods to dislodge monolayers in adherent cell
culture
- Pipeting
- Tapping the layer
-Trypsinizing
Procedure of monolayer sub culture
 Monolayer should reach to confluency in 2-4 days.
 Aspirate medium & floating cells from a confluent
monolayer & discard them.
 Add 4ml of RT complete growth medium to each 25cm2
flask(12 ml to a 75 cm2 flask)
 Resuspend cells by pipetting the medium across the
monolayer with a Pasteur pipette. (Enzymatic
dissociation is not recommended)
 Observe cell monolayer using an inverted microscope to
ensure adequate cell detachment
Contd..
 Perform viable cells count on harvested cells.
 Inoculate cells at 2 x 105 viable cells/ml into respective culture
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vessels.
Inoculate cultures kept at 25-28 C
On day 4 post-planting, aspirate the spent medium from one side
of the monolayer & subculture the flask
With slower growing cell lines, it may be necessary to feed the
flasks on day 3-4 post planting
Subculture the flasks when the monolayer reaches 80-100%
confluency, approx 2-3 days post planting
Working with suspension culture
 Insect cells are not generally anchorage dependent & can be well
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adapted to suspension culture
Prior to establish a spinner culture, cells are maintained firstly as
healthy adherent cells.
Use a spinner flask with a vertical impeller
Culture volume should not exceed half of the volume of the flask
Use of surfactant to decrease shearing e.g. Pluronic F-68
Contd..
 Not necessary to change medium regularly. Sub culturing requires
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the removal of cell suspension & the addition of medium
Impeller should be rotating regularly
Impeller should be submerged 1 cm or more to ensure adequate
aeration
Cell viability of 95% is required
Minimum density of 1 x 106 cells/ml is required
Contd…
 Keep record of the passage number. After 30 passage or more (2-3
months), cells doubling time increased and also loose their
viability and infectivity.
 Keep a cell log, to do so one should have a knowledge of following;
date of initiation of culture, lot number date of passage &
passage number density & viability at passage comment on cell
appearance medium & its lot number
Initiation of culture with freezed
cells
 Thaw the frozen suspension rapidly in a water bath at 28 C
 Seed the cells into a culture flask (1 x 106) containing
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medium 5 ml TC 100 medium
Incubate at 28 C for 5 hrs
Change with fresh medium
Incubate again, until it reach confluence
Subculture it for experimental purpose
Cryopreseravtion of cells
 Freezing cells should be 90% viable and 8090%confluent
 Freezing medium should have 60% Grace’s
insect medium supplemented with 30%FBS &
10% DMSO
Procedure
 Count cells using haemocytometer
 Placed cryovials on ice & label them
 Centrifuge cells at 400-600 g for 10 mts at RT. Remove the
supernatant
 Resuspend the cells to the given density in the freezing
medium
 Transfer 1 ml of the cell suspension to sterile cryovials
 Place at -20 C for 1 hr then transfer to -80 C for 24-48 hrs &
then finally store at Liquid nitrogen
Do and Don'ts
 Check cells daily until a confluent monolayer is formed.
 Passage cells at confluency only, as cells will be easy to
dislodge & shows better viability
 Do not overgrow cells, it results in decreased viability
 Do not splits cells too for. Densities lower than 20%
confluency inhibit growth
 Passage the cells only in log phase, log phase growth can be
maintained by splitting cells in 1:5 dilution
Basic aseptic conditions
 If working on the bench use a Bunsen flame to heat the air
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surrounding the Bunsen
Swab all bottle tops & necks with 70% ethanol
Flame all bottle necks & pipette by passing very quickly
through the hottest part of the flame
Avoiding placing caps & pipettes down on the bench;
practice holding bottle tops with the little finger
Work either left to right or vice versa, so that all material
goes to one side, once finished
Clean up spills immediately & always leave the work place
neat & tidy
Contd..
 Possibly keep cultures free of antibiotics in order to be able
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to recognize the contamination
Never use the same media bottle for different Insect cell
lines. If caps are dropped or bottles unconditionally
touched, replace them with new ones
Necks of glass bottles prefer heat at least for 60 secs at a
temperature of 200 C
Switch on the laminar flow cabinet 20 mts prior to start
working
Cell cultures which are frequently used should be
subcultered & stored as duplicate strains