1.
BACULOVIRUSES are natural viruses that infect
insects in the wild
But we can “unnaturalize” them to turn them into custom
protein-making factories
arly in the infection, the cells make and secrete budded viru
They use this to jump into other cells in the same insectBut the insect gets really sick and can’t support the virusThey have circular, double-stranded DNA genomesTheir goal is to get into insect cells to make more of
them. And they can do this a couple of ways

2.
starts making a sturdier form of the virus that will be able to survive once t

3.
and they can bud fine without it
so they don’t make it until they need it
they only need POLYHEDRIN late in the infection
that sturdy shell’s made up of a protein called
POLYHEDRIN (and some other stuff)
especially since they need a TON of it
to make the occlusion bodies
early infection
very late infection
cell hopping host swapping
so they have to basically devote all
their time & resources to making it
and then they make a TON of it
PolH gene
PolH promoter

4.
we replace the polyH gene with a
gene for our protein
now the cells start making our protein
when they “think” they’re making
polyhedrin
which they only do in the very late phase
but we still need to get it into the cells…
so we have an unneeded protein
in prime spot for expression…

5.
the bacmid DNA needs the coat to get into cells on
its own
+ transfection reagent
some cells take in the
bacmid and start making
budded virus
it has the coat so it can get into the uninfected
cells
and then those can start
making & excreting
virus into the liquid
they’re in
we let the cells make lots of virus
bacmid
we use cells that are
from insects, but we
grow them in dishes
and later flasks or
bags
ated cells because it’s harder to get the bacmid in. late
so we have to help it out

6.
collect the secreted virus
oh no! a cell got in
no worries - we’re going to syringe
filter it
this is the V0. you can keep it in
the fridge until you’re ready to
use it
V0
use it for what? making more
virus!

7.
the virus gets into the cells
we want to collect that virus.
but we need to separate it from
the cells. but since the cells
aren’t stuck down, we need to
centrifuge them to pull them
down
take that V0 & add it
to some cells in a flask
now those cells start making virus
o a lot of virus. so the next step is to make more. but no
V1

8.
there’ll be some
protein in here, so you
can keep the pellet if
you did a biggish V1
but the big yield’s still
to come!
centrifuge to separate
the cells from the virus
what we’re after:
coated virus
V1

9.
use that V1 to infect lots of cells
what we’re after:
protein-stuffed cells

10.
do the same spin -down
but this time focus on the
pellet
what we’re after:
protein-stuffed cells