Transcript Bacterial Transformation Lab

Bacterial Transformation Lab
Just don’t transform them into
sharks. That would be scary.
Introduction
• So here’s the idea:
– Bacteria are really good at collecting DNA.
– So good, in fact, that if you put them near some bits
of DNA, they’ll pick it up and incorporate that DNA
into their cells.
– They have a single circular chromosome that is usually
not involved here, but they also keep a lot of DNA
separate from it.
– That DNA, kept separate from the chromosome but
still used, is in the form of plasmids.
• Plasmids are small loops of DNA used like any other DNA.
Introduction
• In this lab, we’re going to see if we can transform a
sample of bacteria – that is, get them to take up some
DNA of our choosing and express those genes.
– That’s right, you are actually genetically altering a living
organism. Enjoy.
• Which organism?
• E. coli. Yep.
– Most E. coli is harmless, as is this one.
– This E. coli is strain HB101 K-12, which can only grow on
enriched growth media.
– The one that sometimes causes illness is O157 H7.
The Overall Experiment
• How will we know they’re genetically altered?
• In this case, they’re going to glow under a UV
light, since we’re going to try transforming
them with a gene on a plasmid called pGLO.
– The “p” is for plasmid, by the way.
About pGLO
• pGLO encodes a gene that makes GFP.
What’s GFP?
– Green Fluorescent Protein, in this case
found naturally in the jellyfish Aequorea
victoria.
• In addition, the pGLO plasmid also has
a gene for resistance to ampicillin, an
antibiotic (it kills bacteria).
• Finally, there is also an operon in there
that regulates the whole thing.
– Arabinose sugar activates the operon that
makes GFP and ampicillin resistance.
– Much like lactose induces the lac operon.
http://voices.nationalgeographic.com/files/2012/04/Aequorea-477x700.jpg
The pGLO Plasmid
Arabinose Operon
Origin of Replication
[that’s where it starts
copying]
GFP Gene
Ampicillin Resistance
Gene
Plasmids and Bacteria
The Overall Experiment
• To be clear, we’re going to transform bacteria with a
plasmid that makes them:
– Resist ampicillin antibiotic if they have pGLO.
– Glow if they have pGLO and arabinose sugar.
• We’ll grow them in the following environments:
1.
2.
3.
4.
Just nutrient agar (no pGLO plasmid).
Nutrient agar with ampicillin (no pGLO plasmid).
Nutrient agar with ampicillin (with pGLO plasmid).
Nutrient agar with ampicillin and arabinose (with pGLO
plasmid).
• Which one(s) would you expect to glow and resist
ampicillin?
The Expected Results
• Obviously, we’d only expect bacterial growth on
antibiotic-laced plates if they’ve taken up the
plasmid that allows them to be resistant.
• This should closely mimic what happens “in the
real world,” when bacteria mutate to develop
resistance to antibiotics and then spread across
antibiotic-treated regions.
• Here’s a related experiment:
– The Evolution of Bacteria on a Mega-Plate Petri Dish
How we’re going to do this…
• Start by labeling two microtubes:
• +pGLO and –pGLO [and your group names]
• Use a sterile pipet to move 250 μL of transformation
solution (CaCl2) into each microtube.
– This is not a micropipet – these are the disposable kind and
should be discarded once used.
– Why CaCl2? The thinking is that the calcium cations (Ca2+)
help to neutralize the negatively-charged DNA molecule.
– Remember, polar molecules don’t enter cells well. Neutralize
the polarity and DNA can enter more easily.
• Put the tubes on ice in a foam tube holder.
A Note on Pipets
• Though they’re not as adjustable and fancy as
the reusable micropipettes, they do have
important marks:
How we’re going to do this…
• Get an inoculating loop – you may have seen these
in earlier labs – and scoop out one colony of bacteria
from the starter plate.
– The colonies are like little circular pepperoni on your
starter plate pizza.
– One colony represents millions of cells. Getting more
than one colony is actually worse.
• Move the colony into the transformation solution
currently on ice inside your microtubes.
• Spin the loop in your fingers to “blend” the solution
– no chunks!
– Do this procedure twice – once for each microtube –
using a sterile inoculating loop each time.
IMPORTANT NOTE
• We need to avoid contamination at all costs.
• Since other bacteria are everywhere (especially on
you), make sure you never touch the end of the
inoculating loop, the tip of the pipet, or the agar gel
plate.
– That includes not putting it on your lab table or on any
surfaces.
• For the record, E. coli makes small circular colonies.
– Other bacteria grow in different patterns. Keep that in
mind.
How we’re going to do this…
• Next, check to see if your bacteria glow using the
UV light.
– They shouldn’t, obviously, but do it anyway.
– UV light is damaging to skin/eyes. Use it sparingly.
• Get another sterile inoculating loop and transfer
plasmid solution into just the +pGLO microtube.
– It should look like soap film across a bubble wand.
• You know, from your childhood.
– So now you’ve given your bacteria an extracellular
gene. Will they take it up? We’ll see.
How we’re going to do this…
• Tubes go back on ice now for 10 minutes. While
they’re in there…
• …get four nutrient agar plates (mini petri dishes).
• Label these on the bottom as the following:
•
•
•
•
LB/amp: +pGLO
LB/amp/ara: +pGLO
LB/amp: –pGLO
LB: –pGLO
• So you need an LB plate, two LB/amp plates, and one
LB/amp/ara plate.
• LB = agar; amp = ampicillin; ara = arabinose.
About LB
• FYI: LB is a mix of liquid/solid nutrients called
Lysogeny Broth. It was also created by
Giuseppe Bertani and used notably by
Salvador Luria for a fun coincidence in
lettering.
• Also interesting: Salvador Luria’s first graduate
student was James Watson.
– Heard that name before?
Back to
How we’re going to do this…
• Next, heat shock your samples.
– What?
• I said “heat shock your samples.”
– Why?
• Because I said so.
– No, really. Why?
• Because…I don’t know.
• Seriously, the mechanism of heat shock isn’t
understood, but the duration of heat shock is
essential to this experiment working.
• Either way, it is thought to make the membrane more
permeable.
How we’re going to do this…
• So, heat shock your samples by QUICKLY putting
them in the water bath (42°C or 107.6°F) for exactly
50 seconds.
• Immediately afterward, move them back to ice
QUICKLY for two minutes.
• Take out the tubes, then use a sterile pipet to
transfer 250 μL of LB broth to the tubes.
– This is bacteria food.
– It’s like taking a shower in a smoothie or something.
– Use sterile pipets for each transfer as usual.
How we’re going to do this…
• Let your samples sit at room temperature for
another 10 minutes.
– During this time, the bacteria have time to grow
and express their genes.
How we’re going to do this…
• Now that we’ve given the bacteria time to
take up the plasma and grow a little bit, it’s
time to transfer them to their new homes.
– The nutrient agar plates.
• Tap the tubes with your finger to mix them up
and resuspend everything.
• Transfer 100 μL of each tube to their own agar
plates for growth.
• 100 μL to each of the four plates.
How we’re going to do this…
• Using a sterile inoculating loop for each plate, spread
the solutions you just transferred around each plate.
– Be gentle!
– Agar is soft. If you push too hard it’ll puncture the agar
and that’s bad.
• Once you’re done, stack all the plates, tape them
together, and label the bottom of the stack with your
names.
– They’ll go in the incubator overnight.
– The incubator is set at 37°C (98.6°F) for optimal bacteria
growth.
Cleanup
• Day 1 is now done.
• Dispose of all non-sterile materials.
– Keep the foam blocks…
Data Analysis [Day 2]
• With the lights off and while using the UV light,
view your samples.
• Draw your results (observations or results section).
– I know, it’s hard to draw “glowing.”
– Draw the colonies instead, showing which plates had
how much growth…
• Cool, huh?
Other Media
• Franklin and Marshall Evolution Lab Photos
• Bioluminescent Bacteria Christmas Card
Questions to Answer
• The most important questions to answer are those
in the instructions after the procedure:
– Lesson 2: Review Questions
• 1-3
– Lesson 3: Data Collection and Analysis
• 1-3
– B: Analysis of Results
• 1-4
– Lesson 3: Review Questions
• 1-3
• In other words, all the questions on pages 7 and 8.