bacterial transformation lab just don’t transform them into sharks. that would be scary
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Bacterial Transformation Lab
Bacterial Transformation LabJust dont transform them into sharks. That would be scary.IntroductionSo heres the idea:Bacteria are really good at collecting DNA.So good, in fact, that if you put them near some bits of DNA, theyll 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.IntroductionIn this lab, were 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.Thats 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 ExperimentHow will we know theyre genetically altered?In this case, theyre going to glow under a UV light, since were going to try transforming them with a gene on a plasmid called pGLO.The p is for plasmid, by the way.About pGLOpGLO encodes a gene that makes GFP. Whats 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.
The pGLO Plasmid
Origin of Replication[thats where it starts copying]GFP GeneAmpicillin Resistance GeneArabinose OperonPlasmids and Bacteria
The Overall ExperimentTo be clear, were going to transform bacteria with a plasmid that makes them:Resist ampicillin antibiotic.Glow if they have arabinose sugar.Well grow them in the following environments: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?How were going to do thisStart by labeling two microtubes:+pGLO and pGLO [and your group names]Use a sterile pipet to move 250 L 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 dont 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 PipetsThough theyre not as adjustable and fancy as the reusable micropipettes, they do have important marks:
How were going to do thisGet 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 NOTEWe 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 were going to do thisNext, check to see if your bacteria glow using the UV light.They shouldnt, 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 youve given your bacteria an extracellular gene. Will they take it up? Well see.How were going to do thisTubes go back on ice now for 10 minutes. While theyre in thereget four nutrient agar plates (mini petri dishes).Label these on the bottom as the following:LB/amp: +pGLOLB/amp/ara: +pGLOLB/amp: pGLOLB: pGLOSo you need an LB plate, two LB/amp plates, and one LB/amp/ara plate.LB = agar; amp = ampicillin; ara = arabinose.About LBFYI: 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 Lurias first graduate student was James Watson.Heard that name before?Back to How were going to do thisNext, heat shock your samples.What?I said heat shock your samples.Why?Because I said so.No, really. Why?BecauseI dont know.Seriously, the mechanism of heat shock isnt 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 were going to do thisSo, heat shock your samples by QUICKLY putting them in the water bath (42C or 107.6F) 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 L of LB broth to the tubes.This is bacteria food.Its like taking a shower in a smoothie or something.Use sterile pipets for each transfer as usual.
How were going to do thisLet your samples sit at room temperature for another 10 minutes.During this time, the bacteria have time to grow and express their genes.How were going to do thisNow that weve given the bacteria time to take up the plasma and grow a little bit, its 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 L of each tube to their own agar plates for growth.100 L to each of the four plates.
How were going to do thisUsing 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 itll puncture the agar and thats bad.Once youre done, stack all the plates, tape them together, and label the bottom of the stack with your names.Theyll go in the incubator overnight.The incubator is set at 37C or 98.6F for optimal bacteria growth.CleanupDay 1 is now done.Dispose of all non-sterile materials.Keep the foam blocksData Analysis [Day 2]With the lights off and while using the UV light, view your samples.Draw your results.I know, its hard to draw glowing.Draw the colonies instead, showing which plates had how much growthCool, huh?Other MediaFranklin and Marshall Evolution Lab PhotosBioluminescent Bacteria Christmas Card