A few months ago, I was invited by Sara Wylie to talk to some students at RISD about DIY-Bio, specifically, I was invited to show them how I did my DIY transformation a few months ago.  It was an AWESOME experience, and a lot of interesting topics came up in the area of ethics, and most importantly who should be allowed to practice or regulate biology and biotechnology, which are controversial and important.  And too big for this post, which will be about another exciting transformation!

We used the same procedure, as described before in my transformation post.  I wasn’t around in RI for the next week to check on the plates, but Sara sent me some pictures.  It is interesting that this transformation has the same problems as the previous transformation.

Again, there are what look like contaminants/giant satellite colonies.  Again, the GFP expression is very subtle.  I am curious about what the non-green colonies could be.  Maybe the incubation to express the ampicillin resistance is too long, and the b-galactosidase is breaking down the ampicillin when the cells are plated.  I used the same MM294 strain of e. coli and the same pGREEN plasmid as I did last time, but both were ordered fresh from Carolina Biological.  If I don’t see anything that would explain this in my research on the MM294 and satellite colonies, I will probably try this with a different strain.  Maybe that will be a BOSSLAB project for next year (if you are near Boston, and want in, send me a message!).  Anyways, here is a picture of the same plate under a blue LED.

Another protip I have from this, and from yesterdays experiment (TSS transformation) is that you should pour your plates way before you intend to spread on them.  This lets them dry out a little bit before you add a bunch of liquid do them.  You can even put them in an incubator to dry them out a little!  We had some difficulty getting the plates at RISD to absorb 100ul of transformants in broth.  Yesterday a plate I had easily absorbed 300ul of transformants and broth, which was poured the day before, and then pre dried in an incubator for 10-15 minutes.  Just keep an eye on them and don’t let them dry out!  By drying them you can also avoid the ring of growth around the outside as seen below.  This seems to be because bacteria get in condensation/broth/wetness on the plate, and then roll around on the plate.  Surface tension keeps it stuck between the plastic wall and the agar, creating the ring.

Eeew!  So keep your plates dry.  But not too dry.

These bacteria were eventually used in one of the final class projects, here.  There is a pretty swanky picture of a bacto-QR code on that page, definitely check it out.

Coming soon: Dinoflagellates and MORE transformations.

A few months back I worked out how to do a bacterial transformation, and then I (successfully) preformed the procedure.  I did this at BOSSLAB, which is in Sommerville, MA.  If you are in the area, check it out!  This procedure is memorialized in the BOSSLAB lab notebook, which I will scan and upload relevant portions of to the downloads page, but one of the goals of BOSSLAB (??? at best) seems to be to communicate what we do there to the world.  This is my writeup on what I did and how I did it.

What is Transformation?

Transformation is the process of adding genetic material to a bacteria.  The phrase comes from an experiment by Fredrick Griffith in 1928, where he discovered that non-pathogenic strains of Strep. pneumoniae could be “transformed” into pathogenic strains.  This was done by adding dead (heat-killed) pathogenic Strep. pneumoniae to the non-pathogenic strains.  When this mix was injected into mice, the mice died.  Later, the Avery-MacLeod-McCarty experiment showed that DNA caused this transformation.

For bacteria, the most convenient way to add genetic material is with plasmid DNA, which is a little circle-shaped piece of DNA.  There are a few really important things to know about choosing a plasmid, most of which I (fortunately) got to gloss over.

As you can see on the right hand diagram, there are three major parts to a plasmid.  An origin of replication, which is dependent on the target organism (ori-> on the left diagram).  This is where the cell begins the replication of the plasmid, if it is going to make more.  Another important factor in plasmid selection is some kind of selection marker (ampr on the left diagram).  Both of these plasmids use an antibiotic resistance marker.  This is important so that when you have all the transformed and not transformed bacteria mixed up, you can selectively kill all the ones that do not have the antibiotic resistance gene.  The final consideration are the regions that can be cut by restriction enzymes, which are also called restriction enzyme digest sites.  These are used to cut the plasmid open, so you can add genes to an “empty” plasmid vector.  When you have a lot of them together, near each other, it is generally called a polylinker or multiple cloning site.  On the left hand diagram, if you “digested” (cut) the plasmid with EcoR1, you would have two EcoR1 compatible ends on the plasmid.  You could then add a gene with EcoR1 compatible ends.

For me, all I did was buy the plasmid on the left.  The goal was to successfully complete a transformation, not do ligate a gene into a plasmid.  That might be the next step, but trying to do it as an initial foray into genetic engineering or running my own protocol completely independently would add too many uncertainties to the protocol and make it impossible to debug with the materials I had.  For you embedded folks, this would be like trying to hand code assembly with no debugger and no verification of the program memory.

How do you do it?

Bacterial transformation is tricky.  You can put plasmid in a tube with some bacteria and swish it around all day, and probably nothing will happen.  Thats because the outside of bacteria is a membrane which keeps things that should be out, out, and things that should be in, in.  Basically it’s like their skin.  And the plasmid needs to go through it.  To do this, we have to break up the skin so tiny pores open.  When bacteria are in this state, they are called competent.

There are various recipes for making chemically competent cells.  I used CaCl salts, but I hear tell that TSS (Transformation Storage Solution paper by Chung et al. here) is more efficient.  Unfortunately, I had no success with TSS because of (probably) a snafu with a thermocouple, and I didn’t have time or money to do it again (see what went wrong).  Both of these solutions have the same effect; the positive ions in the solution neutralize the (negative) charge on the membrane, and the negative charge on the outside of the DNA.

Once the cells are competent, and chilled, DNA is added.  Later, they are “heat shocked”, which (possibly?) expands the holes in the membrane and allows the DNA to enter the cell.  Afterwards, they are allowed to recuperate, and then a tiny amount of bacteria are spread onto a plate with the antibiotic.  The goal here is that the bacteria without the plasmid will die, leaving you colonies of the transformed bacteria (transformants).

Results (and what went wrong)

First, a picture!

Although I got the procedure to work, I had less than stellar results.  I attempted transformation twice.  The first round had five trials, all of which completely failed.  Some of these used TSS and some of these were CaCl.  The reason these failed is probably because the water bath was not hot enough.  Since we didn’t have a temperature controlled water bath, we used a large beaker of water on a hot plate instead.  The idea is that we would heat it up to about the right temperature (42C) and then turn it off.  since water retains heat pretty well, we thought it would stay near enough to 42C to be used for the transformation.  HOWEVER, on my second attempt at transformation (only two trials this time!) I was measuring the temperature of the water with the same thermocouple we had used before…and I discovered that it was VERY wrong.  I believe the thermometer was on the low side, reading -20C in ice water, so the water for the heat shock was probably around 60C-way too hot.  When I discovered this in the second trial, I had to very quickly figure out a way to get some 42C water!  My bacteria were incubating and I was eager to get on with the heat shock.

I knew from making antibiotic plates that at about 50C, it is safe to add antibiotic and that the media will be hot, but bearable to hold.  So I heated some water until it was uncomfortable to touch, and then cooled it down some.  Estimating that my body temperature was 35, I knew the water would feel very warm, but not too warm to touch.  Using this, I guesstimated that I had a good temperature and preformed the heat shock.

All of this can be avoided by buying a (literally) $3 thermometer from the dollar store.

Another important step that I forgot to figure out before I began was how to verify the GFP was being produced.  It turns out that this variant of GFP fluoresces under blue light.  Since there were batteries and a few blue LEDs handy, I used those.

The other, sneakier problem, was the lack of antibiotic efficiency.  This caused satellite colonies (or maybe just amp-R colonies) to proliferate over the plate.  I have a few theories about why this could have happened:

1. There is some sort of ampR contaminant that got moved from plate to plate
2. The antibiotic was bad.  I tested it before the first attempt, but it could have gone bad in the mean time (sketchy fridge is sketchy), or it could have been damaged in pouring.  I didn’t have enough plates to test if the plates I poured worked, and to do all the transformations.
3. The bacteria incubated too long, and they destroyed enough ampicilin locally to  allow satellite colonies to form.

I think a combination of two and three are most likely, particularly two.  In the first set of trials I also saw slow growth, but no GFP.  This would indicate that there is something wrong with the ampicilin, possibly that it was not concentrated enough, or that it was damaged.  I think three is also likely because  I mostly saw non-GFP growth near the GFP bacteria, or where I pipetted the transformation solution onto.

Procedure (specifically, how do you do it)

The actual procedure I followed, can be found at this link to a pirate pad where I planned the transformation out this summer.  I will also put a nice PDF version (including some of this reference material) on the downloads page.

I did a few biology (specifically bacterial) projects this summer.  The first was an enrichment of V. Fischeri or V. Phosphoreum from squid.  Vibrio Fischeri and Vibrio Phosphoreum are bioluminescent bacteria that are commonly found on fresh fish or other sea animals.  The reason you don’t normally notice them in water is because they are very small, are at a low concentration and only glow when there are a lot of similar bacteria around it (in another post I will explain how that works!).  The trick then, is isolating the target bacteria from all the other bacteria in the ocean.

The first step in any enrichment is to do background research.  The first thing almost anyone stumbles upon is this article from Indiana Biolab.  It turns out that the bacteria I was looking for grew at low (4C) temperature, in salty water.  That is the kind of temperature that is easily achieved with ice and a cooler, and a salinity that is easy to achieve with table salt.  I used a handful of salt and about half an inch of ice (measured from the bottom of my cooler) for experiment.  The “proper” amount of salt would be 30g/L, or 2.8 tsp/L.  The squid I used was from one of the butchers in Haymarket in Boston.  I placed it on the ice so that it would never be completely submerged.  The squid can’t be submerged because the bacteria require oxygen to bioluminesce, and to pick the colonies of bacteria out, you need to be able to see them glowing.

Once the squid is in the cooler, the next thing to do is to let them incubate in a cool room for a few days.  Basements and garages are good places to do this.  You could probably do it in your house/fridge, but there is one caveat: rotting squid smells TERRIBLE.  It also attracts flies like no other.  When you throw the squid away, be sure to double, triple, quadruple bag, do it the day your trash gets picked up, and bleach EVERYTHING, because this will attract flies like a magnet and stink to the high heavens.  Also, be careful of the squid-juices that will form in the cooler.  They smell bad.  The best option may be a disposable cooler that you can just tape up and throw away after.

During those few days, you should check on the squid every 6-12 hours.  Don’t worry about missing the window, but definitely throw it away after 2 days if you haven’t seen anything (see rant above about smell).

Eventually, you may see some glowing.  If you want to continue culturing the Vibrio, I recommend trying to pick off individual colonies and spreading them on plates and continuing the isolation of colonies there.  Here are some pictures I took of the glowing squid!

A note on brightness:  It is hard to capture the light produced by these bacteria in a photograph.  To give you an idea of their luminosity, I would say that a few colonies roughly .25mm in diameter are are comparable to a firefly.  I would also say that in complete darkness, the light from the colonies was enough to illuminate the inside of the ice chest I used, which was surprising.

Even though I never managed to get these growing in culture, it was amazing to see them glow in the dark.  I would recommend this experiment to anyone who is interested in biology.