Hi my name is Dave Micklos.
I'm founder of the DNA Learning Center at the Cold Spring Harbor Laboratory.
Since 2002 the DNA Learning Center has been helping Asian students and teachers
learn new techniques in molecular biology, so we were very concerned
when we learned of the coronavirus and the hardship that it is causing all of you in China, so we thought we'd do this series of laboratories
so you could continue to learn about biology when you're forced to stay at home.
For the first experiment we're going to learn one of the key techniques in molecular biology.
Transformation is the ability to move DNA from one place into another organism
and it's absolutely essential to genetically engineering any species or
producing any genetically engineered drugs or other products
but in the 1980s when I first started doing this it was extremely hard to do and could only be done by the best people in the best laboratories in the country. So I didn't know what to do to bring it to teachers until I talked to my friend Doug Hanahan, and Doug had a really easy way and he knew
the trick of easily getting DNA into bacteria. He shared that with me and now you'll learn that same technique today from Erin McKechnie one of our instructors here. It's really easy and it's really fun and
it's been done by hundreds of thousands of students around the world,
so now you'll be able to do it also. Good evening everyone.
My name is Erin and I'm here to teach you the bacterial transformation lab.
I'm so excited to work with everyone today let's get started.
I'm going to bring up my first slide on the board. So what we are going to be working with DNA, and we're going to be working with bacteria. So I'd love to start off by having a little talk about what DNA is and why it's so interesting. If at any point during our lesson you have questions for me, I would love if you try to type them in. I will do my best to answer some of those
questions and I'll also leave some time at the end so we
can talk about them. Let's talk about what DNA is.
So I have up here a picture of the DNA molecule which is found in all living things. So is anybody able to tell me what the letters DNA stand for? I'll write it up here on the board for you. So DNA stands for deoxyribonucleic acid. This molecule is special and I'd like somebody to tell me, if they can, where we find DNA.
I did say earlier that we find DNA inside all living things. Specifically we find our DNA inside of ourselves. So as humans we're actually considered animals. So we have animal cells. inside our cells.Well we have skin cells. We have various types of blood cells. We have nerve cells and bone cells. We have so many different types of cells. When we're thinking about DNA it's so important for us to understand what the molecule actually looks like.
It allows us to envision that DNA inside of our minds. So that we can better understand how cells access or use that DNA. So let me show you my model that I have here. It looks a little bit like what's on that board. So this model here and what I have on the board they're both in this shape that looks kind
of like a spiral staircase. Does anybody know what the name of this shape is? This is call a double helix and the double helix was discovered in 1953 by two scientists Dr. James Watson and Dr. Francis Crick. This discovery of learning what DNA actually looks like is considered one of the most important discoveries in the history of biology. Now why do you think that might be? Why would we want to know the shape of DNA? Well just like I said before, knowing the shape of something or being able to see it in your mind allows us to study it and think about it in new ways. So let's talk about the information in DNA and why we might want to study that. So DNA has information. I'd like somebody to try to tell me what the
information in DNA is actually for. I'm gonna draw some pictures up here on the board for you. So I'm going to do my best to draw that double helix but it won't be perfect. So here we have our DNA. Inside our DNA we
have sections that carry information. These sections with information or instructions we refer to as genes. Genes are very similar to recipes. These recipes in DNA.. let's me just shrink that a little bit These recipes in DNA.. ultimately .. what do you guys think? What are these recipes in DNA actually for? These recipes in DNA have the potential to
influence various traits within an organism. So what do I mean by traits, this can mean
so many different things. Traits can be what you look like on the outside. Traits can be how your body functions on the inside and it can even affect behaviors. So I left a space in the middle right here. DNA is actually a set of instructions to make molecules that we call proteins. So let me fill that in right here. Proteins can have different shapes and different functions, It just depends on the protein that's made by the cell. So there are proteins that influence, let's
say, what you look like. Different foods that you can digest. Proteins actually do things in cells. Sometimes people are making more of a protein than we expect or less of a protein than we expect and it all relates to how their bodies function. There is actually a way for us to cut out sections of DNA if we want to. We also have the ability to glue sections
of DNA together. These techniques that I'm talking about are a form of genetic engineering. Genetic engineering just refers to our ability to manipulate or change DNA in different ways. One of the really cool things about DNA is
that your DNA although it's not exactly the same as my DNA, there are a lot of similarities in our DNA. For one, your DNA is made of the same genetic code that my DNA is and in fact all living things, our DNA is made of the same genetic code. What I mean by that is that technically your cells could read the information in my DNA and our cells could read any DNA from any other organism as well. What makes our DNA different from other individuals or other organisms is not actually the code itself but it is the sequence or the order of the code. You guys may have heard about this. DNA has a set of chemicals. Let me show you my model. On the inside of the molecule. So here. Let
me untwist this for you. When you untwist it, you can see actually
that it looks kind of like a ladder. Right, all right. So the molecules within
the DNA that I'm talking about are right here. These are our base pairs. So our bas pairs
in the DNA can be adenine, thymine, guanine, and cytosine. Sometimes we just refer to them by their single letters so you might call them A’s T's C’s or G’s. Your DNA is made up of those A's T's C's and G's and so is mine. What I'd like to explain to you is that.
What we'll be doing today is, we're actually going to give living bacteria cells foreign DNA. We're gonna give them DNA that's not theirs.
It comes from a completely different organism. They should be able to take in that DNA if
we do everything correct, and they'll be able to use that DNA, and they should be able to make new proteins. So cool if everything works. They should show us that they have new traits as a result of the experiment. So scientists have actually been doing bacterial transformations for many years now. The bacterial transformations that can be
of highest interest are those that we use to actually help people
who have diabetes. Has anybody ever heard of diabetes bef ore? So diabetes is a genetic disease that has to do with the production of a protein called insulin. So if an individual is not making enough insulin, that protein, what insulin does is insulin actually helps our bodies to regulate or control glucose levels. So if somebody's not making insulin properly
they're not able to control their glucose levels. So they may, it depends on the individual, they may actually have to take injections
of insulin. So where do you get insulin from to give to
somebody who has diabetes? A lot of people think well maybe we could
donate insulin. Could I donate my extra insulin to other people?
But that's not really a possibility. So what scientists have come up with is they have come up with cutting out the gene for human insulin from a human cell and sticking it into bacterial cells. So we actually use bacterial cells as little
factories to make human proteins for us. It's amazing! Let me talk to you about bacteria on the board
for a second. All right. So let me draw you my little bacterium.
Bacteria can have different shapes. So this is just a general picture for you, inside a bacterial cell, they do have their main chromosomes. They have their DNA. In addition, they also have ribosomes.
Does anyone know why ribosomes are so important? So ribosomes, what they do is, ribosomes actually read the information from the DNA and they build or put those proteins together. So really these bacterial cells, although
they don't have as much stuff inside as our cells, they are very successful because they have just what they need to survive. If I were to take, let's say I were to take the gene for human insulin. I'll draw that up here right now. Let's pretend this is from a human cell and
it's the gene for insulin. If I were to put that DNA, let me shrink it
up, right inside of this bacterial cell here. What I suspect may happen is that bacterialcell would probably destroy that DNA because they don't really know what it's for.
Well if we want to introduce new DNA into bacteria we have to take advantage of one
really cool thing about bacteria. Some bacterial cells have additional DNA other
than their main chromosome. This DNA is called plasmid DNA. Plasmid DNA
is actually circular. So let me draw a circular fragment of DNA
right here. I want you to imagine if I had a line of DNA and I took the two ends and stuck them together, I would have a plasmid. I'm going to make this a little bit smaller here, okay. Plasmids can exist inside of bacterial
cells and what bacteria can do is, they can actually
clone their plasmids, just like that. They can make copies of their plasmids. They
can also share their plasmids with other cells. So as scientists, what we do is, we take these
plasmids. Let's take this plasmid, and we're going to
put it right here. Oh I don't know if it'll let me do this. Let's try. Well I guess I'll just draw another one. Okay here's another one. Let's say I have this plasmid. What I can
do is, I have the ability to cut the DNA open. So I can actually take some little molecular
scissors and cut that DNA right open. I can even remove the section here.
I can then take my gene for human insulin and I can glue it in right here.
So look at that. I've created my own plasmid using the bacterial plasmid. I've just mo dified it or adjusted it in some way. If I do it the right way, the bacteria will
not realize that I've messed around with things. So I can then take that plasmid that I've
created and I can put it into a living bacterial cell. If I'm successful, the bacteria should read
that DNA and they should make that protein coded for on
the gene that I inserted. So if I put a gene in for human insulin, that
bacterial cell should be making human insulin that bacterial cell should be making human insulin for us. All we would have to do then is just grow
a lot of that bacteria, which is so simple. We can then break those bacterial cells open and isolate the protein. So this is exactly how they make human insulin today. They use bacterial cells. We are not limited to just making human insulin.
We can actually make any protein that we want. So today, what I'm going to show you guys
how to do this. I'm going to show you how we can actually
take these plasmids, get them inside of the bacterial cells, and have our bacteria make a jellyfish protein. So we'll be working with jellyfish DNA today. The jellyfish that we work with has an amazing
trait the jellyfish has the ability to glow in the dark. So we, let me show you on my board, we are going to take some DNA from our jellyfish. And we haven't already inserted into a plasmid for us. Let me draw my little jellyfish here for you.
So the jellyfish that we're working with, the species is called Aequorea victoria,
and this is a jellyfish that can glow. The jellyfish makes many proteins but the
protein of interest for us today is one that we call GFP.
GFP stands for green fluorescent protein and if we studied the jellyfish DNA
we would find that there is actually a gene within their DNA that codes for that protein.
So that gene is just spec fically for this protein. If a cell makes GFP that cell should have the ability to glow green.
So I have a question for you guys. Okay. Do you think that human cells make GFP? What
do you guys think? Well, I can't see inside of my body right
now, but I would suspect that at least on the outside, I'm not making any GFP. Because I'm not glowing. One of the reasons that we love GFP is
because it's very easy to see when a cell is making this protein.
So naturally humans don't make GFP. But what do you think?
Could we maybe cut out the gene from the jellyfish DNA and inserted into a human cell?
I guess technically we could do that but there are a lot of moral and ethical issues involved with that. About making humans glow. Would any of you guys want to glow? I don't know if I would. So we can actually do this with bacteria and I want to show you that they've done this with other organisms
as well. So let me show you some fun pictures. Over here, I've got a whole bunch of pictures for you.
This one right here is actually a very small worm that we use a microscope to see.
This worm right here is actually the first organism that they transferred the GFP gene into. What about this picture here? You see these mice? well mice do not naturally glow. So when you're looking at these mice right
here, that have the ability to glow, those mice have been genetically engineered with the GFP gene. So when we see experiments in
multicellular organisms like mice, anything that we do in a mouse,
the idea is technically, we should be able to apply that information to humans.
So if they can make mice glow, technically they could make humans glow. Although I don't expect scientists to be
making humans glow anytime soon. The reason that this experiment is so exciting is because, well, it's showing us that it's possible to
take a foreign gene, a new gene, and insert it successfully into these mice. That means we could do that with humans too.
You don't have to make humans glow, but you could have human cells make new proteins.
This is a way of possibly eliminating certain genetic diseases and
even just helping people live better lives. Very exciting!
What I'm going to show you how to do today is introduce plasmid DNA into these bacterial cells. I'd like to just show you the genes that are present on the plasmid we'll be working with
so you understand why everything is important. So let me draw for you our plasmid.
All right. So our plasmid, as I've discussed before, is going to have that gene from the
jellyfish. So one of the sides of my plasmid.
I'm going to draw it over here. One of the sides of this plasmid actually has that jellyfish
gene in there. So our GFP gene. There is a second gene that's
important for this experiment and I'm going to put it on the other side for
you. I just have to shrink up my plasmid a little
bit so I have enough room over here. We have a second gene that is for something
called ampicillin resistance. Has anybody ever heard of ampicillin before?
Ampicillin is what we call an antibiotic. Antibiotics are medicines that can kill or
even inhibit or block the growth of bacterial cells. So generally when we think about how antibiotics work, if I have a whole group of bacterial cells
and I add an antibiotic to them I would expect those bacterial cells to either
be killed off or to not be able to grow. So antibiotics are incredibly useful. Especially if bacteria are making us sick.
This gene is a really special gene because this actually allows our bacterial
cells to resist this antibiotic called ampicillin. Now what do you guys think? What does that mean? If bacterial cells can resist an antibiotic
it means that that bacteria has the ability to protect itself from that antibiotic somehow.
There are different mechanisms that bacteria can use to protect themselves.
In this case, I want to show you just why this is such an important gene for us.
So I'm gonna draw another picture of some bacteria for you guys. Alright. So let me draw my little bacterium
here. We have our DNA again in the center. We have our ribosomes and what I expect is that in our experiment we should have a whole bunch of bacteria.
So I'll draw a lot of cells out here. Even in a successful experiment, what happens is that the bacterial cells most likely will not all take up the DNA.
We want them to, and that's perfectly fine, even if, let's say, I just get this one cell to take up the plasmid that I'm interested in. That one cell should be able to make some GFP. Let me draw some GFP.
That cell should be able to glow. The problem here is that these other cells,
these non transformed cells out here, these ones.
All these ones. they are going to grow on top of my glowing bacteria and
then I'm not going to be able to see if my experiment was a success. So I have given my glowing cell over here.
I've given it the ability to resist the antibiotic ampicillin. So if I apply the antibiotic ampicillin to this environment the other cells here.
What I will expect is that they won't be able to replicate or grow
so I'm just gonna get rid of them. They're not going to be able to grow but this
one, that can glow here, this one, because it has that antibiotic resistance
gene, it's going to be able to replicate and copy itself. All of those little copies are going to be
able to glow as well. So I'm going to have a whole region of bacteria
here that have the ability to glow. We call that region of bacteria, that start
from a single cell, we call that a colony. So I should have a nice green glowing colony,
if this all works. Hopefully at the end of our experiment we
should have way more than one glowing colony. I hope to have hundreds. Let's get started
on the experiments. What I'd like to do is actually
show you some of the instruments that we use for measurement in this lab. This is called a micro pipettor.
We will use different micro pipettors in the lab and they work exactly the same way.
The only thing that's different about them, other than the color on top, i
s that they measure different amounts of liquid. So theymeasure in the same type of liquid
in the same unit. Opps, that's our cat. I wasn't going to show
you that yet, but that's a transformed cat glowing in the
dark. Let's come back to our pipettes so our pipettes
measure in the unit microliters which looks like that. T
here are 1000 microliters in one milliliter. At the start of our lab we will be measuring
250 microliters of a solution called calcium chloride. Before I actually measure anything out, I'm going to label some tubes in this experiment. I will have two tubes. So I will label one
tube with a plus and one tube with a minus. Our plus tube will be our experiment tube
that will have bacteria and plasmid DNA and some
calcium chloride. Our minus tube over here, this one over here,
is actually not going to have any plasmid. So this is going to act as our control. Let
me show you the tubes that we're going to use and I'll show you just how simply I label
them. So I'm going to be using these 1.5 milliliter
tubes right here. Okay. I have two of them set for our experiment
and throughout my work I want to keep everything labeled, easy to identify, and sterile. Does anybody know what sterile means?
Sterile refers to being completely clean or free from contamination.
So I want to keep everything clean and separate. Let me label my tubes right now. I'll show you. So I put a plus on that tube
and I put a minus on that tube. Just like that. Very simple.
Okay. I'm going to measure out now my 250 microliters of the calcium chloride liquid.
So I'll be using the blue pipette because it measures the larger volumes. Our pipette happens to be set on 250 because
I was just using this pipette yesterday. But if at any point you need to adjust the settings
on these instruments, they're so easy to use. There's a dial at the top. You can change
that and it actually I don't know if you guys can see that,
it changes the numbers. So for me I'm going to set mine at 250 microliters. Remember we want to keep everything sterile
so I'll be using this box of plastic tips and
I'll be changing them whenever necessary. so let me place that down there, put a tip on, and I'm going to pick up my tube of calcium chloride. Opening my tube I hold everything up to eye level and I'm going to pipette slowly. Placing the pipette in the liquid. I'm going
to slowly let my thumb come up. I'm going to pick up either tube. I can start
with either one and I'll be adding the 250 microliters of calcium chloride to that tube. At this point I'm going to place my tube on
ice and I'll explain to you shortly why the ice is so important. I'm going to remove another 250 microliters.
Slowly pipette. I can discard my extra. I'm holding up my new tube slowly adding. Closing that up and placing that on the ice as well. At this point, I'm just going to eject the tip and move on to the next step.
I think you guys are gonna find this step pretty exciting. I'm going to transfer some bacteria off of a petri dish. Let me show you. So I actually... let me show you an empty petri
dish first. So this is a petri dish. I don't know if you guys have ever worked
with these or seen these before. But this petri dish, it just has food for
bacteria in it. That's all. Look at this dish. Let's compare. There is also writing on this
dish over here but I want you to see all that smeariness You see that kind of beige colored material that’s swiped or painted all over the plate?
Those are the bacterial cells. So I'm going to do my best to sterilely transfer the bacteria that's on the dish into those two
tubes that I just labeled, and added calcium chloride to. In order to do this sterilely, I'm going to
use some flame. So let me pull my hair back so that everything is safe. I’ve got this great little burner here which
I'm going to turn on. It's just a gas canister and I'm going to
apply a spark. So let's turn the gas on. There it is and
there we go. Alright. I do like to work, oh, let's try that again.
There we go. I do like to work close to the flame to keep
everything as clean as possible. I really consider this area right around the
flame a zone of sterility. The heat is going to push anything up that
might be falling into my experiment. I don't have to work so close to the flame
that I'm gonna get burned. I would never want to do that. But I do work
nice close so that everything is clean. This is an inoculating loop. Can you guys
see that circle? Maybe if I hold it like this. I don't know.
Let's see, oh there it is. I can see it. It's a little tiny circle.
What we do with the inoculating loop is, we're actually going to try to scrape or lift
the bacteria off the petri dish. I don't want to take any of the a ctual gel
that's from the dish. So sometimes, this can be a little tricky.
First thing I'm going to do is, I'm going to sterilize my inoculating loop.
Now keep in mind this is a piece of metal that's basically on fire.
If I take this flaming hot piece of metal and I touch my living bacterial cells,
what do you think might happen to them? They're probably going to die.
So I have a little trick here. I'm going to sterilize my loop and just for a moment, I'm going to press that loop into the gel
to cool it off. At that point I'm going to use my loop to
kind of scrape and lift the bacteria out of the dish. The bacteria sometimes are a little bit slippery and this can be a little bit frustrating.
I think I'm doing a good job though. I'm gonna go with that. I don't know if you
guys can see this amount here. It may not look like a lot but that's actually
a lot of bacteria. So I'm going to pick up one of my tubes with
the calcium chloride, doesn't matter which one. They're both going to get the bacteria. And I'm going to place the bacteria into the tube.
I'm going to do my best now to kind of swirl off the bacteria into that liquid.
I think I did a good job. Let me show that to you. So that looks nice and cloudy.
That's what I want. That's an indication to me that there are a lot of cells in there. So at this point, I'm going to place this
back in the ice. I want to do exactly the same thing for the
other tube. So I'm just going to sterilize my loop. Looks
good. Don't forget to cool it off. Then I'm going
to scrape and lift some bacteria again. I think I got a good amount. Let me find my
other tube. There it is. Open that tube place the bacteria in and swirl it off. Okay. I'm going to sterilize my loop one more
time just to keep everything clean in the lab. At this point I'm going to turn the flame off and push that out of my way for a minute.
Okay. Now I don't know if you guys can see this, but there are actually a few clumps of the bacteria right in the tube. I do prefer if it's more evenly mixed. Oh
yeah. Right on the bottom. Can you guys see that little white clump down
there? What I'd like to do is, I want to break up
that clump. So I'm actually going to use the pipette to
do that. So I'll get myself a new tip. I'm gonna open my tube hold everything up
to eye level and I'm going to just very simply lift the liquid
out and replace it. At no point am I actually going to remove
the liquid from the tube. I'm really just using the pipette as a method
for mixing. We call this step resuspending the cells.
That looks pretty good. I'm going to change my tip and get myself a new one.
I'll be working with the next tube. There we go. It looks pretty well mixed already
but I just want to make sure. Everything is broken up. Alright, great job.
Now I'm going to add the plasmid DNA to one of the tubes.
Do you guys remember which tube we said was the experiment tube and which was the control?
Actually I wrote it on the board, so hopefully you guys can figure that out.
This one is our experiment tube. So I'm going to add the plasmid DNA just to that tube. In fact I want to make a list.
I want to keep track of what I'm adding to the different tubes because it's very easy
to get confused. So whenever I do my lab work, I like to write
my information down. So let's start with this. I've got my plus tube, I got my minus tube.
All right. So at this point they actually have the same stuff inside.
So they both have 250 microliters of calcium chloride and they both have the bacteria.
The type of bacteria that we're working with today is a type of harmless E. coli called
mm294. We like working with this bacteria and we
like that they're safe. We would never want to work with bacteria
that could get us sick and these bacterial cells are great for transformations.
One thing I have to add to the board is what we're about to do.
I am about to add the plasmid to the plus tube.
I'm going to add 10 microliters of the plasmid, and the plasmids name is pGFP.
Let me show you that plasmid again just as a reminder.
So here it is. This is our plasmid. In the center I'm just going to label it pGFP. So as I add this liquid in the next step,
which will look to you and to me just like water.
I want you to envision again that this plasmid, thousands of them are in that liquid,
we are going to add those plasmids into tube with our bacteria.
So I am going to use the gray pipette because I'm only measuring 10 microliters. I'm gonna check that my pipette is set and
it is it's on 10 microliters and now. Let me just organize, got my plus tube right
there and over here. I have my plasmid DNA.
So every time I give these tubes out to my students, they say,
Erin you gave us an empty tube. We need a new one.
But I want you to check that out. I want you to see the volume at the bottom. Oh let me hold it up to my shirt. Maybe that's
a little bit better. Is that good? It's still pretty small, right? It's still
pretty small right there. I promise you, there's at least 10 microliters
of liquid in there. So I'm going to take my pipette, get a new
tip, and draw up that liquid. There's actually a lot of extra DNA in that
tube. Now, am I putting this in my plus tube or
my minus tube? You guys remember? I'm gonna find my plus tube. Open that up.
When I do this step, it's important that I place the tip directly
into the liquid and then push the plasmid in.
I always want to make sure that when I add the plasmid DNA,
it's in direct contact with those cells. Put that back in the ice and I'm going to
change my tip. At this point I like to let the bacterial
cells get nice and cold so I would let these cells sit in ice for
about 15 minutes. What I'm hoping to do in the next step is
I want to actually push the DNA into the cells. I'm going to do a quick drawing for you and
then an animation and then we'll do the other steps. Okay. So let me draw a little bacterium for
you. So many of the cells that we work with,
I would expect that they would have these openings in their cell membrane,
which we call adhesion zones. So now let's say I've got a whole bunch of
these cells up, hold on, let's say I've got a whole bunch of these
cells in there. If I'm talking about the plus tube, I've also
added, I guess let's make it in red here, I've also added some plasmid. Okay. So right now the plasmids are on the outside
of the cells and where I want the plasmids to be, Is…
I want them to go into the cells. In order to do that, I'm actually going to
take advantage of temperature and how temperature impacts the movement of molecules.
Let me show you a cool animation. Okay. So this step is called our heat shock step.
This is a wonderful little bacterium and they have a much better picture here of these
beautiful adhesion zones in the membrane. I'm gonna zoom in on one of those adhesion
zones. So this itself is the adhesion zone right
there. Let me see if I can draw in here. So here's
our adhesion zone. It won't let me. It's right in the center and you'll notice
right outside that in the membrane we have these very fast-moving phospholipid
molecules and they do carry a negative charge. So what I'd like to do is, I would like to
transfer, let's say there is a plasmid out here
I would like to transfer that plasmid right through that adhesion zone into the cell.
But there are a few issues here. So if I try to do that, that was our plasmid.
Did you see it bounced right out? Okay. One of the reasons that this is an issue
for us is that plasmids are DNA. Therefore they are negatively charged.
You'll see that we have these phospholipids bouncing around also negatively charged.
So why is that a problem? If we have these two negatively charged molecules
and we're trying to force the DNA through, unfortunately with this two charges that are
negative the DNA is going to be repelled or bounced
right back out just like we saw. So the way that we handle
this situation is, by using our calcium chloride solution. I
also like to cool the cells down. You'll notice that when we do that, let's
drop that temperature. That all the molecules slow down and we can
have a neutral environment there. If I use temperature to slow the molecules
down, I can also do the reverse. If everything is neutral at this point and
everything is slow. I want you to think about what would happen
if I suddenly warmed up the tubes really quick made them nice and hot.
What I suspect will happen is that, because of the movement of the molecules,
we should have at least some of the plasmids move into the cell.
Okay. So we're gonna warm them up and hopefully we get a transfer of the plasmids into those
cells. At the end of this
I would also like to put the cells in ice to encourage the plasmids to stay where they
are. So let me show you this step. So I have here
,I have my two tubes still on ice. You guys remember. So I've still got my plus
and minus tubes in the ice. When I do the heat shock step, it's important
that I go right over to my water bath, which is at 42 degrees Celsius, and put my
tubes directly from the ice into that water bath.
In fact, what I'd like to do is write this on the board for you.
I just want to make sure everything's nice and clear.
So we have our heat shock, which is really very simple. Three differences in temperature. We're starting
out on ice which is zero degrees Celsius. I like to do that for 15 minutes. The next
step is warming everything up. We find that 42 degrees Celsius works best
and we do that for 90 seconds. At the end of that 90 seconds, if you remember,
I'm going to place the tubes back in the ice. I do that for at least one minute in the ice
at the end. So let me show you this step. Okay. So here's our lovely little water bath
over here. I have it set at 42 degrees Celsius. I'm ready
to go and again, I want to emphasize, we're gonna stay in the ice or
we're gonna be right in here so when I'm ready I have my timer set.
I'll be taking my tubes, they go directly into this water bath and they'll sit there
for 90 seconds. At the end of that 90 seconds,
what I'm going to do is very simply transfer those tubes back into this ice. So we'll pretend that we did 90 seconds. There.
Okay. I'm gonna let my tubes sit in here for at
least a minute. So that means I could let them sit longer during that time.
What I usually like to do is, I usually like to label my petri dishes.
I'm not gonna take my tubes out of ice quite yet because I still want them to sit.
But if I hold the tubes up to you do you expect that they would be glowing just yet?
I do not think that they would be for a couple of reasons. So under ideal conditions, so meaning everything's
perfect, I would think that bacterial cells have the
ability to make a new protein in maybe as little as 20 minutes.
But even if they were making that protein in 20 minutes
I'm not going to be able to see it because the individual bacterial cells are just too
small. I need to take those cells and give them an
opportunity to grow. So one of the ways we can do that is by plating
them on petri dishes. So I showed you the petri dishes before.
I actually have a set of dishes here all labeled for us to work with. Okay, so here's one of my dishes we’ll actually
be using. For dishes for this experiments and most of
the information on the dishes is the same, let me just give you a quick look at what
I put on the dish is there. It is okay, so here you'll see I have four
different petri dishes and in fact most of the information on the dishes
is the same. So I did draw my initials on the dish. On
each dish, there we go, I also put the date, which is important. I want to know when I
did this experiment. Up here you'll notice that I wrote pGFP on
each dish. What is different though on these dishes,
is that some dishes have a minus pGFP and some dishes have a plus pGFP
and that becomes important when we interpret the results.
You'll also notice that I have some other information here on the dishes.
Some of the dishes say LB and some of them say AMP. Let me explain what that's all about.
So in our lab we have so many different types of petri dishes.
Some of our petri dishes just have food for bacteria and some also have antibiotics in
them. So the petri dishes that say LB on them, those
are just petri dishes with food. The petri dishes that say amp on them, those
actually have the antibiotic ampicillin. So I want you to imagine if I take my bacteria
and I plated them onto these dishes, some of the bacteria have added plasmids some
don’t. Some of the plates have just food. Some have
antibiotics. So I want to consider what our possible results
would be. Let me erase these little lines here just
to make sure everything's nice and clear. All right. So if right here, and I'm just
gonna circle this just to emphasize, you can see this is a - plate right here.
Okay. That's a - plate. So if I have an LB plate
right here. This is the plate I'm talking about.
Okay. I have an LB plate and I'm just adding bacteria from my - tube,
well that bacteria is just regular bacteria. No plasmid with DNA was added to that.
So if you put regular bacteria onto a dish with food what do you expect to happen?
I would expect that they're probably just going to grow.
So what I'll draw is a pattern of growth. Let's see if I can do this.
A pattern of growth up one more time up. Let's try one. Alright. Let's try this again.
We'll put this down. One, there we go. Okay it doesn't want to
do it. We'll just do it without. Let's try this. Okay we'll do it like that.
Alright. So I expect the bacteria to grow all over
the dish in a pattern of growth that we consider a lawn.
Or we call a lawn. So there's bacteria all over that dish now.
Let's imagine I take this same bacteria that's in the - tube and what I want to do is,
I want to add it to this ampicillin plate down here.
Okay, I'm going to add just regular bacteria because we have that - right?
Remember that? So I'm just adding regular bacteria to an
ampicillin plate. What do you think happens if you put regular
bacteria on a petri dish with an antibiotic? If you said that you think that the bacteria
would not grow, I would agree. So we will not see growth on that plate. That's
actually a very important plate. No growth on this plate indicates to us that
the bacteria are inhibited by this antibiotic. They're not able to grow in it. Let's consider
our plus bacteria now over here. So this is plus bacteria, which means that
those bacterial cells had the plasmids added to their tubes.
If I did everything, and everything worked properly, and the plasmids went in the cells,
and those cells are making that GFP. I should get some glowing bacteria.
But I have a whole bunch of other bacteria in there that are just not glowing.
Let's try it this way. There we go. So I won't be able to see my glowing bacteria
on this dish because everything can grow on there.
Let's take the same plus bacteria from that same tube. Okay. So we're gonna add it to this plate. I'm going
to expect we should get some glowing bacteria. I am going to be putting that same regular
bacteria. Oh No. Putting that same regular bacteria that was
on this plate, on this plate. They're all together. They're all mixed. So
when I add a little bit of everything. I technically am getting it all over the plate.
This plate though, right here, this plate has the antibiotic ampicillin in it though.
So the regular cells here, these regular cells, I would expect that the ampicillin is going
to block their growth. They're not going to be able to survive on
that plate. The only cells that we'll end up seeing on
that plate should be our amazing green bacteria. They should be our successfully transformed
bacterial cells. So let me show you, just very quickly,
how we transfer the bacteria from the tubes onto the plates.
Then I'll show you our results. So I personally, there are a lot of labels on everything, so
I like to organize my plates. I take a quick look. These are both my plus plates. These are both
my minus plates. I'm realizing I forgot to add one step.
I always like to add a little bit of extra food to my bacterial tubes before I plate
them. So right here I have what's called LB. So
this is basically just like a regular petri dish.
All the nutrients without any of the gel. I'm going to place 250 microliters of this
liquid into each the plus and the minus tube. So let me drop that liquid nice and slow.
I'll pick up either tube. Close that up. I'm gonna change my tip. Keep
everything clean. I don't want to mix or contaminate my samples.
There we go.Throw that out. Here's my - tube. All right. so at this point
I actually don't need the ice anymore. I've just placed my tubes right in the rack
and I'm ready to plate them onto my petri dishes.
One thing I like to do before I add the bacteria, and you could do this before or after you
add the bacteria, is I like to add these little sterile beads
that we have that we use as a mechanism for 0 spreading
the bacteria across thesurface of the plate. So here I've just got a tube of beads.
I'm going to just lift open the plate and dump the beads on. Try not to touch anything and close.
I will add beads to each plate and try to do so in a sterile manner as best
I can. One more. If any of these beads happen to
bounce out onto the table as I was doing that, that would be okay. But I wouldn't want to
add them back into the plate because they wouldn't be sterile at this point.
I'm ready to add my bacteria. So I'm going to add a hundred microliters
of cells to each plate. I'll set my pipette. Now I just want to be
sure that I add the correct bacteria to the correct place.
So again, one trick I use for doing that is just separating my plates out.
I'm using the yellow pipette. Going to get a new tip and let's take this
is my - . So I'm gonna add it directly to my - plates.
I have plenty of liquid for both plates here. I'll be drawing up 100. Lifting the cover
just for a moment. Releasing the liquid on the plate. Getting
another hundred from that same tube. At this point I can throw out that tube. Get
a new tip. I'm going to repeat the process for the plus
tube. I'll drop a hundred microliters, add it to
the plate. Draw up another hundred, I can discard that.
Add this to this plate. All right. Now all I have to do, this is the
fun part, I stack my plates in the same direction and
I shake. You guys hear that clinking noise?
So that clinking noise is the beads bouncing all over the place.
What I expect is that the beads are moving the bacteria evenly across the surface.
If I do this for about a minute we should have bacteria everywhere.
At this point I would take the beads out of the plate and
I'll just do one so you see how I do this. I kind of knock them toward the bottom hold
both sides of the dish and knock them out. I usually check so that there is no sound
and I know they're out. Let me actually show you what the real plates
look like because I did a set yesterday for you guys. The GFP protein it glows green but it glows
super green when it's under a UV light. So I'm actually going to turn the lights down
for a second turn on our UV light and show you the special
plates. You guys still see me all right? It's a little bright in here because it's
morning over here. Okay. we’ll check this out. Wait, let me
turn the light off for a second. All right. I know you can't see very clearly
but you don't see anything glowing right? We don't see anything glowing there.
All right now. Let's turn that light on. You guys see that?
We hold it up to the light. Whoa! Doesn't that look amazing?
Do you guys see that? So these are cells that did not have the ability
to glow yesterday, right? We were able to take these cells, give, here's
another plate, we were able to take these cells.
Give them some jellyfish DNA and now they have two new traits actually. Not only do these cells have the ability to
glow but they actually already have, they have the ability to also survive in the
antibiotic ampicillin and grow. So cool!
All right. So I just want to tell you guys what an absolutely fantastic time I had working
with you and I'd love to know if you guys would be
willing to maybe do a quick survey and tell me a little bit about yourselves. I'd love to know a few details about you.
Like where you guys are coming from or where you're watching.
So let me ask you a few questions and if you guys would be willing to tell me the answers
I just love to know. So first let's start in which school are you currently enrolled. You guys should see a whole bunch of answers
there and hopefully, nope you won’t, yep okay.
I do see a pretty cool question up there as you guys are doing this.
Won't the UV light kill the bacteria? Oh that's a great question.
So yes, I guess if I left the bacteria on there for an extended amount of time.
You are a hundred percent right that too much UV light would kill those bacteria cells.
But if you notice, I just did it very quickly and I can do that you know more than once
there's no problem with that. I could even, now that I have these transformed cells,
I could actually take these cells off the petri dish and grow them on another petri
dish and make more glowing bacteria.
So fantastic question. Oh! You guys have a pen in a UV light? That's
awesome! Great stuff, those are cells, yes those are
cells, so all that green material that you saw, that
was just a lot of glowing cells. Each of those little spots is what we call
a colony. Which if you remember is where we had one
individual transformed cell. So one little cell that took up the plasmid
and divided and divided and divided and produced that larger area of that glowing
bacteria. Oh you guys are, oh great, great. I see you
guys answering so many, great. Oh? how long can it be kept? You mean the
bacteria itself? So for the bacteria, two things. So one person
asked about incubating them overnight. So you can definitely put the bacteria in
an incubator at 37 degrees Celsius and what you would get is, if I do the experiment
right now, and I put my cells in an incubator tonight. When I come in tomorrow I should be able to
see my cells because at 37 degrees Celsius, the cells are gonna grow rapidly. If I don't
have an incubator, I can just leave my cells on the counter at room temperature. They will
grow more slowly. So rather than seeing them in in one day,
I would see them probably in two days. I did see someone ask about whether or not
you could keep the bacteria. We do actually keep our plates.
Sometimes if they look really good we might put a piece of what we call parafilm around
them and put them in the refrigerator. Technically you could keep them outside the
refrigerator but what would happen is everything would get
really dried up pretty soon. Okay. So could you transfer the plasmid gene
to plants or other organisms or maybe through the zygote?
Okay. So all interesting questions. Bacteria, makes it really easy to work with
bacteria because as you can see, we can do some very simple steps in a very
short amount of time. The more complex the organism may require different steps in order to get those genes into those cells,
but you can absolutely get this gene into other organisms as well.
So you guys remember the bacteria. You guys know about the mice and you did see
the cat that I showed you? Right, you saw the cat.
Okay I want to, if I have a few minutes, so I'm gonna ask you another question.
There it is. Okay, there, okay. You guys are answering. You guys are so good.
Thank you so much! So in case you weren’t, would you like to
come to the DNALC to study more in person? I hope so. I would like to tell you guys that
this has been such an incredible opportunity to be able to talk to you guys, being in New
York and you guys are in China. But it's like we're together it's amazing.
I would absolutely love to have an opportunity to meet some of you to work together with
you. I have colleagues, I have people that teach
with me and they are amazing and you guys would have an incredible experience
if you came to work and do our labs with us. Okay. I did see somebody wrote about pigs
on there. Yes, they do do a few with pigs as well. So again when we're inserting the gene, I
see that about mammals, it's just a little more complicated. Okay. You guys did such a great job. Do we have any more questions?
All right thank you everybody.
