This content provides a detailed, step-by-step guide for conducting a basic genetic engineering experiment using the Amino Labs Engineer-it Kit and a DNA Playground, focusing on safety protocols and the initial preparation of bacterial cultures.
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Hello Everybody, over the next couple of days we're going to be doing the Engineer-it Kit
experience by Amino Labs.
And we're going to be using a DNA Playground.
Which is a fun little device to do genetic engineering with.
And we're going to start by talking a little bit about safety.
So there are a couple of things, this is all biosafety level 1, so its generally non-pathogenic.
So we don't need too much safety gear, but we do follow a couple simple rules.
One is shoes - they should cover your toes completely.
And this is to prevent any hot liquids or caustic liquids like bleach from falling directly
on your feet.
The second is a lab coat.
So when you're doing biosafety level 1 stuff, you're typically protecting your experiments
from you and you from your experiments, OK so, if you're covered in cat hair or dust
from your normal everyday life, a lab coat will protect your experiments from getting
that stuff into to it and conversely if you happen to spill some of your experiment on
you, the lab coat will protect you.
OK so, it goes both ways.
Lab coat is pretty important and you should just get used to wearing one.
There is no reason not to.
Um, and then we've got gloves.
These are nitrile gloves.
I typically buy these from Uline because I order so many of them, but you can get them
from your neighbourhood pharmacy really easily.
OK so I'm just going to go put these on right now.
You can wear safety glasses if you like.
Its always great to be extra safe.
The liquids (volumes) in here are quite small and there shouldn't be much splashing, um
but, you can always wear safety glasses.
And if you have long hair it is always good to tie up the hair so that as you're doing
your experiment the hair doesn't go into the samples but also if you're leaning close into
the samples, you're not going to dip your hair into the samples.
OK so we're going to go through what is in an Engineer-it Kit.
It has everything you need in order to do a genetic engineering experiment where you
can program cells to produce a coloured pigment for you.
And we're just going to go ahead and rip this open - its pretty sealed.
Alright so one of the first things you'll notice are these long rods, and these are
inoculation loops.
There are two different kinds that we're going to use throughout the experience.
There is blue and there is yellow - and you can see they have different sized loops at
the end - and we'll see what that is all about shortly.
There is a plate streaking stencil, which we're going to use to guide us to streak cells.
We've got a baggy of DNA, transformation buffer, and recovery media and we're going to be doing
an experiment with Raspberry Red, OK?
We've go another baggy with some cells - these are the E. coli cells that we're going to
be using and again they're a lab strain that is biosafety level 1 - so these are not pathogenic
E. coli - so you don't have to worry about that.
And there is a little tube of antibiotics - which we're going to use to select for our
engineered cells.
And then we've got a little tube of powder - its called LB agar - and this is kind of
like JELL-O powder.
Once you put it into hot water and let it cool it gels and as you'll see, thats how
we're going to be growing our cells.
And we've got some petri dishes and these are going to be the little "houses" for your
cells.
We've got a bottle here of sterile water, and we're going to use this to make the food.
And then lastly we've got a bag in here and this is called an inactivation bag.
So throughout your experiment you're going to be creating some waste and in general you
can put your waste into this bag and at the end we're going to inactivate all of the organisms
on it using bleach.
And uh, just a quick note - the way it works is you put everything in there and then at
the very end add, using tap water generally - fill this bottle six times with water and
dump them into the bag and then you'll add one bottle of bleach and dump it into the
bag and zip it closed and let it sit for 24-48 hours and then you can get rid of the liquid
and put the solid material in the garbage.
So, after we use this bottle today, you'll want to hang onto it.
Ok so thats everything, and I'm going to put my original bag on the side because we're
going to store things in it overnight.
So step one, the steps we're going to be doing today is we're going to making the LB agar
petri dishes with and without antibiotics so that we can start growing bacteria on them
and then we're going to streak some of the cell that you get in this little baggy here
on to that plate and then we're going to grow those overnight.
And we call those blank cells because they haven't been engineered.
They are not containing any "DNA program" and we're going to use those after we grow
them, tomorrow to engineer this Raspberry Red DNA program.
OK? So, today we're not going to need the DNA or any of these buffers here - we're going
to put them back.
And we are going to need all three of these.
And we're going to need three yellow loops.
And I'm going to put the rest of these loops back.
Great.
OK, here we go.
Lets get going.
Step one we're going to make the molten LB agar.
This basically means that the agar is really hot and it is liquid.
So I'm going to start by heating up the water.
You unscrew the lid a little bit so that as you heat it, it doesn't explode - and we'll
put that in for 45 seconds.
And you can keep an eye on it and make sure it doesn't boil over.
And while we're warming that up I'll explain a little more about this stuff.
So right now all we need is the powder.
And this powder contains the sugar, amino acids, the minerals that the bacteria need
to grow.
And second, it contains agar which is a gelling agent.
It is similar in what comes in JELL-O except it comes from ocean creatures rather than
animals.
And we're going to pour this into the boiling water and you'll see that after we pour it,
there is going to be a little bit of powder that is wet and around the rim.
And that is not a big deal as long as you get the vast majority of the powder into the
liquid, we're good to go.
OK, and you can see it is steaming and it is bubbling a little bit, so it is hot enough.
You have to make sure that it is boiling when you do this.
So if it is not boiling just put it in for another 10 seconds and reassess.
Now I have my powder, I'm just going to dump that all in - Tap Tap Tap, and as I said we
get a little bit of powder getting wet from the humidity and sticking to the lip and thats
OK.
And so you can see the powder inside.
I'm just going to put the lid on.
And I'm going to give it a bit of a swirl here.
For about 10 seconds.
You don't want to swirl too vigorously and get the liquid up the sides - it might cool
down and solidify and you'll lose a little bit - so just a gentle swirl.
So you can see, it looks like its well mixed in.
But it doesn't look like it is completely transparent yet.
And thats because the water probably cooled down a little bit.
So we're going to boil it for another few seconds here and after you put the powder
in, it boils over really easy so you never want to go more than 5 seconds increments.
I'm just going to go ahead and heat this for another 5 seconds.
If you do it more than 5 seconds it will boil over and create a little pool and you'll actually
lose your agar and you might not be able to do your full experiments.
So keep that in mind.
It looks pretty good actually after that five seconds.
I don't know if you can notice, but, it is yellow in colour, but it is transparent.
If it looks like it is powdery.
It is hard to see with this bottle because it is opaque, but from my standpoint it looks
like it is fully dissolved now.
So thats all I can really say, you'll have to try it a few times to really get a handle
on it.
Alright, so now that we've got our molten LB agar.
We can start pouring our plates.
And so this is still quite hot.
One other thing I should mention is that when you're swirling, you don't want to swirl too
vigorously and create bubbles.
Because when you create bubbles, they'll pour into your plate and they'll remain its kind
of a nuisance to have them there - OK, so you're going to need a permanent marker here.
And one of the plates we're going to label "NS" for non-selective and this is what we're
going to grow our blank cells on today.
And then the other three we're going to put "S's" which stands for selective.
These are the plates that are going to have antibiotics in them.
And we'll talk about that more a little later.
One of the plates is going to be a negative control plate which we're going to put some
of our blank cells onto tomorrow and that will help to see whether we made these plates
properly.
Similarly we will be doing a positive control (+) and we'll talk about that tomorrow and
then we'll be doing our experimental samples, for our actual engineered cells.
And again, these are all for tomorrow.
We should label everything.
OK, so the first thing we're going to do, because we haven't added any antibiotics in
here yet.
We're going to pour our non-selective plate (NS).
I'm going to pour a little bit back here.
And you just need to cover - cover the bottom - thats good enough.
Now what I'm going to do is I'm going to make them selective (S) and I'm going to do that
by putting in the antibiotics.
And I'm just going to dump that in.
Put the lid on, you should always try to keep things contained so that fungi or other spores
in the environment don't get in there.
I'm in a pretty clean environment so I can leave my plate open.
If your in an environment that has mold floating around in it, you'll probably want to put
the lid back on and you might even want to buy a little HEPA filter that you're running
in your "lab" space.
It doesn't have to be an expensive HEPA filter, it can just be a $50 or $40 one from your
local store.
OK I'm swirling this around and we can see that the little antibiotic pill is starting
to dissolve.
Just as a question, I'm not going to give you the answer - you can post it perhaps in
the comments below, if somebody can do a rough calculation to see how much chloramphenicol
are in here, um, that would be cool.
And then also try and relate that to how much antibiotics a human would take on a given
day - you'd be quite surprised by that answer.
So you can see it dissolving.
The important thing is to dissolve the inside of the capsule, because that is where the
antibiotics are.
I think it is pretty much all dissolved now, we'll swirl it for another 5-10 seconds, but
it looks like the insides were pretty much gone - just a little bit of the gel capsule
is there.
And that will continue to dissolve over the next day - so you don't have to worry about
that.
Ok there were a few bubbles in there, I hope that they don't pour in.
You can see that the contents of the capsule are gone.
Now I'm just going to go ahead and pour this just like the last one.
I just want to cover the bottom.
OK - good.
And you don't need the rest of this, there is a little bit left in there and there is
always a little bit extra stuff and its just there in case you do a little spill or if
something goes awry.
So what I'm going to do, because we need this later for inactivation, I'm just going to
pour the rest of this into the bag as far down as I can.
There we go.
And I can also put some of my other waste in here.
You always want to put your tubes in here OPEN, so that when we add the bleach water,
the bleach water can go into all the tubes.
OK?
often people in labs will throw away their tubes with the lids on and there is no way
for the contents inside to be inactivated.
Lets put it like so, I'm just going to close this for now.
There is still a little bit liquid in there and we don't want it to spill out, I'm going
to lean it against something.
And I'm just going to put the lid back on this and we're going to use it for the inactivation.
OK.
Now the time it will take for these to cool and solidify really depends on the environment.
So if your environment is humid it will happen slower, and if it hot, then it will happen
even slower.
It is probably 19 degrees (C) in here, maybe 20, so its a little on the cool side and they
will cool quite rapidly.
Maybe over the course of...
I don't know... five minutes.
And so you can see, we're looking from the top down and you can see the marker really
well.
These are quite clear right now and thats a good sign, it means we heated the molten
agar enough so that it fully dissolved.
If it looks powdery and a little opaque and you see particles floating around you may
on your next try want to microwave it a little bit more and swirl it a little bit more.
This one, this try seems to have worked well.
So I'm going to come back in about five minutes and we'll have a look at these and they should
have gelatinized, like JELL-O does when it cools and then we can get going with the streaking.
OK, so I'm back and its been about 5 minutes.
Uh, the plates looking pretty gelatinized.
It has become pretty solid.
OK we can see that it is about half way up - maybe a little more if you like, but that
should be OK.
OK and these guys also look like they good.
We did these a little after because we had to add the antibiotics, but it looks like
these are already pretty solid as well.
They are still steaming a little bit, the plates are still steaming a little bit.
So we'll just leave those - actually we can put the lids back on them to prevent any extra
contamination and we'll just leave them out and deal with them later.
OK, so now we're onto the streaking step.
Now streaking is used to isolate individual colonies of bacteria.
OK?
Tomorrow when we do our engineering of the cells, of these blank cells we want these
little individual spots of cells called colonies because they grow fast and they take up DNA
the best.
OK?
And, so streaking is a method where we're going to take a loop, dip it into these cells
and so there are going to be lots of cells on there and we're going to streak it across
the plate along the red line - line 1.
And thats going to deposit lots of cells there - OK?
So when they grow there is going to be a solid line of cells.
Then what we do is get another loop, a clean loop, and we drag it through just a small
part of that line - so we're only grabbing a small amount because it was a clean loop
and then we spread that out.
So now there are less cells here, and you might start to see single bacteria colonies
forming after we incubate it, but surely when you do it the third time, and again you just
get a new loop that is clean, you drag it through so you're only collecting a tiny bit
of this line and you do a zig zag.
We should be spreading out individual bacteria around this area and as they grow they will
form little mounds called colonies.
OK?
So we can get going.
Get your cells from the little baggy.
There's lots of cells in here and so you don't need to dip and you should dip the loop too
far in, you only need to go into the surface and when you pull it out you'll see a little
bit of agar.
And so I've got my loop.
You want to be careful to never touch this end, it has to be clean, you don't want to
put your bacteria on it.
OK, there are going to be lots of cells in here, I'm just going to dip it in a little
bit - again you don't need to go in too far.
When we look at it there are lots of cells.
There is liquid in the middle now and that contains lots of cells.
So I'm just going to tip this side ways, this way, because I'm left handed - and you want
to be as horizontal as you can so you don't puncture the agar and so you can rest the
loop on and for this first one I usually go back and fourth a couple times to make sure
I get quite a bit of liquid on there.
OK, now that I'm done with this I can put it into my inactivation bag or alternatively,
I can put it into a waste container that I have as a temporary storage - and I'll do
that - for now - its just a little easier.
OK now that I have deposited one line which has lots of bacteria in it, I need to get
another clean loop and drag that through to collect a smaller proportion of cells and
spread them out - OK, so I don't want to move this plate because I know my line is there
so turn the whole paper, and similarly you want to be nice and angled here so that you
don't press and accidentally punch in - you want the loop to stay above it.
And I'm just going to go through the line, zig zig zig zig zig.
You can follow it exactly if you want to.
OK great, Its kind of hard to see, I was hoping we could see a little line, but if you don't
press to hard you're not really going to see much happening there.
And for the third one, I'm going to the exact same thing.
I'm going to drag this loop through and do a few more zig zags like that.
OK?
And we're done!
Now our cells are spread throughout the plate and hopefully tomorrow we're going to get
some nice single colonies growing over here that we can collect and program.
OK, so now we're going to grab the DNA Playground here.
OK, so just a little quick intro of a Playground, so this has different temperature stations
and a little incubator on the side that allow you to grow and engineer cells.
And this is made by Amino Labs.
And I think for this, but we have decided yet, but I think we'll go through the whole
process of actually bioproducing pigment, so we might actually transfer to a BioExplorer
in a couple of days, but we'll see if we've got time for that.
I'm going to turn this on.
And actually you can see, its a little chilly in here - 16 degrees (C), its a chilly Fall
day out right now.
OK so now what I'm going to do is turn on the incubator over here.
I'm going to turn it on to 37 (C).
OK?
And 37 C is the temperature that E. coli like to grow at because they have evolved to do
so in the large intestines of animals - like humans are body temperature is 37 C. So I
forgot to mention that this is an incubator paddle and it was just hooked onto the back
and we use this to take things in (err) take things out and put things in the oven (incubator).
And this is a little humidity chamber and this is a little puck.
And the way this all works is, you take your plate that you've streaked and we're going
to flip that over, OK?
The reason you flip it over is because as you heat this, there is going to be some evaporation
happening and you don't want your plate to dry out.
And so one way to help prevent that is flipping your plate and as it heats a lot of the vapour
will rise and will keep the surface of the agar a little bit moist.
So you put your plate on the paddle.
You put this little puck on there.
And then you put your little humidity chamber over it, OK?
And then we just slide that into the oven - the little incubator and then we're going
to close it and lock it.
It is always good to lock it to make sure that this is fully secured and doesn't accidentally
open during operation.
And if it does, the temperature in the incubator is going to cool down and it just isn't going
to work very well for you.
So it is always good to lock it, and we're going to hang up the paddle.
You can see it is heating up - it takes about 20-30 minutes to reach the optimal temperature
- Um and we're going to turn on the timer and we're going to the next step in 16 to
24 hours.
I've got these empty loop packing and I'm just going to put those in a normal garbage.
I may as well put my card back in here.
And I'm going to go ahead and throw my stab of cells into the inactivation bag.
Also I have some loops, I have more loops from other experiments, I'll just throw everything
in here and then inactivate it - great - OK.
And then I can just close that up and leave it for tomorrow.
I can put this baggy in the fridge to keep it cool.
And I'm also going to take the selective plates that I made today and I'm going put them back
in the original petri dish bag and put those in the fridge.
And then we can get them tomorrow for Day 2.
There we go, I'm just going to put these all in the fridge, and I'll see you in 16 to 24
hours.
One thing I forgot to mention, was what to do with your gloves!
And so there are a couple of things you can do - if you've got a box of them you can simply
take them off and you try and grab them by the wrist area.
You don't really want to touch inside or else you're going to contaminate that and then
you just pull them off.
OK?
And the same thing on this side - you shouldn't have really touch much so it is not really
a big deal and so if you've got a box of them you can now just throw them away in the garbage
- you don't need to put them in the inactivation bag because there shouldn't really be anything
on them - thats just a precaution.
The other thing you can do is hang onto them in side out and use them again tomorrow, OK?
And um one technique that you can use to invert them - because you don't want to put your
hand inside - because that is the outside and this is the inside is you can flip them
through.
So what you do is you grab, again right by the wrist - and you're going to push them
through.
Now this part is the outside - and the inside is back there.
We need to get the little fingers out.
Some people just put them up to their mouth and blow, but I don't tend to do that because
I don't really want to put my face near the gloves if I don't have to.
And so what you do is make sure there is a little pocket here - see there is an opening
- and then what I'm going to do is spin this around and pull wide so that it traps the
air inside.. as a little bubble.
You'll see and then we'll pop and the fingers will come out.
Big circle and then as you spin you're going to pull tight and then spin around -OK?
So we go zzzip so now you can see there is a little bubble in there where the air is
trapped.
And I'm going to squish it as close as to the wrist area as I can, because there shouldn't
be any contamination there.
If you clearly had some sort of contamination on your hands, then you can put your gloves
into the inactivation bag.
If that happens then do that, but if not, and the gloves are relatively clean, you can
do this.
OK, so, again.
Zip it around and then I press like that.
Press it away from the fingers because that is generally you're grabbing things with your
fingers.
Right?
Now you can see that all of the fingers are out.
And now I can go ahead and put it back on.
OK?
So I will just demonstrate that with the other glove.
Push it through - again keeping hold of it near the wrist area.
I'm going to make sure there is a nice little bubble there, see there is a big opening.
And as I spin it I'm going to tighten and trap some air in there.
Like so, right?
And I'm going to squish - again, away from the fingers - there we go.
I've got all of my fingers out and I can reuse these gloves.
