Indoor, Outdoor & Kids' Trampolines

Engineer-it Kit & DNA Playground: Day 1


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.

Reader Comments

  1. Excellent teaching video Justin, we will be using this for our next school seminar. Love the updated Amino Lab with incubator underneath, just need a countdown timer with alarms as well.

  2. Leaving the agar plates to cool with the lids off is asking for contamination. Convection will drive large amounts of air over the surface of the hot agar and into the water droplets condensing on the lids partially covering the plates. Placing the lid of the water bottle, bottom down on the lab bench before replacing it on the bottle is also a questionable protocol?

  3. I recommend to watch this tutorial series and try out their simulation at the same time. Really great tutorial. https://chrome.google.com/webstore/detail/virtual-bioengineer/cdpfeajebhfcphlaikhikeabljifofhf

Leave a Reply

Your email address will not be published. Required fields are marked *