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Laboratory Investigation VIII: Genetic Transformation

Lecture Slides are screen-captured images of important points in the lecture. Students can download and print out these lecture slide images to do practice problems as well as take notes while watching the lecture.

  • Intro 0:00
  • Genetic Transformation Introduction 0:06
    • Purpose
    • Materials
    • Time
  • Set-Up 4:18
    • Starter Culture with E. Coli Colonies
    • Just E. Coli
    • Ampicillin with No Plasmid
    • Ampicillin with Plasmid
    • Ampicillin with Plasmid and Arabinose
  • Procedure 8:35
  • Analysis 13:01
  • Genetic Transformation Connection 14:59
    • Easier to Transform Bacteria Than a Multicellular Organism
    • Desired Trait Can be Expressed from the Bacteria
    • Numerous Applications in Medicine

Transcription: Laboratory Investigation VIII: Genetic Transformation

Hi, welcome back to, this is Laboratory Investigation 8, genetic transformation.0000

For introduction, the purpose is to genetically transform E coli bacteria so that it glows green, when it is exposed to UV light.0008

That is pretty awesome. 0018

This is taking advantage of another organism, a kind of sea jelly or jellyfish that has that protein,0019

that allows it to glow naturally.0025

When this is moving around the ocean and absorbs UV radiation from the sun,0027

the way interaction of protein gives him this greenish glow.0032

It serves them a purpose in their environment.0035

The bacterium does not naturally have it.0037

But when you take that DNA out from the sea jelly’s genome and put it in a little plasma, 0041

the little circular piece of DNA and introduce it to the bacterium, you can get it to accept that DNA and express it.0046

That is the key to genetic transformation, literally transforming the genetics in an organisms.0053

Materials, 5 agar plates, this would be per group.0058

Agar is a nutrient source, it is from a kind of seaweed.0062

It looks like plain jello with a yellowish tint, you put in petri dishes.0071

You would usually take the powdered version, dissolve it in liquid, heat up.0077

Pour it, let it cool until it solidifies.0085

You need an E coli starter culture.0087

You would need to do this under the supervision of someone with experience, 0090

a teacher or professor because typically with this lab, you need to order E coli that is not as harmful.0094

There are strains of E coli that typically do not kill a person and make a person sick.0103

That is what you use to this lab.0107

You still do not want to get E coli even from the harmless ones in your mouth.0108

You have to be careful.0113

A starter culture to get the colonies going.0114

Ampicillin which is an antibiotic and a ribonose which is a kind of sugar that helps this lab work.0117

Plasmid, this is not something that is easily found.0123

You have to order this from a company that provides the plasmid.0128

The plasmid has amp resistance.0131

What I mean by amp resistance is resistance against ampicillin, 0134

so it allows the bacterium when exposed to plasmid to not die from this antibiotic.0137

It allows them to make what is called the GFP, which I will mention again, 0142

the green fluorescent protein, from the gene that was originally in the sea jelly.0149

They look like either metal or plastic.0155

They look like long sticks that have a little ring at the end, they are really good for making a starter culture,0158

where you take a little bit of bacteria from an aqueous area, like in a vial and you streak it on the plate.0170

If you were to do this lab in real life, you would follow these directions to maximize the formation of those colonies of bacteria.0179

Pipettes, to suck up the materials from the vials you would be using, get the ampicillin and ribonose, etc.0186

Medicine dropper is a same thing.0195

An incubator, heating up these plates to a certain temperature 0197

will maximize the cell division of bacteria and make this lab come to fruition a little bit faster.0203

UV light for visualizing that green glow.0208

Time required about 5 days from the point where you have your starting culture of E coli,0211

to point where you actually can see the glowing.0221

Sometimes you can have it happen in as little as 3, it really depends .0225

Like I hinted earlier with having a teacher or professor supervise you, there is some risk of getting ill or worse, with this lab.0228

Gloves and goggles would be required.0241

If you do get something on your hands, it is not going to harm you.0244

Before you put your hands in your mouth, dispose the gloves, wash your hands thoroughly.0250

Use a hand sanitizer and make sure that nothing terrible will happen during this lab.0253

Set up, the 5 plates, one of them is going to be the starter culture with E coli colonies.0259

With the E coli, you are going to take that vial, we usually have like preserved E coli.0266

You add some fluid to rehydrate them, shake it up.0275

You take the inoculating loop and streak it all over this plate.0278

You will incubate it for about a day, maybe 2 days.0285

To the point where you actually would have seen just significant amounts of white dots.0289

All of these separate white dots started as a single bacterium that kept dividing and dividing.0302

As the little daughter cells keep dividing, it forms these visible whitish opaque colonies.0311

That is E coli which has at least hundreds of thousands of bacteria, closer to million.0319

When you take one of these colonies from this plate, the starter plate, you can then introduce them to this 4 other plates here.0325

These are the 4 plates that you will use for the actual experiment.0335

One of them which is the control is just E coli.0338

Just E coli, I will use black for that one.0344

A lot of times in class, before we actually streak the plate, when the agar has settled and solidifies, 0351

where you can turn the plate over and upside down without it leaking everywhere, it is solid agar.0359

I have students write in permanent marker on one side of the plate LB, which stands for Liria Britanin, 0365

they are scientists who started the use of agar as a nutrient source for bacteria in this lab.0371

LB means plain agar.0378

Next one, let me use red, ampicillin with no plasmid.0383

We would write amp negative plasmid.0395

It still is LB, meaning it is still agar.0403

All of them we will write LB on them.0406

This has ampicillin in it, no plasmid, you have not inserted the DNA that allows resistance against ampicillin,0408

and the ability to actually make that protein for glowing.0416

If you are wondering, the ampicillin is actually in the agar for these plates.0423

Before you pour the agar, the ampicillin is actually inside of it.0426

Next plate is ampicillin with plasmid.0432

This is still LB, ampicillin + plasmid.0436

Ampicillin is in there, the plasmid is added once the bacteria have started growing on this plate.0445

Finally, we will do this in purple, this is ampicillin with a plasmid and a ribonose which is a sugar.0452

LB amp R + plasmid.0463

This is going to be the one plate that actually should have the glowing at the end.0474

If it is not clear why, I will explain that a little bit later in this lab.0478

These are your 4 plates.0483

When you start the culture or start the colonies growing on these particular plates, 0486

you take it from your starting plate with the inoculating loop.0491

You would streak on the side, rotate them a little bit, take a tiny bit from that streak, streak over here.0494

You want to try to get to the point where you are isolating single colonies of bacteria.0499

It makes the lab happen easier, when you have isolated colonies.0505

The likelihood of them taking up the plasma and expressing it, is going to be greater.0511

After making the plates and letting them set, you streak the plates with the E coli colonies, like I mentioned before.0517

By setting, I mean the agar is set then you take from that starting plate to make these colonies.0525

You add the plasma to the appropriate plates and mark them clearly.0532

You got to make sure that two of the plates do not have the plasmid at all.0534

Three of the plates have the ampicillin in them.0540

Only one of the plate has the ampicillin, that important sugar, and the plasmid together.0542

You incubate the plates for one day at 30 up to 34° C, in the 90° F, bacteria really like that.0549

If you go too far, too hot, closer to 40, it is not going to be ideal.0560

If you are at room temperature, you have to wait closer to 4 -5 days,0567

before you see actual colonies developing, for you to see that glowing happen.0571

Observe the plates after, record the characteristics of the various colonies.0577

Here is an example of what you would see on your starting culture.0580

The one that is actually that was LB, what you typically see on that one is, I’m going to draw it like a big blob.0587

The reason why you end up seeing a blob is, there is nothing controlling bacterial growth here.0598

On the LB plate, it looks almost like a blanket of bacteria.0603

With the one that you had the starting culture, some of it might be a blanket.0607

But when you take little bacteria from it and streak to a plate to get this started, 0611

they say you are trying to take from the colonies that is only a few millimeters in diameter.0616

Each one of these is a colony with a lot of E coli bacteria.0623

You could see they look sort of slimy looking.0628

They have that white, yellowish white opaque look.0632

The one that is LB amp, no plasmid, nothing, no growth at all.0636

I you do have growth, it is probably from contamination from some other bacterium 0648

that is not going to die from ampicillin, maybe it is resistant to ampicillin.0652

This antibiotic got on their somehow.0657

When you do this lab correctly, nothing grows in this plate.0660

The reason why, E coli is not resistant to ampicillin, at least not the strain used for the lab.0663

If you have not introduced the plasmid, on the plasmid is a gene that allows resistance to ampicillin and potential growing.0669

This other one, that was LB amp + plasmid.0681

It is actually going to look like this.0691

The reason why you do not see a blanket of bacteria is because not all the bacteria took up the plasmid.0700

You add it to the plate, the ones that take it up great, the ones that actually got it, awesome, they survived.0708

The others that do not suck it, they do not resist the ampicillin.0716

You are not going to get usually quite as much of a blanket of growth over the whole plate, like you do in this control group.0720

The plasmid have some resistance, the reason why you do not see glowing here 0729

when you shine the UV light on it is, you have not given them a ribonose.0733

A ribonose is what is called a transcription factor that allows expression of that GFP, of that green fluorescent protein.0737

Finally, this is the magical one.0746

I should have used green for this but you will get it using purple.0757

Here is where you are going to see kind of a similar look, in terms of the colonies to this green one over here.0762

The difference being that, when you shine UV on this green one, we do not see glowing.0771

When you shine UV light on this, you are going to see glowing.0775

I will show you on the next slide what that looks like.0779

Look at that, you can see that on this particular plate where you have the ampicillin,0784

a ribonose that has that transcription factor that allows that GFP to be expressed and resistance to ampicillin in that plasmid.0791

Not all of the bacteria are glowing because, for instance, these bacteria they are resistant to ampicillin0799

but they did not actually use the ribonose, potentially.0807

The glowing, maybe if you let it sit for longer, there might be more glowing.0813

You have plenty of glowing in the colonies here.0819

It looks pretty cool when you are shining UV light on it and it looks like that.0822

This lab demonstrates that ampicillin kills bacteria.0825

We saw that kind of negative control that plate 2, which I add ampicillin but no plasmid, 0828

bacteria are going to grow in that environment.0837

They will grow if they are given the plasmid with the protein that allows resistance to the antibiotic ampicillin.0839

Also, the glowing only occurs if you provide the bacteria with a plasmid and a ribonose.0847

That is the transcription factor for the GFP, the green fluorescent protein, to be made.0855

Literally, that sugar is used to dock on the DNA to help RNA polymerase actually go through that gene and make the RNA 0860

that is eventually going to be the protein that will glow in the presence of UV.0873

Unlike some other labs, this lab actually involves students doing lab work, like higher level lab technicians and biologists.0877

That is pretty cool, there are other labs that I have told about in this course where,0886

there is a representative of what is really going on out there in nature.0890

This one, you are really doing it.0896

The connection to genetic transformation on higher level is that, it is easier to transform bacteria than a multicellular organism.0900

You are not going to genetically transform me, at this point in time.0907

With to where technology is at this time, to go into every one of my 100 trillion cells, approximately that much.0910

And add a new gene that is going to be expressed correctly in my cells and 0918

not cause problems or interference with what is already there in the chromosomes, it is too chaotic.0922

There are too many unknowns and actually a lot of times at human genetic transformation, have ended in sickness or death.0929

A single celled being like bacteria E coli, the fact that E coli and bacteria, in general, 0937

have one central chromosome, it is much easier for them to take up DNA from outside of themselves and express it.0943

When exposed to the DNA plasmid and the appropriate transcription factors, the desired trait can be expressed from the bacteria.0952

We can use them for our own ends, for what we want to achieve.0958

This has numerous applications in medicine.0965

One that I believe I mentioned before early on in this course, human insulin can be made from bacteria.0967

Here are little insulin delivery devices.0972

Long time ago, bovine insulin was more commonly used, taken from cows.0975

Some people had allergic reactions to that.0980

When you have bacteria take up DNA that is from humans and you get them to express that,0983

they will crank out human insulin, the exact protein based hormone that is needed to help people with diabetes.0990

That is the amazing application of genetic transformation.0998

Thank you for watching