Genetic Engineering - Xplore Health€¦ · Genetic Engineering - 14 - 4. Using the 200 µl micropipette (with a new tip each time), add the bacteria (100-200 µl) from tubes 1 and

Genetic Engineering Take part in research to combat atherosclerosis

PROTOCOL

Experiment Workshop

Genetic Engineering - 2 -

Genetic Engineering Searching for a target for the treatment of atherosclerosis

Introduction

Biomedical research encompasses the physical and chemical processes which

take place inside living beings and those processes which trigger diseases. One of

the main aims of this field of research is to identify therapeutic targets, i.e. parts of

the body at which new treatments can be directed that will stimulate responses and

help to combat the diseases.

This protocol follows a line of biomedical research which focuses on the study of a

potential therapeutic target that could be recognised by a drug against

atherosclerosis.

What causes atherosclerosis?

Aterosclerosi is a vascular disease caused by the accumulation of fats on the walls of the blood

vessels. There are many different signs and degrees of severity depending on where the affected

vessels are and how far the disease has progressed. In our society, consumption of foods high in

saturated fats has considerably increased the risk of suffering from cardiovascular diseases. This

excess of fat in our bodies can become deposited and accumulate at certain points of the artery walls

in the form of plaque, called atheromatous plaque, which obstructs the blood flow.

-----� Normal artery

-----� Moderate atherosclerosis

-----� Severe atherosclerosis


Cholesterol and the Macrophages

Cholesterol is one of the lipid substances that make up atheromatous plaque. To stop cholesterol from

becoming deposited on the walls, our bodies have a “cleaning” system, the macrophages, which are

cells that circulate in the blood and pick up the harmful cholesterol molecules, known as LDL (low-

density lipoprotein).

The macrophages recognise them thanks to a

receptor on their membrane. This cleaning system

is efficient if the increased cholesterol is not too

excessive.

If the amount of cholesterol is very excessive, the

macrophages continue to pick up the LDL, but, once

they have engulfed large amounts, they turn into what

is known as “foam” cells. These produce substances

which induce inflammation and the proliferation of cells

in the artery wall (endothelials) which results in the

formation of the atheromatous plaque, blocking the

blood flow.

Right now, research groups all over the world,

including Barcelona University Nuclear Receptor

Research Group, are trying to better understand

exactly how macrophages are involved in the

regulation of cholesterol levels and the development of

atherosclerosis.

More specifically, scientists are studying the role of a protein in the macrophages called MYLIP. The

main function of this protein is to break down the macrophage’s membrane receptor that allows it to

recognise LDL. Scientists have seen that if this protein is produced in larger quantities, the

macrophages ingest less cholesterol. However, its role in the context of atherosclerosis is still not fully

understood.

LDL

Oxidised LDL

Oxidation of LDL

Proliferation of endothelial cells

Immune system activation

LDL

Foam cell

Oxidation of LDL


.

Since it has been seen that the MYLIP protein is

associated with the regulation of bad cholesterol,

scientists believe that there could be a new target

for the treatment of atherosclerosis in the

regulation process.

How can we study the MYLIP protein involved in the regulation of cholesterol?

In order to study this potential therapeutic target, researchers need to produce large quantities of it in

the laboratory. To do so, they use a genetic engineering technique called bacterial transformation, with

which they transfer DNA from an organism to a bacterium so that the bacterium will produce large

amounts of DNA, which can then be introduced into other cells so they then produce the therapeutic

target under study.

They start from a purified form of the gene that produces the MYLIP protein. They then join it to a

circular fragment of DNA called plasmid and insert it into bacteria so they will produce replicas of the

genetic material.

In this protocol, we invite you to work as biotechnicians and perform a bacterial transformation!

Oxidació de LDL Oxidació de LDL Macrophage

Oxidised LDL

Oxidation of LDL


Organisation of the workshop:

1. We will start by doing a bacterial transformation to incorporate the DNA responsible for

producing the MYLIP protein, so the bacteria act as bioreactors and manufacture the genetic

material in large quantities.

2. We will allow the bacteria culture to grow in a suitable medium and then select those which have

incorporated the gene.

3. We purify the genetic material that contains the gene responsible for producing the MYLIP

protein so it can be introduced into other types of cells which will produce the protein (*)

(* ) since the growth of bacteria requires one and a half days, the purification will be done from a transformed bacterial culture

that the monitors have prepared beforehand


Equipment and materials required for each group/table

2 tubes with bacteria

on ice marked as

Nos. 1 and 2.

(A) polystyrene box +

Ice gel

1 tube on ice with the

circular DNA, Plasmid

pCR2.1-MYLIP.

(B)

1 tube with “LB”

bacterial growth

medium

(C)

20 µl and

200 µl micropipettes

20 µl and

200 µl micropipette tips

2 Petri dishes with

agar and antibiotic

(D) Ampicillin

Plastic loops

Fluid bath with distilled

water

Float for tubes &

Stopwatch

Beaker for solid waste Beakers for liquid

waste

Adhesive tape &

Indelible marker

1 tube with 1 ml of

bacterial culture (E)

1 tube with Mini-prep

“Solution 1” (F)


“Solution 2” (G)


“Solution 3” (H)

1 tube with

“Chloropan” (I)

1 tube with “Ethanol”

1 tube with

ultrapure water.

(milliQ).


(A) Bacteria which allow the entry of DNA (called XL1-blue competent cells)

(B) Circular DNA called Plasmid (pCR2.1)

(C) LB (Luria-Bertani) culture medium: yeast extract 5 g/l, Tryptone 10 g/l, NaCl 5 g/l. Sterilise in

autoclave. Keep cold.

(D) LB/ampicillin plates: LB culture medium, Agar 1.6%, Ampicillin 100 µg/ml.

(E) Bacteria cultured in 2XYT medium, overnight (16 h)

(F) Solution 1: 50 mM Glucose/25 mM Tris-HCl pH 8.0/10 mM EDTA/Dilute in distilled H2O.

(G) Solution 2: 20 µl SDS detergent (sodium dodecyl sulphate) 10%/4 µl NaOH 10N/176 µl de H2O

mQ/for each sample. Prepare in duplicate on day of practical.

(H) Mini-prep solution 3: 73.60 g Potassium Acetate/28.75 ml Acetic Acid/Dilute in H2O mQ to a final

volume of 250 ml.

(I) Chloropan: 25 ml Equilibrated Phenol/24 ml Chloroform/1 ml Isoamyl Alcohol. Centrifuge for 10 min

at 3000 rpm.


Procedures

1- BACTERIAL TRANSFORMATION

A bacterial transformation is a biotechnological process by which scientists introduce the genetic

material responsible for producing a protein under investigation into a bacterial cell. This cell will then

act as a bioreactor and produce copies of this genetic material, which can then be introduced into

another type of cell in order to produce the protein of interest.(*)

In this workshop, we will be starting from the purified gene of our “MYLIP” protein, which has been

introduced into a plasmid or circular DNA fragment.

Using this genetic material, we are going to perform a bacterial transformation, i.e. we are going to

introduce the gene responsible for producing our protein into the bacteria by heat shock – subjecting

the sample to different temperatures.

(*)In cases where the protein of interest is not so complex, the transformed bacteria themselves can go on to act as bioreactors

to produce the protein.


Protocol for bacterial transformation

1. We have two tubes with bacteria on ice. Each tube contains a buffer solution which will help the

transformation thanks to the Ca2+

cations of the salt CaCl2 in the buffer.

What happens? The Ca2+

cations, under the cold conditions,

prepare the cell membranes so they are permeable to the DNA.

The Ca2+

ions bind to the phospholipids in the cell membrane,

protecting their negative charges and forming small pores in the

membrane of the bacteria.

2. Add the DNA in the form of plasmid to the bacteria : using the 20 µl micropipette, pipette 10 µl of

plasmid (tube P) and add it to tube 2, which contains the bacteria. Cap the tube and mix gently

by tapping the tube with your finger. Tube 1 will be the control since it contains bacteria without

plasmid.


What happens? The Ca2+

ions also interact with the negatively-charged

phosphate groups in the DNA and this makes it possible for them to get close to

the bacteria membranes without being repulsed as a result of their electrical

charges.

3. Leave the tubes to settle for 15 minutes

* Meanwhile, use this time to carry on with the first step of the protocol: “Growth of the transformed bacteria”

4. After the 15 minutes, put tubes 1 and 2 in the fluid bath at 42º for exactly 1 minute 30

seconds.

What happens? The circular DNA or plasmid penetrates

through the pores of some of the bacteria. How? At 42º,

the elasticity of the bacterial membrane increases and this

helps the plasmid to enter through the pores.


5. Put tubes 1 and 2 back on ice for 2 minutes.

What happens? As the temperature drops, the

membranes stabilise and the plasmid which had

passed through the pores remains inside the bacteria.


2- GROWTH OF THE TRANSFORMED BACTERIA

Once the plasmid is incorporated into the bacteria, we need to get the bacteria to grow by providing a

suitable medium and the right temperature. Since bacterial transformation normally produces a

mixture of a very few transformations and lots of untransformed cells, we need a method to identify the

cells which have incorporated the plasmid.

We can make sure we only grow the transformed bacteria by growing them in culture plates that

contain an antibiotic. Because the plasmid contains a gene which makes the bacteria resistant to this

drug, only transformed bacteria will grow in colonies. From these colonies, we will then be able to

continue growing only the bacteria that produce the gene of interest.


Protocol for the growth of the transformed bacteria

1. Mark the plates on which you are going to grow the bacteria. One will be a control plate with

bacteria without plasmid, and the other, where you are going to grow the bacteria with plasmid.

2. Using the 200 µl micropipette, add 500 µl of “LB” bacterial growth medium, which contains

nutrients, to tubes 1 and 2. Cap the tubes and mix gently by tapping them with your finger.

3. Incubate the mixture in the fluid bath at 37º for 30 minutes

What happens? This period gives the bacteria time to

multiply, so that, as they duplicate their DNA, they also

generate a copy of the plasmid DNA which contains the gene

of interest.

* While you are waiting for the bacteria to grow, you can carry on to the third step of this workshop which consists in isolating

the genetic material from the bacteria.


4. Using the 200 µl micropipette (with a new tip each time), add the bacteria (100-200 µl) from

tubes 1 and 2 to the agar plates which also contain the antibiotic, Ampicillin.

5. Spread the bacteria over the surface of each agar plate using a new sterile plastic loop for each

one. Turn the plate as you move the rod back and forth. Seal the plates with strips of adhesive

tape and write your initials on them with the date and the type of bacteria.


6. Incubate the plates inverted at 37ºC and observe the next day for results. If there is no incubator

available, simply leave the bacteria to grow for a couple of days at room temperature.


3- ISOLATING THE RESULTING GENETIC MATERIAL

In order to obtain large quantities of the transformed bacteria which have grown and formed colonies,

scientists take a sample and put it to grow in another culture medium containing nutrients. The genetic

material the bacteria have produced then needs to be isolated, i.e. it has to be separated from the rest

of the components of the bacteria, such as RNA, DNA of the bacteria and proteins. To isolate the

plasmid, scientists follow a protocol known as Mini-prep. This technique makes it possible to separate

the DNA in the form of plasmid by using various solvents and centrifugation cycles which gradually

discard the different cell components.

The purified DNA is then of great use to scientists for conducting further experiments to study its role

in the regulation of cholesterol and in atherosclerosis, and in the search for new drugs.

Since the process of growing large quantities of bacteria takes over a day and a half, for the

workshop, you are going to use a culture that has been previously prepared by the monitors.


Protocol for purifying plasmid DNA from the bacterial culture (Mini-prep)

1. Centrifuge the bacterial culture tube at maximum speed for 30 seconds. Then remove the

supernatant fluid.

What happens? The cells are separated from the

culture medium in which they have grown, which

normally contains cell waste and other molecules that

we want to discard.

2. Then remove the supernatant fluid and, using the 200 µl micropipette, suspend the precipitate

again with 100 µl of solution 1 and use the pipette to mix.

What happens? The bacteria are re-suspended in order to continue the purification process,

but this time the concentration is higher as they are in a smaller volume. Solution 1 is a buffer

solution that will prevent the denaturation of the circular DNA in the form of plasmid which would

occur if the pH rose above 12.6.

3. Add 200 µl of solution 2 and mix gently by inverting.

What happens? Solution 2, which contains a

detergent, destroys the bacterial phospholipid

membranes, releasing all the cell content into the

medium, through a process called bacterial lysis.

This solution also contains a strong base (sodium

hydroxide, NaOH) which denatures the proteins

involved in maintaining the structure of the cell

membrane and the bacteria’s own DNA.

However, the plasmid DNA is not affected since

the pH is below 12.6.


4. Add 150 µl of solution 3 and mix by inverting.

What happens? Solution 3 is an acid solution

of sodium acetate which neutralises the pH of

the solution and halts the lysis process. In this

step, the majority of the cell contents – the

proteins, the membrane phospholipids and the

bacteria’s own DNA – precipitate, forming a

white mucus. The bacteria’s own DNA, now

denatured, forms an insoluble complex which

precipitates because the K+ ions bind to the

phosphate groups of the DNA and thus

neutralise their negative charge.

5. Add 300 µl of chloropan, mix well by inverting and centrifuge for 3 minutes at maximum speed

to separate the plasmid that contains the gene of the MYLIP.

What happens? In this step, the plasmid DNA is

separated from the remaining cell contents – proteins,

lipids and other nucleic acids. The chloropan is a mixture

of organic solvents that contains phenol and chloroform.

These solvent dissolve the hydrophobic molecules such

as the membrane lipids and denature the proteins,

making them insoluble in water. Through centrifugation,

we separate the mixture into two phases: the

phospholipids and the cell proteins remain in solution in the lower chloropan phase or trapped at the

interface between the two phases in the form of a white precipitate. The plasmid DNA will be in the

upper aqueous phase as its electrical charges are not neutralised and it is therefore soluble in water.

6. Transfer the upper transparent aqueous phase to a new tube with the 200 µl pipette


7. Add 900 µl of 100% Ethanol with the 200 µl pipette and mix well. The ethanol makes the

plasmid DNA precipitate from the aqueous solution.

8. Centrifuge for 5 minutes at maximum speed. You will see precipitate, which is the DNA in the

form of plasmid.

9. Take out ALL the ethanol with the 200 µl pipette.

10. Re-suspend the plasmid DNA precipitate in 20 µl of purified H2O.


Results and Discussion

1. Make a diagram of the bacterial transformation process and explain what role it plays in

research into finding new drugs to combat atherosclerosis.

2. With the help of a diagram, explain what a heat shock is.

3. To make the bacteria grow, you used a culture plate that contained an antibiotic. Why?

4. What is a bacteria colony?

5. On which of the two plates you spread, the control or the one with transformed bacteria, will you

obtain colonies? Why?


6. From the colonies that have formed, how do you obtain multiple copies of the gene of interest?

7. How do you make bacterial DNA and the DNA of interest precipitate? Supplement your

explanation with a diagram.

8. With the separation technique called Mini-prep, you have managed to isolate multiple copies of

the DNA of interest. What do scientists do once they have obtained this DNA?

Researchers who have contributed to the writing of this protocol: Theresa León, Jonathan

Matalonga, Universitat de Barcelona

This work is under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported licence. To see a copy of this licence, visit visiteu http://creativecommons.org/licenses/by-nc-nd/3.0/

FUNDED BY: PROJECT PARTNERS: AUTHOR

Documents

Genetic Engineering - Xplore Health€¦ · Genetic Engineering - 14 - 4. Using the 200 µl micropipette (with a new tip each time), add the bacteria (100-200 µl) from tubes 1 and