Take part in research to combat atherosclerosis

Genetic
Engineering
Take part in research to combat atherosclerosis

Experiment Workshop
PROTOCOL

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

Genetic Engineering - 2 -

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
LDL excessive.

Oxidised LDL

Oxidation of LDL

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 Proliferation of
endothelial cells
is known as “foam” cells. These produce substances
which induce inflammation and the proliferation of cells Immune
in the artery wall (endothelials) which results in the system
activation
formation of the atheromatous plaque, blocking the
blood flow.
Foam
cell

Right now, research groups all over the world, LDL
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.
Oxidation of LDL

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.


.
Oxidació de LDL
LDL Macrophage
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.
Oxidised LDL

Oxidation of LDL

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!


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 1 tube on ice with the 1 tube with “LB” 20 µl and
on ice marked as circular DNA, Plasmid bacterial growth 200 µl micropipettes
Nos. 1 and 2. pCR2.1-MYLIP. medium
(A) polystyrene box + (B) (C)
Ice gel

20 µl and 2 Petri dishes with Plastic loops
200 µl micropipette tips agar and antibiotic
(D) Ampicillin

Fluid bath with distilled Float for tubes & Beaker for solid waste Beakers for liquid
water Stopwatch waste

Adhesive tape & 1 tube with 1 ml of 1 tube with Mini-prep 1 tube with Mini-prep
Indelible marker bacterial culture (E) “Solution 1” (F) “Solution 2” (G)

1 tube with Mini-prep 1 tube with 1 tube with “Ethanol” 1 tube with
“Solution 3” (H) “Chloropan” (I) 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.
2+
The Ca 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.


2+
What happens? The Ca 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

AUTHOR FUNDED BY: PROJECT PARTNERS:

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/


Take part in research to combat atherosclerosis

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