Protocol for youngsters to carry out a bacterial transformation in a lab. The 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. The experiment protocol is an opportunity for science centres, museums and schools to replicate a real experiment done in a real lab doing research on drug discovery.

1. Genetic Engineering
Searching for a target for the treatment of
atherosclerosis

2. RISK FACTORS
Consumption of foods high in saturated fats and a sedentary lifestyle increase the
risk of suffering from cardiovascular diseases such as atherosclerosis.

3. WHAT IS ATHEROSCLEROSIS?
Atherosclerosis 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.
Normal artery
Moderate
atherosclerosis
Severe atherosclerosis
Formation of atheromatous plaque, which obstructs the blood flow.

4. “BAD” OR LDL CHOLESTEROL
• When there is too much cholesterol
in the diet, it either ends up being
stored as fat or continues circulating
in the blood in the form of LDL (low-
density lipoprotein) or what is more
commonly known as “bad
cholesterol”.
• LDL is one of the components of
the atheromatous plaque.

5. ACCORDING TO THE WORLD HEALTH ORGANISATION (WHO)
•Although heart attacks and strokes are major killers in all parts of the world,
80% of premature deaths from these causes could be avoided by controlling the
main risk factors
•Every year, an estimated 17 million people globally die of cardiovascular
diseases (CVD), particularly heart attacks and strokes.
•CVDs occur almost equally in men and women.
•Are the leading cause of death in developing countries, as well as developed
ones.

6. THE MACROPHAGES, A “CLEAN-UP SYSTEM”
• The macrophages act as a “cleaning” system to stop the cholesterol from being
deposited on the walls.
• This clean-up system is efficient if the increased cholesterol is not too excessive.
LDL
Macrophage
Oxidised LDL
Oxidation of LDL

7. HOW DOES AN ATHEROMATOUS PLAQUE FORM?
Proliferation
of endothelial
If the quantities of cholesterol are very cells
excessive:
Immune
system
activation
The macrophages continue to pick up the LDL
Foam
But, once they have engulfed large amounts, they turn cell
into what is known as “foam” cells.
LDL
Macrophage
These induce inflammation and the proliferation of cells in
the artery wall
Oxidised LDL
Oxidation of LDL
Formation of the atheromatous plaque

8. RESEARCH INTO ATHEROSCLEROSIS
One of the objectives is:
To understand how macrophages are involved in the regulation of cholesterol levels and what role they play in
the development of atherosclerosis.
?

9. STUDY OF THE LDL RECEPTOR AND MYLIP
We know that the macrophages recognise oxidised LDL thanks to some receptors.
We know that a protein in the macrophages called MYLIP breaks down the LDL
receptor.
Macrophage
LDL receptor
Oxidised LDL
If the macrophages produce MYLIP in
large quantities, they ingest less
Oxidation of LDL cholesterol.

10. MYLIP, A POTENTIAL THERAPEUTIC TARGET
Scientists are studying the MYLIP protein in depth because they think that it could be
regulated by a drug and thus stop the macrophages from ingesting less cholesterol.
Macrophage
Therapeutic target
Oxidised LDL
Oxidation of LDL

11. HOW CAN WE STUDY THE PROTEINS?
• Large quantities of a protein are needed to
study its function.
•One of the tools in molecular biology which
allows us to study the proteins is genetic
engineering.
•Genetic engineering is the technology of the
manipulation and transfer of DNA from one
organism to another.

12. FIRST WE CLONE THE GENE OF THE PROTEIN OF INTEREST:
MYLIP
We insert it into a fragment of
Human cell ADN MYLIP gene
circular DNA called plasmid

13. WE PERFORM A BACTERIAL TRANSFORMATION WITH THE
GENE OF THE PROTEIN OF INTEREST: MYLIP
1. Bacterial 2. Selection of transformed 3. Bacterial growth
transformation bacteria

14. THE GENE OF THE MYLIP PROTEIN IS THEN INTRODUCED
INTO CELLS IN ORDER TO PRODUCE MORE
Protein
MYLIP
3. Bacterial 4. We isolate 5. Production of
growth the DNA the protein
(introducing the DNA
into eukaryotic cells)

15. HOW DO WE DO A BACTERIAL TRANSFORMATION?
With drastic changes in
? temperature and by adding
cations, we help the entry of
the DNA in the form of
plasmid into the cells.

16. HOW CAN WE MAKE SURE THAT TRANSFORMED BACTERIA
WILL GROW?
?
The plasmid has a gene
that is resistant to an
antibiotic.

17. HOW CAN WE MAKE SURE THAT TRANSFORMED BACTERIA
WILL GROW?

18. HOW DO WE ISOLATE THE PLASMID DNA FROM THE
BACTERIAL CULTURE?
?
Doing a Mini-prep:
Using various solvents and centrifugation cycles, we gradually discard the
different components

19. WITH THE DNA ISOLATED, WE CAN GET ON WITH THE
RESEARCH!
5. Production of the protein: (introducing the DNA into
eukaryotic cells).
6. Once the scientists obtain the
protein, they can then:
• Study its role in cholesterol regulation
• Search for new drugs for atherosclerosis.

20. Put on a lab coat!!
You too can be a researcher!!
Researchers who have contributed to the writing of this presentation: Theresa León, Jonathan
Matalonga, Barcelona University
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 http://creativecommons.org/licenses/by-nc-nd/3.0/