Genetic Engineering. Take part in research to combat atherosclerosis. Experiment Workshop PROTOCOL

Transcription

1 Genetic Engineering Take part in research to combat atherosclerosis Experiment Workshop PROTOCOL

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 Genetic Engineering - 2 -

3 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 (lowdensity lipoprotein). LDL The macrophages recognise them thanks to a receptor on their membrane. This cleaning system is efficient if the increased cholesterol is not too excessive. Oxidation of LDL Oxidised 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 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. Proliferation of endothelial cells Immune system activation Foam cell 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. LDL 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. Genetic Engineering - 3 -

4 DL. Macrophage Oxidació de 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 Oxidised LDL regulation process. 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! Genetic Engineering - 4 -

5 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 Genetic Engineering - 5 -

6 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 200 µl micropipette tips 2 Petri dishes with agar and antibiotic Plastic loops (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 Solution 3 (H) 1 tube with Chloropan (I) 1 tube with Ethanol 1 tube with ultrapure water. (milliq). Genetic Engineering - 6 -

7 (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: 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. Genetic Engineering - 7 -

8 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. Genetic Engineering - 8 -

9 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 Ca 2+ cations of the salt CaCl 2 in the buffer. What happens? The Ca 2+ cations, under the cold conditions, prepare the cell membranes so they are permeable to the DNA. The Ca 2+ 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. Genetic Engineering - 9 -

10 What happens? The Ca 2+ 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. Genetic Engineering

11 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. Genetic Engineering

12 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. Genetic Engineering

13 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. Genetic Engineering

14 4. Using the 200 µl micropipette (with a new tip each time), add the bacteria ( 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. Genetic Engineering

15 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. Genetic Engineering

16 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. Genetic Engineering

17 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 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 Genetic Engineering

18 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 Genetic Engineering

19 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 H 2 O. Genetic Engineering

20 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? Genetic Engineering

21 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 Genetic Engineering