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Team:Heidelberg/Project/Science Communication/Open day
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... we will soon report on it in detail | ... we will soon report on it in detail | ||
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| + | == '''Material to carry out an Open Day on Synthetic Biology''' == | ||
| + | |||
| + | We provide the idea and the material needed to carry out an open day for a school class, presenting synthetic biology (and optionally iGEM). | ||
| + | |||
| + | Number of visitors: 20 pupils | ||
| + | Time needed: 5 hours | ||
| + | |||
| + | Schedule: | ||
| + | • Introduction and Presentation on iGEM, Synthetic Biology and your project | ||
| + | • Explaining the idea of BioBricks and the labwork in greater detail | ||
| + | • Lab parcour including the stations: Microscopy, Miniprep, Gel electrophoresis | ||
| + | |||
| + | |||
| + | == '''Minipreparation''' == | ||
| + | |||
| + | After being given a theoritcal introduction, the pupils carry out a plasmid minipreparation using a standard kit. | ||
| + | |||
| + | '''Theoritcal background:''' | ||
| + | |||
| + | All synthetic biologists in the lab need plasmids as very important tools for their work. You can find them in bacterial cells – either naturally, or put in there by scientists. The bacteria amplify the plasmids in their cells (they replicate them, meaning they make identical copies) and of course they multiply the total amount of plasmids by growing – more bacteria means more plasmids on the whole. If we want to work with these plasmids, like checking whether they are the right ones or engineering them, we need to get them out of the bacteria. Therefore we need plasmid preparation. | ||
| + | |||
| + | The procedure is simple, but honestly, it is not very convenient for the bacteria: We need to destroy them; scientists say to lyse them, to set the plasmids free together with the other contents of the cell. Then you will have a mixture of DNA (chromosome and plasmids), RNA, proteins and others cell components. But you only need the plasmid DNA in this case, everything else has to be separated from it, because the rest can have a negative effect on further applications. | ||
| + | |||
| + | The method we mostly use in the laboratory to isolate plasmids is called alkaline lysis. This classical method is today standardized. Like the name implicates, you need alkaline agents for the lysis of the cell. Additionally, you use detergents, to break the cell wall and lyse the cells. | ||
| + | |||
| + | [[Image:Miniprep_eng.jpg]] | ||
| + | |||
| + | '''1. Lysis''' | ||
| + | |||
| + | You can, for example, also find detergents in washing agents. There they help to remove grease spots. This works as follows: Detergents have a very similar structure to lipids. They therefore can mix with them and loose their connection to fibres of the clothes. In Bacteria, they act in a very similar way: They mix with the lipids that form the bilayer of the plasma membrane and destroy there ordered structure. This is how they make holes in the plasma membrane, which leads to the lysis of the cell. | ||
| + | |||
| + | '''2. Precipitation and separation of proteins and chromosomal DNA''' | ||
| + | |||
| + | Now we come to the alkaline part of this procedure: In adding sodium hydroxide (NaOH) to the solution, the pH is increased from roughly 7 to 12. Because this is a huge contrast to the normal physiological pH, proteins and DNA denature and change their conformation. RNA is degraded. Proteins and DNA, which are denatured cannot be soluted any more and precipitate. You all know the phenomenon of denatured proteins from making fried eggs: The egg changes its consistency if you heat it and gets hard – this is due to the denaturising proteins. The same effect as heat, have acids or bases (like NaOH). In denatured DNA, the two strands get separated from each other. | ||
| + | |||
| + | The trick the alkaline method uses, to separate the plasmid DNA from the chromosomal DNA and the proteins, is to lower the pH again by adding acid (for example potassium acetate) to the solution. If the pH approximates the physiological one, the plasmids are able to renature first, because they are small and the two DNA strands find each other with a much higher possibility than the ones of the big chromosome. Therefore if you keep the renaturising step short, only the plasmids are able to build double helices and solute again. The chromosomal DNA and the proteins stay precipitated. They can be separated from the plasmids by centrifugation. | ||
| + | (The white solids on the tube after centrifugation are the precipitated proteins, DNA and other cell components. The plasmid DNA is soluted in the clear supernatant.) | ||
| + | |||
| + | '''3. Binding of DNA''' | ||
| + | |||
| + | Now how do we get the plasmids out of this salty solution (remember we put a lot of salts to the solution to first rise, then lower the pH) into a clear one that can be used for further applications? You could precipitate the plasmids like we did with the proteins before, but there is another possibility, which is widely used today: | ||
| + | You bind the DNA to a solid phase. This often consists of silica, which can be used in a bead like form, but in most cases, it is included in a column integrated in an eppendorf tube. The DNA can bind to the silica column, if the solution is very salty. If you centrifuge, the salty solution is drawn through the column into the tip of the tube and the DNA remains bound to the column. | ||
| + | |||
| + | '''4. Eluation of the plasmids''' | ||
| + | |||
| + | In the last step, you eluate the DNA from the column, meaning you loosen the bonds between DNA and silica and solute the DNA again. This is possible with every solution containing low or no salt at all. You can take water, for example. You put it on the column, centrifuge again, but this time the DNA will also be drawn through the column into the tip of the tube together with the water. | ||
| + | You can then proceed to work with your purified plasmids: you can, for example, check, whether you have isolated the right one using agarose gel electrophoresis. | ||
Revision as of 16:15, 25 October 2008
Open Day
On the 27th of October, we will carry out an Open Day for school classes. We will present Synthetic Biology, iGEM and our project. We also let the pupils experience to do lab work on their own and therefore set up a little parcours to visualize, isolate and proof the existence of Biobricks.
... we will soon report on it in detail
Material to carry out an Open Day on Synthetic Biology
We provide the idea and the material needed to carry out an open day for a school class, presenting synthetic biology (and optionally iGEM).
Number of visitors: 20 pupils Time needed: 5 hours
Schedule: • Introduction and Presentation on iGEM, Synthetic Biology and your project • Explaining the idea of BioBricks and the labwork in greater detail • Lab parcour including the stations: Microscopy, Miniprep, Gel electrophoresis
Minipreparation
After being given a theoritcal introduction, the pupils carry out a plasmid minipreparation using a standard kit.
Theoritcal background:
All synthetic biologists in the lab need plasmids as very important tools for their work. You can find them in bacterial cells – either naturally, or put in there by scientists. The bacteria amplify the plasmids in their cells (they replicate them, meaning they make identical copies) and of course they multiply the total amount of plasmids by growing – more bacteria means more plasmids on the whole. If we want to work with these plasmids, like checking whether they are the right ones or engineering them, we need to get them out of the bacteria. Therefore we need plasmid preparation.
The procedure is simple, but honestly, it is not very convenient for the bacteria: We need to destroy them; scientists say to lyse them, to set the plasmids free together with the other contents of the cell. Then you will have a mixture of DNA (chromosome and plasmids), RNA, proteins and others cell components. But you only need the plasmid DNA in this case, everything else has to be separated from it, because the rest can have a negative effect on further applications.
The method we mostly use in the laboratory to isolate plasmids is called alkaline lysis. This classical method is today standardized. Like the name implicates, you need alkaline agents for the lysis of the cell. Additionally, you use detergents, to break the cell wall and lyse the cells.
1. Lysis
You can, for example, also find detergents in washing agents. There they help to remove grease spots. This works as follows: Detergents have a very similar structure to lipids. They therefore can mix with them and loose their connection to fibres of the clothes. In Bacteria, they act in a very similar way: They mix with the lipids that form the bilayer of the plasma membrane and destroy there ordered structure. This is how they make holes in the plasma membrane, which leads to the lysis of the cell.
2. Precipitation and separation of proteins and chromosomal DNA
Now we come to the alkaline part of this procedure: In adding sodium hydroxide (NaOH) to the solution, the pH is increased from roughly 7 to 12. Because this is a huge contrast to the normal physiological pH, proteins and DNA denature and change their conformation. RNA is degraded. Proteins and DNA, which are denatured cannot be soluted any more and precipitate. You all know the phenomenon of denatured proteins from making fried eggs: The egg changes its consistency if you heat it and gets hard – this is due to the denaturising proteins. The same effect as heat, have acids or bases (like NaOH). In denatured DNA, the two strands get separated from each other.
The trick the alkaline method uses, to separate the plasmid DNA from the chromosomal DNA and the proteins, is to lower the pH again by adding acid (for example potassium acetate) to the solution. If the pH approximates the physiological one, the plasmids are able to renature first, because they are small and the two DNA strands find each other with a much higher possibility than the ones of the big chromosome. Therefore if you keep the renaturising step short, only the plasmids are able to build double helices and solute again. The chromosomal DNA and the proteins stay precipitated. They can be separated from the plasmids by centrifugation. (The white solids on the tube after centrifugation are the precipitated proteins, DNA and other cell components. The plasmid DNA is soluted in the clear supernatant.)
3. Binding of DNA
Now how do we get the plasmids out of this salty solution (remember we put a lot of salts to the solution to first rise, then lower the pH) into a clear one that can be used for further applications? You could precipitate the plasmids like we did with the proteins before, but there is another possibility, which is widely used today: You bind the DNA to a solid phase. This often consists of silica, which can be used in a bead like form, but in most cases, it is included in a column integrated in an eppendorf tube. The DNA can bind to the silica column, if the solution is very salty. If you centrifuge, the salty solution is drawn through the column into the tip of the tube and the DNA remains bound to the column.
4. Eluation of the plasmids
In the last step, you eluate the DNA from the column, meaning you loosen the bonds between DNA and silica and solute the DNA again. This is possible with every solution containing low or no salt at all. You can take water, for example. You put it on the column, centrifuge again, but this time the DNA will also be drawn through the column into the tip of the tube together with the water. You can then proceed to work with your purified plasmids: you can, for example, check, whether you have isolated the right one using agarose gel electrophoresis.
