Genetic engineering from A to Z part 3
Summary of the previous series :
Scientists have discovered the gene blue glow. We read about this gene lit up to make a glowing transgenic tree. We found its name and sequence in the specialized resources, knocked out a business trip from the boss and went to where the animal lives - the buttery containing the gene.
Through various tricks with the use of special equipment, we obtained pure DNA molecules of the bl1 gene.
To these DNA molecules, utility sequences were placed to work inside the cell, and transgenic E. coli bacteria were created based on them.
On the transformation of plants.
Ideally, while we were doing all the gene manipulations, our comrade worked intently on what he was looking for, how to transform the Christmas tree. If we just pour it with the bl1 DNA solution, even with all the service areas, nothing will change. It is necessary to somehow put these molecules inside the cell, and not just inside the cell, but inside the DNA molecule located in the cell nucleus.
')
And this is a very difficult task, which strongly depends on what we transform into an organism.
The agent that delivers the DNA molecules into the cell is called a vector . A vector may simply be a DNA solution, some organisms (bacteria), viruses, special particles.
Delivery method
Most often, plants are transformed with a vector based on Agrobacterium ( Agrobacterium tumefaciens ). This is such a parasitic bacterium that carries a giant plasmid called Ti. This plasmid transfers genes into the host-khziyan genome that allow this bacterium to exist comfortably. That is, Bactria itself is able to embed pieces of its DNA into the plant's DNA.
So, a bacterium can be deceived and a Ti plasmid constructed for it, so that everything necessary for transfer remains there, but replace the portable part with the one we need, for example, the bl1 gene with the 35S promoter from tobacco mosaic virus. This trick is the basis of the so-called agrobacterial transformation.
Unfortunately, the Christmas tree is not so simple. To be honest, I did not hear at all that they could transform it. For such difficult objects, often the only way is direct ballistic transformation or gene gun .
This is an expensive and complicated, but universal way of delivering DNA into the cell. It lies in the fact that DNA molecules stick on gold microparticles, which are then fired at high speed into cell culture. These particles enter a part of the cells and leave the DNA molecules, which with a rather low probability are inserted into the genome.
What exactly are we transforming?
But, in fact, even if the act of embedding occurs, it will occur in one cell! She must somehow survive among the masses of others.
To do this, use the genetic marker that is contained in the DNA that we shot. This marker can be a herbicide resistance gene (a substance that is toxic to plants).
The culture of the cells after shooting is placed on the herbicide medium, to which only transformed cells are resistant. The picture shows that part of the calluses is yellow - dead, and part is green. This is how the action of the herbicide is manifested. Callus is the unorganized mass of undifferentiated plant cells.
Have you noticed that everywhere I am not talking about the whole plant, but about the culture of its cells? This is an important addition, it is impossible to “mutate”, that is, to transform the whole body at once (by the way, a stone in the garden of illiterate sci-fi about mutants). It is necessary to work with individual cells. It comes - because it is difficult and uncomfortable, the cells outside the body are very capricious and often do not want to grow as we need.
Obtaining a living cell culture is a separate biotechnological problem, which will not be discussed further, since it is too complicated to be included in this article.
But in any case, working with cell cultures is also sterile conditions that can be achieved only by purchasing a laminar box and an autoclave. Cell cultures grow on special media in Petri dishes. Different substances are added to the medium for growth - salt, sugar and, most importantly, a certain composition of hormones that induces cells to divide or form adult plants.
Concrete steps.
It was a lyrical introduction, and now the prose goes. The whole process is divided into several stages.
Plasmid
We take our plasmid with the existing utility sequences, cut out the desired piece from there (using the methods described in part 2 ) and place it in the purchased vector plasmid for transfer to plants. We transform agrobacteria with this plasmid (methods are also described in the previous article). We do not work with agrobacteria right away because they are much less convenient than E.coli and grow worse.
Cell culture
At the same time, we are trying to obtain a sterile cell culture of a plant, using our own literature and our own data on which nutrient medium the cells of this species grow best of all. As a starting material for cell culture, seeds or cuttings are used, which grow on the medium with the addition of hormones into an unorganized mass of cells - callus. Usually the process of obtaining cell culture takes 1-2 months (as they usually grow slowly).
Bacteria culture
After that, we grow agrobacterium culture or prepare gold particles with DNA sputtering for transformation, depending on which method is suitable for this type of plant.
Transformation
Now we have everything to transform. We are incubating with bacteria (about an hour), or a shot of gold particles using a gene gun.
Selection of transformants
We place on the environment with antibiotics to kill bacterial residues and herbicide so that only transformed cells grow.
Regeneration
After some time, the transformed cells form colonies - calli. These calli are sown on the medium with the addition of plant hormones (auxins and cytokinins) for regeneration.
We get transgenic plants (in our case - a glowing Christmas tree)!
Next we have to test them for the presence of the insert, its performance, but this is a separate conversation.
Here is a process diagram for better understanding:
As in any scientific experiment, in the transformation it is imperative to set controls, that is, experiments showing us that everything is done correctly.
A positive control for obtaining a transgenic plant is the transformation using the purchased test vector with the presence of certain markers. If it happens, it will show us that we are doing everything correctly and our methodology is appropriate.
Negative control is the lack of transformation and placing the cells directly on the medium with the herbicide. What is it for? In order to avoid, for example, such a dose of herbicide is insufficient to completely suppress the growth of non-transformed plants.
If a positive and negative control is carried out, and the transformation has failed, then this means that the problems in our design and everything needs to be redone
Well, as you would like, genetic engineering is not an easy thing :)
Remember, setting up control reactions at each stage of the experiment is a necessary condition in order to be sure that everything goes as it should.
That's all, in my opinion, I described how could the process of obtaining transgenic plants in the laboratory. I hope you found the information interesting :)
Conclusion
I remember that I promised to calculate the cost of equipment (based on the real cost of purchasing used ones) and reagents for carrying out this process.
Amplifier - 30 thousand rubles
Centrifuge table - 20 thousand
Reagents (in the form of ready-made kits) - 30 thousand
Current source, phoresis chamber, UV lamp (transilluminator) - 30 thousand
Laminar box - 90 thousand
Desktop autoclave - 50 thousand
Automatic dispensers from 0.5 to 1000 μl - 25 thousand
Laboratory balance - 20 thousand
Consumable plastic (test tubes, dispenser tips, petri dishes, etc.) - 7 thousand
Software for working with DNA sequences can cost more than $ 1000, but you can do without it or use trial versions.
Room cost, etc. I do not consider, because it is too individual.
In principle, it can be seen that quite many people can equip a genetic laboratory; there would be a desire and skill. At this stage, the equipment is expensive, but not worth the space money.
In general, I have a serious feeling that genetic engineering is now only at the beginning of my long journey, so knowledge of it will not be superfluous to anyone :) It is unlikely that laboratories will be distributed “on the knee”, but there is no doubt that genetic technologies firmly enter into the medical, food industry, criminology. And who knows how it will go on.
2all I apologize for such a delay in the release of the third part, it was written long ago, but there was no time to bring it to mind and publish it.
Links to previous parts of the article:
The first part: https://geektimes.ru/post/48533/
The second part: https://geektimes.ru/post/48846/
Scientists have discovered the gene blue glow. We read about this gene lit up to make a glowing transgenic tree. We found its name and sequence in the specialized resources, knocked out a business trip from the boss and went to where the animal lives - the buttery containing the gene.
Through various tricks with the use of special equipment, we obtained pure DNA molecules of the bl1 gene.
To these DNA molecules, utility sequences were placed to work inside the cell, and transgenic E. coli bacteria were created based on them.
On the transformation of plants.
Ideally, while we were doing all the gene manipulations, our comrade worked intently on what he was looking for, how to transform the Christmas tree. If we just pour it with the bl1 DNA solution, even with all the service areas, nothing will change. It is necessary to somehow put these molecules inside the cell, and not just inside the cell, but inside the DNA molecule located in the cell nucleus.
')
And this is a very difficult task, which strongly depends on what we transform into an organism.
The agent that delivers the DNA molecules into the cell is called a vector . A vector may simply be a DNA solution, some organisms (bacteria), viruses, special particles.
Delivery method
Most often, plants are transformed with a vector based on Agrobacterium ( Agrobacterium tumefaciens ). This is such a parasitic bacterium that carries a giant plasmid called Ti. This plasmid transfers genes into the host-khziyan genome that allow this bacterium to exist comfortably. That is, Bactria itself is able to embed pieces of its DNA into the plant's DNA.
So, a bacterium can be deceived and a Ti plasmid constructed for it, so that everything necessary for transfer remains there, but replace the portable part with the one we need, for example, the bl1 gene with the 35S promoter from tobacco mosaic virus. This trick is the basis of the so-called agrobacterial transformation.
Unfortunately, the Christmas tree is not so simple. To be honest, I did not hear at all that they could transform it. For such difficult objects, often the only way is direct ballistic transformation or gene gun .
This is an expensive and complicated, but universal way of delivering DNA into the cell. It lies in the fact that DNA molecules stick on gold microparticles, which are then fired at high speed into cell culture. These particles enter a part of the cells and leave the DNA molecules, which with a rather low probability are inserted into the genome.
What exactly are we transforming?
But, in fact, even if the act of embedding occurs, it will occur in one cell! She must somehow survive among the masses of others.
To do this, use the genetic marker that is contained in the DNA that we shot. This marker can be a herbicide resistance gene (a substance that is toxic to plants).
The culture of the cells after shooting is placed on the herbicide medium, to which only transformed cells are resistant. The picture shows that part of the calluses is yellow - dead, and part is green. This is how the action of the herbicide is manifested. Callus is the unorganized mass of undifferentiated plant cells.
Have you noticed that everywhere I am not talking about the whole plant, but about the culture of its cells? This is an important addition, it is impossible to “mutate”, that is, to transform the whole body at once (by the way, a stone in the garden of illiterate sci-fi about mutants). It is necessary to work with individual cells. It comes - because it is difficult and uncomfortable, the cells outside the body are very capricious and often do not want to grow as we need.
Obtaining a living cell culture is a separate biotechnological problem, which will not be discussed further, since it is too complicated to be included in this article.
But in any case, working with cell cultures is also sterile conditions that can be achieved only by purchasing a laminar box and an autoclave. Cell cultures grow on special media in Petri dishes. Different substances are added to the medium for growth - salt, sugar and, most importantly, a certain composition of hormones that induces cells to divide or form adult plants.
Concrete steps.
It was a lyrical introduction, and now the prose goes. The whole process is divided into several stages.
Plasmid
We take our plasmid with the existing utility sequences, cut out the desired piece from there (using the methods described in part 2 ) and place it in the purchased vector plasmid for transfer to plants. We transform agrobacteria with this plasmid (methods are also described in the previous article). We do not work with agrobacteria right away because they are much less convenient than E.coli and grow worse.
Cell culture
At the same time, we are trying to obtain a sterile cell culture of a plant, using our own literature and our own data on which nutrient medium the cells of this species grow best of all. As a starting material for cell culture, seeds or cuttings are used, which grow on the medium with the addition of hormones into an unorganized mass of cells - callus. Usually the process of obtaining cell culture takes 1-2 months (as they usually grow slowly).
Bacteria culture
After that, we grow agrobacterium culture or prepare gold particles with DNA sputtering for transformation, depending on which method is suitable for this type of plant.
Transformation
Now we have everything to transform. We are incubating with bacteria (about an hour), or a shot of gold particles using a gene gun.
Selection of transformants
We place on the environment with antibiotics to kill bacterial residues and herbicide so that only transformed cells grow.
Regeneration
After some time, the transformed cells form colonies - calli. These calli are sown on the medium with the addition of plant hormones (auxins and cytokinins) for regeneration.
We get transgenic plants (in our case - a glowing Christmas tree)!
Next we have to test them for the presence of the insert, its performance, but this is a separate conversation.
Here is a process diagram for better understanding:
As in any scientific experiment, in the transformation it is imperative to set controls, that is, experiments showing us that everything is done correctly.
A positive control for obtaining a transgenic plant is the transformation using the purchased test vector with the presence of certain markers. If it happens, it will show us that we are doing everything correctly and our methodology is appropriate.
Negative control is the lack of transformation and placing the cells directly on the medium with the herbicide. What is it for? In order to avoid, for example, such a dose of herbicide is insufficient to completely suppress the growth of non-transformed plants.
If a positive and negative control is carried out, and the transformation has failed, then this means that the problems in our design and everything needs to be redone
Well, as you would like, genetic engineering is not an easy thing :)
Remember, setting up control reactions at each stage of the experiment is a necessary condition in order to be sure that everything goes as it should.
That's all, in my opinion, I described how could the process of obtaining transgenic plants in the laboratory. I hope you found the information interesting :)
Conclusion
I remember that I promised to calculate the cost of equipment (based on the real cost of purchasing used ones) and reagents for carrying out this process.
Amplifier - 30 thousand rubles
Centrifuge table - 20 thousand
Reagents (in the form of ready-made kits) - 30 thousand
Current source, phoresis chamber, UV lamp (transilluminator) - 30 thousand
Laminar box - 90 thousand
Desktop autoclave - 50 thousand
Automatic dispensers from 0.5 to 1000 μl - 25 thousand
Laboratory balance - 20 thousand
Consumable plastic (test tubes, dispenser tips, petri dishes, etc.) - 7 thousand
Software for working with DNA sequences can cost more than $ 1000, but you can do without it or use trial versions.
Room cost, etc. I do not consider, because it is too individual.
In principle, it can be seen that quite many people can equip a genetic laboratory; there would be a desire and skill. At this stage, the equipment is expensive, but not worth the space money.
In general, I have a serious feeling that genetic engineering is now only at the beginning of my long journey, so knowledge of it will not be superfluous to anyone :) It is unlikely that laboratories will be distributed “on the knee”, but there is no doubt that genetic technologies firmly enter into the medical, food industry, criminology. And who knows how it will go on.
2all I apologize for such a delay in the release of the third part, it was written long ago, but there was no time to bring it to mind and publish it.
Links to previous parts of the article:
The first part: https://geektimes.ru/post/48533/
The second part: https://geektimes.ru/post/48846/
Source: https://habr.com/ru/post/52862/
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