chapter 21 part 3 Flashcards
Vectors: part 1
The most commonly used vectors in genetic engineering are bacterial plasmids - small circular molecules of DNA separate from the chromosomal DNA that can replicate independently.
Once a plasmid gets into a new host cell it can combine with the host DNA to form what is called recombinant DNA.
vectors part 2
Plasmids are particularly effective in the formation of genetically engineered bacteria used, for example, to make human proteins.
The plasmids that are used as vectors are often chosen because they contain what is known as a marker gene.
For example they may have been engineered to have a gene for antibiotic resistance.
This gene enables scientists to determine that the bacteria have taken up the plasmid, by growing the bacteria in media containing the antibiotic.
How To insert a DNA fragment into a plasmid: part 1
first it must be cut open.
The same restriction endonuclease as used to isolate the DNA fragment is used to cut the plasmid.
This results in the plasmid having complementary sticky ends to the sticky ends of the DNA fragment.
Once the complementary bases of the two sticky ends are lined up, the enzyme DNA ligase forms phosphodiester bonds between the sugar and the phosphate groups on the two strands of DNA, joining them together
How To insert a DNA fragment into a plasmid: part 2
The plasmids used as vectors are usually given a second marker gene, which is used to show that the plasmid contains the recombinant gene.
This marker gene is itself often placed in the plasmid by genetic engineering methods.
The plasmid is then cut by a restriction enzyme within this marker gene to insert the desired gene.
If the DNA fragment is inserted successfully, the marker gene will not function.
concerns about antibiotic resistance:
In the early days of genetic engineering, these marker genes were often for antibiotic resistance.
There have, however, been many concerns about antibiotic resistance in genetically engineered organisms.
As a result, genes producing fluorescence or an enzyme that causes a colour change in a particular medium are now more widely used as marker genes.
If a bacterium does not fluoresce, or change the colour of the medium, then it has been engineered successfully and can be grown on
diagram of engineering a desired gene into a plasmid vector
Transferring the vector: part 1
The plasmid with the recombinant DNA must be transferred into the host cell in a process called transformation.
One method is to culture the bacterial cells and plasmids in a calcium-rich solution and increase the temperature.
This causes the bacterial membrane to become permeable and the plasmids can enter.
Transferring the vector: part 2
Another method of transformation is electroporation. A small electrical current is applied to the bacteria.
This makes the membranes very porous and so the plasmids move into the cells.
Electroporation can also be used to get DNA fragments directly into eukaryotic cells.
The new DNA will pass through the cell membrane and the nuclear membrane to fuse with the nuclear DNA.
Although this technique is effective, the power of the electric current has to be carefully controlled or the membrane is permanently damaged or destroyed, which in turn destroys the whole cell.
It is less useful in whole organisms.
Electrofusion: part 1
Another way of producing genetically modified (GM) cells is electrofusion.
In electrofusion, tiny electric currents are applied to the membranes of two different cells.
This fuses the cell and nuclear membranes of the two different cells together to form a hybrid or polyploid cell, containing DNA from both.
It is used successfully to produce GM plants.
Electrofusion: part 2
Electrofusion is used differently in animal cells, which do not fuse as easily and effectively as plant cells.
Their membranes have different properties and polyploid animal cells - especially polyploid mammalian cells - do not usually survive in the body of a living organism.
However, electrofusion is important in the production of monoclonal antibodies.
A monoclonal antibody is produced by a combination of a cell producing one single type of antibody with a tumour cell, which means it divides rapidly in culture.
Monoclonal antibodies are now used to identify pathogens in both animals and plants, and in the treatment of a number of diseases including some forms of cancer.
Engineering in different organisms:
the techniques of genetic engineering vary between different types of organisms but the principles are the same.
It is much easier to carry out genetic modification of prokaryotes than eukaryotes, and among the eukaryotes, plants are easier to work with than animals.
Engineering prokaryotes:
Bacteria and other microorganisms have been genetically modified to produce many different substances that are useful to people
These include hormones, for example insulin and human growth hormone, clotting factors for haemophiliacs, antibodies, pure vaccines and many of the enzymes used in industry.
Engineering plants: part 1
One method of genetically modifying plants uses Agrobacterium tumefaciens, a bacterium that causes tumours in healthy plants.
A desired gene - for example, for pesticide production, herbicide-resistance, drought-resistance, or higher yield - is placed in the Ti plasmid of A. tumefaciens along with a marker gene, for example, for antibiotic resistance or fluorescence.
This is then carried directly into the plant cell DNA.
The transgenic plant cells form a callus, which is a mass of GM plant cells, each of which can be grown into a new transgenic plant.
Engineering plants: part 2
Transgenic plant cells can also be produced by electrofusion.
The cells produced have chromosomes from both of the original cells and so are polyploid.
The cells that are fused may be from similar species, or very different ones.
The main stages in this process involve removal of the plant cell wall by cellulases, electrofusion to form a new polyploid cell, the use of plant hormones to stimulate the growth of a new cell wall, followed by callus formation and the production of many cloned, transgenic plants.
Engineering animals:
It is much harder to engineer the DNA of eukaryotic animals, especially mammals, than it is to modify bacteria or plants.
This is partly because animal cell membranes are less easy to manipulate than plant cell membranes.
However, it is an important technique both to enable animals to produce some medically important proteins and to try and cure human genetic diseases such as CF and Huntington’s disease.
genetic engineering in plants - diagram
