Flashcards in Genetic engineering Deck (26):
What is recombinant DNA?
DNA which is composed of DNA from two species, (e.g. when foreign DNA is put into a bacterium's plasmids).
What are transgenic organisms?
Organisms which contain DNA from another species as well as its own (they contain recombinant DNA).
Applications of genetic engineering?
Human genes into bacteria so they make useful human proteins.
Genes into plants and animals so they acquire new characteristics.
Functional genes into humans with non-functional genes so they no longer suffer genetic diseases.
What is a gene?
A sequence of bases on the DNA that codes for the production of one polypeptide.
Overview of genetic engineering??
1. Obtain the human gene that codes for human insulin.
2. Insert this human gene into a bacterial plasmid (vector).
3. Bacterial cells take up the plasmids (transformation).
4. Identify the bacterial cells that have taken up the insulin gene by the use of gene markers.
5. Growth or cloning of the transformed bacterial cells.
6. Isolation and purification of the protein insulin made by the bacteria.
1. obtain the insulin gene using gene probes?
DNA with wanted gene heated to make single stranded and gene pool add.
Gene probe is labelled with either fluorescence or radioactivity.
Gene probe attaches to gene by complementary base pairing.
Desired gene identified by labelled gene probe.
What is a gene probe?
A short, single-stranded piece of DNA that is complementary to a known sequence of bases within the desired gene.
1. Or make the insulin gene using reverse transcription?
Get some cytoplasm that contains insulin mRNA.
mRNA is mixed with free DNA nucleotides and reverse transcriptase.
(enzyme is from retroviruses which use to make DNA from their RNA).
Reverse transcriptase uses the mRNA as a template to synthesise a single strand.
The cDNA is made double-stranded by DNA polymerase which builds up complementary nucleotides.
Double strand DNA fragment contains a copy of the insulin gene.
What does a restriction enzyme do?
Insulin gene is cut using it to form sticky ends.
Cut DNA at specific base sequences (recognition site).
Hydrolyses the backbone of DNA.
Gives a staggered cut, leaves some bases exposed (SE).
What are sticky ends?
They are short sequences of unpaired DNA bases
2. Inserting the gene into a plasmid
Plasmids used have 2 genes in, resistance to tetracycline and ampicillin (used as gene markers later).
Treat with both so you only have ones with resistance to them.
Treat plasmids with chemicals to dissolve their cell walls.
Use ultracentrifugation to separate plasmids.
Add same restriction enzyme used to cut gene, to form complementary sticky ends to the gene.
Human genes, cut plasmids and DNA ligase placed together. As both are complementary gene should enter plasmid and DNA ligase will join them together to form the backbone.
CREATED RECOMBINANT DNA.
Problems with step 2?
When you mix plasmids, gene and Ligase together you get two types of plasmid. Some with the gene (want) and some sealed plasmids back up without the gene (don't want).
3. Transferring the plasmids into bacterial cells (host)?
Plasmid with recombinant DNA is used to transfer gene into bacterial cells, if plasmid is used, host cell needs to be persuaded to take it up.
Host cells placed into ice-cold calcium chloride to make cell walls permeable.
Plasmids added and mixture is heat-shocked (42 degrees) encouraging cells to take up the plasmid.
Cells that take up the plasmids with gene are said to be transformed.
What is transformation?
The taking up of DNA from outside the cell.
What is a masterplate?
An agar plate where a sample of all bacterial cells are grown into colonies.
Problems with transformation?
You now potentially have 3 types of bacteria!
Bacteria which have no plasmids.
Bacteria with plasmid with no gene.
AND bacteria which have the plasmid and human gene.
HOW DO YOU SEECT ONLY 3??
4. Identification of host cells that have taken up DNA using gene markers?
Gene markers are used (in R plasmids).
When plasmids cut to insert gene, specific restriction enzyme was used whose restriction site is found in middle of tetracycline resistance.
If insulin gene in plasmid, tetracycline gene no longer works.
So... master plate of colonies kept safe, replica of bacteria cells is made and grown on ampicillin (no plasmids killed),
Tetracycline added which kills bacteria which have the gene in middle of plasmid.
You want these cells, but they are on the master plate, so these can be identified and grown on a large scale.
Bacterial colonies that contain the gene can be grown on a large scale in a fermenter.
Bacteria will produce human insulin,(PS)
Insulin can be isolated from the cells and purified.
Advantages of recombinant DNA technology?
No limit to the amount of protein that can be synthesised.
Complicated structure of many proteins cannot artificially synthesised except by living cells.
No need to extract proteins from mammalian organs (issues of disease).
Disadvantages of recombinant DNA technology?
Complicated, experienced staff and equipment required.
Expensive on an industrial scale.
Difficult to identify genes from entire human genome.
Some proteins require 1 gene for their synthesis.
Not all eukaryotic genes can be expressed in prokaryotic cells.
What are genetically modified plants?
Plants are genetically modified to give them desirable characteristics such as disease resistance (to bacteria, fungal..), higher yield and longer shelf life.
Benefits of GM plants?
Superior keeping qualities and flavour.
Reduction of pesticide use on GM plants resistance to insect attack.
Addition of N fixing genes into crops to reduce use of fertilizers.
Disadvantages to GM plants?
Risk of pollen from GM pollinating wild natural plants, could result in unknown consequences.
Concerns about unknown long-term health impacts from eating products derived from GM crops.
Increased use of GM crops could lead to reduction in biodiversity.
How to produce GM crops?
Use of bacteria.
Certain bacteria attack plants and stimulate tumours grow.
Gene to form tumours is removed from bacterial plasmids, replaced with desirable characteristics.
Allow bacteria with these new plasmids to invade the plants.
e.g. soya and tomatoes.
Genetically modified tomatoes??
Gene introduced into tomato plants that has complementary base sequence to that of the gene producing enzyme that breaks down pectin in wall.
mRNA produced by this gene is complementary to the original genes.
Two combine to produce double strand, preventing translation of original mRNA and production of enzyme.
Gives tomato longer shelf life and better taste.