Genetic Engineering Flashcards
What is genetic engineering?
Allows genes to be manipulated, altered and transferred from one species to another making a GM organism.
What can genes be transferred to?
- Bacteria to make useful things
- Plants and animals to make useful characteristics
- Humans to reduce effects of genetic diseases.
define recombinant DNA and transgenic.
Recombinant DNA = genetic material from 2 species combined
Transgenic = organism with DNA from other species in its cells. The introduced DNA is donor DNA and the organism is the host.
Define transformed.
When an organism has incorporated a plasmid containing a foreign gene
What are the 5 stages of producing a protein using genetic engineering?
- Isolating the DNA fragments
- Inserting the DNA into a vector
- Transfer of DNA to a host cell
- Identification of the host cells that have taken up the gene using gene markers
- Cloning transformed host cells
How is the gene located?
Donor molecule of human DNA containing gene coding for insulin used. Gene hard to find but can be identified using a gene probe ie a specific segment of a single stranded DNA Complimentary to a section of the gene.
How is the gene isolated.
Using one of 2 enzymes: restriction endonuclease or reverse transcriptase.
Describe how restriction endonucleases work.
Cut DNA at specific nucleotide sequences. The sentences occur in many places so DNA cut to many fragments and individual genes isolated. Some cut straight through = blunt cut but many make a staggered cut.
Why is a staggered cut better with restriction endonucleases?
Leaves inspired bases at both ends that pair with complimentary bases easily (called sticky ends).
E. coli produces a restriction endonuclease called EcoR1. What does this do?
Catalyses the formation of breaks in DNA backbone in a specific sequence of nucleotides where a nucleotide containing guanine is next to one with adenine. The line of cut leaves sticky Ends when the cut strands are seperated.
Explain the sticky ends made by ECOR1.
The 4 unpaired bases are in reverse order so form a palindrome. If ECOR1 made a cut either side of the insulin gene the gene would be isolated from the rest of the genome.
What are the 2 drawbacks of using restriction endonucleases?
- If the recognition sequence occurs within the gene of interest the gene will be broken into fragments and have no function
- Using the whole gene means introns will be incorporated and because bacteria don’t have introns their genome might not have the enzymes to process RNA to remove them. protein will make extra amino acids and not function.
Describe how reverse transcriptase isolated a gene.
There are 2 insulin genes but many MRNA strands made from it. reverse transcriptase makes DNA from RNA templates. It synthesis DNA called copy DNA which is complimentary to the RNA. many copies can be made and there are no issues with introns. DNA polymerase than makes DNA complimentary to the CDNA making a double stranded molecule containing the insulin gene.
What is the vector that the gene is carried into the cell by in this instance?
A plasmid ie a small double stranded circle of DNA found in bacteria. Much smaller than a bacterial chromosome and contains only a few genes. They can move in and out of cells
How is a plasmid isolated?
Bacteria treated with EDTA which destabilises cell walls, detergent which dissolved the phospholipid membrane and sodium hydroxide to make an alkaline environment and denature membrane proteins. They are then seperated from cell debris.
How is the recombinant plasmid made?
Endonucleases cut the plasmid up. They are the same ones used to isolate the gene so the same nucleotide sequence on the sticky ends. The vector and gene are mixed and the gene is now loosely bound to the plasmid. DNA ligament makes the join permanent. The gene has now been ‘spliced’ into the vector. The plasmid is now recombinant DNA.
What makes a good vector?
- Self replicating
- Small
- Not broken down by host cell
- Not stimulate immune response
- Screened to confirm that the gene was inserted
- Hade markers to allow host cells that have taken it up to be identified
How does calcium chloride increase the amount of bacterial cells that take up the plasmid?
The positive charge on calcium ions bonds with the negatively charged DNA backbone of the plasmid and the membrane lipopolysaccharides.
How is the DNA transferred to the host cell?
Plasmid DNA passes into the cells with a heat shock, in which cells are chilled to 4 degrees and briefly heated to 42 degrees.
How are genetic markers used?
The vector can be sequenced. A vector that hasn’t taken up the gene is empty
Plasmids with antibiotic resistant genes are used.
How do plasmids with antiobiotic resistant genes act as markers?
Cells cultured in a growth medium containing the antibiotic. If the bacteria breaks down the antibiotic and it grows it is resistant and contains the plasmid. If it dies it doesn’t contain the plasmid bc it isn’t resistant.
What is ‘blue white screening’?
Bacterial cells grown on a medium containing lactose analogue X-gal. They turn white if they contain the plasmid with the gene but blue if the plasmid is empty.
How are the bacterial cells cloned with the insulin gene in?
Cultured in large volumes in fermenters. Each culture forms a clone. Cloning produces multiple colonies so multiple plasmids. The bacterial enzymes then transcribe the insulin gene and translate the MRNA they produce. Insulin is made in large quantities.
5 pros of genetic engineering.
1: medical products like insulin, clotting factors for haemophilia
2. Tooth decay: modified structures of oral bacteria don’t produce lactic acid which contribute to tooth decay
3. Prevention and treatment of disease ie made to produce vaccines and hinder tumours
4. Enhance crop growth
5. Environments use eg detecting environmental hazards
4 cons of genetically engineering bacteria
- Plasmids easily transferred eg antibiotic resistance easily transferred
- Fragments of human DNA used to make gene samples and cytoplasmic MRNA used to make CDNA may contain oncogenes or gene switches for proto oncogenes that could contaminate the final product
- A micro organism with a new gene may be a threat
- A newly introduced gene might disrupt function of other genes
