11: into to GE Flashcards
What are the two key techniques in plant GE?
Two key methods in plant GE are the use of A. tumefaciens and of microprojectiles.
What are promoters? (in GE)
Promoters control expression in foreign gene construction.
- Serve to ensure strong or tissue-specific or timely expression of genes.
What is CoMV35s?
CoMV35s is a promotor used in many GE constructs because it is constitutative (always on) leading to high levels of transgene expression.
What is the biggest issue in plant GE vs animal GE?
Plant GE is difficult because the transgene has to pass through a tough cellulose cell wall barrier.
How is a ligase enzyme used in GE?
DNA ligase is used to combine fragments cut by restriction enzymes in cloning, to create simple recombinant DNA. This means you can merge pieces of DNA from different sources.
What makes restriction fragments able to be joined together using ligase?
DNA restriction fragments (double-stranded) can be joined together with ligase because they have single-stranded regions at the end called sticky ends. When bases are complementary in these sticky ends it is possible to “stick” fragments together bc bps will readily connect.
What are the most common cloning vectors?
Plasmids and BACs (Bacterial Artificial Chromosomes) are the most common cloning vectors.
How are plasmids used in GE?
Plasmids can have a transgene and two marker genes added to them (lacZ and AMPr). Can be made into a recombinant plasmid using RE and ligase technique then cultured in a media by putting them into E. Coli where only bacteria with modified plasmids can grow due to presence of antibiotic (need AMPr for antibiotic resistance) and grown in presence of X-gal (lactose substitute). If the colony is blue this shows it can hydrolyse X gal and so the lacZ gene is NOT interrupted by the transgene, while white colonies have an interrupted lacZ
Thus white surviving colonies have a successful transgene added but blue have no transgene.
These cultures are then grown at large scale and extracted by a process that destroys bacterial chromosomal DNA but leaves plasmid intact, and then the desired transgene can be cut from the plasmid using a restriction enzyme. Can check the process has been successful by checking the size of the gene using gel eletrophoresis.
How can microprojectiles be used in plant GE?
Microprojectiles are used in the form of small gold pellets covered in transgene.
Plant tissue cultures (usually embryonic callus) are bombarded with transgene-coated microprojectiles and rely on a small number of these penetrating into cell nuclei into the plant DNA and being incorporated into the plant genome using natural repair mechanisms, resulting in a modified organism.
–> EXTREMELY low success rate: getting right conditions is the limiting factor. Need to use MANY MANY cells in a large tissue culture.
How is A. tumefaciens used in plant GE?
A. tumefacians is evolved to cause crown gall disease by injecting a Ti plasmid into a plant cell and essentially incorporating some of its own DNA into the host plant, which then makes proteins accordingly. Genetic engineers can modify this plasmid to remove the disease-causing components can add genes coding for desired traits with selectable markers and then use A. tumefaciens to surpass cell wall and inject into the plant. The plant is then regrown from a single modified cell.
GE of eudicots vs monocots?
Monocots are less susceptible to the A. tumefaciens disease so are better GE’d with microprojectiles, while A. tumefaciens method is common for eudicots.
Why are selectable markers important in GE?
Selectable markers are used to confirm if the modified plasmid is present.
What are the 5 steps to crease a new GE crop?
Steps in creating a GE crop:
1) Insert transgene into a plasmid
2) Transformation of target organism with transgene
3) Control expression of transgene
4) Safety testing
5) Field trials to show effectiveness + worth
What is expression?
Expression is the process by which genetic info is used to make proteins
basically the extent of how the genotype interacts with the phenotype.
Are foreign genes incorporated into a known location of host chromosome?
Transgenes are incorporated randomly into the chromosome: may disrupt a gene and lead to a faulty plant so many tissues with the transgene may not function especially well: need field trials to select out individuals without important sequences evidently disrupted.
