Chapter 18 Flashcards
Why is the Ti plasmid from A. tumefaciens well suited for developing a vector to transfer foreign genes into plant chromosomal DNA?
Vir genes encode the genes necessary for transfer and integration of T-DNA region into genome of plant cell. Can integrate DNA without microbombardment. Includes selectable marker, ORI for E. coli and A. tumefacients, right border which is necessary for integration and transfer, a MCS in the T-DNA region and a killer gene downstream of left border to prevent unwanted DNA past left border.
How do 1) binary and 2) cointegrate Ti plasmid based vector systems for plant transformation differ from one another
Binary cloning vector system produces an E.coli – A.tumefaciens shuttle vector where as using the cointegrate Ti plasmid-based vector system will produce a recombinant Ti plasmid.
The binary system has all the cloning steps done in E.coli before introduced into a disarmed strain of A.Tumefaciens. Therefore, the T-DNA cannot transfer because it is disrupted or removed. The vector is then inserted into the plant chromosomal DNA by enabling the T-DNA with the synthesis of the vir gene products.
On the other hand, the cointegrate vector system relies on in vivo homologous recombination for integrating the target gene into the disarmed Ti plasmid. By recombination, the Ti plasmid is able provided with the vir genes necessary to transfer the T-DNA to the host plants.
The plant selectable marker is placed next to the left border. In the case of the recombinant Ti plasmid, it is before the homologous DNA sequence, but still precedes the left border.
What are the reporter genes and how are they used when plant cells are transformed?
Reporter genes contain genes whose proteins produce a detectable response to a specific assay. They are used to quantify level of expression of a gene.
How are plants transformed by microprojectile bombardment?
Spherical cold particles coated with DNA are accelerated to high speed by a particle gun. High pressure helium is used as a source of particle propulsion. The projectiles can penetrate plant cell walls and membranes while not significantly damaging the cells. Once inside the cell, the DNA is removed and integrated into the plant DNA. This method allows introduction of foreign DNA into plant cell suspensions, callus cultures, and monocots (which are less susceptible to Agrobacterium-mediated DNA transfer.
How is foreign DNA targeted for integration into chloroplast DNA?
Foreign DNA is typically introduced by microprojectile bombardment into the chloroplast genome on a plasmid vector with both the foreign DNA and a selectable marker flanked by specific chloroplast DNA sequences.
Homologous recombination is the normal mode of DNA integration into the chloroplast genome.
How would you produce a transgenic plant that does not contain a marker gene?
Method 1 – can cotransform plants with two plasmids, one containing marker gene and one with target gene. Traditionally breed until plant with selectable marker is removed.
Method 2 – incorporate selectable marker between transposase gene in T-DNA
How would you ensure that a foreign gene that has been inserted into the chloroplasat is expressed at a high level?
Naturally, foreign gene would be expressed at high levels because there are multiple copies of the chloroplast DNA. Also, chloroplast-borne genes are organized into operons and produce polycistronic mRNAs. This makes it easier to engineer and express several genes that are regulated together as part of a metabolic pathway than coordinate several genes under different promoter control.
What is the advantage of introducing foreign genes into chloroplast rather than nuclear DNA?
Pollen cannot transmit contents of chloroplasts and therefore prevent spread of foreign genes in chloroplast. More chloroplasts than cells and therefore have a higher rate of expression.
What is rizosecretion? Why is it useful? How can it be engineered
Rhizosecretion: secretion of foreign protein through the roots into culture medium.
This is useful for protein purification. It avoids the process of harvesting proteins by disrupting plant cells before purifying the recombinant protein.
This is done by directing the protein to the root apoplast by a secretion signal. As long as the protein DNA fragment encodes a signal peptide, the transgenic plant will excrete the protein product.
How can chromosomal marker genes be removed without using sexual crossing to segregate the selectable marker from the gene of interest.
The chromosomal marker can be remobed using the enzyme recombinase.
The selectable marker is paired with a recombinase gene which mediates the excision of sequences between recombinase recognition sequences.
Have the gene directly upstream of the selectable marker and have the pair flanked by recombinase recognition sequences to have the selectable marker and recombinase genes excised.
Describe a strategy that would be used to remove antibiotic resistnace genes from chloroplast DNA
Transformants are selected by their resistance. Once selection is complete, the transformants are grown without antibiotics which causes the selectable marker gene to be lost as a result of homologous recombination between the 174 bp repeat elements.
Suggest a strategy that would facilitate the large-scale purification of soluble proteins such as antibody fragments in plants
Engineer a protein so that it binds to the C-terminal end of an oleosin molecule
Attach a secretion signal onto a protein that targets the protein to the apoplast of the roots which will eventually be secreted via rhizosecretion
A strategy that would facilitate the large-scale purification of soluble proteins such as antibody fragments is glycosylation of the proteins without the trouble carbohydrates.
How can oleosins be used to facilitate the purification of a target protein synthesized in a transgenic plant?
Oleosins are found in oil droplets in seeds and they can be fused to foreign protein for purification. Since N terminus and C terminus are hydrophilic, target proteins can fuse on C-terminus and be subsequently cleaved off via cleavable linker between the target protein and oleosin.
How would you modify the glycosylation pattern of a mammalian protein produced in plants?
Modify the plant so that it does not add problem carbohydrate residues and incorrectly glycosylate mammalian proteins. For example, using RNAi to down regulate certain enzymes that add problem carbohydrate residues.
