BLGY1211 Genetic Engineering case studies
Common application of genetic engineering
99% for research (the vast majority, quite sophisticated changes) to study genes
and gene function
Applied biology (much less, and usually simpler changes) To generate hybrid
(recombinant) genes for protein production (i.e. human proteins in microorganisms),
To modify properties of proteins (protein engineering), To modify biochemical
pathways (pathway engineering), To modify the characteristics of whole organisms
(i.e. Pest/stress resistant crops)
Applied biology
Placing a human coding region under control of bacterial promoter (easy); e.g. make
human insulin in e. coli but must be careful of codon usage, introns, protein folding
and inducible promoter
Protein engineering; from protein structure to function; Random mutagenesis and
selection (Molecular evolution), Site-directed mutagenesis based on educated guess
(see what happens) purposeful change in enzyme properties (mainly industrial
purposes); heat stability, active under high sale, heavy metals, resistance to
proteolysis
Pathway engineering; take a typical biochemical pathway but we want a certain
product from this pathway which is usually then metabolised into something else so
they engineer the pathway to get accumulation of this intermediate usually by taking
the enzyme that turns it into the other product (this does not tend to work and
usually another pathway will take over – worked when it was found by accident)
Analysis of transgenic plants; usually lab experiments not crop improvement;
complementation of a mutant, promoter fusions, experiments with modified genes
to study gene function, introduction of traits into crops
Plant transformation
Insertion of the gene of interest (or DNA fragment of interest) into binary vector;
Restriction digestion
Gel purification
Ligation
E.coli transformation + analysis of recombinants
, Isolation of high quality DNA
Agrobacterium transformation with new recombinant binary vector
Plant transformation by tissue culture and co-cultivation
Generate sterile plant material
Co-cultivation with Agrobacterium strain
Induction of cell divisions under selective conditions
Regeneration of plant (apical meristem, shoots, then roots)
Agrobacterium infiltration in entire plants or flowers
Avoids the need for tissue culture and sterile work practice
Fool-proof (not much you can improve, not much that can go wrong)
The “floral-dip” transformation method
Dip Arabidopsis flowers into Agrobacterium solution for 1-2 minutes.
Cover them with plastic foil for a few days to keep high humidity to
encourage proliferation of
Agrobacterium in planta.
Let uncover and them grow in the greenhouse and collect dry seeds (2
months).
Since one Arabidopsis plant has many flowers, seeds from various
independent transformation events can be obtained (=independent T-DNA
insertion lines).
Grow seeds and select or screen for transgenics.
Score the phenotype if you do a complementation assay.
Of course the methods works also for other purposes.
Some plant species can’t be transformed with Agrobacterium
Alternatives are based on naked DNA transfer, this is when you have the
genes of interest and selectable markers on a plasmid, and you transfer
purified plasmid DNA (no ampicillin resistance) directly into plant cells, either
via electroporation OR chemical cell permeabilisation (PEG, Calcium) OR
ballistic methods, particle bombardment (plasmid-coated gold particles
propelled by an airgun with the plant tissue under vacuum)
Some of this DNA ends up in the nucleus of target plant cells, and a portion of
this can be integrated into the genome by illegitimate recombination.
It is unpredictable how much of it is integrated
Incorporation of genetic engineering into plant breeding
Specific transformation of plants using known genes for a known reason (but
random insertion into the genome)
Establishment of predicted trait (hypothesis-driven but usually well informed)
Extensive back-crossing with parent strain; Clearing out any possible unexpected
mutations, Maintaining desired trait, Re-establish general properties
Crossing with related variety to create hybrid seed; Heterosis effect (hybrid vigor),
Yield, quality, appearance….
Case studies - tomatoes
Tomato with modified ripening programmes; Fruit softening during ripening is
caused by the enzyme endo-polygalacturonase (involved in pectin degradation,
mediates fruit softening, helps rotting and liberating the seeds in nature, decreases
Common application of genetic engineering
99% for research (the vast majority, quite sophisticated changes) to study genes
and gene function
Applied biology (much less, and usually simpler changes) To generate hybrid
(recombinant) genes for protein production (i.e. human proteins in microorganisms),
To modify properties of proteins (protein engineering), To modify biochemical
pathways (pathway engineering), To modify the characteristics of whole organisms
(i.e. Pest/stress resistant crops)
Applied biology
Placing a human coding region under control of bacterial promoter (easy); e.g. make
human insulin in e. coli but must be careful of codon usage, introns, protein folding
and inducible promoter
Protein engineering; from protein structure to function; Random mutagenesis and
selection (Molecular evolution), Site-directed mutagenesis based on educated guess
(see what happens) purposeful change in enzyme properties (mainly industrial
purposes); heat stability, active under high sale, heavy metals, resistance to
proteolysis
Pathway engineering; take a typical biochemical pathway but we want a certain
product from this pathway which is usually then metabolised into something else so
they engineer the pathway to get accumulation of this intermediate usually by taking
the enzyme that turns it into the other product (this does not tend to work and
usually another pathway will take over – worked when it was found by accident)
Analysis of transgenic plants; usually lab experiments not crop improvement;
complementation of a mutant, promoter fusions, experiments with modified genes
to study gene function, introduction of traits into crops
Plant transformation
Insertion of the gene of interest (or DNA fragment of interest) into binary vector;
Restriction digestion
Gel purification
Ligation
E.coli transformation + analysis of recombinants
, Isolation of high quality DNA
Agrobacterium transformation with new recombinant binary vector
Plant transformation by tissue culture and co-cultivation
Generate sterile plant material
Co-cultivation with Agrobacterium strain
Induction of cell divisions under selective conditions
Regeneration of plant (apical meristem, shoots, then roots)
Agrobacterium infiltration in entire plants or flowers
Avoids the need for tissue culture and sterile work practice
Fool-proof (not much you can improve, not much that can go wrong)
The “floral-dip” transformation method
Dip Arabidopsis flowers into Agrobacterium solution for 1-2 minutes.
Cover them with plastic foil for a few days to keep high humidity to
encourage proliferation of
Agrobacterium in planta.
Let uncover and them grow in the greenhouse and collect dry seeds (2
months).
Since one Arabidopsis plant has many flowers, seeds from various
independent transformation events can be obtained (=independent T-DNA
insertion lines).
Grow seeds and select or screen for transgenics.
Score the phenotype if you do a complementation assay.
Of course the methods works also for other purposes.
Some plant species can’t be transformed with Agrobacterium
Alternatives are based on naked DNA transfer, this is when you have the
genes of interest and selectable markers on a plasmid, and you transfer
purified plasmid DNA (no ampicillin resistance) directly into plant cells, either
via electroporation OR chemical cell permeabilisation (PEG, Calcium) OR
ballistic methods, particle bombardment (plasmid-coated gold particles
propelled by an airgun with the plant tissue under vacuum)
Some of this DNA ends up in the nucleus of target plant cells, and a portion of
this can be integrated into the genome by illegitimate recombination.
It is unpredictable how much of it is integrated
Incorporation of genetic engineering into plant breeding
Specific transformation of plants using known genes for a known reason (but
random insertion into the genome)
Establishment of predicted trait (hypothesis-driven but usually well informed)
Extensive back-crossing with parent strain; Clearing out any possible unexpected
mutations, Maintaining desired trait, Re-establish general properties
Crossing with related variety to create hybrid seed; Heterosis effect (hybrid vigor),
Yield, quality, appearance….
Case studies - tomatoes
Tomato with modified ripening programmes; Fruit softening during ripening is
caused by the enzyme endo-polygalacturonase (involved in pectin degradation,
mediates fruit softening, helps rotting and liberating the seeds in nature, decreases
