Practical summary term 2, 2024-2025
The practical is build up from different steps, these steps are broadly:
1. Isolation of the Gene Of Interest (GOI)
2. Making the entry vector
3. Making the expression vector
4. Transformation of A. tumefaciens
5. Transient plant transformation
6. Analysis of transgene expression in plants
Isolation of the GOI
During this step we identified the gene that will enable the red colouration in the
tobacco leaves.
This red colouration is usually anthochyanins or carotenoids. For both the
biochemical pathway has been elucidated and responsible enzymes and
transcription factors controlling the expression of these enzymes have been
characterized in many plants.
Knowledge on biochemical pathways and the genes involved allow a person to
activate such pathways in plants or plant parts under conditions in which the
biochemical pathway is normally not active.
Gene delivery methods in plants:
- Direct DNA delivery
Particle bombardment: DNA is physically shot into plant cells
Electroporation/PEG-mediated transformation: DNA is
delivered into protoplasts (plant cells without cell walls) via
electrical pulses or chemical treatments
- Indirect DNA delivery via A. tumefaciens
Agrobacterium transfers part of its plasmid (T-DNA) into the
plant genome
T-DNA carries genes for plant hormones and metabolites that
introduce tumors (tumor-inducing or Ti-plasmid), but there
tumor-causing genes are replaced with the gene of interest
(GOI)
The Ti-plasmid becomes a modified expression vector for
plant transformation
Gateway cloning system:
, - Efficiently transfers DNA fragments between bacterial vectors using
recombinases LR clonase and BP clonase
1. Att-site recombination
DNA fragments are flanked
by attB sites in PCR
products
BP clonase facilitates
recombination between
attB sites (in PCR) and
attP sites (in donor
vector) to create an entry
vector
The ccdB gene in the
donor vector kills bacteria
that do not undergo successful recombination
2. Transformation and selection
E.coli is transformed
with the recombination
mix, selecting for
bacteria containing the entry vector via an antibiotic
resistance gene in the backbone of the donor vector
The ccdB gene in the donor vector kills bacteria that did not
recombine
3. Confirming entry vector
After selecting a single E.coli clone, the DNA sequence of
the GOPI in the entry vector is confirmed to ensure no
mutations occurred during PCR
4. Shuttling to expression vector
The GOI from the donor vector can be recombined into an
expression vector (destination vector) using LR clonase
LR clonase recombines attL sties (from entry vector) with
attR sites (in the expression vector), and the process creates
attB and attP sites making the reaction reversible
5. Transformation and selection of expression vector
The recombinant expression vector is transferred to E.coli.
Selection is done using a different antibiotic resistance
gene compared to the other entry vector
The ccdB gene in the destination vector kills bacteria that do
not carry the correct recombinant vector
6. Confirmation of expression vector
No need to sequence the GOI again since the recombination
process is highly accurate. A restriction enzyme digest is
used to confirm the correct insertion
Design isolation primers:
The practical is build up from different steps, these steps are broadly:
1. Isolation of the Gene Of Interest (GOI)
2. Making the entry vector
3. Making the expression vector
4. Transformation of A. tumefaciens
5. Transient plant transformation
6. Analysis of transgene expression in plants
Isolation of the GOI
During this step we identified the gene that will enable the red colouration in the
tobacco leaves.
This red colouration is usually anthochyanins or carotenoids. For both the
biochemical pathway has been elucidated and responsible enzymes and
transcription factors controlling the expression of these enzymes have been
characterized in many plants.
Knowledge on biochemical pathways and the genes involved allow a person to
activate such pathways in plants or plant parts under conditions in which the
biochemical pathway is normally not active.
Gene delivery methods in plants:
- Direct DNA delivery
Particle bombardment: DNA is physically shot into plant cells
Electroporation/PEG-mediated transformation: DNA is
delivered into protoplasts (plant cells without cell walls) via
electrical pulses or chemical treatments
- Indirect DNA delivery via A. tumefaciens
Agrobacterium transfers part of its plasmid (T-DNA) into the
plant genome
T-DNA carries genes for plant hormones and metabolites that
introduce tumors (tumor-inducing or Ti-plasmid), but there
tumor-causing genes are replaced with the gene of interest
(GOI)
The Ti-plasmid becomes a modified expression vector for
plant transformation
Gateway cloning system:
, - Efficiently transfers DNA fragments between bacterial vectors using
recombinases LR clonase and BP clonase
1. Att-site recombination
DNA fragments are flanked
by attB sites in PCR
products
BP clonase facilitates
recombination between
attB sites (in PCR) and
attP sites (in donor
vector) to create an entry
vector
The ccdB gene in the
donor vector kills bacteria
that do not undergo successful recombination
2. Transformation and selection
E.coli is transformed
with the recombination
mix, selecting for
bacteria containing the entry vector via an antibiotic
resistance gene in the backbone of the donor vector
The ccdB gene in the donor vector kills bacteria that did not
recombine
3. Confirming entry vector
After selecting a single E.coli clone, the DNA sequence of
the GOPI in the entry vector is confirmed to ensure no
mutations occurred during PCR
4. Shuttling to expression vector
The GOI from the donor vector can be recombined into an
expression vector (destination vector) using LR clonase
LR clonase recombines attL sties (from entry vector) with
attR sites (in the expression vector), and the process creates
attB and attP sites making the reaction reversible
5. Transformation and selection of expression vector
The recombinant expression vector is transferred to E.coli.
Selection is done using a different antibiotic resistance
gene compared to the other entry vector
The ccdB gene in the destination vector kills bacteria that do
not carry the correct recombinant vector
6. Confirmation of expression vector
No need to sequence the GOI again since the recombination
process is highly accurate. A restriction enzyme digest is
used to confirm the correct insertion
Design isolation primers:
