2. Review
• Which enzyme is used as a scissor in
genetic engineering ? Which one is used
as glue?
Sciessor: Restriction Enzyme
Glue: DNA ligase
• What’s the function of gel
electrophoresis?
Separate DNA fragments by their
different size
3. 13-3 Cell transformation
• Cell transformation= change the cell’s
DNA
• A cell takes DNA from outside. The
external DNA becomes part of the
cell’s DNA
4. 1) Transforming bacteria cells
– Using plasmid
• small circle of extra bacterial DNA, double strand
• Foreign DNA can be inserted into plasmid
– Forms recombinant plasmids
– Plasmids used in genetic engineering are called vectors.
– Can deliver DNA into another cell
6. 1) Transforming bacteria cells
– Using plasmid
• Plasmid has a origin site for replication
• Plasmid has a genetic marker (usually a gene for
resistance to antibiotics)
• two types of plasmid integration into a host
bacteria
7. Restriction enzyme DNA or cDNA Foreign DNA, plasmid
cuts molecule fragments with DNA, and modification
of chromosomal DNA sticky ends enzymes are mixed
or cDNA
recombinant
plasmids
containing
foreign DNA
Same enzyme cuts plasmid DNA
same sequence in with sticky ends
plasmid DNA
host cells containing
recombinant plasmids
8. What if insertion eukaryotic DNA
into bacteria ?
Will there be any problems ???
9. Transcription
RNA polymerase mRNA splicing
DNA Pre-RNA mRNA
No introns
Bacteria don’t know
how to deal with introns
USE cDNA!!!
10. Reverse transcription=
Making cDNA mRNA transcript
Reverse transcriptase
cDNA -- A DNA reverse
transcription from a mRNA–cDNA hybrid
matured mRNA
`
“c” -- complementary
single-stranded cDNA
DNA polymerase
double-stranded cDNA
11.
12. 2) Transforming plant cells
• Recombinant plasmid is transformed
to plant
– By using bacterium Agropbacterium
tumefaciens
– By gene gun injection
13. By using Agrobacterium
Plasmids carrying the gene
of interest is transferred
to agrobacterium
The agrobacterium
inserts the foreign
DNA into the plant
cell to form
transgenic cell
14. By using gene gun
To coat metal particles
with gene containing
plasmids
Gene gun injects
the DNA covered
particles into the
plant cell
15.
16.
17.
18.
19. 3) Transforming animal
cells
• Animal cell
– Inject into egg nucleus
some enzymes may help to insert
foreign DNA into chromosomes
– Gene replacement ( gene “knockout”)
