Transcript Ligation and Transformation

Bacterial Transformation
RET Summer 2009
Overall Picture
Bio-Rad pGLO Transformation
Need:
1. Good host
2. Self-replicating vector to carry our gene
3. Selection marker
Insertion of GFP gene into HB101 E. coli
Transformation
• The process of transferring foreign DNA
fragments into a recipient (host) cell for
growth and replication
• Our host cells: HB101 E. coli
• Our foreign DNA: GFP & b-lactamase
genes (contained in the pGLO plasmid)
• Plasmids
Plasmids
– small (1-200,000 kb)
– circular
– extrachromosomal
DNA
• “Relaxed” plasmids replicate independently of
the host’s cell cycle; amplification of gene
product
• A type of cloning vector used to carry a gene
not found in the bacterial host’s chromosome
Overall Transformation
Process
1. The plasmid vector must be cut with a
restriction endonuclease (aka: restriction
enzyme)
2. DNA ligase joins the DNA fragment & vector
DNA
3. Host cell is made competent so can plasmid
can enter
4. Transformed cells are grown on selection
media
Overall Transformation
Process
1. The plasmid vector must be cut with a
restriction endonuclease (aka: restriction
enzyme)
2. DNA ligase joins the DNA fragment & vector
DNA
3. Host cell is made competent so can plasmid
can enter
4. Transformed cells are grown on selection
media
Restriction Enzymes
• Endonucleases:
–protect bacteria from intruding
DNA in nature
–cut up (restrict) the viral DNA
–can leave blunt or sticky ends
–cut only at very specific
nucleotide sequences
• Restriction site:
recognition sequence for a
particular restriction enzyme
• Restriction fragments:
segments of DNA cut by
restriction enzymes in a
reproducible way
Restriction Enzymes
• Insertion of a gene
Cut gene of interest with same
restriction enzymes to make
complementary “sticky ends”
• DNA ligase:
joins the sticky ends of DNA
fragments
Ideally, you would cut
opposite ends of the gene
with different restriction
enzymes.
Overall Transformation Process
1. The plasmid vector must be cut with a
restriction endonuclease (aka: restriction
enzyme)
2. DNA ligase joins the DNA fragment & vector
DNA
3. Host cell is made competent so can plasmid
can enter
4. Transformed cells are grown on selection
media
Transformation of Bacteria
• Generally occurs through heat shock &
addition of a divalent cation (Ca++)
• Ca++ stabilizes negatively charged
phosphates
• Heat shock briefly permeabilizes the
membrane
• Competent cells are those capable of taking
up the plasmid
• Cells most likely to become competent are
in log growth phase
Overall Transformation Process
1. The plasmid vector must be cut with a
restriction endonuclease (aka: restriction
enzyme)
2. DNA ligase joins the DNA fragment & vector
DNA
3. Host cell is made competent so can plasmid
can enter
4. Transformed cells are grown on selection
media
Selection
• Selective medium - used to determine
which bacterial cells contain the antibiotic
resistant plasmid insert & which do not
• Example: bacterium containing a plasmid
with resistance to a particular antibiotic
(ampicillin) will grow on medium that
contains that antibiotic
• In addition, our plasmid contains a
regulatory element that activates the GFP
gene only in the presence of arabinose
Selection Media
LB plates: Control (-pGLO)
LB + amp: Should contain only cells with the
amp-resistant pGLO plasmid; colonies
appear white (-pGLO, + pGLO)
LB + amp + ara: Should contain only cells with
the amp-resistant pGLO
plasmid; colonies floresce green
(+pGLO)
Factors that Affect Yield &
Quality of Plasmid DNA
• Plasmid copy number
• Host strain used, carbohydrate
production
• Culture medium, selection, and culture
time
– Want to harvest during log growth phase
Transformation Applications
GFP Uses
• Use as a reporter molecule
to follow changes in gene
expression over time
• Nondestructive, nontoxic
• Coding sequence can be
cloned into a variety of
vectors
• GFP keeps its fluorescence
in cells from different
species
• Can be tracked in living
cells over to time to study
development
• Can be directed to specific
subcellular compartments
• Can combine GFP coding
region with the regulatory
region for another gene
and observe changes in
gene expression
• Can be used to make a
fusion protein to study
localization, turnover &
intracellular associations of
native protein
• GFP gene is switched on
when cells are grown in the
presence of arabinose