Bacterial Transformation

RET Summer 2007

Overall Picture

Bio-Rad pGLO Transformation

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 & -lactamase
genes (contained in the pGLO plasmid)

Plasmids
Plasmids
small (1-1000 kb)
circular
extrachromosomal DNA

Growth is independent of the hosts cell cycle;

amplification of gene product
A type of cloning vector used to carry a gene not
found in the bacterial hosts 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:
in nature, they protect bacteria
from intruding DNA
cut up (restrict) the viral DNA
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
DNA ligase:
joins the sticky ends of DNA
fragments

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 and
addition of a divalent cation to permeabilize
the membrane
Competent cells are those capable of taking up
the plasmid

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
A selective medium is used to determine which
bacterial cells contain the antibiotic resistant
plasmid insert and which do not
For example, a 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 ampresistant 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 and

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