Transcript Ligation and Transformation

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 & b-lactamase
genes (contained in the pGLO plasmid)
Plasmids
• Plasmids
– small (1-1000 kb)
– circular
– extrachromosomal DNA
• Growth is independent 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:
– 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