Sterile Technique and
Bacterial Transformation
October 7, 2015
Molecular Cloning
• Manipulation of DNA sequences to create
recombinant DNA
• Recombinant DNA (rDNA)- DNA constructed
from multiple sequences in a laboratory
setting and therefore do not exist in nature
– Also referred to as DNA constructs
• Used to amplify and study specific gene(s) of
interest
How do we create recombinant DNA?
• Amplify DNA
sequences from
original organism
(ex. PCR)
• Synthesize artificial
sequences in the lab
www.flmnh.ufl.edu
How do we create recombinant DNA?
• Ligate (or paste) fragments of DNA together to
connect sequences into single reading frame or
region of gene transcription and expression
• This typically generates circular DNA, also known as a
plasmid, that contains all of the sequences required
to transcribe and express your DNA of interest
www.di.uq.edu.au
Bacterial use in rDNA amplification
• Use bacteria as “factories” to rapidly and efficiently
generate large amounts of DNA
• Take advantage of bacterial growth rate: population
doubles every ~20min
• Isolate DNA from bacteria for subsequent analysis
https://upload.wikimedia.org/wikipedia/commons/4/42/Plas
mid_replication_%28english%29.svg
How do we get our plasmid DNA into the
bacteria?
• Bacterial Transformation- procedure to
facilitate uptake of foreign DNA into bacteria
• Use “competent” cells - treated to improve
transfer of plasmid DNA
– Chemically competent cells (CaCl2) + heat shock
– Electroporation
Growing transformed cells
• Luria (or lysogeny) broth (LB) or agar
– Commonly used to grow bacteria
– Contains tryptone (peptides), yeast extract
(organic compounds) and sodium chloride (ions,
osmotic balance)
• However, these are requirements for any
organism to grow
• How do we selectively grow bacteria that
contain our gene/plasmid of interest?
Antibiotic resistance
• The plasmids with our
gene also contain a
sequence that code for
naturally occurring genes
that confer antibiotic
resistance
• Therefore, only bacteria
containing plasmids with
resistance gene will grow
on a medium containing
the antibiotic while other
cells will die
Antibiotic resistance in medicine
http://www.niaid.nih.gov/topics/antimicrobialResistance/Understanding/Pages/mutation.aspx
Antibiotic resistance in medicine
http://www.niaid.nih.gov/topics/antimicrobialResistance/Understanding/Pages/mutation.aspx
How do we prevent contamination of
our transformed bacteria?
Sterile Technique
Sterile Technique
• Use antimicrobial reagents to sterilize work area and
equipment
– 70% ethanol, 10% bleach, sometimes flame
• Use clean, pre-sterilized reagents and plasticware
– No open fires at JABSOM so will use all disposable plastics
and filter tips for pipets
• Minimize manipulation of cells
– Don’t handle cells or plates unnecessarily
• Minimize exposure to environment
– Limit time tubes and plates are open
– Conduct experiments in locations with minimum traffic
• Properly dispose of all contaminated samples in
biohazardous waste
Our experiment
• Transform bacteria with two different
plasmids containing different antibiotic
resistance genes
• Assess growth of transformed cells on
different antibiotic-selective LB agar plates
Our plasmids
Next steps…
• Check growth of transformed cells in the
morning
• Confirm presence of cloned gene of interest in
plasmid by restriction enzyme digest