Download Exp.9 Bacterial Transformation

Transformation of
Escherichia coli
Exercise 9
Experiment Goals

Understand the transformation procedure using
the heat shock method.
 Understand how DNA can be transferred to an
organism and the change in phenotype that may
result from such a transfer.
 Transformation of a gene for resistance to the
antibiotic ampicillin into a bacterial strain (E.
Coli) that is sensitive to ampicillin.
Bacterial Transformation

Definition:
 Transformation is the genetic alteration of
a cell resulting from the uptake and
expression of foreign genetic material
(DNA). i.e. the act of putting foreign DNA
into a bacterial cell
 Occurs in nature, but rarely
 If the foreign DNA has an origin of replication
recognized by the host cell DNA
polymerases, the bacteria will replicate the
foreign DNA along with their own DNA.
Escherichia coli (E. coli)

Lives in the human gut
 Relatively simple and well understood organism
 Its genetic material consists of:



DNA 3-5 million base pairs in length,
Plasmids, usually ranging from 5,000-10,000 base
pairs in length
It is these plasmids that bacteria can transfer
back and forth, allowing them to share genes
among one another and thus to naturally adapt to
new environments.
Plasmids
 Plasmids



small
circular
extrachromosomal DNA
 Self-replicating
DNA molecule separate
from chromosome
Plasmids

Plasmid DNA usually contains
genes for one or more traits that
may be beneficial to bacterial
survival.
 In nature, bacteria can transfer
plasmids back and forth allowing
them to share these beneficial
genes.
 This natural mechanism allows
bacteria to adapt to new
environments.
 The occurrence of bacterial
resistance to antibiotics is due to the
transmission of plasmids.
Transformation Applications
How ?

Use enzymes to
cut plasmid DNA
 Insert a DNA
sequence for a
desired protein
 Insert plasmid into
bacteria and
grow…grows fast!
 Isolate protein
made by bacteria
Competency
 Not
all bacteria take up free-floating DNA
in the environment
 Bacteria which are able to uptake DNA are
called "competent"


Some bacterial strains have NATURAL
COMPETENCE. Ex. Bacillus, Streptococcus,
Haemophilus and Neisseria.
Some bacterial strains, such as E. coli, can
be made ARTIFICIALLY COMPETENT.
Artificial Competency

Since DNA is a very hydrophilic molecule, it won't normally pass
through a bacterial cell's membrane.

In order to make bacteria take in the plasmid, they must first be made
"competent" to take up DNA.

This is done by creating small holes in the bacterial cells by different
methods.

Cells that are undergoing very rapid growth are made competent
more easily than cells in other stages of growth.
Experiment
 In
this experiment, we will introduce a
gene for resistance to the antibiotic
ampicillin into a bacterial strain that is
sensitive to ampicillin
 If the sensitive bacteria incorporate the
foreign DNA, they will become ampicillin
resistant.
Sterile Technique





It is important not to contaminate E. Coli plates
with external bacteria from hands or the table,
etc
The plates without ampicillin are especially
prone to contamination, since anything can grow
on those plates.
Wearing gloves can help avoid contaminating
plates and solutions
Keep the lids on the plates whenever not adding
things to the plates
All of the solutions and plastic ware will be using
have been sterilized
Steps of Bacterial Transformation
1.
2.
3.
4.
Competency
Transformation
Recovery period
Growth & Isolation
1- Competency


Label two sterile microtubes:
one “+”and the other “-”.
Using a disposable pipette,
add 250 μl of 50mM CaCl2
solution to each tube (“+” and
“-”) and place them both on
ice.
1- Competency

Use a sterile plastic loop to transfer one or two 3
mm bacterial colonies to the “+” tube, return tube
to ice (Do not pick up an agar as it may inhibit
the transformation process)
 Transfer a mass of cells to the “-” tube
 Add 10 µl of ampicillin resistant plasmid directly
into the CaCl2 in “+” tube.
 Return the “+” tube to the ice. DO NOT add the
plasmid to your "-" tube. Incubate both tubes on
ice for 15 minutes.
1- Competency
• Ca2+ neutralizes the negative charges on the phosphate
backbone of the DNA, so that the DNA is not repulsed
from the phospholipids of the cell membrane.
• This allows the DNA to more easily pass
2- Transformation

Insertion of a plasmid
that contains a gene for
the resistance to
ampicillin
 Heat shock - helps
plasmid enter the cells
2- Transformation
3- Recovery period

Allow the cell to regain strength and start to
multiply
 Necessary to allow expression of the antibiotic
resistance gene on the plasmid
 Add 250 µl sterile Luria Broth to both tubes
using a sterile pipette
 Move tubes to water bath at 37oC for 5 min.
4- Growth & Isolation

Mark four Luria Broth agar plates
Transformation procedure
CaCl2
cold!
E. coli
Plasmid
E. coli
+
pAmp
42oC
Recover
at 37oC
LB + Amp
Incubate at
37oC overnight
LB + Amp
E. coli
4- Growth & Isolation
Transformation Efficiency
 After
incubation count the colonies on each
plate.
 Calculate the transformation efficiency
 Transformation Efficiency = no. of
colonies observed/ µg Plasmid