Transcript File - Down the Rabbit Hole

Bacterial
Transformation
AP Biology Transformation Lab
What is Transformation?

Changing the genes and phenotype of a
bacteria by uptake of foreign/new DNA
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a natural process that bacteria have evolved in
order to obtain DNA from their environment.
enables scientists to insert genes by
recombinant techniques and place the plasmid
into a bacteria for expression
What is Transformation?

In order to transform bacteria we need to
overcome two problems
Disadvantage –cells that contain plasmids grow more
slowly
1.
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There is pressure on cells to get rid of their plasmids
Needs to be an Advantage to keep plasmids

Antibiotic resistance
How do we tell which cells have the plasmid?
2.
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We use a marker

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Grow the bacteria on plates that contain the antibiotic
Use a color pigment marker that is present when a particular
enzyme is present
Let’s Review: Bacterial genome

Bacteria are
prokaryotes—no
nucleus.
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The area where DNA
is located is called
the nucleoid
DNA is organized in
one double stranded
circular molecule
Bacterial DNA
Bacterial cell
Plasmid DNA
Genomic DNA
Size
Which bacteria will
we be using?
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Escherichia coli is the most common bacterium in the
human gut. It has been extensively studied in the
laboratory and is an important research organism for
molecular biology.
E. coli reproduce very rapidly; a single microscopic cell
can divide to form a visible colony with millions of cells
overnight.
Like all bacteria, E. coli has no nuclear envelope
surrounding the bacterial chromosome and thus no
true nucleus.
All of the genes required for basic survival and
reproduction are found in the single chromosome.
What is a Plasmid?

A circular piece of
autonomously replicating
DNA
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exists outside the main bacterial
chromosome
Originally evolved by bacteria
Carries separate genes for
specialized functions.
In genetic engineering,
plasmids are one means used
to introduce foreign genes
into a bacterial cell.
Scanning electron micrograph
What is carried on the Plasmid?

The plasmid contains genes necessary
for survival and can be passed from one
bacteria to another

ampR gene
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confers resistance to the antibiotic ampicillin.
E. coli cells containing this plasmid, can
survive and form colonies on LB agar that has
been supplemented with ampicillin.
Cells lacking the ampR plasmid are sensitive
to the antibiotic, which kills them.
An ampicillin-sensitive cell can be
transformed to an ampicillin-resistant cell by
its uptake of a foreign plasmid containing the
ampR gene.
The same can be said for the lac gene, which
codes for lactose. I
If this gene is taken in, the organism can
break down lactose.
Transformation has 4 main steps
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Prepare cells
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Incubate with plasmid
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Plasmid associates with membrane of cells
Shock the cells
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Make them competent
To initiate plasmid uptake
Allow cells to recover and plate on agar
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Allow cells to recover in rich medium for 0.5 – 1 h (without antibiotic
to avoid stressing the cells)
Plate on agar with antibiotic for selection
Next day can pick single colonies or clones
The Transformation Lab…
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Our plasmid: pBlu plasmid
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Into E. coli (scary?…no!)
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Our plasmid contains
genes for:
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AMP= ampicillin (an
antibiotic) resistance
Beta-galactosidase-an
enzyme that converts X-Gal
 Indo Blu
RNA
Protein that
allows for
antibiotic
resistance
RNA
Enzyme that breaks down
X-Gal to make Indo Blu
How do we get the plasmid inside
of the bacteria?
1.
2.
To transform cells, you first
need to make them
competent to take up
extracellular DNA.
Obtain E. Coli bacteria cells
+ Add to ice cold CaCl2
1.
2.
3.
Helps plasmid attach to
bacteria
Makes the cell competent
Add plasmid to same
microtube
1. E. Coli
2. pBlu
plasmid
How It Works
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Transformation solution
CaCI2
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Positive charge of Ca++ ions shields
negative charge of DNA
phosphates
DNA becomes “neutral” and can
pass through the cell membrane
Ca++
Ca++
O
O P O
O
CH2
Base
O
Sugar
O
Ca++
O P O
Base
O
CH2
O
Sugar
OH
How do we get the plasmid inside
the bacteria?

Wait…and then
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Heat shock! This
temporarily opens
pores to allow the
plasmid to enter the
bacteria…timing is
critical!!!
Chemical transformation

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Ice-cold CaCl2
 To make competent
 Slows the fluid cell
membrane
Heat shock
 Increases permeability
of membranes by
opening pores
 Plasmid DNA is taken
up
Nutrient broth incubation
 Allows beta-lactamase
expression
Bacterial
chromosomal
DNA
Cell wall
GFP
Beta lactamase
(ampicillin resistance)
pBLU plasmids
What is Nutrient Broth?
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Luria-Bertani (LB) broth
Medium that contains nutrients
for bacterial growth and gene
expression
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Carbohydrates
Amino acids
Nucleotides
Salts
Vitamins
How will we know if the bacteria
actually got into the plasmid?
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Any ideas?

We can grow the bacteria on a plate:
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That contains ampicillin and X-Gal
Regular bacterial medium
What do you predict will happen in each?
Predict
What will we observe???
pBlu
Amp
X-Gal
pBlu
Regular
Control
Amp
X-Gal
Control
Regular
Growing the bacteria
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After they have
received the plasmid…
Place on a growth
media and allowed to
grow.
~ 100 µl
spread
evenly
overnight
37 ºC
discrete
colonies
(~106 cells)