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Bacterial Transformation
AP Biology Transformation Lab
What is Transformation?
Changing the genes and phenotype of a bacteria by uptake of foreign/new DNA 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 problems1. Disadvantage –cells that contain plasmids grow more slowly
There is pressure on cells to get rid of their plasmids Needs to be an Advantage to keep plasmids
Antibiotic resistance
2. How do we tell which cells have the plasmid? We use a marker
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 DNA first…
Let’s Review: Bacterial genome
Bacteria are prokaryotes—no nucleus. The area where DNA
is located is called the nucleoid
DNA is organized in one double stranded circular molecule
Bacterial DNA
Plasmid DNA
Bacterial cell
Genomic DNA
Size
Which bacteria will we be using?
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 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 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
Prepare cells Make them competent
Incubate with plasmid Plasmid associates with membrane of cells
Shock the cells To initiate plasmid uptake
Allow cells to recover and plate on agar 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…
Our plasmid: pBlu plasmid
Into E. coli (scary?…no!)
Our plasmid contains genes for: AMP= ampicillin (an
antibiotic) resistance Beta-galactosidase-an
enzyme that converts X-Gal Indo Blu
Protein that allows for antibiotic resistance
Enzyme that breaks down X-Gal to make Indo Blu
RNA
RNA
How do we get the plasmid inside of the bacteria?
1. To transform cells, you first need to make them competent to take up extracellular DNA.
2. Obtain E. Coli bacteria cells + Add to ice cold CaCl2
1. Helps plasmid attach to bacteria
2. Makes the cell competent3. Add plasmid to same
microtube
1. E. Coli 2. pBlu plasmid
How It Works
Transformation solution CaCI2 Positive charge of Ca++ ions shields
negative charge of DNA phosphates
DNA becomes “neutral” and can pass through the cell membrane
Ca++
Ca++
OCH2
O
P O
O
OBase
CH2
O
P
O
O
O
Base
OH
Sugar
Sugar
OCa++
How do we get the plasmid inside the bacteria?
Wait…and then
Heat shock! This temporarily opens pores to allow the plasmid to enter the bacteria…timing is critical!!!
Chemical transformation
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
expressionBeta lactamase(ampicillin resistance)
pBLU plasmids
Bacterial chromosomal DNA Cell wall
GFP
What is Nutrient Broth?
Luria-Bertani (LB) broth Medium that contains nutrients
for bacterial growth and gene expression Carbohydrates Amino acids Nucleotides Salts Vitamins
How will we know if the bacteria actually got into the plasmid?
Any ideas?
We can grow the bacteria on a plate: That contains ampicillin and X-Gal Regular bacterial medium
What do you predict will happen in each?
Predict
pBlu
pBlu
Control
Control
Amp
X-Gal
Regular
Amp
X-Gal
Regular
What will we observe???
Growing the bacteria
After they have received the plasmid…
Place on a growth media and allowed to grow.
~ 100 µlspread evenly
discrete colonies
(~106 cells)overnight37 ºC