Download Bacterial Growth and Transformation

Bacterial Growth and Transformation
Please note that today's work will be under S1 conditions. Make sure that you follow the
S1 rules, that you learned in week 1, at all times.
Bacteria, such as Escherichia coli (E. coli), reproduce through binary fission. That means
that a single cell divides into two identical daughter cells. The time required for this division
is known as the generation time. If E. coli cells are grown in optimal conditions [Luria Broth
(LB) medium at 37 °C], the generation time can be very fast – on the order of 20 minutes.
After E. coli cells are placed in fresh LB at 37 °C, they pass through 4 stages of growth.
1. Lag Phase – Cells prepare to divide by synthesizing RNA and protein
2. Log Phase – Cells divide at their maximum rate
3. Stationary Phase – Cells reproduce and die at equal rates
4. Death Phase – Cells die faster than they reproduce
During log phase, growth is exponential. That is, the cell number doubles at each generation.
The expected number of cells is:
Number of starting cells x 2n
where n is the number of generations. Thus, if we start with one cell, we expect 2, 4, 8, 16, 32,
64, and, 128 cells after 1, 2, 3, 4, 5, and 6 generations. After 10 generations we expect 1024
cells. Growth continues at this rate until the food source becomes limiting and toxic waste
products from metabolism accumulate in the medium. The number of cells in a bacterial
culture can be estimated by reading the absorbance at 600 nm (OD 600). For E. coli cells, an
OD600 of 1.0 ≈ 5 x 108 cells/ml.
Because bacteria reproduce so rapidly, they are often used for molecular cloning. Bacteria
can be transformed with plasmid DNA, which means they take plasmid DNA from their
environment into the cell and then replicate it to high copy number. Thus, researchers can
insert a DNA sequence of their choice into a plasmid vector, and use the bacteria to make
many copies of the selected DNA. Typically, vector plasmids also contain a gene conferring
antibiotic resistance. Normally, E. coli cells are killed by antibiotics, such as ampicillin.
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However, cells transformed with an antibiotic resistance plasmid can survive and reproduce.
By growing the bacteria on plates containing ampicillin, one can ensure that all cells contain
the plasmid of interest. After the cells have been grown to high concentration (for example,
overnight in LB-Amp medium), the plasmid DNA can be purified using standard techniques,
such as the mini-prep procedure.
In today’s lab, we will do the following:
- Plot a time course of bacterial growth
- Transform cells with plasmid DNA containing an ampicillin resistance gene
- Plate transformed cells on LB-agar containing ampicillin
LB
to make 1 liter
980 ml water
10 g Bacto-Tryptone (or peptone)
5 g Bacto Yeast Extract
10 g NaCl
(For LB plates: add 15 g Bacto-agar)
Mix and sterilize by autoclaving.
For LB-Amp plates: allow to cool to ≈ 65 °C, then add 50 mg Ampicillin per liter
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Bacterial Growth
1. Using sterile technique, pipet 6 ml of LB into a culture tube.
2. Vortex the overnight E. coli culture and add 50 µl of it to the LB. Mix by finger
tapping.
3. Pipet 500 µl of the new culture into a cuvette for the time 0 reading (we will use sterile
LB to “blank” the spectrophotometer).
4. Place the lid on the tube and incubate at 37 °C with shaking.
5. After 1 hour, pipet 500 µl of your culture into a cuvette and read the OD600.
6. Return your culture to the incubator.
7. Repeat steps 5 and 6 every hour until the end of the lab.
Afterwards, convert your OD600 readings to cells/ml and plot a time course of bacterial
growth. Did your experiment follow the expected growth pattern? Why? Why not? What is
the generation time during log phase?
Bacterial Transformation
1. Add 2 µl of miniprep plasmid DNA to 98 µl sterile water.
2. Add 2 µl of this 1/50 dilution to 1 tube of competent cells, mix gently, and quickly
return the cells to ice.
3. As a control, add 2 µl of sterile water to 1 tube of competent cells, mix gently, and
quickly return the cells to ice.
4. Incubate cells on ice for 20 minutes.
5. “Heat shock” the bacteria for exactly 45 seconds at 37 °C (water bath), then return
them to ice for 2 min.
6. Add 250 µl LB to each tube, mix gently, then incubate at 37 °C for 1 hour.
7. Pipet 50 µl of the plasmid cells onto an LB-Amp plate and spread the cells evenly over
the surface.
8. Pipet 50 µl of the control cells onto an LB-Amp plate and spread the cells evenly over
the surface.
9. Incubate plates overnight at 37 °C.
Afterwards, count (or estimate) the number of bacterial colonies on your experimental plate.
Each colony represents a single cell that was transformed in our experiment. Are there any
colonies on the control plate? Why? Why not?
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