Lab Exercise 10 – Transformation of Bacterial DNA
Lab Exercise 10 – Transformation of
Bacterial DNA
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Objectives:
Following this exercise the student should
be able to:
compare and contrast bacterial mechanisms of
genetic variation
describe the lab methods used to bioengineer
traits
perform a bacterial transformation
evaluate the role of bacterial transformation and
use of DNA to modify organisms in light current information on the biotech
industry, biological warfare, and medical applications.
Background
DNA represents the blueprint (genotype) for the physical attributes (phenotype)
expressed in organisms and their offspring. Bacteria reproduce via binary fission unlike
Eukaryotic cells, which undergo mitosis. Binary fission produces identical daughter
cells, or clones. This contrasts with the products of meiosis that produce genetic
variability and offspring with completely new combinations of genes. Genetic changes in
bacteria changes are often associated with factors that increase pathogenicity by
equipping the bacteria with additional abilities to produce toxins, evade the immune
system, or resist antibiotics. While eukaryotic cells have most of their genetic material in
chromosomes within the nucleus and a small amount in organelles such as the
mitochondria, bacterial DNA exists in the nucleoid as a single circular chromosome and
as small circular extra-chromosomal DNA called plasmids.
Plasmids are not necessary for bacteria to survive. They reproduce at a rate
different from the chromosome, and can be transferred to other bacteria altering their
phenotype and abilities. Genetic variation in bacteria takes place through three
methods: conjugation, transformation, and transduction. In this laboratory exercise you
will perform a bacterial transformation. Before looking into the procedure, fill out the
chart below to describe the three mechanisms for genetic variation in bacteria.
Type of mechanism for
Description of factors necessary for this
genetic variation
to occur
Conjugation
Transformation
Transduction
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http://www.animalnetwork.com/fish2/aqfm/1999/dec/wb/wbfig6.asp
Extract jellyfish
GFP gene
Create a plasmid
with an Ampicillin
resistance gene and
activator
pGLO
Ampicillin
resistance
pGLO plasmid
with arabinose
activator
Insert the plasmid
into E.coli K-12
icbxs.ethz.ch/members/ leu/gfp_home.html
In this lab you will use biotechnology to transform
bacteria (alter their phenotype) using a gene isolated
from jellyfish. The gene, called GFP (Green Fluorescent
Protein), is naturally found in a bioluminescent jellyfish
called Aequorea victoria. The GFP gene has been
removed and used to create a plasmid called pGLO. The
plasmid will be inserted into E.coli transforming the
bacterial genome resulting in bacteria with the capacity to
produce GFP. In addition to coding for the fluorescent
protein the plasmid also codes for resistance to ampicillin
and has a gene-regulating mechanism that switches the
gene on in the presence of arabinose (a sugar).
A special strain of E.coli is being used called E.coli
K-12. This strain does not produce any toxins and can
not survive outside of the lab. The transformed cells are
selected from the parent cells by culturing on agar made
with ampicillin. The susceptible parent cells will not
survive, but the new transformed cells are resistant to
ampicillin and will grow. The transformed cells grown on
media with arabinose will switch the GFP gene on.
The procedure will require three main steps. In
step one the transformation fluid (CaCl2) neutralizes
the phosphate charges in the phospholipid cell
membrane and DNA backbone allowing the plasmid
(pGLO DNA) to enter the cell. In the second step you will
heat shock the cell which further enhances DNA uptake
but is highly time sensitive. In the third step the bacteria
will be incubated with the appropriate ingredients to: a)
select the transformed strain and b) allow transformed
cells to express the trait.
Materials:
Ampicillin
resistant glowing
E. coli K-12
stock cultures of E.coli K-12
plasmid pGLO
transformation fluid (CaCl2)
LB broth
5 pipettes
loops and incinerators
2 lab groups work together – 6 people each setup
1 LB (Luria & Bertani) plate
2 LB plates with ampicillin
1 LB plates with amp & arabinose
microtubes
tube holders
stop watch
ice bath
water bath at 42ºC
Gram stain equipment and microscope
Lab Exercise 10 – Transformation of Bacterial DNA
Procedures:
1. Read 230-234 in the text before coming to the lab.
2. Label one microtube +pGLO and another –pGLO. Place them in the microtube
rack.
3. Using a sterile pipette transfer 250µl of transformation fluid (CaCl2) into both the
+pGLO and –pGLO microtubes.
4. Place the tubes on ice.
5. Using the loop and aseptic technique, pick one isolated colony from the stock
plate and place it in the +pGLO microtube. Use the loop to gently mix.
6. Using the loop and aseptic technique, pick one isolated colony from the stock
plate and place it in the -pGLO microtube. Use the loop to gently mix.
7. Use the sterilized loop to add a loop of the pGLO plasmid to the +pGLO
microtube ONLY. Use the loop to stir the plasmid and stock culture. DO NOT
ADD anything to the –pGLO microtube.
8. Incubate the tubes on the ice for 10 minutes.
9. While the tubes are incubating, label the LB plates as follows

LB plate: -pGLO: LB

LB/amp plate: +pGLO:LB/amp

LB/amp plate: -pGLO:LB/amp

LB/amp/ara plate: +pGLO:LB/amp/ara
10. Heat Shock: Using the foam microtube holder, transfer the +pGLO and –pGLO
microtubes into the 42ºC water bath for exactly 50 seconds. Make sure to push
the tubes all the way down in the rack so that they make contact with the water.
11. After 50 seconds, rapidly transfer the microtubes back to the ice bath for 2
minutes.
12. After the 2 minute ice incubation, using a new sterile pipette for each tube,
place 250µl of LB nutrient broth in each microtube. Close the tubes.
13. Incubate the tubes 10 minutes at room temperature.
14. Tap the closed tubes with your fingers to mix the contents, then using a new
sterile pipette for each tube, pipette
Experimental Plates

100µl of the +pGLO tube to the LB/amp plate: +pGLO:LB/amp

100µl of the +pGLO tube to the LB/amp plate: +pGLO: LB/amp/ara
Control Plates

100µl of the -pGLO tube to the LB/amp plate: -pGLO:LB/amp

100µl of the -pGLO tube to the LB/amp plate: -pGLO:LB
15. Using aseptic technique, streak the suspensions on the plates. Be sure to
incinerate between plates.
16. Stack your plates tape them together and label them with your group name and
AM or PM lab. Incubate them, upside down, in the 37ºC incubator.
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Exercise 10 – Transformation Lab Report
Name_________________
Control Plates
1. Observe the plates & count the colonies. Then observe the
colonies under UV light. Record the results of your experiment in
the table below.
Plate
Approximate
Observations under UV light
Number of
colonies
-pGLO:LB
-pGLO:LB/amp
Transformation
Plates
+pGLO:LB/amp
+pGLO:LB/amp/ara
2. Under regular light describe any difference between the various colonies?
3. What do we call bacteria of the same species that have different genetic abilities
but are still biochemically the same or very similar? (circle all that apply)
a. a new species
b. a different genus
c. a strain of the original species
d. a mutant organism
e. a transformed organism
4. Ask the instructor if you should Gram stain all four plates and note any
differences in the Gram stain.
5. What was the purpose of:

the ice bath

the 42ºC water baths

the transformation fluid (CaCl2)
6. Why was the ampicillin resistance gene added to the plasmid?
7. Why was the activator gene added to the plasmid?
Last Updated 2/12/2016
©Janet Fulks