Julia Sevco

Ms. Williams
3 June 2016
Academic Biology

Academic Biology Transformation Lab

Introduction
Bacterial transformation is the process where foreign DNA is introduced into a bacterium

which can then clone the DNA. Transformation occurs naturally in some species of bacteria, but

scientists found ways to make transformation occur artificially. This lab uses heat shock as a

form of artificial transformation. The type of DNA most commonly used in bacterial

transformation is a plasmid. A plasmid is a small circular piece of DNA that contains important

genetic information for the growth of bacteria. A plasmid can reduce its size by supercoiling so it

can easily pass through pores in a cell membrane. A plasmid is a gene that is resistant to

antibiotics. Recombinant DNA or rDNA is an artificially made DNA strand that is formed by the

combination of two or more gene sequences (Smith 1).

The purpose of this lab was to understand the transformation procedure by artificially

transforming DNA into bacterial cells to see what effect this would have on the growth of the

cells. 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 the DNA. This is accomplished by treating the bacteria with calcium

chloride which causes water to enter into the cells and make them swell. These swollen bacteria

are competent bacteria.

The hypothesis for this lab was that the transformed E.coli with the ampicillin resistance

gene would grow in the ampicillin plates, but the non-transformed E.coli would not grow. Also,
the E. coli would grow in the plates with or without Luria broth because there is no antibiotic

present on those plates.

Natural transformation is useful to bacteria because bacteria grow in the same habitat as

molds and fungi which make toxins that kill the bacteria. As a result, bacteria make proteins to

inactivate the toxins (“Bacterial Transformation” 1). Natural transformation helps bacteria

survive.

Artificial transformation is useful to humans. For example, artificial transformation can

be used to make large amounts of certain human proteins, such as insulin, which can be used to

treat people with Type I diabetes. Millions of diabetics use synthetic insulin to control their

blood sugar.

Materials
 2 15-mL test tubes
 Microtube rack
 Ice
 Sterile plastic inoculating loops
 500 µL of ice cold .05 M Calcium chloride (CaCl2) solution
 2 Sterile micropipets
 Water bath
 Glass beads
 Tape
 Timer
 Streak plates of E.coli
 4 media plates
 P-GREEN plasmid
 500 µL of Luria broth
 Spreading rod
 Incubator
Procedures
1. Mark one sterile 15 mL tube “+ plasmid” (experimental) and one “- plasmid” (control)
2. Using a sterile transfer pipet, add 250 µL of ice cold CaCl2 (Calcium Chloride) to each
tube
3. Put both tubes in ice
4. Use a sterile plastic inoculating loop to transfer isolated colonies of E.coli from the starter
plate to the + plasmid tube.
4a. Do not pick up any agar as it may inhibit the transformation process.
4b. Immerse the cells on the loop in the CaCl2 solution in the “+” tube and
vigorously spin the loop to dislodge the cell mass. Hold the tube up to the light to
observe that the cell mass has fallen off the loop.
5. Immediately suspend the cells by repeatedly pipetting in and out with a sterile transfer
pipet. Examine the tube against the light to confirm that no visible clumps of cells remain
in the tube or are lost in the bulb on the transfer pipet. The suspension should appear
milky white.
6. Return the “+” tube to the ice.
7. Repeat steps 4 and 5 using the “-“ tube.
8. Return the “-“ tube to the ice.
9. Use a sterile plastic inoculating loop to add one loopful of plasmid DNA to the + plasmid
tube. (When the DNA solution forms a bubble across the loop opening, its volume is 10
µL). Immerse the loopful of plasmid DNA directly into the cell suspension and spin the
loop to mix the DNA with the cells.
10. Return the + plasmid tube to ice. Incubate both tubes on ice for 15 minutes.
11. Label the bottom of media plates as follows:
a. LB/Amp plate - “+ plasmid” This is an experimental plate
b. LB/Amp plate - “- plasmid” This is a negative control
c. LB plate – “+ plasmid”
d. LB plate – “- plasmid”

12. Heat shock the cells. Remove both tubes directly from the ice and immediately immerse
them in 42°C water bath for 90 seconds. Gently agitate the tubes while they are in the
water bath. Return both tubes directly to the ice for 1 or more minutes.
13. Use a sterile transfer pipet to add 250 µL Luria broth (LB) to each tube. Gently tap the
tubes with your finger to mix the LB with the cell suspension. Place the tubes in the test
tube rack at room temperature from a 5 to 15 minute recovery.
14. Use a sterile transfer pipet to add 100 µL of cell suspension from the “-“ plasmid
transformation tube to each appropriate plate. Immediately spread the cells over the
surface of the plate as follows:
a. Slightly open (“clam shell”) the lids and carefully pour 4 – 6 glass
beads onto each plate
b. Use a back and forth shaking motion to move the glass beads across
the entire surface of the plate, evenly spreading the cell suspension all
over the agar surface
c. Let the plates rest for a several minutes to allow the cell suspension to
be absorbed into the agar.
d. To remove the glass beads, hold each plate vertically over a container,
clam shell the lower part of the plate, and tap out the glass beads into
the container.
 15. Use another sterile transfer pipet to add 100 µL of cell suspension form the + plasmid
tube to each appropriate plate
16. Immediately spread the cell suspension as detailed in step 14
17. Wrap the plates together with tape and place the plates upside down either in the
incubator or at room temperature. Incubate them for approximately 24 – 36 hours in a
37°C incubator or 48-72 hours at room temperature.

Results
The LB “–“ plasmid and the LBAmp “-“ plasmid plates did not show any growth. The
observed result on the LB “+” plasmid and LBAmp “+” plates showed no growth as well. The
LB “-“ plasmid and the LB “+” plasmid plates are positive controls. The LBAmp “-“ is a
negative control. The LBAmp “+” plasmid has the transformed bacteria.
Discussion
The LB “-” plasmid plate should have had bacterial growth because there was no

antibiotic. The LB “+ “ plasmid plate should have bacterial growth because the transformed

bacteria will grow since there is no antibiotic to inhibit growth. The LBAmp “-“ plasmid plate

should have no bacterial growth because the untransformed bacteria is unable to grow in the

presence of bacteria. The LBAmp “+” plasmid plate the transformed bacteria is able to grow

even in the presence of the antibiotic because the plasmid contains the gene allowing for

antibiotic resistance.

My hypothesis was correct, but my results were incorrect except for the LBAmp “-“

plasmid plate. Some sources of error could have included the accidental picking up of agar when

transferring the E. coli from the starter plate to the + plasmid tube. (See Step 4) This would

inhibit the transformation process. Another source of error would include having visible clumps

of cells remain (See Step 5). Also, it was necessary to add the Luria broth to “-“ first to avoid

cross contamination of plasmid. Not doing this correctly would be a source of error. Errors could

also have happened if the temperature and incubation periods used were not as directed.
 Works Cited

“Bacterial Transformation.” Biotech learn. n.d. Web. 29 May 2016.

Rapoza, M. & Kreuzer, H. (2012). Transformations: A Teacher’s Manual. Burlington: Carolina
Biological Supply Company
Smith, Yolanda. “What is recombinant DNA?” News-medical. n.d. Web. 29 May 2016.