BACTERIAL TRANSFORMATION

The purpose of this lab is to demonstrate visible changes in E. coli bacteria that have been
transformed with a gene that codes for a green fluorescent protein, the source of this gene
being bioluminescent jellyfish. In real life, the gene provides the jellyfish with a glow-in-the-
dark feature in the deep, dark ocean. You will be using a plasmid called pGLO (p = plasmid)
because the gene being carried on is the code for the fluorescent protein.

There is another important gene on this pGLO plasmid---an antibiotic resistant gene that
confers resistance to ampicillin (the bacterium now makes beta-lactamase enzyme). Cells
that inherit the pGLO take on fluorescence and ampicillin resistance. There is a molecular
switch on the plasmid, and it is activated by arabinose sugar. When the transformed cells are
grown in a medium with arabinose the pGLO genes are on: when not grown on arabinose
medium, the genes are off.

The lab protocol can be conceptualized as four major steps.

* PRE-INCUBATION
The recipent E. coli cells will be exposed to positively charged calcium chloride (CaCl2) ions.
This treatment is meant to stress the bacterium in order to render its cell membrane and cell
wall permeable to the donor plasmid. This process will make the recipient E. coli "competent"
to uptake the plasmid. Bacteria are not naturally receptive to plasmid uptake.
* INCUBATION
The plasmid (with amp+ gene) is added to a recipient E. coli suspension, which will now be
called E. coli + because it is the one which is being transformed. Another E. coli suspension
will act as a control, called E. coli - because it will not be exposed to the plasmid; therefore, it
will NOT inherit the gene.
* HEAT SHOCK
The recipient cells plus plasmids and the control cells not exposed to the plasmids are briefly
exposed to 42 degrees C. This step will maximize the uptake of the plasmid through the wall
and membrane of the cells.

GO TO BIO-RAD TO GET MORE INFO---diagrams, pictures, explanations, etc.

http://WWW.BIO-RAD.COM/ (go to Life Science Research)
The following procedure is from the Bio-Rad pGLO kit.

OBJECTIVES:

Transform a wild-type E. coli into a ampicillin-resistant strain that can produce fluorescent
protein.
Learn a relatively simple method used in biotechology.

MATERIALS NEEDED:

per table
E. coli starter plate with large isolated colonies
LB broth
42o C water bath
CaCl2 tubes in ice
pGLO plasmid (shared by class)
foam rack for microtubes
UV lamp
sterile glass beads in a container
disinfectant solution for contaminated beads at side of room
pre-calibrated inoculation loops (calibrated for 10 microliters/10ul)
poured agar plates: LB, LB/ampicillin, LB/amp/arabinose

THE PROCEDURE:

per table

Make sure that you always mix the tube contents WELL: hitting the microtubes sharply on the
table top will do that.
When incubating the microtubes in the foam rack, be sure that the tube bottoms are all the
way into the water below the rack.

1. Label 2 microtubes---+DNA (pGLO) and -DNA (no plasmid). Place them both in the
foam rack.
2. Transfer 0.25ml of COLD CaCl2 into each tube and place them both in ice.
3. Using the calibrated, sterile, plastic inoculating loops, pick up one single colony from
the starter plate and immerse it into the CaCl2. Do this for each tube.
o Be sure that all of inoculum comes off of the inoculating loop.
o Repeatedly pipetting IN AND OUT, using the sterile transfer pipet.
o Be sure that solution is homogenous and equally distributed in solution. No
clumps of cells should be in the tube or in pipet.
4. Return both E. coli tubes to the ice.
5. Use another sterile inoculating loop to transfer one loopful (10 ul) of the plasmid DNA
solution to the +DNA tube. Carefully remove the DNA plasmid from the solution
WITHOUT touching the pGLO tube sides (otherwise, you will have lost important
DNA information on the sides of the tube) The DNA will look like a bubble across loop
opening. Be sure to knock the loop around in solution in order to get as much DNA off
as possible.
6. Incubate both tubes on ice for 10 minutes.
7. While tubes are incubating, label plates with group name and date. For each pair, one
should be labelled +DNA and the other -DNA. -----2 LB plates-----2 LB/amp------2
LB/amp/arab-----
8. Following 10 minute incubation on ice, heat shock the cells by removing both tubes
from the ice and IMMEDIATELY immersing them in 42 degrees C water bath for 50
seconds . Place them directly back into the ice after this heat shock period .

Carry the tubes over to the water bath in the ice and replace them when done. In other words, AT NO
TIME SHOULD THE TUBES BE OUT OF EITHER THE ICE OR THE WATER
 BATH.

9. copyright Bio-Rad Corporation

10. After both tubes have sat in the ice again for 2 minutes, transfer 0.25ml LB broth to
each tube. Gently tap tubes with finger to mix and set tubes in test tube rack at ROOM
TEMPERATURE for a 10 minute recovery period.
11. Use a sterile pipet to transfer 0.1ml of the E. coli/pGLO suspension onto the 3 agar
plates marked +DNA and make spread plates. Pour a few of the STERILE glass
beads---about 5---onto each agar plate with the inoculum, with the top on, and
furiously shake the beads all around the agar plate. This will equally distribute the
inoculum around the plate.

Place the used, contaminated glass beads in the disinfectant solution container.
Minimize the exposure time of agar plate to the open air to avoid contamination.
Do NOT allow cell suspension to sit on the agar too long before being spread: this will
maximize equal distribution on the agar.

12. Do the same for the E. coli without pGLO ( -DNA), using about 5 small glass beads for
each plate.
13. Wrap the 6 plates together with tape, and place them upside down in the 30 o C
incubator.

Pick up the pGLO DNA picrotube that you originally got your plasmid
sample out of and take it to the UV lamp. Does the tube fluoresce?
WHY?

INTERPRETATION:

You will need to observe each of the 6 plates both in the white light and under the UV
lamp.

1. What was the purpose of the LB plates run on both the -DNA and +DNA E. coli?

2. Look at the 3 plates with the E. coli without the pGLO plasmid.
Did you find growth on the LB/amp? WHY?

3. Look at the 3 plates with the E. coli with the pGLO plasmid.
Did you find equal numbers of transformed cells on the LB/amp and LB/amp/arab?

4. Which plate---LB/amp or LB/amp/arab--- produced glowing colonies? WHY?

5. What was the purpose of the arabinose?

6. You could actually determine the percentage of E. coli cells that were transformed. Here is
how you could do it:

The formula is:

total number of cells growing on the LB/amp = # of transformants per mg of DNA
amount of DNA spread on the agar plate

1. First, count the number of cells on either the LB/amp or the LB/amp/arab.
2. To determine the amount of DNA:
total amount of DNA [mg] = concentration [mg/ml] X volume [ml] = 10mg/ml X
0.03ml = 0.3 mg
3. To determine fraction of DNA solution spread:
volume of solution spread on plate divided by total amount in tube = 100 ml/510 ml
= 0.2
4. To determine the mass of DNA spread on the plates:
total mass of DNA [mg] X fraction spread = 0.2 X 0.3 = 0.6 mg
5. number of colonies = # of transformants per mg of DNA = ?
0.6 mg

QUESTIONS:

1. Describe the 2 traits on the pGlo plasmid. What activates the genes on this plasmid?

2. What is the purose of the exposure to high and low temperatures?

LAB MANUAL: TABLE OF CONTENTS

8/2009, Jackie Reynolds, Richland College