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 donar plasmid. This process will make the recipient E. coli "competent" to uptake the plasmid.
* 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

42^o 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:

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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.
   • Be sure that all of inoculum comes off of the inoculating loop.
   • Repeatedly pipetting IN AND OUT, using the sterile transfer pipet.
   • 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.

   copyright Bio-Rad Corporation
9. 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.
10. 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.

11. Do the same for the E. coli without pGLO ( -DNA), using about 5 small glass beads for each plate.
12. 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. Determine the transformation efficiency.   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

  1/2004, Jackie Reynolds, Richland College