Genetic transformation of the green florescent protein (GFP) gene into the bacterial cells of Escherichia Coli by use of the heat shock method of transformation.

Lab report by: Sonja Silva

Date: 10/31/11

Group Members: Janalee Thompson, Cody Thompson, Cassidy Kruger.

LIFE 102- L57

Supervisor: John Fitts

Introduction:

Heat shock transformation is used in cells to increase their permeability and cause them to uptake DNA contained in either a phage or plasmid. In this experiment, we are using the plasmid pGLO. A plasmid is a circular piece of DNA that is found in bacterial cells, also called a vector because of its nature of being used for transportation of DNA into a cell. A plasmid usually contains genes that are beneficial to the cell, and so a cell will readily take it in during transformation. (Weedman2009). Knowing how this process works will help us to make an accurate hypothesis about what is going to happen in this experiment.

Heat shock treatment must be performed after the cell is submerged in a transformation solution (in this case CaCl2). Bacterial cells take up the calcium which carries water into the cell and causes the cell to expand and be “competent” to take up DNA during heat shock (Roe). The cell’s permeability to take up DNA is best achieved through the combination of calcium and heat. (Chawla 2002).

Heat shock only works under certain conditions. The heat applied should not be over the optimum temperature for the organism, otherwise the cells will be damaged. During the heating period is when the cell becomes permeable. The cells will recover in ice and later in nutrient agar plates at room temperature so that the cell can operate normally with the new gene after transformation. (Chawla 2002). During recovery, heat shock proteins are activated to repair the cell (Henzler 2000).

In this experiment we took E. Coli bacterial cells which contain the plasmid, pGLO. We transformed it with a pGLO plasmid containing the gene for GFP and a gene that codes for resistance to ampicillin. We included the gene for ampicillin resistance so that when we introduced a media containing ampicillin, the cells that did not contain the pGLO would die off (Weedman 2009).

The GFP gene is activated by the presence of arabinose, so in order to create a plasmid with an active GFP gene, the DNA for pGLO has been engineered to contain the arabinose operon (araC). In the presence of arabinose, the araC aids in the binding of RNA polymerase, and GFP is produced (Weedman 2009).

Based on the information we have read and researched about heat shock transformation and the nature of the pGLO plasmid, when the experiment is done, it is expected that the bacterial cells that take up the plasmid will express the GFP gene, causing the bacteria to glow in the dark. Only cells that take up the plasmid will survive when exposed to ampicillin, and, that way, we will be able to test the success of heat shock as a method of transformation.

Materials and Methods:

To begin the experiment, we obtained two microcentrifuge tubes. We used a micropipetter to transfer 250 microliters of transformation solution (CaCl2) into each of the two tubes and labeled them +pGLO and –pGLO. (The –pGLO signified the control group that will not receive the pGLO plasmid). We closed the lids and set both tubes in a beaker of ice. We obtained a colony of E. Coli bacteria by swiping it out of a starter plate with a sterile loop and submerged the loop in the transformation solution contained in the tube labeled +pGLO and swirled it around until the colony was completely mixed into the transformation liquid. We returned the tube back to the beaker of ice. We did the same thing for the tube labeled –pGLO, dispersing the bacteria into the solution.

Next, we obtained the pGLO plasmid and mixed it into the solution labeled +pGLO using a sterile loop. We allowed both tubes to sit in the ice bath for 10 minutes.

We then took plates of a media containing LB (Luria Broth) nutrient agar, ampicillin, and arabinose. One plate contained just the LB (for the control), two plates contained LB and ampicillin, and the fourth plate contained LB, ampicillin and arabinose. The plate containing LB only was labeled with –pGLO, meaning it would not receive the plasmid containing bacteria. The plates with LB and ampicillin were labeled one with –pGLO and one with +pGLO, meaning one would receive the plasmid and one would not. The plate with LB, ampicillin, and arabinose was labeled +pGLO.

After the ten minutes on ice, we took the tubes out of the ice and, immediately, placed them on floating racks in a 42°C water bath for 50 seconds exactly. We placed the tubes directly back into the ice, and allowed them to recover for 2 minutes.

After 2 minutes, we took the tubes out of the ice and placed them in a rack. Using a pipette (changing tips each time) we added 250 microliters of the LB nutrient broth to the +pGLO tube and to the –pGLO tube. We allowed the tubes to sit closed for 10 minutes at room temperature.

After 10 minutes of recovery, we measured 100 microliters of –pGLO and +pGLO into the appropriately labeled nutrient agar plates (using different tips on the pipette for each plate). We then mixed the solutions gently into the nutrient agar plates using sterile loops. We closed the lids on the plates and stacked them upside down to incubate at 37°C for seven days (only 24 hours required for this process).

After waiting for the bacteria to grow and recover, we tested them under black light to see which ones glowed. We also examined the area of where the bacteria were spread out to see which of the plates of bacteria had grown.

Results:

The expected result was that the plate marked LB would grow naturally because it contained normal E. Coli cells. The plate marked LB/amp with the pGLO would grow because it contained the gene for ampicillin resistance, and the plate marked LB/amp without the pGLO plasmid would get killed off because it did not contain the gene for ampicillin resistance. The plate marked LB/amp/ara was supposed to grow and glow because it contained the gene for ampicillin resistance and the arabinose would trigger the glow of the GFP gene.

Expected results:

Grow Glow Die-pGLO LB X+pGLO LB/amp X (only cells that did not

receive the plasmid)-pGLO LB/amp X+pGLO LB/amp/ara X X (only cells that did not

receive the plasmid)

In our experiment, the only plate that survived was the plate marked LB. The other plates did not contain the bacteria at all. It all died off.

Actual results:

Grow Glow Die-pGLO LB X+pGLO LB/amp X-pGLO LB/amp X+pGLO LB/amp/ara X

Discussion:

Our hypothesis was that the plate containing the pGLO plasmid would glow and grow in the presence of arabinose and ampicillin. Our results did not support our hypothesis because all the plates containing E.Coli that were heat shocked with the pGLO plasmid did not survive with exposure to ampicillin.

We know that our hypothesis was correct because when tested in another situation, the pGLO plasmid was taken up and cause the plate labeled +pGLO LB/amp/ara to grow and glow. Our experiment could have gone wrong for several reasons. Something may have gone wrong in any of the steps to cause the cells to not become permeable enough to uptake the pGLO plasmid. Also, if the heat applied was too much, the proteins within the cells could have denatured, or the DNA, for that matter could have denatured, and caused the cells to die off.

Our results conflict with other results of what has been done before with the pGLO plasmid, so we know it should have worked, but there were some possible sources of error, such as mismeasurement, or we may have overlooked changing the micropipetter tip and contaminated the DNA. A number of things could have happened, but I am unsure as to what exactly it was.

Literature Cited:

Weedman D. 2009. Genetic transformation. LIFE 102 Lab Manual. 6th ed. Minnesota: bluedoor, LLC. p. 105-113.

Chawla HS. 2002. Introduction to plant biotechnology. 2nd ed. New Hampshire: Science Publishers Inc. p.187

Roe, Bruce. Bacterial Transformation and Transfection. http://www.genome.ou.edu/protocol_book/protocol_adxF.html. 6 November 2011.

Henzler HJ. 2000. Influence of stress on cell growth and product formation. Advances in Biochemical Engineering in Biotechnology. New York: Springer. p. 2-7.