AP Bio pGLO Transformation Formal Lab Report

Topics: Bacteria, Plasmid, DNA Pages: 5 (1330 words) Published: May 28, 2006

The topic of this research involved the occurrence of genetic transformation in bacteria (E. Coli). More specifically, a previously prepared pGLO plasmid--which consisted of the gene to be cloned--was used to transform non-pathogenic bacteria. The pGLO plasmid contained a gene for the Green Fluorescent Protein (GFP) from a bioluminescent jellyfish and a gene for resistance to ampicillin, an antibiotic. Essentially, we wanted to determine the conditions of the bacteria that would glow. Our hypothesis was that the transformed solution with no plasmid DNA and ampicillin would produce no bacteria colonies, as it wouldn't be able to grow without the gene for ampicillin resistance. Also, the transformed solution with just LB and ampicillin would produce bacteria colonies but the transformed solution with LB/ampicillin/Arabinose would produce glowing bacteria colonies (as Arabinose allows the GFP gene to be expressed, but in both cases bacteria colonies would be present because of the gene of resistance to the antibiotic, ampicillin). We essentially made the required transformed solutions--and the controls--swiped them on the agar plate, and then observed to see whether or not bacteria colonies grew and whether or not they glowed. Our data fully supported our hypothesis. We can thus conclude that bacteria can take in foreign DNA through the process of transformation and that this foreign DNA can fundamentally change the bacteria (ex: making it glow). Future research can involve inserting other pieces of DNA into bacteria from different organisms, making the bacteria take on various phenotypic characteristics.

Background Information:

Genetic transformation is one of the most important processes in biotechnology. Essentially, genetic transformation involves the process where a cell (in this lab, a bacterial cell) takes up foreign DNA from its surroundings and incorporates it into its own DNA. This gene transfer is accomplished with the aid of a plasmid, a naturally-occuring small piece of circular DNA in bacteria. Bacteria can efficiently adapt to new environments by transferring plasmids with beneficial genes aiding survival to other bacterial cells.

In biotechnology laboratories, plasmids are transformed. Essentially, restriction enzymes are used to make cuts in the plasmid--the 'vector'--and in the DNA sequence containing the gene to be transferred to the bacteria. Then, this gene to be transferred is forged into the plasmid (in this lab we used previously made plasmids called pGLO, containing a bioluminescent gene and a gene for ampicillin resistance). Plasmids are then 'forced' into bacterial cells using a process called "heat shock," where small pores in the bacteria open up. In the end--in order to find out which bacteria received the new gene--the transformed solution can be incubated on agar gel. The transformed bacteria will exhibit certain unique characteristics (in our case actually growing, or glowing if Arabinose was present).

Materials and Methods:

For this experiment, the following materials were used: two micro test tubes, sterile transfer pipettes, 500 µl of Transformation Solution (CaCl2), an ice bath, a foam rack for the tubes to keep them afloat in the ice-bath, non-pathogenic E. Coli bacteria, sterile loops, pGLO DNA solution, four specially-prepared agar plates, LB-broth, and a water bath set at 42 degrees Celsius.

First we labeled one micro test tube '+DNA' and the other '-DNA.' Then we transferred 250 µl of Transformation Solution (CaCl2)--using a sterile pipette--into each micro test tube, and placed the tubes in a foam rack, which floated on an icy water bath. Next, we used two sterile loops to transfer E. Coli. Bacteria from the starter plate to each of the tubes ('+DNA' and '-DNA' tubes). Now that we had added the required bacteria, we proceeded in our experiment by adding the pGLO DNA solution--using a new sterile loop--to the micro test tube labeled '+DNA.'

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