KEY CONCEPTS: Genetic transformation, plasmid DNA, cloning ...

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KEY CONCEPTS: Genetic transformation, plasmid DNA, cloning, restriction enzymes, antibiotic selection, gene regulation, transcription, protein expression INTRODUCTION Genetic transformation is the process by which a gene or genes from one organism are transferred to another organism via an engineered molecule of DNA. If the procedure is successful, the organism is capable of producing the protein encoded by the transformed gene, thus creating a genetic change. Genetic transformation is commonly used in biotechnology. In agriculture, transformation is used to confer genes for pest, frost and spoilage resistance. Transformation of the human insulin gene into bacteria has allowed for production of the protein on a large scale.1 To aid in bioremediation of oil spills, bacteria are transformed with genes that allow them to digest toxic components of the oil.2 The procedure contained in this lab will allow for the transformation of bacteria with a gene from the bioluminescent jellyfish, Aequorea victoria. A successful transformation will result in the expression of the green fluorescent protein (GFP) in the bacteria, causing them to glow bright green under long-wave UV light. Transformation and Antibiotic Selection: Genetic transformation in this laboratory will be facilitated by using the pGLO plasmid (see below). A plasmid is a circular, self-replicating DNA molecule which can be contained in a bacterial host cell without interfering with the function of the bacterial chromosome. Bacteria are capable, on their own, of randomly acquiring small pieces of DNA from their environment, but the process is inefficient. The transformation protocol in this lab uses a chemical, calcium chloride (CaCl2), plus heat to increase the efficiency of DNA uptake by the bacterial cell.

Even with the chemical transformation procedure, not every bacterial cell will incorporate the pGLO plasmid into the bacteria, not every cell will receive a copy of the plasmid. To isolate only the cells containing the pGLO DNA, the plasmid contains the beta-lactamase gene which encodes for an ampicillin resistance (Ampr) protein. After the transformation, the cells are grown on a solid medium called an agar plate. This

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medium will contain the antibiotic ampicillin. In the presence of the ampicillin, only the bacteria containing the pGLO plasmid will have the Ampr protein which will break down the antibiotic, and be able to grow (Fig. 2). This process is called antibiotic selection.

Figure 2. Transformed Bacterium: This is a diagram of what is occurring inside a bacterium transformed with the pGLO plasmid. The bacterium is plated on agar medium containing ampicillin. To grow, the bacterium must contain a pGLO plasmid and be expressing the ampicillin resistance protein, β-lactamase (Ampr). The ampicillin is inactivated by the β-lactamase protein, allowing the bacterium to grow on medium containing the antibiotic. Cloning a Gene: Plasmids can be engineered to carry a variety of genes that are not endogenous to the host cell, like the GFP gene. A plasmid usually starts out as a very small piece of DNA that contains a replication origin, an antibiotic resistance gene and a cloning region, an area of the DNA that has multiple unique restriction enzyme sites. Restriction enzymes are proteins which recognize specific DNA sequences and will cleave, or cut, the DNA backbone at these sequences. Once the DNA backbone is cleaved, it is possible to add, or clone, new DNA into this site. The diagram of the pGLO plasmid (Fig. 1) shows restriction sites for the enzymes NdeI, EcoRI, and HindIII, enzymes that are part of the original cloning site on the plasmid. The pGLO plasmid also carries the gene araC, which produces a protein needed for transcription of genes in the presence of arabinose sugar. The purpose of this protein is discussed below. Transcriptional Gene Regulation: In many cases, a researcher may want to control when a cloned gene is producing mRNA, and the corresponding protein. Proteins called transcription factors are frequently used by cells to turn transcription “on” or “off” depending on environmental conditions. The transcription, or production of mRNA, of the pGLO gene is controlled by using a promoter that is only active in the presence of the sugar arabinose (Fig. 3). The AraC protein, encoded by the araC gene on the pGLO plasmid, is the transcription factor necessary for this control. This protein is

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bound at the pGLO promoter site, but without arabinose is in the incorrect conformation, or shape, to recruit RNA polymerase and initiate transcription. (See figure below) In the presence of arabinose, the sugar binds to the AraC protein and changes its conformation so that in combination with RNA polymerase, transcription is initiated and an mRNA transcript is produced. In bacteria, transcription and translation, or protein synthesis, occurs simultaneously, and the GFP protein is produced.

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In this lab, it is important to confirm w hich cells have received the plasmid, and under which conditions the β-lactamase and GFP proteins are being produced (Fig. 4). When the pGLO transformations are plated on agar medium containing ampicillin and arabinose, a series of controls will be plated as well (Fig. 5). Two transformations will be performed: one with pGLO plasmid (+pGLO) and one without the plasmid present (-pGLO). A portion of the –pGLO transformation is plated on an agar medium without ampicillin or arabinose. This control is to be sure the bacteria are viable after the chemical and heat transformation procedure. This plate should be covered with a bacterial “lawn”. Another portion of the –pGLO transformation is plated on an agar plate containing ampicillin. No bacteria should grow on this plate. If it does, it means the bacterial culture has acquired resistance and is no longer suitable for use in this experiment. The +pGLO transformation is also plated on two different types of media. The first portion of the transformation is plated on agar containing ampicillin only. This control proves that the transcriptional control of the GFP gene is intact, and no GFP protein is produced in the absence of the arabinose sugar. The final plate is the experimental plate, containing both ampicillin and arabinose. The bacteria on this plate are the only ones that should glow when exposed to long-wave UV light.

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