Genetic Transformation

Introduction

In this week’s laboratory period students had the opportunity to perform a common procedure preformed by many if not all microbiologists known as genetic transformation. Genetic transformation is the ability to move DNA into an organism and thereby altering its genotypic and genetic makeup (2). Genetic transformation has shown to have a wide variety of uses in many scientific studies. In agriculture, gene coding for traits such as frost, pest, or spoilage resistance have been genetically transformed in plants. In the pharmaceutical industry bacteria and yeast are transformed with human genes of interest in order to produce therapeutics for human disorders. An example of pharmaceutical industries use of transformation is seen in the development of insulin, which is used to treat some forms of diabetes mellitus (2).

In nature, many strands of bacteria genetically exchange genetic information during a process known as conjugation, and the new information is passed on subsequent generations. The advantage of using bacteria relates to the single-celled nature of the organisms. Only one cell needs to be changed in order to integrate the new genetic information and allow for its transmission to the next generation. The exchange process of genetic information allows the organism to increase in its capacity to adapt to its environment.(2)

Genetic transformation is also seen in organisms that are multicellular. However, the process is more difficult and in some cases can be particularly challenging. The multicellular nature of most plants introduces the complication of transforming each cell of the plant in order to fully integrate the new information (2). Often the approach taken in higher organisms, such as plants, involves transforming an individual plant cell and then regenerating it into a whole organism (2).

Genetic transformation is not only naturally occurring in plants, but bacteria and viruses are able to carry this out routinely as well. One of these bacteria is Escherichia coli.

E. coli is a strand of bacteria that is commonly found in the lower intestine of warm-blooded animals (3). Most E. coli bacteria strains are harmless, but some are capable of causing food poisoning in humans (3). In addition, the bacteria can also be grown easily and because of its simplicity it can be easily manipulated, thus making it one of the best-studied prokaryotic model organisms, and an important species in biotechnology (3). For this reason, E.coli was the choice of bacteria used for genetic transformation during the laboratory experiment.

This process involved the insertion of a new DNA into the E. coli cells. In this case the new DNA was a gene that coded for Green Fluorescent Protein (GFP) which, following the transformation procedure would be expressed in the bacteria causing them to glow a brilliant green under an ultraviolet light. Furthermore, the purpose of this experiment was also to teach students the process of moving genes from one organism to another with the aid of a plasmid. In addition to have a chromosome, bacteria also contain one or more small round pieces of DNA called plasmids (1). Plasmid DNA usually contains genes for more than one trait and through genetic engineering, scientists have been able to insert genes coding for new traits into the plasmid. In lab, students used a plasmid called pGLO which carries the GFP protein which can be switched on in transformed cells by adding sugar arabinose to the cells’ nutrient medium and a gene that allows E.coli to be resistant to ampicillin (1). Cells that have been successfully transformed with pGLO DNA are seen by the amount of cellular growth observed on the antibiotic plates (1).

The laboratory experiment required four different LB nutrient agar plates. Each plate contained a different combination of either (+) or (-) pGLO, ampicillin and arabinose. Each plate contained the following: Plate 1 contained LB, ampicillin, and +pGLO, Plate 2 contained LB,ampicillin, arabinose, and +pGLO, Plate 3 contained LB and ampicillin and Plate 4 contained only LB and вЂ"pGLO. Based on these environments it was predicted that the plates containing ampicillin would show bacterial growth thereby indicating E. coli’s resistance to ampicillin. In addition, the bacteria grown in plate 2 would also glow a green fluorescence when put under a ultraviolet light, however, plate 1 would not have this characteristic because of the absence of arabinose, the transformation solution. Lastly, it was also predicted that plate 3 would show a lawn of grown while plate 4 would show no growth since it did not provide any nutrients needed for bacterial growth, thus making plates 3 and 4 the control plates.

Materials and Methods

Supplies and Materials

• 1 E. coli starter plate

• 4 poured agar plates (1 LB, 2 LB/amp, 1 LB/amp/ara)

• 1 Transformation solution

• 1 LB Nutrient Broth

• 7 Inoculation loops

• 5 Pipets

• 1 Foam microtube holder/float

• Container full of crushed ice

• Permanent Marker

• 1 vial of rehydrated pGLO plasmid

• 42Ð'oC water bath

• Thermometer

• 37Ð'oC incubator

Prior to the beginning of the laboratory session, several needed materials were

prepared for students by the teaching assistants and by the professor. These included the preparation of the E. coli starter plate from which students were to obtain their bacterial sample, the transformation solution, LB nutrient broth, and (+) and (-) pGLO solutions. The two samples of pGLO solutions were present in the microtubules and foam rack upon arrival to lab.

The first thing that students needed to do was to obtain all necessary materials and label everything properly with their names/initials, dates, and laboratory period. This was done in order to avoid any type of error and confusion from occurring during the experiment. Next, one of the pGLO tubes was obtained and using a sterile pipet, 250 Ојl of transformation solution (CaCl2). When completing this part of the procedure the students were told to transfer the solution