Transformation is the transfers of virulence from one cell to another, through the transferring of genetic material. It was originally postulated in 1928 through the works of Federick Griffith, a British microbiologist. Griffith observed that the mutant form, non-virulent form, of the bacteria Streptococcus Pnumoniae could be transformed into the normal, virulent form, when injected into mice along with heat killed normal forms. He concluded that somehow the information the dead virulent form had transformed the mutant form into a virulent form.
Later on through the works of Avery, Macleod, and McCarty in 1944, it became obvious that DNA is the transforming property and the substance transferred during transformation, between cells. Furthermore, Hershey and Chase, in 1952, hypothesize that DNA and not protein is the genetic material in bacteriophages and after experimenting, concluded this theory and found that DNA must be the molecule used to reprogram cells.
DNA, shorthanded for Deoxyribonucleic acid is a nucleic acid contains instructions for the development, functionality, and maintenance of new cells. DNA consists as chains of nucleotides, in two antiparallel strands in a double helix, connected by hydrogen bonds between complementary nitrogenous bases. Segments of DNA carrying genetic information are called genes, which mostly code for a specific type of protein. This lab will focus on the gene pGLO.
Thus transformation becomes specifically expressed as the intake and influence of new genetic material in the form of DNA. It involves a foreign gene being inserted into a cell, and causing changes in the organism’s traits. The trait changes are often caused by the new genetic material causing a change in protein construction and composition. The changes in proteins then influences the traits expressed by the particular proteins and influence the organism’s phenotype, or physical expression of traits.
This lab uses a gene called pGLO to transform fecal bacterium. The pGLO codes for a Green Fluorescent Protein (GFP), which is often observed naturally in jellyfish. The goal of the lab is to get the bacteria to intake and express the pGLO gene and produce the protein, which will fluoresces green under the presence of ultraviolet light. Furthermore we will require plasmid in order to aid in the transformation process. Plasmids are additional circular units of genetic Material contained in bacteria and codes for trait that assist in bacterial survival.
The pGLO genes stored in the bacteria plasmids in this experiment have been modified to resist ampicillin, an antibiotic, and to be “turned on” or express in the presence of arabinose, a sugar and cell nutrient. After bacterium growth on antibiotic plates, cells will appear in circular colonies, or as a strand of lawns, and if the transformation is a success they will fluoresces green under ultraviolet light.
Before undergoing the transformation lab, confirmation that the substance being added to the bacterium is DNA must be acquired. This is done through electrophoresis. This process creates a uniform electrical field that allows motion of particles of various sizes towards a positively charged end. A larger particles move slower in the charged viscous gel so we will compare water, a small molecule, to what we believe is a plasmid solution, macromolecule.
First, load the electrophoresis with algerose, Jell-O like solution that is liquid unless charged with an electrical current. Comb wells near the negative end of the electrophoresis and load samples of Plasmid Solution and DNA into the wells. Before loading 10 microliters of each, the substance must be mixed with a loading dye on Para film to identify it in the charged algerose gel. Also add a 1 KB ladder marker to be able to determine size of plasmid molecules.
As for the main experiment of transformation, start by looking at the same plasmid...