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, McLeod, 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 biographies 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 antiparticle 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 pogo. 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 rotten 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 pogo to transform fecal bacterium. The pogo codes for a Green Fluorescent Protein (GAP), which is often observed naturally in jellyfish.
The goal of the lab is to get the bacteria to intake and express the pogo 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 ramification process. Plasmids are additional circular units of genetic Material contained in bacteria and codes for trait that assist in bacterial survival. The pogo genes stored in the bacteria plasmids in this experiment have been modified to resist inclining, an antibiotic, and to be “turned on” or express in the presence of rabbinate, 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. [pick] Methods 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 allegros, 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 micrometers of each, the substance must be mixed with a loading dye on Para film to identify it in the charged allegros gel.
Also add a 1 KGB 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 DNA solution under ultraviolet light to see if it fluoresces. Then obtain a solution of fecal bacterium, and divide equally in two centrifuge tubes. Spin he two tubes in a centrifuge for 5 minutes on opposite side of the centrifuge. The bacterium will collect at the bottom of the tube, so pour out the extraneous supine. Then, add 250 micrometers of buffer.
The Ca+ cationic of the buffer neutralizes the repulsive negative charges of the phosphate backbone of the DNA and the phosphoric of the cell membrane allowing the DNA to pass through the cell wall and enter the cells. Place both tubes on ice. Then add 10 micrometers of water into one tube and 10 micrometers of plasmid DNA into another tube labeling the one with DNA with a + and the one with water -, and place on CE for 10 minutes. Next heat shocks the tubes for 50 seconds, followed by icing for 10 more minutes. The heat shock increases the permeability of the cell membrane to DNA. Then add 250 millimeters of LB and incubate for 20 minutes.
The 20 minute incubation following the addition of LB broth allows the cells to grow and express the inclining resistance protein, beta-lactose, so that the transformed cells survive the subsequent inclining selection plates. Plate 100 micrometers the + tubes evenly on two plates; 1 of LB and Amp, and one of LB, AMP, and AR. Plate 00 micrometers of the – tubes evenly on two plates; 1 of LB and AMP, and one on LB only. Results Calculations for number of molecules of Plasmid DNA Molecules For initial inflorescence of water and plasmid DNA they did not fluoresce under ultraviolet light.