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 DNA solution under ultraviolent light to see if it fluoresces.
Then obtain a solution of fecal bacterium, and divide equally in two centrifuge tubes. Spin the 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 supinate. Then, add 250 microliters of buffer. The Ca2+ cation of the buffer neutralizes the repulsive negative charges of the phosphate backbone of the DNA and the phospholipids 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 microliters of water into one tube and 10 microliters of plasmid DNA into another tube labeling the one with DNA with a + and the one with water -, and place on ice 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 milliliters 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 ampicillin resistance protein, beta-lactamase, so that the transformed cells survive the subsequent ampicillin selection plates.
Plate 100 microliters the + tubes evenly on two plates; 1 of LB and Amp, and one of LB, AMP, and ARA. Plate 100 microliters 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 ultraviolent light. For the electrophoresis we see that plasmid DNA loaded in lane 3 migrated a lesser distance than the water loaded in lane 2, showing the plasmid DNA solution contained larger molecules. See picture below. LB/AMP/ARA (+) |LB/AMP (+) LB/AMP (-) |LB (-) | |Type of Reaction |COLONY |COLONY |NO REACTION |LAWN | |Drawing of reaction Colony numbers |35 |23 |0 |Lawn |
The plate of bacteria and water grown in LB (LB-) showed a lawn growth but didn’t fluoresce, while the plate of bacteria and water grown in LB and AMP (LB/AMP-) didn’t have any growth. The LB/AMP- once again didn’t show growth while the plate of bacteria and pGLO grown in LB and AMP (LB/AMP+) did show growth but didn’t fluoresce. And finally the plate with bacteria and pGLO grown in LB, AMP, and ARA (LB/AMP/ARA+) also had growth but it was considerably more than the growth and fluoresced in comparison with the LB/AMP+ plate. Calculations of Transformation efficiency Transformation efficiency = Total number of cells growing on the agar plate/Amount of DNA spread on the agar plate. For LB/AMP/ARA+ = 35/. 06 µg= 583. 33 transformants µg For LB/AMP+= 23/. 06 µg= 383. 33 transformants µg
Our results show that pGLO plasmid DNA can transform fecal bacterium under the addition of LB, AMP, and ARA. The pGLO codes for a Green Fluorescent Protein (GFP) which made this particular plate fluoresce under ultraviolet light. The pGLO genes stored in LB/AMP/ARA+ bacteria plasmids in this experiment have been modified resist ampicillin, by incorporating a B-lactamase gene, and was “turned on” or expressed in the presence of arabinose. In comparing LB- and LB/AMP- a lawn grew on the LB- because there wasn’t any antibacterial AMP to kill the bacteria. In comparing the LB/AMP- and LB/AMP+, the bacteria grew into colonies on the LB/AMP+ because the pGLO genes that transformed the bacteria.
The pGLO causes the expression of B-lactamase gene which gives the bacteria AMP resistance the bacteria isn’t killed. In comparing the LB/AMP+ and LB/AMP/ARA+ both had colonies but only the LB/AMP/ARA+ fluoresced because it contain arabinose (ARA), which turns on the expression of the GFP genes by binding to a regulatory protein, araC. When arabinose is present in the environment of the bacteria, it binds to araC and cause transcription and creation of the Green Fluoresce Protein by ribosomal units and tRNA. Ultraviolent light exposure is absorbed by the protein and slowly phosflouresces, emits absorbed light slowly while transforming some of it.
Part of the transformed phosflouresces is in the form of the green light we witness. Finally, transformation has man practical uses mostly in the area of biotechnology. Scientist can use the genes coding for a particular resistance, such as B-lactamase, or an expression, such as arabinose, to inject in plants or animals in order to modify or introduce new traits. Another use of transformation is to create bacteria that can make insulin. BP also used bacteria, transformed to consume oil, in cleaning up the gulf oil spill. And certain genes, such as sickle cell anemia, are being eradicated in receptive humans by the use of gene therapy. This involves transforming the genes of a sick individual with healthy genes from a donor.