Confirmation of Bacterial Transformation and DNA as the Inducing Material
Biol2000Lab5: Randall Barley
April 15 2014 (late)
Confirmation of Transformation and DNA as the Inducing Material
The experiments in this report are a recreation the famous Griffith and Avery experiments which discovered the transformation process that bacteria can undertake and that DNA is the genetic material, respectively (Griffth, 1928. Avery 1944). Bacteria can incorporate foreign DNA found in their medium into their genomes in a process known as transformation. This process can be accelerated in CaCl solution at colder temperatures. The first part of this report deals with confirming that transformation does occur following a standard procedure, and the second of the report uses a similar procedure to confirm that DNA is the particle that induces transformation.
In the following procedures Escherichia coli was used:
(Part 1) For the first set of experiments a solution of half live ampicillin sensitive strain and half of a heat killed ampicillin resistant strain was mixed and incubated at 37°C for forty five minutes. The resulting fluid was spread onto a prewarmed LB and a prewarmed LB Amp100 medium, respectively. In addition six controls were set up which shared a LB and a LB Amp100 medium. These controls were given their own area on the media and streak plated. The controls consisted of the live ampicillin sensitive strain, the dead ampicillin resistant strain and the live ampicillin resistant strain plated onto each medium. Aseptic technique was used in treating all solutions, media and transfers between.
(Part 2) In the second set of experiments 1.0µL of kanamycin sensitive bacteria was added to two test tubes, both subjected to the same treatment. The bacteria in the tubes were then pelleted by centrifugation at 3000rpm for seven minutes. Most of the supernatant was discarded and the pellet suspended in 250µL of prechilled CaCl2 solution. Next the bacteria were repelleted by centrifugation at 2000rpm for two minutes after which the supernatant was discarded and the pellets resuspended in 100µL of chilled CaCl2 solution. The 100µL of now competent in each tube were combined, and then 50µL of the 200µL was placed into three separate tubes.
Sets of controls and treatments of various hydrolysing enzymes were prepared according to Table 3. The experimental treatments had 1µL of cell extract added, namely blenderized heat-killed ampicillin resistant cells, 1µL of an enzyme added and 3µL of buffer. The first controls contained 1µL of cell extract and 4µL of buffer. The second controls had 5µL of buffer prepared. These mixtures were incubated for fifteen minutes at 37°C, then incubated at 65°C for ten minutes. 0.5µL of 25mM EDTA was added to the DNAse and RNAse treatments prior to the ten minute incubation.
The 5µL mixtures were mixed with their own 50µL of competent cells then incubated on ice for ten minutes. Next...