Bacterial Transformation
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Bacterial Transformation
Introduction
Biotechnology has to do with the manipulation of organisms to get useful products. One of the basis of biotechnology is genetic transformation. Genetic transformation occurs when DNA is taken in and expressed by a cell from a living organism. Three basic thing are needed to perform genetic transformation: a host, a vector, and a method to select and isolate the transformed organisms. The host is the organism that will take in the DNA. The vector is the means of transporting the DNA into the host. A common method for selecting the transformed organisms is to introduce them to an environment containing what they are resistant to (once they transform) because only the transformed organisms will be able to survive in that environment.
In this experiment, we will perform genetic transformation of the bacteria Escherichia coli (E. coli), therefore it is properly defined as bacterial transformation. E. coli will be our host. It is a good host for transformation because it contains one chromosome and it divides and grows very quickly. The vector in this case will be a plasmid, the simplest bacterial vector. Plasmids are circular DNA that contain important information for the growth of bacteria, in this case genetic information vital for their survival. In this experiment, the plasmid will enter the bacteria cell and insert the gene of interest: resistance to the antibiotic ampicillin. Therefore, the purpose of the experiment is to verify that only transformed bacteria (containing plasmid DNA) will be able to survive and grow in a medium containing the antibiotic ampicillin. To fasten the uptake of plasmid by the bacterial cells, the procedure of heat shock will be performed. The cells will be transferred suddenly from ice to a water bath at a temperature of 42oC. This sudden change in temperature will cause the pores in the cell wall of the bacteria to open, so that the plasmid DNA can enter the cell. Then, the bacteria will be transferred to the ice again so that the pores can be closed and the new DNA synthesized.
Ampicillin is an antibiotic fatal to many bacteria. It affects Gram-positive (largest group of bacteria; blue or violet under a microscope) and some Gram-negative bacteria (smaller group; red appearance) by penetrating their cell walls and causing cell lysis. As studied in previous sections, cell lysis means that the bacteria cells will burst because the ampicillin concentration will be too high inside the cell. This will induce water uptake by the cell, which will end up dying. E. coli belongs to the Gram-negative bacteria group and it is intolerant to ampicillin. E. coli is a disease causing bacteria. (common meningitis, pneumonia)
Four plates will be used in total for this experiment. The “LB/Amp +”, the experimental plate, will contain Luria broth (nutrients), ampicillin, DNA plasmid, E. coli. Hypothetically, because the bacteria cells should be transformed by the vector (plasmid), there should be a bacteria growth in this plate. In the presence of ampicillin and nutrients, the ampicillin resistant E. coli should be able to survive. “LB/Amp -” will be the negative control for transformation. It contains LB, ampicillin, E. coli, but no plasmid. In the absence of a vector, there should be no growth because the cells are not transformed, and will not tolerate the antibiotic. “LB +”: contains LB and E. coli with plasmid, and therefore ampicillin resistant. In this plate, there should be a big growth because the transformed bacteria are in a nutrient rich medium. Even if some of the cells are not transformed, they would still be viable because there is no ampicillin in the plate. The “LB-” plate contains luria broth and the non-transformed bacteria. There should be regular growth in this plate because normal E. coli cells without ampicillin are able to survive. All these hypothetical assumptions will be tested by the experiment.
Materials
For this experiment, the following materials were used:
– Two eppendorf tubes
– Transfer pipettes
– A container with ice
– Ice-cold calcium chloride
– Escherichia coli (E. coli) sample
– Plasmid DNA sample
– Plastic loop
– 4 starter plates (Petri dishes)
– A microcentrifuge
– Luria broth (LB)
– Ethanol
– A cell spreader
– A Bunsen burner
– Parafilm
Method
1. Two eppendorf tubes were marked “+ DNA” and “- DNA”, respectively.
2. 250 uL of cold calcium chloride was added to each tube using a transfer pipet. Then the tubes were placed on ice for about 2 minutes.
3. A small cell mass of E. coli colonies was transferred to the “+ DNA” tube using a plastic loop. Then the tube was tapped until the cell mass disintegrated. After that, the cells were suspended by pipetting the solution in and out with the transfer pipette. Then the tube was returned to ice.
4. The same procedure (as in step 3) was performed for the “- DNA” plate. The tube was returned to ice with the “+ DNA” plate.
5. 10 uL of plasmid was added to the “+ DNA” tube. The contents were mixed by tapping the tube gently and returned to ice. Then, both tubes were incubated for 30 minutes.
6. During the incubation time, the plates were labeled with the group date, section and date. The plates were labeled “LB/Amp +”, “LB/Amp -” ( Luria broth and ampicillin in both), “LB +” (one half of the class) and “LB -” (the rest of the class. Assigned to our group)
7. After the incubation time was up, a heat shock was performed to the E. coli cells. The tubes were taken out of the ice and placed in a 42oC water bath for about 90 seconds, where they were agitated every 10 seconds. Then, the tubes were placed in the ice container for 2 more minutes.
8. The test tubes were centrifuged for 1 Ð minutes at 6000 rpm. The liquid in the tubes, the supernatant, was poured out without moving the cell pellet at the bottom.
9. Then 250 uL of Luria broth (LB) was added to each tube. The cells were re-suspended