The purpose of this lab experiment was to see if a person could use a plasmid (R plasmid) vector containing certain fragments of foreign DNA for ampicillin resistance to be used to transform E. coli (Escherichia coli) cells to give the E. coli cells ampicillin resistance. Ampicillin resistance is needed in order for the E. coli to have the ability to survive in an ampicillin environment. Plasmid is a small ring of DNA that carries accessory genes separate from those of bacterial chromosome. Ampicillin is an antibiotic that is derived from penicillin that prevents bacterial growth by interfering with cell wall synthesis. E. coli is a commensal bacterium inhabiting the human colon that is widely used in biology, both as a simple model of cell biochemical function and as a host for molecular cloning experiments. In nature genes can be transferred between bacteria in different ways like transformation, conjugation, and transduction. One-way is bacterial transformation, it is when transfer of genetic information into a cell by the direct uptake of the DNA. Then the DNA is used to transform the cells from the original DNA to take a certain trait. However these bacteria can take up DNA only during the period at the end of logarithmic growth. Conjugation is performed between to bacterial cells of different mating types and genetic information is exchanged between the two through pili. Transduction is conducted between two bacteria cells, but this process requires the presence of a virus that acts as a vector in the process.
One could hypothesize that plasmid would be able to successfully be incorporated in the E. coli cell. One would be able to see this by both agar plates without ampicillin and also the agar plate with ampicillin and plasmid would have bacteria growth. However the agar plate with ampicillin, but no plasmid will have no bacteria growth. One could hypothesize this because if the plasmid were incorporated into the E. coli cell correctly the E. coli would have ampicillin resistance.
2 Luria agar plates with Ampicillin (control)
1) First get two microc-netrifuge tubes and mark one of the tubes + and then the other tube -. The tubes with the + is the one that the plasmid will be added to it. Then add 250ml of cold calcium chloride (control) to both tubes.
2) Get a starter plate and use a sterile inoculating loop to move a large colony of bacteria from the starter plated into the – tube of calcium chloride. Then put the transfer loop into the tubes and spin rapidly.
3) Then pass the transfer loop over the flame of the Bunsen burner to clean it and repeat procedure 2 for the + tube.
4) After that, use the capillary micropipets to add 10ml of the plasmid pUC8 solution to the + tube. Carefully and gently tap the pipet to mix the plasmid into the solution.
5) Put both tubes on ice for 15 min (control). Then went the tubes are in the ice get two Luria agar plates and two Luria agar plates with ampicillin. Mark one of the plates + and the other – and then do the same for the plates with ampicillin.In order for the plasmid to be able to enter the cell it must be heat shocked, so remove the tubes from ice and right away place them in to a 420C hot water bath for 60 to 90 seconds (control).
6) Next take the tubes out of the water bath and right away put them on ice for two minutes. (This part is critical to get right if not the plasmid would not of enter into the cells.)
7) After the two minutes, add 250ml Luria broth (control) to each tube with a disposable pipet (make sure the pipet is sterile). Tap the tubes to mix the solution. One can leave the tubes temp. At room temp.
8) Add 100ml Luria broth (control) of the solution in the + tube to the two + plates with a disposable pipet (make sure the pipet is sterile). Then add 100ml of the solution in the – tube to the two – plates with a new disposable pipet (make sure the pipet is sterile). So now one plate has ampicillin and plasmid (variable), another with ampicillin and no plasmid (control), the next one without ampicillin and with plasmid (variable), and the last one without ampicillin and no plasmid (control).
9) The get the bacteria spreader and sterilize it by passing it through a flame. Once the bacteria spreader has been sterilized let it completely cool down. Gently spread the cells over the entire surface of the plates using the bacteria spreader (Flame the bacteria spreader after putting the cells on the agar plates each time.)
10) Let the plates sit for five minutes and then place the plates in a 370C incubator, upside down, and overnight.
Some of this data might not be completely accurate due to some errors. One error could be due to the fact that the temperature of the water bath could have been too high and killed or denatured the plasmids. One could also not of put the + and – tubes in the water bath or ice for the right amount of time. Also that there could have been contamination between the different substance that was used during the experiment or when spreading the E. coli on the different agar plates the bacteria spreader could of not been sterilized properly and contaminated the rest of the agar plates. As well when spreading the E. coli one could have broken up the agar in the agar plates and it could have changed the results. In addition one could have rushed and did not put the right amount of plasmids or Calcium Chloride into the solution, which might of change the results. There could be many things that could be the cause for the incorrect information.
R plasmids and antibiotic resistance is found in the real life not just in laboratories or controlled experiments. For example if a type of penicillin that has a broader antibacterial spectrum is constantly used in a hospital to clean, a bacteria could eventually get a resistance to that antibiotic. E. coli is very adaptable to its environment. This is because it is able of producing large numbers of mutant cells, to make sure that there is a large chance that some cells capable of surviving new environmental stresses like an antibiotic that might arise. For E. coli gene the probability of mutation is 1 in 10 million per cell division. When some new stress is introduced into the environment there is a high chance that some of the mutants will be able to survive and in some times reproduce and make more of its kind in the new environment, letting the population to adapt to the new conditions. This is due to the short life span of the E. coli and its extremely fast rates of reproduction and multiplication. As a result there is bacteria that has a resistance to certain antibiotics, which could be lethal in a hospital. Many hospital epidemics have been due to the spread of an epidemic strain of bacteria under over use of a broad spectrum penicillin. Therefore over use of an antibiotic in the world is a dangerous.
The hypothesis that was made was not completely supported by the data that was collected in this lab. This was discovered because the agar plate that had ampicillin and the plasmid did not grow any bacteria. Which means that the plasmid was not correctly incorporated into the E. coli cells and did not have the ampicillin resistance. In view of the fact that the original prediction that the plasmid would be able to be incorporated into the E. coli cells the data did not support the hypothesis.
The expected results were that any of the agar plates without ampicillin would have bacteria growth and also any agar plates that have plasmid would also have bacteria growth. Yet the data that was found from the experiment showed that the procedure had an error in it because the plasmid did not give the E. coli ampicillin resistance. There were bacteria growth on two of the agar plates out of the three agar plates that were supposed to have bacterial growth. The two agar plates that did not have the ampicillin had bacteria growth. However the plate that had ampicillin and the plasmid did not have bacteria growth. Therefore it is logical to think that an error played a role in this experiment, it could be the procedure or some other things that could have gone wrong. However many errors are ruled out due to the fact that there was bacteria growth in two of the agar plates.
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