Bacterial transformation is the process of bacteria taking in and expressing exogenous DNA. This has led to many other discoveries. In order for bacterial transformation to occur the bacteria must be in a certain physical state to be able to take in DNA. This is called competency and it allows the cell membrane to be permeable so DNA can pass through. Currently researchers are studying the transformation of E. Coli, which was used in this experiment. It is not normally in a state of competency so the researchers treat it with chloride salts and heat shocking to induce competency. EDVOTEK, 5-6)
The GFP gene is the green fluorescent protein gene. This gene codes for a protein that produces a green fluorescent light. This gene is being studied because it is often fused to proteins to study many biochemical processes. It is important because it is a biological tag and can be used to determine whether bacterial transformation occurred or not depending on if a fluorescent light was produced. The GFP protein can also demonstrate the effect of amino acid changes on the structure and function of a protein. (EDVOTEK, 5) The GFP/AMP plasmid DNA is injected into the E.
Coli and altered to become competent so the bacteria can be transformed. This plasmid contains nucleotides that if expressed would code for a GFP protein that would produce a fluorescent light and an AMP resistance protein that would kill any ampicillin the bacteria was exposed to. Researchers use the GFP/AMP plasmid for studies on bacterial transformation to understand how to accurately transform bacteria. (EDVOTEK, 5) The GFP gene is an inducible gene, meaning that the gene is usually not expressed until it is “turned on” by and inducer.
The inducer of the GFP gene is a small molecule called IPTG, which binds to the repressor inhibiting the expression of the GFP gene therefore inactivating it. This allows for RNA polymerase to transcribe the gene. When IPTG detaches from the repressor it stops transcription of the gene. This is important because it shows that it isn’t enough to transform the bacteria because the gene injected into it must be allowed to be expressed. (EDVOTEK, 7)
Methods: In order to set up for this experiment two microcentrifuge tubes had to be labeled; one with “+DNA” and the other with “-DNA”. . 5 ml of 0. 05 M CaCl? solution was added to the “-DNA” tube. E. Coli colonies were then scraped off the surface of the source plate using a sterile loop. This was inserted into the “-DNA” tube and then the cells were suspended. 250 µl was extracted from the “-DNA” tube and added to the “+DNA” tube and both tubes were placed in ice water. The “+DNA” tube was removed from the ice water and 10 µl of pGFP was added. The “+DNA” tube was returned to the ice water and both tubes remained there for 10 minutes. Immediately after, both tubes were placed in water at 42? for 90 seconds.
Both tubes are returned to the ice water for 2 minutes. Once removed 250 µl of recovery broth was added to both tubes. Then, both tubes were incubated in a waterbath at 37? for 30 minutes. Each of the four agar plates are labeled during this time. To plate the cells a 1 ml sterile pipet was used to transfer the DNA onto the plates. 0. 25 ml of the “-DNA” was added to each of the agar plates labeled with “(-DNA)”. The cells were spread around the plate using a sterile inoculating loop and the plates were covered so the liquid could be absorbed. These were the control plates because no pGFP was added to them. . 25 ml of the “+DNA” was added to each of the agar plates labeled with “(+DNA)”.
The cells were then spread and the plate was covered. All the plates were stacked and then taped together with our group labeled on the tape. Once the cells were completely absorbed by the agar the plates were inverted and left in a 37? bacterial incubation overnight (16-18 hours). After incubation the room is darkened and a UV light is held underneath the plates to visualize the transformed cells. This was done to show which plates contained transformed bacteria and which plates contained bacteria that as unable to transform.
Results: (Figure 1) (Figure 2) Example (-DNA)/(-AMP) (+DNA)/(+AMP) Expresses Expresses GFP regular bacteria gene and growth. contains roughly 1000 colonies. (Figure 3) (Figure 4) (+DNA)/(+AMP) (-DNA)/(+AMP) Doesn’t show signs Few bacterial of expressing the growth present GFP gene,which due to the could mean presence of experimental ampicillin and errors occurred, no resistance and contains only 1 gene to destroy colony. It. (Figure 5) (+DNA)/(-AMP) Regular bacterial growth, however the gene is not expressed.
Transformation Efficiency Calculations: •Equation: Number of transformants/ µg of DNA) X (final volume at recovery(ml)/volume plated(ml) = number of transformants per µg •Example (+DNA)/(+AMP) (Figure 1) (1000 transformants/ 0. 05 µg) X (0. 50 ml/0. 25 ml) 20,000 X 2= 40,000 (4 X 10^4) transformants per µg •(+DNA)/(+AMP) (Figure 3) (1 transformant/ 0. 05 µg) X (0. 50 ml/0. 25 ml) 20 X 2 = 40 (4 X 10? ) transformants per µg Discussion: In this experiment bacterial transformation was tested and only one of the four plates was expected to express signs of bacterial transformation. Plates (-DNA)/(-AMP) and (-DNA)/(+AMP) were the controls of this experiment.
This is because there was no DNA added to them so they would show the difference between bacterial transformation and no bacterial transformation. Plate (-DNA)/(-AMP) had regular bacterial growth, while plate (-DNA)/(+AMP) had no growth. Plates (+DNA)/(+AMP) and (+DNA)/(-AMP) were the plates being tested because they were the ones that were given the DNA. This means that they could potentially transform the bacteria depending on the whether the bacteria is competent or not. (EDVOTEK, 5) Plate (+DNA)/(+AMP) showed no signs of bacterial transformation because the GFP gene was not being expressed and few bacterial colonies grew.
Plate (+DNA)/(-AMP) showed normal bacterial growth with no signs of bacterial transformation. Both the DNA and the AMP had an effect on the growth of the bacteria. A plate with AMP most likely would not grow because the ampicillin would destroy all the bacteria. This would happen unless the DNA was added and expressed because it contains an ampicillin repressor gene along with the GFP gene. (EDVOTEK, 7) The plates without the AMP would grow normally because there is no substance there to attack or destroy the bacteria. The (-DNA)/(-AMP) plate and the (+DNA)/(-AMP) plate grew as expected.
The bacteria was expected to grow as normal bacteria would and that is what occurred on these two plates. The (-DNA)/(+AMP) plaste also grew as expected. No growth at all was expected to occur on this plate due the presence of ampicillin and no ampicillin resistance gene to kill it before the ampicillin destroys the bacteria. (EDVOTEK, 7) The plate had no growth in this experiment as expected. The (+DNA)/(+AMP) plate did not go as expected. This plate was suppose to be transformed because the cells were suspended in the DNA along with CaCl? to induce competency. Then they were heat shocked to further induce competency.
Finally, the presence of the AMP should have activated the transformed piece of DNA because it contains an ampicillin repressor gene as well as the GFP gene. (EDVOTEK, 5/7) The plate should have had many (the example contained 1,000) bacterial colonies and a fluorescent light should be emitted. However, the plate from this experiment had only 1 bacterial colonies and expressed no fluorescent light, which means errors occurred in the bacterial transformation process. Since the (+DNA)/(+AMP) plates were the only ones expected to have been transformed they were the only that were calculated.
The example (Figure 1) had a transformation efficiency of 40,000 transformants per µg. This is a relatively high transformation efficiency and is what is typically expected as a result for this particular experiment. However, the (+DNA)/(+AMP) plate from this experiment (Figure 3) as much lower than what was considered accurate. The transformation efficiency of this plate was 40 transformants per µg. Due to the huge gap between the expected transformation efficiency and the one that was shown from this experiment it can be assumed that many errors occurred throughout the experiment inhibiting the bacterial transformation process.
Based on the results i conclude that the experiment was carried out unsuccessfully because the only plate that was suppose to exhibit the bacterial transformation didn’t turn out as expected. This means that many errors occurred. One such error could have been how the heat shocking was not continuous because since there were other groups the microcentrifuge tubes were exposed to room temperature first rather than going straight from hot to cold.
This could have inhibited the bacteria’s ability to become competent, therefore inhibiting bacterial transformation. Also, another error could be that this experiment was carried out over multiple days in only roughly one hour intervals because the only time to conduct the experiment was during class. This could have resulting in many errors that inhibited bacterial transformation. This experiment might have been more successful if it wasn’t done only during class time along with multiple groups conducting the same experiment at the same time.