Transfer of Plasmid Puc18 Into Escherichia. Coli Strain
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The aim of the practical is to study the transfer of plasmid pUC18 into Escherichia. coli strain DH5α via the process of artificial transformation. Plasmid pUC18 is a 2.7kb genetically engineered plasmid, which consists of an ampicillin resistance gene (ampR) and a gene for the enzyme beta-galactosidase (lac Z). Artificial transformation is the process by which competent bacterial cells take up extracellular DNA with the help of special techniques. The growth of these E. coli colonies is then monitored by using 2 different indicator plates. Successfully transformed colonies, E. coli DH5α (pUC18), can survive in media containing ampicillin. On the other hand, E. coli DH5α will not survive in media containing ampicillin as it is sensitive to ampicillin.
In this practical, the major techniques for transformation are applied, which includes extracting the plasmid DNA from E. coli (pUC18) culture, transformation of the plasmid into competent E. coli cells and finally, the screening for successfully transformed cells.
Materials and Methods
- High-Speed Plasmid Mini Kit: Buffer PD1, Buffer PD2, Buffer PD3, W1 Buffer, Wash Buffer, Elution Buffer
- E. coli (pUC18) culture
- Competent E. coli cells
- Double distilled sterile water
- LB broth
- Indicator plates: Luria-Bertani, Luria-Bertani + ampicillin
Plasmid pUC18 was extracted and purified from E. coli (pUC18) culture by using the High-Speed Plasmid Mini Kit. Transformation is subsequently carried out using the chemical method: competent cells are left on ice then subjected to a heat-shock. The transformed cells are then further diluted and plated to the different indicator plates. By comparing the results, proliferation of E. coli cells could be studied.
The experiment was modified at the transformation level. The following changes were made to the protocol: 1) Leaving the mixture of competent cells and plasmid on ice for 15 minutes and 2) subjecting it to heat shock at 42°C for 5 minutes. (The standard protocol that the control experiment followed was to leave the mixture on ice for 30 minutes and to heat shock it for 90 seconds.)
Based on the number of colonies observed, the plates were ranked accordingly. The plate with the greatest number of colonies is given rank 1, while the plate with the least number is given rank 6. The control LB plate (Plate 1) has the most number of colonies as the E. coli cells were able to thrive and multiply in the absence of ampicillin, forming a lawn of colonies. In contrast, the control LBA plate (Plate 2) had no colonies. This is because E. coli DH5α (without addition of plasmid pUC18) is ampicillin sensitive and thus, will not survive on media containing the antibiotic. As for Plates 3 - 6, growth of bacterial colonies could be observed as they have taken in plasmid pUC18 and is hence ampicillin resistant. It is also found that the number of colonies is directly proportional to the amount of DNA added; the higher the amount of plasmid DNA added, the greater the number of colonies observed.
By comparing the results in Table 1, both experiments show the same trend in terms of ranking. For Plates 3 - 6, Plate 5 has the most number of colonies as 15 μl (neat) of plasmid was added. This is followed by Plate 3, with 5 μl (neat) of plasmid. Next, Plate 6 has 15 μl of plasmid added, but since it was diluted 10 times, the effective amount of plasmid added was 1.5 μl. Lastly, 5 μl of plasmid was added to Plate 4 and diluted by 10 times, which means the effective amount of plasmid was 0.5 μl, which also explains the ranking of the plates.
However, by comparing the transformation efficiency for both experiments, the values obtained from the control experiment was significantly greater than the manipulated experiment. This difference in the results shall be further discussed below.
In this practical, the gene of interest is the gene coding for β-lactamase, which can be found in plasmid pUC18. The enzyme, β-lactamase, can hydrolyse the beta-lactam ring of the antibiotic, thus inactivating it. This confers the ampicillin resistant characteristic in bacteria . This is shown in the results as only E. coli cells carrying the gene are able to grow and form colonies on plates containing ampicillin. For example, Plate 2 is the only LBA plate without any colonies formed. No DNA plasmid was added to the competent cells; hence they are ampicillin sensitive and cannot survive in the presence of ampicillin.
Plasmid pUC18 was first extracted and purified from E. coli (pUC18) culture using the High-Speed Plasmid Mini Kit. Various buffers were used for the extraction and their functions shall be explained briefly: 1) Buffer PD1 was used to digest RNA and inactivate nucleases that are protecting plasmid DNA; 2) Buffer PD2 was used to lyse the cells and denature most chromosomal DNAs and proteins using strong alkaline; 3) Buffer PD3 helps to bring down the pH rapidly so that the small plasmid DNA can re-anneal quickly and also to denature proteins; 4) W1 Buffer was used to denature any protein left; 5) Wash Buffer was used to remove salt to ease the elution step and 6) Elution Buffer was used to elute the purified Plasmid pUC18. The Geneaid Spin column was used to selectively adsorb the plasmid DNA, using the glass fiber matrix present. It ensures that RNA, cellular proteins and other metabolites are not retained, and a purified sample of plasmid DNA could be obtained.
The next part of the practical involves the transformation of the E. coli cells. In artificial transformation, E. coli cells are first made competent so that the plasmid DNA could be taken up from the external environment. Transformation efficiency is the number of cells transformed per microgram of DNA. By comparing the values obtained from the modified experiment and the control experiment, the transformation efficiency for the control experiment was much higher than that of the modified experiment. This could be due to one of the changes made in the modified experiment.
During bacterial transformation, the tubes were placed on ice for only 15 minutes, instead of 30 minutes. This is expected to result in a three-fold loss in transformation efficiency . And by comparing the results, there was an average of a four-fold loss in transformation efficiency. Hence, this change was not the sole factor for the loss in results.
The second change that was made was to increase the duration of heat shock at 42°C from 90 seconds to 5 minutes (300 seconds). The expected results was that it could a fall in the value for transformation efficiency as the prolonged duration may have caused heat-degradation to occur. Thus, this could also justify why the loss in transformation efficiency was more than three-fold.
In addition, another point that could have affected the results of the manipulated experiment was that the tubes were not inverted at 10 minute intervals (of the incubation step) to mix. Mixing the plasmid DNA with the E. coli should result in higher transformation efficiency.
A negative control was used in the practical to ascertain that E. coli DH5α used does not carry plasmid pUC18 in the first place. It was proven true as no colonies were found on the LBA plates. In contrast, in the absence of ampicillin (LB plate), the E. coli cells were able to grow rapidly, as seen in Plate 1. Double distilled sterile water was added to this negative control, as a replacement for the plasmid DNA, to eliminate the possibility of contamination.
However, the results could have been affected by limitations or uncertainties. One such uncertainty is the chance that two Colony-Forming Units are growing so close to each other that they combine and form one colony instead of two. Hence, this would lower the transformation efficiency as lesser colonies were counted.
Furthermore, this experiment was using the chemical method for bacterial transformation. Another alternative would be using the physical method, which is creating the pores by electroporation. It has a much higher efficiency as compared to the chemical method, and it could also introduce larger DNA fragments (e.g. chromosomal DNA) into the bacteria.
Nonetheless, there are also tips to enhance the transformation efficiencies using the chemical method. As seen in the results, incubation of plasmid DNA with cells on ice should be for 30 minutes instead of shortening the time to 15 minutes. The duration of heat shock also matters as the long exposure to heat might degrade the DNA. Also, the duration for incubation, (at which the E. coli cells are incubated at 37°C), could be increased to 1 hour instead of just 20 minutes. Alternatively, SOB or SOC broth could also be used to replace LB broth as they contain more nutrients and could eventually lead to higher transformation efficiency.
In conclusion, both changes that were made in the modified experiment have contributed to the drop in transformation efficiency. The extent as to which change have affected the results more could be further studied by conducting a separate experiment for each change.
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