Discussion
The depletion of ozone in the earth’s atmosphere has led to
the concern of higher levels of UV light reaching the earth’s atmosphere. This concern arises because of the known
damage that UV light can incur in DNA in humans and other organisms. In the bacteria Bacillus subtilis, a specific small acid-soluble spore protein
(SASP) is produced by the SspC gene. The protein is released in spores created
by the bacteria. This protein has been
shown to protect the bacteria from death due to UV light. While B.
subtilis naturally produces this protein when it is undergoing sporulation,
E. coli does not undergo sporulation
or produce any SASP’s. If the gene
coding for the production of SASP’s in B.
subtilis is transformed into E. coli
via plasmid, the gene should continue to be expressed even in E. coli, a non-spore producing bacteria.
When exposed to UV light, SASP’s should continue to be produced and E. coli containing the SspC gene should
see a greater survival rate than those without it.
Varying experiments were performed on the many different
methods of DNA purification and PCR techniques.
PCR was performed successfully on E.
coli containing the λ virus to yield the correct band at 900 base pairs.
This is evidence that the methods chosen for PCR and gel electrophoresis could
yield appropriate results. DNA
purification was then attempted for B.
subtilis using a Qiagen purification kit, but the gel electrophoresis
yielded no DNA results. Once it was
evident that the DNA purification was not effective, the step was skipped and
bacteria was placed directly in the PCR cocktail. The annealing temperature was also lowered,
and DNA eventually appeared on the gel electrophoresis, although not at the
desired base pair range.
The results shown in figure 4 are evidence that by using the
methods chosen PCR can be accomplished successfully, and the desired length of
bands can be shown on a gel electrophoresis.
While another successful gel was not completed in the remainder of the
research project, it is evidence that PCR and gel electrophoresis were
completed successfully at least once.
The results obtained do not necessarily support the original
hypothesis, however there were many improvements made in the methods used as
the experiment progressed. In
experimentation with the DNA purification, it was determined that the Qiagen
kit in the labs was not practical for use on gram-positive bacteria. Once this was learned, it was evident that a
different method was necessary to lyse the gram-positive bacteria and obtain
the DNA inside. Unfortunately, this
determination was made too late to obtain a gram-positive DNA purification kit,
and no further improvements were made as far as DNA purification. Instead, the DNA step was skipped and the
bacteria were added directly to the PCR cocktail. Immediately, this returned similar results as
using the DNA purification kit. The
annealing temperature was lowered from 48°C to 35°C as a possible correction for
the lack of DNA evidence, and as a result, DNA was present in the gel electrophoresis. While there was DNA present, the band was not
present in the appropriate range of base pairs. Most likely, the annealing
temperature was lowered too low, so that the primers annealed to some DNA non
specifically and created blurry, non specific bands on the gel.
From previous work done on gram-positive bacteria, there are
many different ways to lyse them for DNA extraction. While they are more difficult to lyse then
gram-negative bacteria, it is not impossible.
Possible methods include lyophilization, microwave oven treatments, or a
gram-positive DNA extraction kit. While
the DNA extraction kit is the most obvious and probably the most effective, it
might have been useful to experiment with methods other than the heating used
in the PCR tests. In similar experiments
with B. subtilis, lyophilization was
used as a method (Setlow, 1991). While
this method would have been the most likely to work in our experiment since it
was used in previous similar experiments, it would have been too costly for the
use in the Biology-145 lab. It would have also been more time and cost
efficient to use a DNA extraction kit.
There was also previous research done on lysing using treatments in a
microwave oven (Bollet et al.,
1995). While this would have been a very
time and cost efficient method, there was still the danger that the DNA could
be damaged due to the microwaves.
Because this research heavily depends on the survival rate of the
beacteria and the preservation of the DNA, this method is not desirable. The DNA purification kit would have been the
safest and easiest way to ensure the proper extraction of DNA from the
gram-positive bacteria.
The main difficulty that arose in the research project was that the bacteria were gram-positive. This created obstacles for DNA extraction that could not be solved in the time allotted. However, even if DNA had been successfully extracted before the end of the semester it would have been difficult to accomplish the entire research project before the end of the semester. As the end of the semester neared, it was evident that the project originally designed would take much longer than 15 weeks of 4 hour labs each week. While there may have been other small human errors that contributed to the lack of DNA evidence, the main cause was the bacteria being gram-positive.
Future Directions:
In order to completely answer the question originally posed and prove the hypothesis, there is still much more research that needs to be done. The first step to take would be to isolate the B. subtilis SspC gene using PCR and the previously created primers. To remove the DNA from the gram-positive bacteria, a DNA purification kit would need to be purchased from an online supplier. The DNA would be purified and the SspC gene would be successfully isolated using PCR and proven using gel electrophoresis. This would be proven if a band appeared around 700 base pairs long. The resulting DNA pieces can be cut using a restriction enzyme and ligated into a plasmid. The restriction enzyme that would be needed depends on the plasmid that is cheapest and easiest to use. A restriction enzyme would be chosen based on ligation sites present on the plasmid and the enzymes that would work with the SspC gene. When the plasmid and the gene have been cut using the correct restriction enzyme, the gene would be ligated into the plasmid. The plasmid would then be transformed into the E. coli bacteria using a heat transformation protocol. To be sure that the SspC gene made it successfully into the E. coli, PCR and gel electrophoresis would be performed as well. The primers used in the B. subtilis would be used again. Once it was proven that the SspC gene made it into the E. coli successfully, the UV light treatments could begin. There would be 3 different liquid cultures produced. One would have the wild-type E. coli with no genetic alterations, one with E. coli with the SspC gene transformed inside on a plasmid, and one with the wild-type B. subtilis. There would be 4 cultures made for each type. Each tube would be exposed to UV light for a different amount of time: 0 seconds to be used as a constant, 1 second, 10 seconds, and 60 seconds. Each tube would be swiped on a plate and grown. Once the bacteria had enough time to create cultures, the cultures would be counted and compared. It would be expected that B. subtilis would have the highest survival rate, then E. coli with SspC transformed would have the next highest, and wild-type E. coli would have the lowest. The results of this experiment would allow final conclusions to be made for the original question and hypothesis.
