Abstract
The purpose of this study is to examine induced beneficial genetic mutations in Chlamydomonas reinhardtii’s carbon acclimation regulatory genes in a concentrated carbon dioxide environment by identifying the beneficial Pmp1 mutation of the LciB gene. The initial hypothesis was that over the period of the study, beneficial mutations would occur within the LciB gene amongst all other induced mutations, allowing C. reinhardtii to proliferate healthier in an elevated CO2 environment. Algae are a major producer of Earth’s oxygen, and maintain a static level of atmospheric carbon dioxide, which is why it is important that the survival of these creatures in an elevated carbon dioxide environment is evaluated and fully understood. This study is demonstrating the effects of increased atmospheric carbon dioxide on photosynthetic life and examining potential beneficial mutations that may occur within a photosynthetic genome. The EPA predicts the atmospheric carbon dioxide to steadily increase over the next fifty years which is predicted to affect photosynthetic organisms across the globe (Gerrard, 2008). The Algae colonies were examined over multiple generations in one, five, and ten percent volume to volume CO2 environments. The high concentrations of CO2 act as a selecting agent. UV radiation was applied as a mutation catalyst, utilizing UV illumination in addition to artificial growth illumination. Although UV induced mutations are expected to occur throughout the genome, mutations within the genes of the Carbon Concentrating Mechanism (CCM) could be extremely beneficial because they regulate the amount of inorganic carbon within a cell to support photosynthesis. CCM mutations are predicted to assist C. reinhardtii’s survival within an extreme carbon dioxide condition. This study examined the LciB gene, a gene which directly regulates carbon dioxide acclimation through the production of multiple transport systems (Wang and Spalding, 2006). The Pmp1 deletion which is a common mutation of the LciB gene was identified using PCR to detect beneficial mutations of C. reinhardtii because expression of this mutation successfully allows mutant strains of C. reinhardtii to thrive in very high CO2 concentrations (Wang and Spalding, 2006). This study will attempt to induce selection of CCM mutations throughout the cultures utilizing high concentrations of CO2. We predict the high CO2 environment coupled with ultraviolet radiation will select beneficial CCM carbon acclimation mutants which will be identified analyzing the LciB gene for the Pmp1 deletion because this mutation is one of the many mutations required for survival within an extreme CO2 environment allowing the mutant C. reinhardtii strains to competitively exclude the wild type strains (Fukuzawa et. al, 2001) (Moroney and Ynalvez, 2007) (Wang and Spalding, 2006). The experimental results of this study found no evidence to support these predictions; however a virtual model generated from Avida-ED led to the conclusion that in order for sustainable photosynthetic life to exist in an extreme CO2 enviroment, a very high mutation rate is required. The virtual model predicts an approximately 5% fixed mutation rate is most beneficial in an environment for which there is an evolutionary advantage to evolve such as elevated CO2.
..Significance of Experimentation
Since algae is a major component in the world’s photosynthesis and oxygen production it is important to determine that they are able to survive and thrive in the increasing trend of carbon dioxide concentrations in the environment. If the experiment was done correctly and showed positive results for beneficial mutations, it demonstrates the C. reinhardtii’s ability to evolve in an elevated carbon dioxide environment in short periods of time.
The examination of the selected induced genetic mutations of C. reinhardtii should allow us and future researchers to predict a time line of evolution and also predict future genetic trends in simple photosynthetic organisms in response to rising CO2 emissions. Several investigations have observed that over time, plants grown at elevated CO2 levels tend to show a decline in fitness. The reasoning behind this, however, still remains unknown but there are several proposals which strive to explain this phenomenon. One such example is the prediction that a decline of carboxylation efficiency may be caused by a decrease in the amount and activity of rubisco, which is the enzyme which initiates photosynthetic carbon reduction in photosynthesis (Bazzaz, 1990) (Bowes, 2008). We predict that the only way plants will be able to survive is if genetic mutations occurred. These genetic mutations would have to enhance a plant’s ability to sufficiently use the excess carbon dioxide. During experimentation, we attempted to identify a beneficial mutation (Pmp1), which is an evolutionary non UV induced mutation found in C. reinhardtii, which would allow us to determine if any beneficial CCM mutations have occurred with C. reinhardtii’s genome (Tural, 2005) (Wang and Spalding, 2006). The only way that the C. reinhardtii can survive in high carbon dioxide environments is by mutating genes which will allow the species to sustain itself in high carbon dioxide environments. Because the only chance of survival will be from genetic mutations to compensate for the reduced rubisco activity during plant acclimation, under evolutionary principals the C. reinhardtii will mutate and only the fittest ones which are capable of mutating to acclimate to the high carbon dioxide levels will survive (Bowes, 2008).
C. reinhardtii is a unicellular, photosynthetic algae very commonly used in photosynthetic studies. They are ideal for experimentation due to their short gestation periods, growth simplicity, fully sequenced genome, and their abundance on Earth (Tural, 2005). Since Algae are responsible for half the Earth’s photosynthesis there is certainly concern for how they will fare experiencing an increasing trend of carbon dioxide levels due to fossil fuel consumption (Moroney, 2007). To evaluate the effects of increased carbon dioxide, we examined the C. reinhardtii over several gestation periods in concentrated carbon dioxide environments of one, five, and ten percent, by volume. To accelerated the mutation rate, thus also accelerating the process of evolution, C. reinhardtii will also be exposed to low amounts UV radiation in order to catalyze the genetic mutations.
C. reinhardtii has a complex carbon concentrating mechanism (CCM) which is regulated by approximately 63 genes (Tural, 2005). The CCM allows photosynthesis to support cellular metabolism because of the CCM’s ability to concentrate carbon for fixation and the production of sugar. Rubisco, the enzyme that converts carbon dioxide to glucose, is only effective when there are high concentrations of inorganic carbon in the cell because Rubisco can utilize both oxygen and carbon dioxide which are competitive substrates (Moroney and Ynalvez, 2007). Not much is known about the CCM of simple photosynthetic life forms. The CCM is known to produce multiple carbon transport systems for carbon transport between nearly every membrane including the cell wall. If an abundance of carbon dioxide is present in the cell’s surroundings, then the cell will accumulate mate a proportional amount thus increasing photosynthetic output (Bowes, 2008). However, the pH will decrease due to the increase in carbon dioxide because C. reinhardtii accumulates carbon dioxide and carbonate which will decrease cytosol pH (Bowes, 2008).
Stress will also be induced on the active transport systems due to the cell’s large concentration gradient between periplasmic space and outer environment. We predicted that the cells will either evolve and compensate for the elevated concentration of carbon dioxide or die due to increased intracellular pH. We predict that the beneficial mutations will occur in the CCM genes of C. reinhardtii eventually due to magnitudes UV exposure (Fukuzawa et. al, 2001). The wild type C. reinhardtii will struggle to survive the new environment but will eventually be outcompeted, demonstrating an artificial selection process. In order to insure the mutations are not already present, we will be using a wild type primer. If mutations occur over time, then the DNA is tested from the cells with PCR, the DNA will not anneal itself to the wild-type primers. When annealing occurs, bands can be seen when DNA is tested through gel electrophoresis. When no bands appear, it can be concluded that the gene has mutated.
Previous experiments have found that the evolution of beneficial traits is predicted to be identifiable utilizing the Pmp1 mutation within the LciB gene, which has been predetermined to play a major role in carbon acclimation and the CCM, as a model beneficial mutation should be able to aide in the survival of C. reinhardtii in high concentrations of carbon dioxide (Moroney and Ynalvez, 2007). The selection of this gene throughout the cultures of C. reinhardtii will be traced using PCR to track the changes of CCM in the gene pool. The LciB gene is regulated by the CCM1 gene, a carbon concentration mechanism regulator gene (Fukuzawa et. al, 2001). LciB has been determined as a carbon dioxide uptake gene and plays a major role in carbon acclimation. Using UV light as a mutation catalyst, we expect mutations to occur within the LciB gene, we also predict to discover Pmp1 mutations within the cultures of C. reinhardtii which signifies that a beneficial mutation has occurred after a several weeks time since it has been predetermined that the Pmp1 mutation will be beneficial to plants in high carbon dioxide concentrations (Wang and Spalding, 2006).
In our experiment, three different cultures of C. reinhardtii were grown in three varying carbon dioxide levels: one percent, five percent, and ten percent; however, the cultures only grew in the one percent and ten percent carbon dioxide levels. When DNA from each was collected and ran though PCR, none of the DNA properly annealed. Bands were discovered through gel electrophoresis; however, the bands were determined to be approximately 300 base pairs when the proper bands should have been approximately 1030 bases (Figure 2, Figure 4, and Figure 5). It was concluded that the bands that appeared are primer dimers, which are undesirable PCR products that result when one primer binds to the other primer and are extended. Consequently, when primer dimers form, the primers can become unavailable for binding to the desired target sequence. However, in one attempt to extract the DNA from the C. reinhardtii cells, the extraction process did not work and no bands were created at all (Figure 3). It is concluded that the methods of DNA extraction did not properly work and no pure DNA was able to be extracted from the cell wall and call membrane due to primer dimers forming and in all samples. Overall, the experimental results from PCR, DNA extraction, and gel electrophoresis were inconclusive.