Embedded Files
Yeast Sunburns
Yeast Sunburns
Inducing mutations in Saccharomyces cerevisiae adenine biosynthesis pathway with UV radiation, creating adenine auxotrophic yeast, to then run complementation analysis in order to identify the specific gene in which the mutation occurred.
Video Abstract:
Video Abstract:
ABSTRACTVIDEO.mov
Written Description:
Written Description:
Why did you do it:
Why did you do it:
We were enticed by the protocol of the lab itself. While we were not testing a specified bivariate relationship within the realm of genetic mutations in yeast, we accomplished our objective of creating, identifying, and cultivating specific mutations. The lab tested not only our ability to induce mutations in yeast, select and screen for certain types of yeast phenotypes but also our ability to adapt: utilizing the scientific method, trial and error, and an excess amount of time in the lab spreading close to a hundred plates of yeast. Our reasoning for embarking on such a lab is as simple as enjoying the scientific lab process as aspiring biologists and chemists. More specifically, the lab also enabled us to investigate genetics. Genetics has and will continue to be the underlying basis of most if not all biological experiments. As a result, given the chance to practice, we were able to further our understanding of genetics with a specific focus on mutations and their effect on certain biosynthesis pathways.
What did you do:
What did you do:
The first segment of our experiment was an extensive hunt for yeast colonies that had developed a mutation that causes them to present a red or pink pigmentation. Starting with HA0, or wild-type yeast, which always grew into uniform white colonies, we used UV light as a tool for causing mutagenesis. Our preliminary testing established the optimal amount of exposure to balance survival with maximum mutations. We exposed dozens of agar plates containing individual HA0 cells to optimal amounts of UV light. We allowed them to grow with their new mutations for 2 days, and then observed carefully under a microscope, searching for the tiny red colonies among an agar plate environment that had become much more diverse.
Although it took many trials, eventually we discovered 11 candidate colonies which demonstrated the red/pink phenotype. We transferred these pigmented colonies to a liquid culture, where the population was vastly expanded, and subsequently re-plated onto agar plates. These new plates contained a much higher proportion of red/pink colonies which we could use in our complementation test.
The complementation test we did allowed us to pinpoint, which mutation in the yeast cell had occurred. Each candidate colony was mated with 3 different testing colonies of the opposite mating type and was allowed to grow as diploid colonies for 2 days. Depending on which, if any, of the mated colonies retained their red color, we could determine the location of the mutation.
What did you learn:
What did you learn:
In general, we learned that lab procedures don’t necessarily go as planned, and that’s the way it should be. The point of the lab, at least from our perspective, was to set out to induce and isolate a specific mutation in yeast. While we knew that this mutation occurred in about 1 out 35000 yeast colonies after UV radiation, the first set of plates looked bleak in terms of achieving our goal. We learned how to refigure our protocol to best achieve the intended mutation based on our preliminary results.
Specifically, we learned how to induce mutations in yeast; how to choose specific radiation levels, how to choose specific dilutions based off of preliminary dilutions; how to organize “mass” amounts of replicates through plate, liquid, and plate stages; how to screen and pick colonies; and how to run a complementation test in order to determine what specific mutations occurred.
Why is this important:
Why is this important:
Upon first reading over the initial protocol, we joked that we were reverse sunburning yeast. While we essentially reversed the protocol, the effect was the same: we were irradiating the cells of living organisms in order to change their pigmentation. Though the mechanism of actual sunburning diverges from the type of “sunburning” we worked with, the mutations occurring under UV radiation are essentially the same. UV radiation, at high enough levels and or exposure, causes deletions and base-pair mutations within cells. These mutations can lead to the development of cancerous cells on skin which is why sunscreen absorbs and protects our skin from UV rays. Our yeast weren’t as lucky, but the relevance between our “sunburning” and sunburning remains.
What does this mean:
What does this mean:
Scientifically, this project represents an introduction to many other investigations of the mutagenesis of yeast cells. We were able to create and isolate two mutations that interested us, but we do not have substantive data to determine the likelihood of these specific mutations. Are they related in terms of their base-pair length in the yeast genome? Further investigations could answer questions regarding the reduced growth rate of mutant colonies or explore the possibility of developing yeast which are resistant to mutagenesis by UV radiation.
Personally, this project was significant because it represented an opportunity where we were responsible for the uncertainties of the experiment and were in control of the experiment’s timeline. Mr. Edgar provided us only a platform, with the stock cultures and lab equipment, and his occasional advice. Each time we showed up to the lab, during free periods or in the evening, it was because we felt compelled to always be pushing our experiment forward.
Plate Picture Slideshow (with music!)
Plate Picture Slideshow (with music!)
PlateMontage.movFirst Stage: Mutating Yeast
First Stage: Mutating Yeast
Background and Goal
Background and Goal
Saccharomyces cerevisiae, commonly known as brewer's yeast, is able to synthesize adenosine monophosphate (AMP), a molecule which contains adenine, one of the four constituent bases on nucleic acids (DNA and RNA). This biosynthesis pathway begins with a sugar-phosphate compound called phosphoribosylpyrophosphate (PRPP). With 12 enzymatic steps (see figure), the yeast makes AMP.
We used ultraviolet radiation to induce mutations in, or mutagenize, the HAO strain of S. cerevisiae. UV radiation causes mutations through several mechanisms like dimerizing and oxidizing (both of which prevent DNA replication) The random mutations which are caused by the UV radiation may be in any part of the genome, but specifically we are interested in causing mutations in genes which encode the enzymes of the adenine biosynthesis pathway.
Mutations that disrupt the pathway by making the cell unable to produce enzymes will cause a buildup of an intermediate product. In particular, disruptions in the genes of two enzymes, named Ade1 and Ade2, will cause a buildup of either phosphorribosylaminoimidazole (AIR) or phosphorribosylaminoimidazole carboxylate (CAIR). These intermediate product are adjacent in the synthesis pathway, but more importantly, when oxidized they become a red pigment. Therefore, yeast colonies with a mutation in either the Ade1 or Ade2 gene will be red when grown aerobically in media with minimal amounts of adenine. We search for these two specific adenine-auxotrophic mutations within our mutagenized yeast using this red phenotype.
Preparing Solid Media
Preparing Solid Media
First Batch of Liquid Media
First Batch of Liquid Media
Preliminary Testing Plates
Preliminary Testing Plates
Plating Yeast onto Agar Plates
Plating Yeast onto Agar Plates
Spreading Yeast on Plate
Spreading Yeast on Plate
Preliminary Testing
Preliminary Testing
Preliminary testing, the first 15 plates of 4 equal groups of UV radiation, failed to produce any of the visible mutations in the intended Adenine biosynthesis pathway. Because of the multiple stages of UV radiation trialled in preliminary testing, the best levels of radiation for the factors of individual yeast colony growth, concentration of yeast throughout the plate, and the relative presence of mutants within the plates. Preliminary testing informed how we should proceed in order to maximize our chances of getting red mutants.
Main Procedure:
Main Procedure:
Inoculating HAO colony (Wild-Type Yeast) from Origin Plate
Inoculating HAO colony (Wild-Type Yeast) from Origin Plate
At the beginning of the experiment, a single HAO (wild type) yeast colony was taken from the origin plate, and grown for two days in a shaking culture tube containing 3 mL of YEPAD media, a very rich media containing excess amounts of all necessary nutrients.
Diluting Liquid Yeast Culture
Diluting Liquid Yeast Culture
Then, a very small sample with cells in suspension (5 uL) was taken from the liquid culture, and diluted in 10 mL of YEPAD media, for a 2000-fold dilution. These were stored in a refrigerator at 4 C, until ready for immediate use.
Mutagenizing Yeast from Diluted Culture Tube on YED Plates
Mutagenizing Yeast from Diluted Culture Tube on YED Plates
Next, yeast cells were transferred to solid media (agar) plates, to prepare them to be mutagenized. These plates contained YED media, a slightly less rich mixture, which importantly does not contain the excess amounts of adenine that YEPAD does. On these places, colonies with ade1 or ade2 mutants will show their red phenotype (if grown on YEPAD, the cells ignore the adenine synthesis pathway, and mutants will be indistinguishable), but will grow at a slower rate. For each plate, 100 uL of the diluted yeast liquid culture was transferred, and spread evenly with a sterilized plate spreader.
Identifying and Growing Red-Phenotypes Mutants in YEPAD
Identifying and Growing Red-Phenotypes Mutants in YEPAD
All mutagenized yeast plates were incubated for 2 days before being examined under a microscope for red-pigmented colonies. Those were marked with a pen, and if necessary, allowed to continue expanding until they reached a sufficient size to transfer. Each red-pigmented colony was transferred to a liquid YEPAD culture with the precise end of the inoculation loop, and grown for 2 days on an orbital shaker.
Plating Red Mutants from Diluted YEPAD Yeast Culture Tube
Plating Red Mutants from Diluted YEPAD Yeast Culture Tube
Finally, these liquid cultures were diluted to achieve a proper cell concentration, and like before, 100 uL of diluted cultures were grown on YED plates for 2 days. These individual colonies, of which a larger portion presented the red phenotype, would be used in the complementation test.
Results
Results
The second and third "rotation" of YED plates radiated at half 6000 mJ and half 12000 mJ produced 11 individual colonies of visible red and pink. Though the chance of these types of mutants were limited (1/10000 - 1/15000), the procedure used produced several viable colonies that we would then use to multiply and generate red phenotype mutants which would then be able to be run in a complementation test with known Ade-1 and Ade-2 mutants. Although the colonies themselves were varied with some having less pigmentation, lesser size, and undesirable locations for plucking, the colonies would be able to be multiplies in the next leg of the experiment.
Through background research, we expect these red mutants to be a product of mutations in the Ade-1 or Ade-2 genes which rendered them non-functional, and, thus, this synthesis pathway produced build-up, pigmented red, at the enzymes these genes code for. Although, this experiment has been run beforehand with these mutants indeed being a result of the Adenine synthesis pathway, this experiment works entirely in the realm of random mutations, so red pigmentation could be the result of other random mutations which cause build-up or even production of red-pigmented substance.
All red phenotype yeast colonies were noticeably smaller than the neighboring white colonies (see figure 1). The mutation induced not only affects the color of the yeast but also the growth of the yeast. Without the ability to efficiently produce adenine, an essential nucleobase, the yeast will not be able to grow and duplicate, especially in a plate with limited Adenine. The red pigment is evidence of the lack of adenine in the plate as the red mutant attempts to produce adenine, which is broken. Other white mutants also didn't grow as much as the average white colony (see figure 2). Some of these colonies may have also been mutated within the adenine synthesis pathway but not a stage where the buildup of red-pigment would happen. Others may, as Mr. Edgar suggests, result from mitochondrial mutations which commonly cause little growth in yeast.
Fun fact: we estimate that we screened between 45,000 and 50,000 individual yeast colonies for Ade1 and Ade2 mutations after exposing them to UV radiation. That isn't even counting the cells that suffered fatal mutations!
Fun fact: we estimate that we screened between 45,000 and 50,000 individual yeast colonies for Ade1 and Ade2 mutations after exposing them to UV radiation. That isn't even counting the cells that suffered fatal mutations!
Second Stage: Complementation
Second Stage: Complementation
Background and Goal
Background and Goal
After successfully finding several red colonies on our mutagenized HA0 yeast cultures, we sought to confirm their status as adenine auxotrophs, and furthermore, determine whether the mutation occurred in the Ade1 gene, Ade2 gene, or both. A complementation test allows us to locate each mutation. It involves mating each of our red candidate colonies with several "tester" strains of the opposite (B) mating type. This is done in a matrix grid on a YED plate, by placing a small dot of cells from both strains next to each other, and physically mixing them such that the different cells can be in proximity to each other. The cells, because they are of opposite mating types, will combine their DNA to form a diploid yeast cell. It allows us to locate the mutation because the resulting diploid colony will only be red (from the interruption/buildup within the adenine synthesis pathway) if both parents contained the same mutation. If only one parent has the mutation, it will still be able to access the functioning DNA from the other parent, and continue the adenine synthesis. Additionally, we wanted to confirm the adenine-auxotroph status of our candidate yeast colonies, by testing their viability on MV media, which contains the minimum level of nutrients required for yeast growth.
Procedure
Procedure
Complementation Test: Mating Mutagenized Plates with HB0/1/2 Mutants
Complementation Test: Mating Mutagenized Plates with HB0/1/2 Mutants
For our complementation test, the mutagenized plates that we had created were mated in a grid on a YED agar plate. Once this plate had been incubated, we would be able to identify and match the mutations that we had induced with known Ade1 and Ade2 mutants, as well as with wild-type yeast for a negative control (0). The x column represents unmated mutants from our mutagenized plates, functioning as a positive control.
Complementation Test: Verifying adenine auxotrophy with MV/MV+Adenine Plates
Complementation Test: Verifying adenine auxotrophy with MV/MV+Adenine Plates
In order to verify adenine auxotrophy, the inability to produce Adenine by oneself, we picked colonies from the complementation test plates into Eppendorf tubes with 1 ml of PBS solution. By vortexing and only pipetting 10 uL of solution on the MV/MV+Adenine plates, we mostly limit the concentration of yeast in these plates. Although we had MV plates, we had to create the MV+Adenine plates by pouring Adenine stock solution so that the MV plates could absorb the Adenine.
Results
Results
Within the 10 candidate colonies which were included in the complementation test (the 11th one did not show any red pigment when plated individually on YED), 5 colonies had a confirmed mutation in the Ade2 gene, 2 colonies had a confirmed mutation in the Ade1 gene, and 3 colonies had an inconclusive result (see figure 5). The Ade2 mutants could be identified from a red colony in the unmated column, white diploid colonies in the HB0 and HB1 columns, and red colony in the HB2 column. The HB2 column's diploid colonies had two copies of a non-functional Ade2 gene, while the HB1 column's diploid colonies had one functioning and one non-functioning in both the Ade1 and Ade2 gene. The Ade1 mutants could be identified from a red colony in the unmated column and the HB1 column, and white colonies in the other two. From observing the unmated red colonies, there was no difference in their pigment's hue or intensity between confirmed Ade1 and Ade2 mutants. This is in disagreement with our hypothesis, that the Ade1 mutant would have a lighter pigmentation, due to its later placement in the biosynthesis pathway. Our experimental result is in agreement with other studies, which show that the two intermediate products that are built up as a result of Ade1 and Ade2 mutations are highly similar, and have the same red pigment when oxidized. Although our data appears to demonstrate that Ade2 mutants are more common than Ade1 mutants, the number of samples is much too small to make a determination. Growing each of these diploid colonies from the complementation tests on MV and MV+Ade media yielded results that were mostly as expected in terms of adenine auxotrophy (see figure 6). For example, candidate #1 appeared to be an Ade2 mutant from the complementation test. As expected, it was only viable on MV media after it had been mated with either HB0 or HB1 yeast. All offspring from this colony were viable on MV+Ade media. Unexpected results were still present, such as candidates #2 and #8, whom we believed to be Ade2 mutants from the complementation test. However, they were still viable on MV media after being mated with HB2 yeast. The only possible explanation for this result, given that the unmated "X" group was nonviable, is that those diploid yeast cells had mutated again, and regained their ability to synthesize adenine. Another anomaly was candidate #10, whose unmated colony was non-viable, even on MV+Ade media. This suggests that it is auxotrophic for a different nutrient.
Page updated
Report abuse