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This project will be a blog-style final that goes through the semester with me! So, first we started with introductions (to the course and each other)

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I may have submitted it in the wrong section so who knows if I showed up in another class's presentation :) oops

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The first week of class went by so fast, and I was so ready to attend out first in-person lab! This was my first 'real' lab class after all.

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Then I got COVID and couldn't attend any classes :( My roommates and I just played Stardew Valley for 5 days straight...

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While I was unfortunately absent :/ My classmates were just getting started in the lab! They went over safety protocol, did a micro pipetting activity, and looked at how to analyze and make a graph out of rising rate data. They also got all their lab equipment together such as goggles, lab coats, masks, and gloves!

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During the first class, everyone also began experimenting! What were we experimenting with? Well, since this course was a Course-Based Research Experiment, we were working all together as a big research group! Since it was a CRE, there were a lot of people, but everyone was making an impact! The project was based around sourdough starters and the process of breadmaking.

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Breadmaking requires a lot of specific ingredients and conditions that can be particularly stressful to many microbes. This could include conditions with lots of salt, or high osmolarity. The main question of the experiment was defined by asking how increased salt concentration will impact the microbial community in sourdough starters.

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Then, if we take it one step further we can ask if we can use the genome sequences of the microbes to determine what that microbe contributes to the starter through its metabolism.

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Well, sourdough starters need yeast to rise and make the bread that we love! Then, the bacteria and acids within the starter contribute to the taste. So, for this experiment we will need to first make our sourdough starters. We will have one control sample, and one salt sample. Then, everyone in the class will get a different salt concentration. Mine was .125M of NaCl. Since I was not there, my lab partner did the first lab experiment on his own!

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During the first lab class, we had to label both of our tubes. One was labeled as control and the other was 0.125 NaCl. This ensured that we did not get anything mixed up in the process. We then poured 6g of flour and pipetted 6ml of salt concentration into the tubes. We then mixed this up super well to ensure that our starter was all ready. Then, we drew a line in sharpie to mark where the starter 'started!'

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We also streaked some plates this week in order to grow some microbes for testing! This will allow us to isolate microbes of interest and do some further experiments on them.

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I was finally done with COVID and so thankful that it was an online week :) During week 3, we looked at a whole bunch of notes regarding PCR. This was also when we submitted our first rising rate graph!

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Our first rising rate experiment established a basis on what we could maybe expect. It allowed us to see that sometimes the salt concentrations were preforming better than the controls. Other, higher, salt concentrations were not doing quite as well and did not rise as much. Since I was testing the .125M of salt, I was actually surprised to see that it preformed a bit better than the control at the end. I was not too surprised to see that the 2.0M and 3.0M were not preforming as well since that much salt is probably a huge environmental stressor. We can make predictions on if we think any adaptations will occur throughout our experiment. Will the smaller concentrations keep out-preforming the controls? Will the larger concentrations show any change?

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Course Averages_ Effect of NaCl on Sourdough Starter Rising Rate

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Came to my first in-person lab and had a wonderfully-streaked colony plate! This week we began isolating some interesting colonies for further experimentation.

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We start every lab from now on by making more starters! So, today we labeled our tubes of sourdough, added 6g of flour, and pipetted 6ml of .125M NaCl concentrated water. We mixed it all up and put it under the camera to be recorded over 20 hours. These results will give us our next rising rate graph.

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We took our streak plate from week 2 and began working with it in this lab. We took our tiny strip of 4 PCR tubes and labeled the sides with our initials. I then circled 4 of my favorite colonies and began isolating them. I put each colony on a separate pipette tip and began to streak it on a new plate that was separated into 4 quadrants. Then, I took all of those pipette tips, put them in their own PCR tube and microwaved them to get the colonies warm. After the microwave, I took all the pipette tips out and began working with the tubes.

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I took my four PCR tubes and added 100μL of sterile water, 12.5 μl of ITS-1F primer, and 12.5μl ITS4 primer. I took a p200 (bigger) pipette and began mixing it all up and down. They then went into the thermal cycler! Our cycling conditions were defined as the following:

Initial denature step: 98 C – 3 min

35 cycles of

Denature: 98 C – 10 sec

Anneal: 55 C – 30 sec

Extend: 72 C – 30 sec

Final extension step: 72 C – 5 min

Hold at 4 C

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DNA sequencing determines the order of the chemical building blocks (bases) that make up a DNA molecule. The bases include Adenine, Thymine, Guanine, and Cytosine. There are different method to sequencing and we started with examining sanger sequencing.

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We looked at sanger sequencing which is just a method of determining a DNA sequence. It deals with electrophoresis and chain-termination. The fluorescent chain-terminating ddNTPs glow so that the machine can determine which protein is next by the color of its florescence.

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This graph just observes my concentration of NaCl with the control groups. As we can see, the salt groups were not doing to great in this rising rate graph. They only briefly spiked to out-preform the control and then quickly went back down. This just tells us that they are not really adapting quite yet but that was expected.

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New week, new starter! We repeated the previous steps to make another starter to observe four our next rising rate graph.

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To determine the success of our ITS PCR we preformed last week, we will be doing an electrophoresis on the samples. My partner and I both added 8 20-ul dots of loading dye onto a piece of Parafilm. We then each added 5ul of our samples to a separate dot of loading dye. We plugged in our E-gel and loaded up our samples.

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We then were able to take a picture of our electrophoresis results. I had one very bright line in my first PCR tube! It got about 1/3 the way down the tray and we had to clean it all up.

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In order to sequence the PCR product that we just found in the gel electrophoresis, we need to clean it up. This just means that we are removing any remaining primer, polymerase, and unwanted salts from the buffer. Since we only had one band, we had one clean up.  In the clean up process, we added our PCR product to a DNA Binding Buffer and then put it in a centrifuge to be spun. We then took out the remaining waste, added an elution buffer, and spun it again.

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This time we had 8 PCR tubes to fill with colonies. Four would come from the control and the other four would come from our salt sample group. We then continued with the same streaking procedure as last lab.

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Not much to do on our 7th week online, so all we did was make our rising rate #3! As we can see, our salt concentrations are still not doing great. They are pretty much staying at the bottom and not rising too much while our control groups are rising a lot more! This just means that the salt concentrations are not adapting that much.

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Course Averages_ 0.125M NaCl Treatment (2)

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More starters this week! More flour, more salt concentration, and more mixing.

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We took our samples from last week and began gel electrophoresis with this set as well! In this run of our gel electrophoresis, we had 3 bright bands this time. This means 3 clean ups!

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Went to the mountains over spring break and came back to campus with a new puppy, so here's a little puppy break with Winnie!

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More notes of sequencing methods! This times we looked at High Throughput Sequencing which instead of copying the sequence from one short piece of DNA, the company sequences the entire genome.

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Rising rate 4 looks much different from our previous ones! The salt concentrated groups were beginning to rise at greater rates than before which could symbolize an adaptation to the environment.

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Course Averages_ 0.125M NaCl Treatment (3)

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What was found in the original starter? - Saccromyces Cerevisiae (bakers yeast), Candida Stellata, and Starmarella Stellata (budding yeast)

Which one do I think mine was? - Probably Saccromyces Cerevisiae because of colony characteristics and the DNA sequencing result done by FinchTV. My colonies were very shiny, bumpy and a yellowy-white color which matched the description of a lot of the other colonies found!

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Organisms still found - Saccromyces Cerevisae with no new ones!

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New organism found!!
- Saccromyces Cerevisiae
- Starmerella stellata
- Phaemonellia ascomycese
- Aspergillus sp.

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When looking at our rising rate 1 graph, we can see that the .5 or 1.0M are the highest concentrations that a sourdough starter might be able to handle before being significantly reduced in rising rate. It was interesting to see how at times the lower concentrations like .125 or .5 did as well or better than our control groups. Lasty, the 1 or 1.5M would be best used for adaptation observation since they still have a high stress environment but are still preforming well.

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Since our experiment was about osmotic stress, we wanted to see if the starters would adapt over time to the environments. Did they? Not really! If they did we would have seen the sample groups matching the control groups in their rising rates after the weeks, but we did not. That is ok though because we did observe the .125 and .5 outperforming the control which was very interesting to see! We also have to not that our .5 starter died and we had to begin using .125 instead which means less salt concentration.

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If we were to continue this experiment we could learn from our mistakes in the following weeks. We could isolate one salt concentration and see if we could form some sort of adaptation. We could make sure out .5M concentration does not die so that we have consistent results. Also, we could make sure our plates do not grow mold somehow since some classes had that happen. There is so much room for improvement and so many things we could do in the future to further the experiment on isolating gene sequences in sourdough starters!