General Overview Of Our Experiment

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Our Audio Choices

Frequency	Example Song	How it may help algae growth
Low tones (~60 – 250 Hz)	"Cello Suite No. 1 in G Major" – Bach "Elgar Cello Concerto" – Elgar "Song of the Birds" – Pablo Casals “慢冷” — 梁靜茹	Deep, resonant vibrations can stimulate growth and cell activity.
Mid tones (~250 – 2,000 Hz)	“告白氣球” — 周杰倫 “突然好想你” — 五月天 Canon in D – Pachelbel Clair de Lune – Debussy The Four Seasons: Spring – Vivaldi River Flows in You – Yiruma Gymnopédies No. 1 – Erik Satie All of You — John Legend Photograph — Ed Sheeran	Balanced, rhythmic vibrations may promote stable growth environments.
High Tones (~2,000 – 5,000 Hz)	Comptine d’un autre été – Yann Tiersen Spring Allegro – Vivaldi “小幸運” — 田馥甄	Higher pitches can gently stimulate surface activity but may need moderation.

We chose to use audio as a factor to see if different tones can enhance algae growth. Our song choices are based on how sensitive our algae (Emiliania huxleyi) is. Our song choices have softer tones and larger vibrations to stimulate the algae without causing distubances that can harm them.

Experimental Design: OD vs. Cell Concentration

1. Use E. huxleyi culture with appropriate growth medium.
2. Measure OD with spectrophotometer (start at 750 nm).
3. Count cells using hemocytometer or microfluidic counter.
4. Track data across lag, log, and stationary phases.
5. Repeat across wavelengths (550/600/680/750 nm).
6. Plot OD vs. cell concentration and fit linear regression.
7. Calculate R² to assess fit strength.

Experimental Procedure for Transforming a Plasmid Into E. coli Using Chemical Methods (Calcium Chloride)

Background

How CaCl₂ increases the E. coli’s competence in a heat shock: The positively charged Ca²⁺ can interact with the negatively charged components of the cell wall and the phospholipids of the membranes, reducing the negative charge on the bacterial surface. This lessens the repulsion between the negatively charged DNA and the negatively charged surface, allowing DNA to get closer to the cell.

Source: Asif, Azka, et al. “Revisiting the Mechanisms Involved in Calcium Chloride Induced Bacterial Transformation.” National Institutes of Health, 7 Nov. 2017, https://pmc.ncbi.nlm.nih.gov/articles/PMC5681917. Accessed 11 June 2025.

Materials

• E. coli competent cells (e.g., DH5α or other strains)
• Plasmid DNA to transform into the E. coli
• 0.1 M CaCl₂ sterile solution
• LB Broth:
  • 10 g tryptone
  • 5 g yeast extract
  • 10 g NaCl
  • Per liter of distilled water. Use an autoclave to sterilize the broth.
• LB agar plates with antibiotic:
  • Mix 15 g of agar per liter of LB broth
  • Autoclave to sterilize
  • Cool to 50°C
  • Add the appropriate antibiotic
  • Pour into Petri dishes and solidify before storing at 4°C

Preparation of Competent Cells

1. Select a single colony of E. coli to ensure genetic uniformity and no contamination.
2. Add to 5 mL of LB broth and incubate overnight at 37°C with shaking at 200–250 rpm.
3. Dilute the overnight culture 1:100 in a sterile flask to enter log phase.
4. Incubate at 37°C with shaking until OD₆₀₀ reaches 0.4–0.6.
5. Transfer to conical tubes and place on ice for 10–15 minutes.
6. Centrifuge at 4000 × g for 10 minutes at 4°C to pellet the cells.
7. Decant the LB medium carefully without disturbing the pellet.
8. Resuspend the pellet in ice-cold 0.1 M CaCl₂ (half of original volume).
9. Repeat centrifugation and decanting.
10. Resuspend in 5–10% of original culture volume with 0.1 M CaCl₂ + 15% glycerol.
11. Aliquot into tubes (50–100 µL each) and freeze at -80°C.

Transformation Procedure

1. Thaw competent cells on ice.
2. Label two tubes: “+ Plasmid” and “− Plasmid”.
3. Add 50–100 µL of competent cells to each tube.
4. Add 1–5 µL of plasmid DNA (10–100 ng) to “+ Plasmid” tube; add equal volume of sterile water to “− Plasmid”.
5. Mix gently and incubate on ice for 30 minutes.
6. Heat shock at 42°C for 45–60 seconds.
7. Immediately return to ice for 2 minutes.
8. Add 900 µL of sterile LB broth to each tube (10x dilution).
9. Incubate at 37°C with shaking for 45–60 minutes (recovery period).
10. Plate 100 µL from each tube onto agar plates:
    • “+ Plasmid / + Antibiotic”
    • “− Plasmid / + Antibiotic” (negative control)
11. Invert and incubate plates at 37°C for 12–24 hours.

Expected Results

Condition	Expected Outcome
+ Plasmid / + Antibiotic	Numerous colonies from transformed cells
− Plasmid / + Antibiotic	Ideally, no colonies (control)

Calculation: Transformation Efficiency

Formula:
Transformation Efficiency (CFU/µg) = Number of Colonies Counted / Mass of Plasmid DNA Plated (µg)

Kluyveromyces marxianus vs. Saccharomyces cerevisiae

Overview of Differences

Substance Utilization

Saccharomyces cerevisiae can effectively use C6 sugars (e.g., glucose, fructose, and sucrose). It cannot utilize C5 sugars (e.g., xylose), lactose, and inulin.

Kluyveromyces marxianus can process a wider range of sugars and other substances, such as glucose, lactose, fructose, galactose, xylose, cellobiose, arabinose, lactic acid, and malic acid.

Source: Ha-Tran, Dung Minh, et al. MDPI, 11 Dec. 2020

Temperature Tolerance

Saccharomyces cerevisiae flourishes optimally between 20 and 30°C.

Kluyveromyces marxianus is temperature resistant and can grow even at temperatures up to 52°C.

Growth Rate

Though both grow relatively quickly, Kluyveromyces marxianus has an extremely fast growth rate among eukaryotic microbes.

Differences in Metabolic Characteristics

Saccharomyces cerevisiae is a “crabtree-positive” yeast. It favors fermentation over respiration when sugar concentrations are high, which is a disadvantage when biomass production is the goal.

Kluyveromyces marxianus is largely “crabtree-negative.” It favors respiration over fermentation in the presence of oxygen, leading to more biomass accumulation and less ethanol production.

Source: Guo, Yalin, et al. Oxford Academic, 22 Mar. 2024

Differences in Lipid Production

Oleaginous vs. Non-Oleaginous

Saccharomyces cerevisiae is non-oleaginous, producing lipids mainly for membranes and basic functions, usually comprising under 20% of the dry weight.

Source: Barik, Amita, et al. Oxford Academic, 24 Mar. 2015

Kluyveromyces marxianus is moderately oleaginous and can accumulate more than 20% of lipids by dry weight.

Source: Arkin, Adam P, et al. National Library of Medicine, 25 Sept. 2018

How the DGA1 Gene Affects Lipid Synthesis

DGA1 encodes diacylglycerol acyltransferase (DGAT), which catalyzes:

Diacylglycerol (DAG) + Acyl-CoA → Triacylglycerol (TAG) + CoA

Source: Banas, Antoni, et al. National Library of Medicine, 30 May 2000

Saccharomyces cerevisiae

• Both DGA1 and LRO1 contribute to TAG synthesis.
• Deleting DGA1 reduces triglycerides and alters lipid particle morphology.
• Overexpressing DGA1 increases lipid content.
• Dga1p is localized to lipid particles and the endoplasmic reticulum.

Sources: Fernandez-Moya & Da Silva, 2017, SGD, Kainou et al., 2007

Kluyveromyces marxianus

• Overexpression of DGA1 increases fatty acid and lipid levels.
• More promising for lipid synthesis than S. cerevisiae due to inherent oleaginous nature (compared to 5–7% lipid content in S. cerevisiae).
• Industrial advantages include: higher temperature tolerance, broader substrate utilization, and rapid growth rate.

Source: Arkin, Adam P, et al. National Library of Medicine, 25 Sept. 2018

Algae Culture

• CCMP 371
  • Culture: Sterile artificial seawater with f/2 nutrients + 0.01 μM selenium.
  • Temperature: 20°C; Light: 100 μmol m−2 s−1 constant illumination.
  • Manual agitation twice daily.
• CCMP 379
  • 250 mL flasks, 150 mL modified f/2-si medium.
  • Filtered, autoclaved seawater, salinity adjusted to 30 psu.
  • Light: 40 μmol photons m−2 s−1, 18:6 h light:dark cycle.
  • Temperature: 15°C.
• f/2 Medium Preparation
  • 950 mL filtered seawater + NaNO₃, NaH₂PO₄, Na₂SiO₃, trace metals, vitamins.
  • Top up to 1L with seawater.