Growth Curve and Cell Count

Cell count via Hemocytonomer

1. Clean the cover glass mounting support and the coverslip of the hemocytometer with a lens paper and some ethanol.
   Note: Coverslips for counting chambers are specially made and are thicker than those for conventional microscopy, since they must be heavy enough to overcome the surface tension of a drop of liquid.

2. Place the coverslip over the counting surface prior to putting on the cell suspension.

3. Introduce the suspension into one of the V-shaped wells with a pasteur or other type of pipet.
   Note: The area under the coverslip fills by capillary action. Enough liquid should be introduced so that the mirrored surface is just covered.

4. Place the counting chamber on the microscope stage and bring the counting grid is into focus at low power.

Growth Curve

1. With the use of a hemocytometer, count the number of cells present in a small sample of your culture

2. With this number, calculate the cell density (cells/ml) of your culture

3. For the same culture, take cell count measurements on a regular basis (every 5 hours) for an entire week

4. Record all the data you collect and the time at which you took the measurement!

5. Once the week is over, plot cell density versus time in order to obtain a growth curve

6. You must be able to see the different growth phases the species goes through (log, stationary)

Antibiotic Spot Test

Gibson Assembly

1. The DNA you wish to be assembled and the Gibson Master Mix should be combined with a volumetric ratio of 1:3 in a PCR tube.

   1. Note: we need 300 femtomoles of each part for the reaction
   2. The total volume can be from 20-50µl.

2. The PCR tube should then be incubated for 1 hour at 50°C

Gibson Master Mix:
Taq Ligase (40 u/µl) -- 50 µl
5x isothermal buffer -- 100 µl
T5 exonuclease (1u/µl) -- 2 µl
Phusion polymerase – 6.25 µl
Nuclease-free water – 216.75 µl
Total Volume → 375 µl

5x Isothermal buffer: 25% PEG-8000 – 0.75 g
500 mM Tris-HCl pH 7.5 -- 1500 µl
50mM MgCl2 -- 75 µl
50mM DTT -- 150 µl
1 mM dATP – 30 µl
1 mM dTTP -- 30 µl
1 mM dCTP -- 30 µl
1 mM dGTP -- 30 µl
5mM NAD -- 300 µl
Nuclease-free water – remainder
Total Volume → 3000 µl

Transformation and Genomic DNA Extraction

Electroporation

Vulgaris, Kessleri, Saccharophila, Ellipsoidea, UVM1

1. Calculate OD of sample of interest to determine desired total cell count. Densities may range from: 1 x 10^6 cells - 1 x 10^8 cells (Use gamma radiated centrifugation tubes)

2. Calculate OD [750nm] of culture and compare with growth curve to determine cells/mL and determine total volume of culture required to get desired total number of cells.

   1. Note: aim for cells in mid log phase of the growth cycle.

3. Take note of values used in the lab book.

   1. Note: You will require both a -ve and +ve control. Take this into account when preparing cells for centrifugation.

4. Obtain plasmid of interest

   1. i.e. CrGFP, No linker GFP, etc.

5. Ensure that the part is Gibson assembled.

6. Set up restriction digest. All pieces to mixed together in 1.5ml centrifuge tube.

7. Determine volume required to obtain 5 ug of plasmid DNA. [Calculation example: Part Concentration [µg/µL] x unknown volume [µL] = 5ug of plasmid DNA

8. Set up requirements for a 30 µL digest:

   1. 5 µg Plasmid DNA [Volume as per calculation above]

   2. 2 µL SwaI [Last piece to be added to tube]

   3. 3 µL NEBuffer (10X)

   4. 0.5 µL 100X BSA

   5. TBD dH20 [Top up to 30µL with dH20] **30µL Total**

9. Incubate each tube at 25 °C for 1-2 hours. This may require incubation in water bath or other climate controlled apparatus.

   1. Optional: Run gel to confirm cassette is assembled properly. With the except of the ladder [5µL], each well should contain the following:

      1. 1.2 µL DNA [~200ng]

      2. 3.3 µL dye [Following a 5:1 ratio of total reaction: dye]

      3. 15.5 µl dH20

      4. 20 µL Total/well

10. Harvest the cells via centrifugation at 2500 rpm for 10 minutes at room temperature. Discard the supernatant by decanting. Remove the remaining supernatant using a pipette.

11. Re-suspend the cells in ~80 µL of dH20. Pipette up and down multiple times to ensure homogeneity.

12. May want to resuspend in osmotic buffer [Tap] for 1hr and then centrifuge again and use a different electroporation buffer, not water.

13. Add the entire content of the RE digest to the cells + dH20. Pipette up and down once again.

    1. Note: it is highly desirable to have the RE digest complete at the same time as the cells are resuspended in dH20

    2. Note 2: May be advantageous to add 25µg of Salmon sperm DNA at this point to act as carrier DNA. [or 200µg/ml]

14. Transfer the contents of the centrifugation tube to a 0.2cm electroporation cuvette. Place the cuvettes on ice for 5-10 minutes prior to incubation.

15. Use Bio-Rad 2 for electroporation with the following parameters:

    C.Vulgaris, C.Kessleri, C.Saccharophila, C. Ellipsoidea

    • Field Strength: 1800 V/cm
    • Voltage: 360 V
    • Ohm: 200Ω
    • Capacity: 25 µF
    • Number of Pulses: >8

    UVM1

    • Field Strength: 1800 V/cm
    • Voltage: 300V
    • Ohm: ∞
    • Capacity: 50 µF
    • Number of Pulses: 4-5



16. Prior to applying voltage, tap the cuvettes to mix contents

    1. Note: The voltage may be adjusted. Take note of the health/number of cells post electroporation. The species may require a decrease in voltage &/or pluses.

17. Once electroplated, place the cuvettes on ice for an additional 5-10 minutes.

18. Make 3mL aliquots of Tap + 40 mM Sucrose into 6 well plates. Wash each cuvette with ~1mL of distilled water and evenly divide the contents of the cuvette into 2 separate wells.

    1. Note: It is advisable to have these plates ready prior to performing electroporation.

19. Label Clearly!!!

20. Place the 6-well plate in the dark at room temperature and let incubate for 24 hours.

21. Once incubation is complete, gently agitate, ~100-150 rpm, for 5-10 minutes to allow for homogeneity within the culture.

22. Pipette contents of sister wells into gamma radiated centrifugation tubes.

23. Centrifuge at 2500 rpm for 10 minutes at room temperature.

24. Remove supernatant via decanting.

25. Re-suspend pellets in 200 µL of distilled water.

26. Plate 100 µL x2 onto appropriate plates.

    1. positive control = Tap

    2. negative control = Tap + 50µg/ml hygromycin

    3. Experimental = Tap + 50µg/ml hygromycin

    **Note: Ensure that the plates are free from condensation**

27. Place plates agar side down in incubator.

    1. Note: Photoperiod may be adjusted once heterotrophic growth ends.

28. Check for successful transformations in 8-10 days.

29. Restreak transformed colonies on sister plates and allow to grow for 3 days.

30. Inoculate in 3mL Tap + appropriate [Hyg]. Let stand.

31. Extract Chlorella DNA.

Cryofreezing Cells

Materials Needed:

1. 2 Spill-proof stryofoam box

2. Metal Rack

3. white gloves

4. Isopropanol

5. Methanol

6. Liquid Nitrogen

7. 1.8 mL crytubes

8. Cryocontainer

Liquid Nitrogen Storage & Transportation:

• Disinfect the bench by spraying ethanol
• Prepare the foam box + metal rack to hold the tubes for N2 freezing
• Wear white gloves + rubber to do N2 freeze & retrieve tubes
• Open & leave N2 out to evaporate (end of the day, somewhere where it won’t spill)

Conditions:

1. 2 sets of culture: Log & Stationary

§ (Note: stationary phase→ higher lipid profile, cryoprotectant cells

2. Vary methanol percentages: 6% methanol (3% final conc.) worked best*

3. Species/Strains: UVM1, Kessleri, Ellipsoidea, Vulgaris, Saccharophila

PROTOCOL:

1. Grow cells to approximately 1,000,000 cells per ml in TAP (strain 2137) or TAP plus Arg (CC-425). Pellet cells and resuspend in 1/10 volume fresh growth media.

2. To a 1.8 ml Nunc style cryotube add 250 µl of appropriate growth media containing 2 - 10% (v/v) methanol or DSMO. We find 6% methanol (3% final concentration) works best for strain 2137 and CC-425 (see below).

3. Add 250 µl(equal volume) of 10X cells to the tube. This gives a final cryoprotectant concentration range of between 1-5% (v/v).

4. Place the tubes in a Nalgene Cryo 1C Freezing Container (esssentially an isopropanol bath and place the Cryo-container in a minus 80 C freezer. The cryo-container slowly freezes the cells at about 0.9-1.0 C/min. Leave Cryo-container in freezer until isopropanol reaches - 40 C; about 65-72 min.

5. Remove tubes and immediately freeze in liquid nitrogen. Store at liquid nitrogen temperatures.

6. Thaw cells by placing in a 35 C water bath for two minutes with gentle shaking.

7. Transfer cells to 10 ml of appropriate growth medium and grow 6-18 hours before plating.

Results

On the left, one of the earliest cassettes built can be seen showing an intense band, and to its immediate right there is a faint, but still present band of one of the later cassettes using PSAD gene’s promoter/terminator flanking a GFP with a nuclear localization signal. To the far right, there is a GFP control which has all the same reagents (including primers) of the other reactions, differing only in the template (pCrGFP) - the template we used to amplify our GFP to build our cassettes.