Insertional Mutagenesis of Chlamydomonas reinhardtii

Materials and Reagents

1. 15 and 50 ml conical tubes
   
2. Petri dishes (90 mm in diameter)
   
3. pHYG3 plasmid (http://chlamycollection.org/plasmid/phyg3/) or plasmid carrying other selection markers
   
4. C. reinhardtii cell wall-less strain such as dw15 (cw15, nit1, mt⁺; http://chlamycollection.org/strain/cc-4619-cw15-nit1-mt-dw15-1/)
   
5. Agar (Caisson Laboratories, Phytoblend)
   
6. TAP medium (Harris, 1989)
   
7. Restriction enzyme for linearizing the plasmid DNA, such as PvuII (New England Biolabs)
   
8. Hygromycin (Life Technologies) or other selection means
   
9. TAP agar plates (see Recipes)
   
10. Top agar (see Recipes)
    

Equipment

1. Common bench-top vortexer
   
2. Spectrophotometer for optical density (OD) measurement, Z2 Coulter Counter (Beckman Coulter) or a hemocytometer
   
3. 250 ml flask
   
4. Bench-top centrifuge capable of centrifugation at 1,500 x g and accommodating 50 ml conical tubes
   
5. Shaker
   
6. Z2 Coulter Counter or hemocytometer
   
7. Glass beads 425-600 μm (Sigma-Aldrich, catalog number: G8772 )
   
8. Glass tubes or round bottom plastic tubes with or without cap (~10 mm in diameter)
   

Procedure

1. DNA preparation (assumes use of pHYG3)
   
   1. Digest pHYG3 plasmid (http://www.biologie.uni-regensburg.de/Genetik/Mages/pHyg3.html) DNA with PvuII. PvuII was chosen specifically for pHYG3 plasmid because it releases a DNA fragment that contains the hygromycin B resistance gene aph7 with short flanking sequences. Based on our experience and others, linear DNAs are often shortened on each end after entering the cells of C. reinhardtii-ranging from dozens to hundreds of nucleotides-due to the exonuclease activity. It makes it harder to map the disrupted loci because the precise sequence of the insertion is unpredictable and different in each clone. Hence, we recommend using a DNA fragment that is just enough to cover the selectable marker (i.e. promoter, coding sequence, and terminator), so if the transformed DNAs are shortened to an extent that compromises the integrity of the marker, the clones harboring them will not be pulled out of the screen. In other words, the clones in the mutant library have the insertion whose length lies between the PvuII-digested fragment and the combined length of promoter, coding sequence, and terminator.
      
   2. Gel-excise the shorter, 2,012-bp fragments of the pHYG3 plasmid.
   
   
2. Growth condition
   In general, cells were grown in liquid TAP medium under continuous light (70-80 μmol/m^-2 s^-1) at 22 °C, with shaking at 100 rpm; or ambient room temperature (~22 °C) for solid media.
   
   
3. Glass bead transformation
   
   1. Grow cultures in TAP media (50 ml in 250 ml flask) until cell density reaches 1-2 x 10⁶ cells/ml determined by Z2 Coulter Counter or hemocytometer, or approximately 0.2~0.3 OD₅₅₀ by spectrophotometer.
      
   2. While cells are growing, prepare hardware: add ~0.3 g (~300 µl) glass beads to test tubes, autoclave.
      
   3. Pellet the cells by centrifugation (1,500 x g for 2 min), and resuspend in TAP to a concentration of ~2 x 10⁷ cells/ml.
      
   4. Transfer 0.3 ml of cells to the glass bead-containing test tube, add ~1 µg linearized plasmid DNA prepared earlier (see Procedure A DNA preparation), and vortex 15 sec at top speed (Note 2).
      
   5. Add 6 ml TAP to the test tube, carefully transfer the cells (6 ml + 0.3 ml) to a new sterile 15 ml conical tube, and shake under growth conditions overnight (Note 3) at 100 rpm.
      
   6. After overnight recovery, add 6 ml of top agar (microwave to melt the top agar, cool to ~40 °C before pouring into the overnight culture) into the overnight culture (1:1 mixture). Mix gently.
      
   7. Immediately pour the 1:1 mixture onto TAP plates (6 ml for one regular Petri dish, 2 Petri dishes needed), let set (Note 4).
      
   8. Wrap plates with parafilm (cut slits to allow gas exchange), and place under lights. Colonies should start appearing in 5 to 7 days on the plate surface or embedded within the top agar layer. Generally speaking, 50 ml of culture (step C1) produces approximately 500 colonies after transformation. To create a mutant library ranging the entire C. reinhardtii genome (about 18,000 genes), it would require ~100 transformations in order to get a 3 fold coverage. Use sterile toothpicks or pipet tips to pick up the colonies.

Notes

1. Transformation by glass beads is only applicable to cell wall-less strains (e.g. dw15, cw15) (Kindle, 1990). For the transformation of cell-wall strains, electroporation is more commonly used (https://www.thermofisher.com/order/catalog/product/A14258).
   
2. Do not vortex the cells too vigorously otherwise DNA fragments tend to break, especially for longer fragments.
   
3. If cell duplication during the overnight shaking (Procedure C, step C5) is a concern (e.g. to avoid duplicated clones for mutant screen), do 8-12 h shaking instead.
   
4. Optional: Pre-warm the agar plates at 37 °C before pouring the 1:1 mixture (Procedure C, step C7).
   

Recipes

1. TAP agar plates
   0.8% agar with 10 μg/ml hygromycin
   
2. Top agar
   TAP medium with 0.4% agar
   

Acknowledgments

This work was supported by the US Air Force Office of Scientific Research [Grant FA9550-11-1-0264 (to C. B.)], by a Strategic Partnership grant from the MSU Foundation (to C. B.), and by MSU AgBioResearch (C. B.).

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