High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

1. UV Mutagenesis

1. Prepare a batch of 100 – 200 rectangular agar plates with Tris-Acetate-Phosphate (TAP) medium^9,10. Prepare these plates a few days ahead and keep them on the bench to dry to ensure rapid absorption of the suspended cells in the next steps.
2. Culture Chlamydomonas cells up to 0.2 – 0.5 optical density (OD₇₅₀; ~ 2 days) in 100 ml of liquid TAP under light, at 25 °C and shaking at 100 rpm.
   NOTE: UV mutagenesis is performed independently in two genetic backgrounds: Mat- Hygro^r (confers resistance to Hygromycin B) and Mat+ Paro^r (confers resistance to Paromomycin). Antibiotic choice is arbitrary, provided the two mating types have complementary drug resistances.
3. Check a sample of each of the cultures under the microscope to ensure that the cells are viable, healthy (swimming and intact), and without contamination.
   NOTE: Overgrown cultures "crash" and lose viability, appearing as "ghosts" in phase contrast microscopy. Do not keep shaker cultures going once they reach saturation. The "ghost" phenomenon is easily detectable in step 1.3.
4. Dilute the culture to 0.003 OD₇₅₀. Wrap the bottle with aluminum foil to ensure homogenous density, as the strain is motile and swims directionally in response to light.
   1. Adjust the density of the suspension based on the planned UV dose, so that accounting for cell killing, 200 – 600 survivor colonies will form on the plates (Figure 1). See Table 1 for information on UV exposure times.
5. Attach a small-tube cassette that fits a liquid dispenser and perform a series of washes for sterilization, according to the manufacturer's instructions, to prevent contamination.
6. Using an 8 x 12 liquid dispenser, dispense 4 x 96 drops of 2 µl each onto rectangular plates (Figure 1). Tap mildly at the edge of the plate to ensure the merging of all drops into a thin sheet of liquid and immediately cover the plates to prevent exposure to light.
   1. To ensure very even single cell dispersal, use dry plates, as mentioned above, and quickly cover the plates to prevent the cells from swimming in response to light. Keep the covered plates level and in the dark until all liquid is absorbed.
7. Place the plates under a germicidal UV lamp (30 watt germicidal UV tube) for periods of time determined empirically to give an optimal yield of ts- mutants among the survivors. Here, use times of 0.5 – 1.5 min at a distance of 40 – 50 cm to result in 90 – 99.9% killing. Survivors contain 100 – 1,000 UV-induced point mutations, of which ~ 10% change coding sequences.
   1. In order to ensure maximum potency of UV irradiation with no reversion due to light-dependent DNA repair^11,12, work in the dark at this step and immediately pack the plates in a dark box after irradiation.
8. Keep the plates in the dark for 8 – 24 hr at room temperature.
9. Place the plates in a 21 °C incubator with illumination to form colonies. Colony formation takes ~10 days. Make sure to wrap the plates with plastic bags and add absorbing paper inside to blot up liquid condensation. Evaporation and condensation cycles otherwise can provide liquid film routes for contaminants to enter the plates.
10. Load the plates in the relevant stacks as sources for robotic colony picking (Figure 2). Pick colonies for 384-arrays on rectangular plates, and grow them at 21 °C with illumination (~ 1 week).
11. Condense the 384-arrays into a 1,536-arrays (4:1) using a replica-plating robot (Figure 3), and allow the plates to grow for ~ 3 days in the 21 °C incubator.
12. Replicate the 1,536-arrays to two plates each and place one in the 21 °C incubator (permissive temperature) and the other in a 33 °C incubator (restrictive temperature). Following 24 hr, replicate the plates in 33 °C to a new set of pre-warmed plates, and place them in the 33 °C incubator.
    NOTE: The reason for this secondary plating is that ts lethal mutants can in some cases accumulate substantial biomass, even if the cells are arrested in the first cell cycle after plating. The secondary replica eliminates this signal and greatly increases sensitivity.
13. Photograph the plates with a digital camera following 3 days of growth at 33 °C and 5 days of growth at 21 °C. Hold the plates in a fixed frame. Use a "grid-plate" marked with nine alignment indicators that is photographed together with the culture plates (Figure 4). Photograph culture plates as alternating paired 21 °C and 33 °C (21/33) images.
    NOTE: Different incubation times are used to equalize growth since the wild type grows significantly faster at 33 °C than at 21 °C.

2. Identification of ts Mutant Candidates: First Screen

1. Process the paired 21/33 plate images with a custom Matlab image analysis software to eliminate the background and to segment the images into a 1,536-array. The program will determine the detected biomass (total pixel intensity) in each position (Figure 5).
   NOTE: The software (provided in the S.I. along with instructions) will use the grid-plate image to determine the locations of cells (individual entries) in a 1,536-array at the magnification and plate alignment used and calculate total pixel intensity in each cell. These values for each mutant are then compared against adjustable parameters to determine required growth at 21 °C relative to a ts+ standard and the degree of temperature-sensitivity, defined as: S(Mut₃₃)/S(WT₃₃)/S(Mut₂₁)/S(WT₂₁), where S is signal (pixel intensity after background subtraction), Mut is an individual mutant, and "WT" is a randomly chosen non-temperature-sensitive colony (mutagenized strain that had ts+ phenotype). Growth at 21 °C is defined as S(Mut₂₁)/S(WT₂₁). In this first screen, apply relaxed selection criteria (allowing relatively low growth at 21 °C and a relatively low degree of temperature sensitivity) to keep the false negative rate low.
2. Load the list of selected colonies generated by the software as an instruction file for the single-colony picking robotics (typically into a 384-array). Prepare the source and target plates according to the robotics instructions and pick colonies to array (Figure 6).
   NOTE: Conventional pickers require an instruction file in some format, such as .csv, .txt, or .xls. All pickers will have the capacity to be file-driven; different formats will require minor editing of the MATLAB code (source code provided).
3. Place the target plates in the 21 °C incubator for ~ 5 days to grow a stock plate.

3. Identifying ts Mutants: Second Screen

1. Repeat step 2.1 to retest picked colonies. Typically 30 – 50% of colonies will retest as clear ts lethals, for a yield of 2% – 5% ts lethals relative to initial colonies surviving UV mutagenesis.
2. Repeat step 2.2 for picking the twice-screened ts lethals, but with a modified instruction file designed to array colonies into blocks of 100 colonies on rectangular plates at a 384-density. This is for convenient microscopic examination in the next step. The instruction file format is specified at runtime by the MATLAB code.
3. Make sure to include several WT colonies as a control and place the plates at 21 °C for ~ 5 days.

4. Initial Phenotype Determination

1. Replicate the 100-block plates arrayed in step 3.2 and place them in the 21 °C incubator for ~ 2 days to obtain freshly growing colonies.
2. Replicate the fresh copy of the 100-block plates (4.1) to three copies. Place one copy in the 21 °C incubator and one in the 33 °C incubator as controls.
3. Place the third copy ("screening plates") in a robotic setup, and spot the colonies with long pins touched with sterilized water.
   NOTE: Chlamydomonas cells pinned to agar robotically tend to land initially as a rather dense spot of cells, with few single cells available for microscopic inspection. To optimize the colonies for microscopic inspection, spot the initially transferred cells with a drop of water (the volume of water transferred by the robotic long pins is ~ 100 nl). This results in dispersion of the cells in a small radius (~ 1 mm) about the initial pinning center, with many isolated single cells.
4. Take photomicrographs of a region of each spot of the screening plates at time 0 (while still single, undivided cells) (Figure 7) and place the plates at 33 °C for incubation. Determine image location by the manual stage control of a modified tetrad dissection microscope set to give stops at the 4.5-mm center-to-center spacing of a 384-density array.
5. Remove the screening plates from the 33 °C incubator and take photomicrographs, as in step 4.4, at varying time points (10 hr, 20 hr, 48 hr). Make sure the stage, the plate holder, and the stage controller are precisely calibrated to get images of the same cells in every time point. Perform quick photomicrograph acquisition (~ 10 min).
6. Use the second copy in the 33 °C incubator to verify the ts phenotype and to make sure that temperature fluctuations during image acquisition have no major effect on ts phenotype.
   1. Analyze microscopic images and select for mutants based on the desired criteria (Figure 8). Spot the final selected set in a 96-arrayed agar plate. Make sure that each plate contains mutants of the same mating type and drug resistance.
   2. For each plate, incorporate the last two columns a positive (query mutation) and a negative (WT) control for the complementation assay.

5. Complementation and Linkage Testing of New Mutants to "Frequent Flyers"

1. Transfer large amounts of the arrayed colonies to nitrogen-free gamete-induction medium¹⁰ in 96-well microplates (density of each colony should be around 0.2 O.D.). Incubate the plates under light (13 – 20 W mercury bulbs) for ~ 5 hr to allow gametogenesis.
   NOTE: This step can be conducted either with a replicating robotic setup or manually with a simple plating device.
2. Suspend queries with the opposite mating types harboring the alternative resistance cassettes into tubes with nitrogen-free gamete-induction medium¹⁰ for gametogenesis.
3. Mix the samples in a target plate in a mating mixture volume of 20 µl (Figure 9). Following ~10 min under the light, spot 5 µl from each well twice: once on a TAP plate for linkage testing and once on TAP + 5 µM Paro + 9 µM Hygro for complementation testing.
4. Incubate the linkage plates in the 21 °C incubator overnight, and then wrap them in foil. Keep them in the dark for 5 days to allow zygospore formation.
5. Incubate complementation-testing plates for ~10 days in the light at 21 °C.
   NOTE: The drug amounts are calibrated to allow survival of doubly heterozygous diploids, but they are still enough to eliminate the mating haploids. These amounts were calibrated for standard resistance cassette integrations used in this specific project; with new integrations, doses should be recalibrated. Most biomass is verified to be diploids by flow cytometry (Figure 9), although a variable level of haploids (probably from meiosis and growth of doubly resistant segregants) is usually observed as well.
6. Replicate the complementation-testing plates into two copies for ts- phenotype identification. Place one copy at 21 °C and one copy at 33 °C for ~ 5 days.
7. Test colonies for the ts- phenotype, as described in section 2.1 (Figure 5). Colonies that are not complementing with the query likely represent alleles of the same gene as the query (diploids hetero-allelic for mutations in the same gene).
8. For linkage testing, following step 5.4, shift the plates back to light to allow meiosis and outgrowth of haploid segregants for ~7 days.
9. Replicate linkage testing plates to TAP + 10 µM Paro and 10 µM Hygro to select for double-resistant progenies (predicted to be 25% of the haploid progeny, since the cassettes are unlinked) and incubate at 21 °C for a week.
   NOTE: Increase the drug dosage for doubly resistant haploids.
10. Test colonies for the ts- phenotype, as in step 2.1.
    NOTE: A ts- phenotype is expected (and observed) for mutants in the same complementation group as the query. In addition, a ts- phenotype in the linkage assay, for a mutant that complements the query, can reflect tight linkage between the query and the new mutation. Mutants that complement the tested queries are likely new genes that will enter the pipeline for bioinformatic identification of the causative lesion, and ultimately for further phenotypic analysis.